202
Cells were inoculated at 5xl05 cells/ml into 200 ml medium contained in a spinner flask
which was agitated at a selected speed for 5 h. The viable cell concentration was determined
at intervals during this period. (A) The change in viable cell concentration for cultures
agitated at 470 rpm. (B) The half-life ofthe viable cell population at various agitation speeds.
Values are means ±SEM for n=3
Conclusions
Supplementation of a serum-free culture of a hybridoma with the unsaturated fatty acids,
oleic and linoleic causes :-
- a substantial growth enhancement (300%)
- an initial enhancement of Mab yield (60%)
- a significant reduction in glutamine uptake (60%)
- an increased tolerance to culture agitation
For maximal productivity of the hybridomas, the fatty acid composition of the cell requires
to be finely balanced between a lipid-starved and lipid-loaded state. As there appears to
be no regulatory mechanism for fatty acid uptake, the lipid-balanced state of the cells in
culture must be maintained by a controlled feeding regime
References
1. Butler, M. and Huzel, N. (1995) J. Biotechnol. 39, 165-173
2. Butler,M., Huzel,N. and Barnabé,N. (1997) Biochem J. 322, 615-623.
3. Karsten, S., Schafer, G. and Schauder, P. (1994) J. Cell Physiol. 161, 15-22
4. Schmid, G., Zilg, H., Eberhard,U. and Johaiinsen, R. (1991) J. Biotechnol. 17, 155-167
5. Rockwell, G.A., Sato, G.H. and McClure, D.B. (1980) J. Cell Physiol. 103, 323-331
6. Rintoul, D.A, Sklar, L.A. and Simoni, R.D. (1978) J. Biol. Chem. 253, 7447-7452
7. Calder, P.C., Yaqoob, P., Harvey, D.J, Watts, A. andNewsholme, E.A. (1994) Biochem. J. 300, 509-518
8. Butler, M. and Huzel, N. (1997) ESACT 14, 657-662.
Discussion 203
Grammatikos:
Butler: How do you add the fatty acids to the medium?
Grammatikos: We attached the fatty acids to de-lipidated BSA.
When you incorporated linoleic acid into cells you showed a much
Butler: lower arachidonic acid level. It is known, theoretically, that
linoleic acid can lead to increases in arachidonic acid. Can you
Jordan: speculate on your result - is it just not processed to arachidonic
Butler: acid?
The figures may have been misleading. I quoted moles percent to
show that the proportion of linoleic acid goes up dramatically. The
amount of arachidonic remains about the same.
Do you have air bubbles in the bioreactor at 470 rpm?
We were not sparging; it was a spinner flask, so we had a vortex
and head space.
LIPID REQUIREMENTS OF A RECOMBINANT CHINESE HAMSTER
OVARY CELL LINE (CHO)
JEFFREY T. MCGREW, CHERYL L. RICHARDS, PAULINE SMIDT,
BRADLEY DELL, AND VIRGINIA PRICE
Immunex Corporation, Department of Cell Sciences,
51 University Street, Seattle, WA 98101 USA
1. Abstract
We have tested the effect of a wide variety of lipid supplements and precursors on the
growth of a recombinant Chinese Hamster Ovary (CHO) cell line. This cell line is adapted
to grow in protein-free media and grows slowly in the absence of any exogenous lipid
source. Several exogenous lipid sources stimulated robust growth including Intralipids.
Intralipid (Kabi Pharmacia) is a filterable lipid emulsion consisting of 10% soybean oil,
1.2% egg yolk phospholipids, and 2.25% glycerin in water. Soybean oil consists of a
mixture of neutral triglycerides. In order to determine if the growth promoting activity
could be due to one of these components, purified triglycerides and phospholipids were
tested alone and in combination. We found that the growth promoting activity required
both triglycerides and phospholipids. We also found that the phospholipid growth factor
lysophosphatidic acid stimulated CHO cell growth.
2. Introduction
Growth of mammalian cells in culture typically requires the addition of exogenous lipids.
The requirement for lipids can be met with serum, partially purified serum fractions, lipid
rich albumin, or protein-free lipid emulsions. Exogenous lipids become incorporated in the
cell membranes and play a nutritional role for cell growth [1]. Lipids are also involved in
signaling intracellularly and extracellularly [2]. Previous studies demonstrated that addition
of exogenous lipids to Chinese Hamster Ovary (CHO) cells improved growth and
production of recombinant proteins [3,4]. For manufacturing purposes lipid supplements
should be chemically defined, filterable, and easy to prepare [5]. In addition, a lipid
supplement would preferably not be derived from serum thus avoiding adventitious
contaminants. The objective of this study is to identify commercially available lipid
supplements that support robust growth of CHO cells and are suitable for manufacturing.
3. Results and Discussion
3.1 GROWTH OF CHO CELLS IN RESPONSE LIPIDS
Table 1 shows the results of a growth stimulation assay of several lipids and lipid
precursors. Consistent with previous results, both Ex-cyte and cholesterol rich lipids
stimulated growth of CHO cells [4]. Intralipid, a protein-free lipid emulsion also supported
robust growth of CHO cells. Figure 1 shows the stimulation of cell growth in response to
increasing concentrations of Intralipid. Intralipid is an emulsion of 10% soybean oil,
1.2% egg yolk phospholipids.
205
O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 205-207.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
206
Since Intralipid supported cell growth, we sought to determine if either soybean oil or
phosphatidyl choline, the primary component of egg yolk phospholipids, would stimulate
CHO cell growth independently. Soybean oil or phosphatidyl choline on their own
supported little, if any, cell growth (data not shown). An emulsion was prepared
consisting of soybean oil, phosphatidyl choline, and F68. This emulsion did stimulate
some cell growth, but not as much as Intralipid (data not shown).
207
Intralipid is a potentially useful lipid supplement for manufacturing. It is chemically well
defined and relatively inexpensive. The mean particle size of Intralipid is 0.37 microns
which is larger that the pore size of a 0.2µ filter [6]. Since emulsion particles should be
somewhat flexible, we pressure filtered Intralipid prior to adding it to media and found that
Intralipid easily penetrated a 0.2µ filter. If, however, Intralipid was added to media prior
to filtration, we found that the filter would clog suggesting that the particle size of the
emulsion increases in the presence of media. Filtering Intralipid prior to adding it to media
did not influence performance of CHO cells in shake flask or bioreactor cultures (data not
shown).
3.2 LYSOPHOSPHATIDIC ACID STIMULATES GROWTH OF CHO CELLS
Lysophosphatidic acid (LPA) is a simple phospholipid that stimulates growth of many cell
types when added to cells extracellulary [2]. LPA bound with albumin is a component of
serum, and many of the growth factor effects of serum can be accounted for by LPA [2].
Since some serum-free media does not contain any polypeptide growth factors, LPA in or
produced from lipid supplements could potentially account for some of the growth
stimulatory effects of exogenous lipids. LPA was added in increasing concentration to
CHO cells in the presence of fatty acid free-BSA as a lipid carrier (Figure 1B). LPA
stimulated modest growth of CHO cells with a saturating concentration of about 20 µg/ml.
In Figure 1B, a saturating concentration of Intralipid supported indicating
the lysophosphatidic acid was not as potent as Intralipid. This suggests that some of the
effects of exogenously added lipids may be due to growth factor like properties rather than
a nutritional properties. LPA also stimulated growth of cells in the absence of BSA, but
less than that in the presence (data not shown).
4. References
1. Schimd, G. (1991) Lipid metabolism of animal cells in culture-A review, in Production of
Biological from Animal Cells in Culture: Research, Development and Achievements. R. E.
Spier et al., (Eds.) Butterworth Oxford, pp61-65
2. Moolenaar, W.H.: Lysophosphatidic Acid, a Multifunctional Phospholipid Messenger, J.
Biol. Chem. 270 (1995), 12949-12952
3. Darfler, F. J. : Preparation and use of lipid microemulsions as nutrition supplements for
culturing mammalian cells, In Vitro Cell Dev. Biol. 26 (1990), 779-783
4. Jenkins, N., Castro, P., Menon, S., Ison, A., and Bull, A.: Effect of lipid supplements on the
production and glycosylation of recombinant interferon-g expressed in CHO cells,
Cytotechnology 15 (1994), 209-215
5. Seamans, T.C., Gould, S.L., Distefano, D.J., Silberklang, M., and Robinson, D.K.: Use of
Lipid Emulsions as Nutritional Supplements in Mammalian Cell Culture, Ann. N.Y. Acad.
Sci. 245 (1994) 240-243
6. Mehta, R.C., Head L.F., Hazrati, A.M., Parr, M., Rapp,R.P., DeLuca,P.P.:Fat emulsion
particle -size distribution in total nutrient admixtures, Am. J. Hosp. Pharm. 49 (1992) 2749-
2755
SUSTAINED EXPRESSION IN PROLIFERATION CONTROLED
BHK-21 CELLS
Keywords: BHK-21, IRF-1, proliferation control, growth regulation, expressions
PETER P. MÜLLER, SABINE KIRCHHOFF
AND HANSJÖRG HAUSER
GBF - National Center for Biotechnological Research,
Mascheroder Weg 1, 38124 Braunschweig, FRG
Tel.: ++49 (0)531 6181 250
Fax.: ++49 (0)531 6181 262
E.mail: [email protected]
Abstract
We have established proliferation controled BHK cell lines employing IRF-1 (Interferon
Regulatory Factor 1), a transcriptional activator that inhibits cell growth. The activity of
IRF-1 has been made hormone dependent by fusing IRF-1 to the hormone-binding
domain of the estrogen receptor. The growth rate of cells expressing the IRF-l-estrogen
receptor fusion protein can be reduced by adding estradiol to the medium. Productivity of
constitutively expressed proteins decreases when growth is downregulated. Expression
during both, unrestricted growth and the growth controled phase is obtained by using two
separate transcription units. Long-term stability of the regulation has been achieved by
coupling the expression of IRF-1 with a selection marker on a dicistronic mRNA
construct. The results demonstrate that stable proliferation control and sustained
expression can be achieved in a biotechnologically relevant cell line.
Introduction
Cell culture processes are initiated from a small number of cells that are expanded
manyfold to achieve high densitiy. In an ideal continuous culture process initial cell
growth is rapid, followed by a production phase in which proliferation is downregulated.
After reaching an optimal high cell density proliferation is no longer required, only the
production is of interest. Proliferation of most cell lines is lower at a high densities,
however, not sufficiently low for continuous culture. In a proliferation inhibited state,
protein synthesis and metabolic activity of the cell could be devoted largely to the
production of the desired recombinant protein. Advantages of a proliferation inhibited
production phase would be
- lower release of intracellular hydrolizing enzymes, leading to a higher uniformity and
stability of the product,
- higher consistency of the product by keeping the cells in an identical state of
proliferation during the whole production period,
- genetic drift is reduced due to the reduced proliferation, resulting in a higher stability of
the production
- reduced media consumption as a consequence of lower metabolic turnover.
Understanding molecular processes that control cell proliferation and progress in the
technology of genetic manipulation of mammalian cells (Hauser, 1997) has allowed us to
209
O.-W. Merten et al. (eds), New Developements and New Applications in Animal Cell Technology, 209-214.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
210
influence the proliferation of cell lines which are of biotechnological interest.
IRF-1 overexpression inhibits the proliferation of a number of cell lines (Kirchhoff et al.,
1993, 1995, 1996). To demonstrate the feasability of growth control in a
biotechnologically relevant cell line, IRF-l-estrogen receptor fusion protein encoding
gene has been transfected into a producer BHK cell line. Upon estrogen addition to the
growth medium, cell proliferation is reduced to various degrees depending on the estrogen
concentration. After three days in the presence of low estrogen concentrations (30nM)
inhibition is about five- to tenfold. After five days of IRF-1 activation the cells are still
viable as determined by trypan blue staining (Kirchhoff, unpublished). Cell morphology
as examined by microscopic observation shows membrane blebbing as well as the
accumulation of granula within the cytoplasm in most of the cells. Whereas native
BHK-21 cell nuclei are not uniformely sized and often show a typical kidney form, IRF-1
activity leads to a rounded nuclear morphology. Indications for apoptosis were not
detected (Kirchhoff, unpublished).
IRF-1 does not lead to a cell cycle specific arrest. Instead, there is a slow-down of all cell
cycle phases (Kirchhoff et al., in preparation). The slight accumulation of the cells in Gl
phase cannot account for the drastic inhibition of cell proliferation. The most likely cause
is interferon secretion induced by IRF-1 imposes the weak Gl arrest. A proportional
extension of other cell cycle phases could explain the reduced proliferation.
IRF-1 activation leads to reduced gene expression, depending on the promoter used.
Productivity of a recombinant immunoglobulin decreases after three days of growth arrest
(Kirchhoff et al 1996). Since IRF-1 is a DNA sequence specific transcriptional activator,
transcription from promoters containing IRF-1 binding sites is enhanced in IRF-1
proliferation controled cells. Both constitutive expression during unrestricted growth and
during the controled growth phase are obtained by employing two separate expression
constructs, one with a constitutive promoter and a second one with an IRF-1 inducible
promoter. Since growth control imposes a strong selection pressure, stability of the
regulatory control is essential. Long-term stability of this regulatory system has been
obtained by tightly coupling expression of the IRF-1 fusion gene to a selection marker on
a dicistronic construct.
Material and Methods
DNA constructs
Constructions were carried out by standard procedures (Sambrook et al., 1989). The
IRF-1 fusion with the human estrogen receptor fragment has been described (Kirchhoff
et al., 1993). An EcoRI-NheI restriction fragment from the plasmid pMC-2P carrying
an IRES element from Polio Virus and the pac gene as a selection marker has been
inserted downstream of the IRF-1-hER gene to create a dicistronic expression cassette
(Dirks et al., 1994). The dicistronic transcript is driven by the constitutive MPSV
promoter. Luciferase is constitutively expressed from the SV40 promoter or IRF-1
inducible expressed from a from a synthetic promoter that contains IRF-1 binding sites.
Cell culture and gene transfer
BHK-21 (ATCC CCL10) cells maintained in Dulbecco’s modified Eagle’s medium
(DMEM) with 10% fetal calf serum were stably transfected by calcium phosphate
coprecipitation method. 5 µg plasmid, 5 µg of high molecular weight DNA and 0,5 µg
of selection plasmid pSV2pac (Vara et al., 1986) or pAG60 (Colbère-Garapin et al.
1980) per cells was used. Cells from a stable cell clone were transfected with the
expression plasmid encoding the fusion protein IRF-1-hER and single cell clones were
isolated and expanded. Clones were cultured in the presence of 5 µ g/ml puromycin and
800 µ g/ml G418 (Gibco). Luciferase activity was measured using the Luclite kit
(Packard) and normalized against LDH activity. Other procedures were performed as
described earlier (Kirchhoff et al., 1996).
211
Results
1. Establishment of stable proliferation control in BHK cells
No obvious phenotype on cell growth was found in cells constitutively expressing 1RF-
1 fusion protein in the absence of estrogen. Nevertheless, long-term cultivation of such
cell clones leads to a loss of estrogen dependent growth regulation. Possibly subtle
growth advantages of cells that have lost the 1RF-1 fusion gene expression lead to
overgrowth by these non-expressing cells. To stabilize the growth regulatory system
we coupled the expression of the IRF-1 fusion gene to the expression of a puromycin
resistance marker gene on a dicistronic construct (Fig. 1). The regulation has been
stable in all experiments and no reversion of the growth controled phenotype has been
observed. This selection pressure has been required during cloning and manipulation of
cells, but could be relieved during actual production periods.
2. Recombinant gene expression in proliferation controled cells
Recombinant genes are commonly expressed from strong constitutive promoters. Cell
growth and expression from the highly active MPSV promoter determined 3, 4 and 5
days after inducing growth restriction showed that productivity is not significantly
altered during this period but decreases from 40 % to 60 % during the fourth day and to
about 20 % at the fifth day (Kirchhoff et al. 1996). The concentrations of estrogen used
do not significantly influence the productivity from these constructs whereas cell
growth is further reduced at higher estrogen concentrations.
To improve expression in growth controled cells we have made use of the fact that
IRF-1 activates transcription of promoters with IRF-1 inducible elements (Kirchhoff et
al., 1993, Tamura et al., 1995). Although basal expression is low, induced expression
levels are in the same range as from strong viral promoters. That these promoters are
inducible by estrogen in BHK-21 cells that express the IRF-1 fusion protein shows that
it functions at the same time to reduce the growth rate and activate transcription of
IRF-1 dependent promoters. Both, constitutive expression and sustained expression
during growth restriction is achieved when cells contain two different expression
constructs, one driven by the constitutively active SV40 promoter and a second
construct with an IRF-1 inducible promoter (Fig. 2).
212
Discussion
We demonstrate the feasability of IRF-1 mediated proliferation control by i) controling
cell growth in the biotechnologically relevant BHK-21 cell line using an estrogen
activatable IRF-1 fusion protein ii) stabilizing the growth regulatory system by coupling
the expression of the IRF-1 fusion protein to the expression of a selectable marker gene
on a dicistronic mRNA, and iii) expression of a recombinant protein could be sustained
during both unrestricted growth and the growth control phase by expression from two
separate constructs that are driven by a constitutively active promoter and and IRF-1
inducible promoter, respectively. Carvalhal et al. (this volume) show that high viability
in 1RF proliferation controled cultures can be maintained. The combination of genetic
manipulation with cultivation techniques moves this regulatory system from an
academic interest oriented research project towards an applicable biotechnology project.
Acknowledgements
We thank H. Conradt, L. Kongerslev, K. Scharfenberg, R. Wagner, D. Wirth and M.
Wirth for suppport and discussions. The work was supported financially by an EC-grant
BIO4-CT95-0291.
213
References
Colbère-Garapin, F., Horodniceau, F., Khourilsky, P. and Garapin, A.C. (1981). A new dominant selective
marker for higher eukaryotic cells J. Mol. Biol. 150, 1-13.
Dirks, W., Schaper, F. Kirchhoff, S., Morelle, C, and Hauser, H.. (1994). A multifunctional vector family for
gene expression in mammalian cells. Gene 149, 387-388
Hauser, H., (1997) in Mammalian cell biotechnology in protein production (Hauser, H. and Wagner, R., eds.).
Walter de Gruiter, Berlin, 3-27.
Kirchhoff. S., Schaper, F., and Hauser, H. (1993). Interferon regulatory factor 1 (IRF-1) mediates cell growth
inhibition by transactivation of downstream target genes. Nucleic Acids Res. 21: 2881-2889.
Kirchhoff. S., Koromilas, A., Schaper. F., Grashoff, M., Sonenberg, N. and Hauser, H. (1995). IRF-1 induced
cell growth inhibition and interferon induction requires the activity of the protein kinase PKR., Oncogene 1 1 ,
439-445
Kirchhoff. S., Kröger, A., Cruz. H., Tümmler, M., Schaper, F. and Hauser, H. (1996). Regulation of cell
growth by IRF-1 in BHK-21 cells. Cytotechnology 22, 147-156.
Sambrook, J., Frisch, E. F., and Maniatis, T. (1989). Molecular Cloning: A laboratory manual. Cold Spring
Harbor Press (New York)
Tamura, T., Ishihara, M., Lamphier, M S., Tanaka, N., Oishi, I , Aizawa, S., Matsuyama, T., Mak, T W.,
Taki, S. and Taniguchi, T. (1995). An IRF-1 dependent pathway of DNA-damage-induced apoptosis in
mitogen-cativated T lymphocytes. Nature 376, 596-599.
Vara J, Portela A, Ortin J and Jimenez A (1986). Expression in mammalian cells of a gene from Streptomyces
alboniger conferring puromycin resistance. Nucleic Acids. Res. 14: 4617-4624.
214 When your cell cycle growth slows down and you look at viability,
Discussion do you see any induction of apoptosis?
Massie:
Müller: We have tried to find signs but there is no indication that the cells
died of apoptosis. The cells accumulate a lot of intracellular
Massie: vesicles and grow larger. After prolonged exposure to estradiol
Müller: they burst.
Al-Rubeai: Do you think that this is a side-effect of estradiol, or regulation of
Müller: other genes by your transcription factors?
Konstantinov:
I think that it is an effect of over-activation of IRF-1 because cells
Müiler: which lose expression of IRF-1 fusion protein do not show any
effect to the action of estradiol.
Kostantinov:
Müller: Have you looked at these vesicles?
We do not know what is in them.
I think that your system is flexible but complex. What is its
advantage compared with a simple decrease of temperature in the
fermenter?
Can tell me how decreasing the temperature can adjust the growth
of stromophilia to the growth of haemopoetic stem cells? You
need to control the growth specifically of one particular cell line.
You cannot just change the overall conditions by taking away one
limiting medium component, or by changing the temperature.
Some groups have successfully shown modulation of BHK cell
metabolism by adjusting the temperature.
You can do that within a certain range.
CELL GROWTH INHIBITION BY THE IRF-1 SYSTEM
A.V. CARVALHAL1 ; J.L. MOREIRA1; P. MÜLLER2, H. HAUSER2;
M.J.T. CARRONDO1,3
1 - IBET/ITQB, Apartado 12, 2780 Oeiras, Portugal
2 - GBF, Mascheroder Weg 1, 38124 Braunschweig, Germany
3 - Lab. Eng. Bioq., FCT/UNL, 2825 Monte da Caparica, Portugal
1. Abstract
A genetic approach, based on the growth regulatory protein interferon-regulated-factor-
1 (IRF-1), activated by the addition of estradiol, has been applied to regulate BHK cell
growth. With the addition of 100 nM of estradiol 48 hours after inoculation, growth
inhibition occurs within 24 hours but is followed by a significant decrease in cell
viability, whereas rec-protein specific productivity is not significantly altered. Viability
decrease is not due to estradiol effect and is independent upon the culture system.
In order to define strategies to extend the stationary growth phase, several parameters
have been studied: estradiol concentration, time post inoculation for estradiol addition
and time span of estradiol exposure. When the time of contact between the cells and
estradiol is reduced the cell viability increases, achieving similar values of the control,
without the estradiol, and leading to a stationary growth phase extension.
2. Materials and Methods
Cell line and medium: BHK clones were obtained from Dr H. Hauser (GBF,
Braunschwig, Germany) and grown in DMEM supplemented with 10% (v/v) FBS,
4.5g/l of glucose, 5 mg/1 of puromycin and 5 mg/1 of vitamin Kl.
Culture system: The cells were grown in T-flasks and inoculated with a seeding
density of . Cell concentration and viability were measured by the
trypan blue dye exclusion method.
Estradiol was directly added to the culture medium using a 100 fold stock solution and,
unless indicated, the final concentration was 100 nM. Estradiol removal from the
culture medium (whenever indicated) was performed by replacing the total supernatant
by fresh medium.
215
O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 215-217.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
216
3. Results and Discussion
3.1. IRF-1 OVEREXPRESSION INDUCTION EFFECT ON:
3.1.1. Cell growth
Estradiol addition to the culture medium 48
hours after inoculation leads to an effective
growth inhibition (Figure 1A) but this is
followed by a significant decrease in cell
viability (Figure 1B) (up to 50% five days after
estradiol addition). This decrease is faster than
the normal pattern of a static batch culture, as
can be seen by the control test, where no
estradiol was added.
The results presented in Figure 1 are
independent on the clone under test and the
culture method (static or stirred cultures).
3.1.2. Rec-protein productivity
Rec-protein productivity is not significantly
affected by the estradiol addition (specific
productivity of 130 ± 20 I.U.).
3.2. PARAMETERS AFFECTING CELL GROWTH INHIBITION
In order to eliminate the cell viability decrease 48 hours after estradiol addition, the
effect of several parameters on cell growth inhibition were evaluated.
3.2.1 Estradiol concentration
There is a clear difference in cell growth pattern between 10 nM and 100 nM: cell
growth inhibition is more efficient when 100 nM is used. However, in both cases a
similar sharp decrease in cell viability is observed, similar to Figure 1B and independent
upon estradiol concentration.
3.2.2. Time post inoculation for estradiol addition
100 nM estradiol was added at two different times, 48 or 96 hours after inoculation; at
the end of the exponential phase (96 hours), a similar pattern to the control was
observed (the cell viability pattern did not decrease differently from the control).
217
3.2.3. Time of contact between the cells
and the estradiol
In the previous reported experiments there is no
estradiol removal after its addition. However, as
presented in Figure 2B, cell viability increases
when the time of contact between the cells and
estradiol is reduced to 72 hours.
The recovery of viability is due to the removal of
estradiol and not to the exchange to fresh medium
(data not shown).
Similar results were obtained with the producer
clone (data not shown).
If the media is changed again 3 days after estradiol
removal by fresh estradiol-free medium, the cells
start to grow again (Figure 2A), clearly indicating
that the cell growth inhibition process can be
reversed.
4. Conclusions
• The induction of IRF-1 overexpression leads to a clear growth inhibition by the
addition of estradiol (100 nM) at the beginning of the exponential growth phase, but is
followed ..bydn:eocrseiagsneifiincacenltl
viability (faster than in the control);
change in rec-protein specific productivity.
• The significant decrease in cell viability can be avoided when the time of contact
between the cells and estradiol is reduced.
• IRF-1 control proliferation system can be reversed by the removal of the estradiol
from the medium.
References
Kirchhoff S , Schaper F; Hauser H (1993), Nuclei Acids Res 21:2881-2889.
Kirchhoff S; Kroger A; Cruz H; Tümmler M; Schaper F; Köster M; Hauser H (1996), Cytotechnology 22:147-
156.
Acknowledgements
The authors acknowledge and appreciate the financial support received from the European Commission
(B1O4-CT95-0291) and from Junta Nacional de Investigação Cientifica-Portugal (BIC 1788). The authors are
grateful to Ms. Maria do Rosário Clemente for technical support.
CORRELATION BETWEEN BHK CELL SPECIFIC PRODUCTIVITY AND
METABOLISM
H. J. CRUZ1, J. L. MOREIRA1, E. M. DIAS1, C. M, PEIXOTO1,
A. S. FERREIRA1 & M. J. T. CARRONDO1,2
1 - IBET/ITQB, Ap. 12, 2780 Oeiras, Portugal
2 - Lab. Eng. Bioq., FCT/UNL, 2825 Monte da Caparica, Portugal
1. Abstract
A rBHK21 clone producing an antibody/cytokine fusion protein was used to study the
dependence of cell metabolism on the glucose and glutamine levels in the culture
medium. Results indicate that glucose and glutamine consumptions and lactate and
ammonia productions show hyperbolic behaviors. The estimated values for were
1.39 ± 0.11 mM for lactate production as a function of glucose and 0.59 ± 0.17 and 0.15
± 0.04 mM for ammonia production as a function of glutamine, at glucose
concentrations of 0.05 and 1.0 respectively. At very low glucose concentrations the
glucose to lactate yield decreased markedly, showing a metabolic shift towards lower
glucose fermentation. At very low glutamine concentrations, the glutamine to ammonia
yield increases, showing a more efficient glutamine metabolism. The cell specific
productivity can be increased significantly by manipulating nutrient concentrations.
2. Introduction
The metabolism of transformed cells shows new properties like high glycolysis and
glutaminolysis rates, glucose and glutamine being the main nutrients utilized by
mammalian cells [1]. Mammalian cells grown in culture excrete lactic acid and
ammonia in quantities that may inhibit cell growth and thus decrease final product
liters. High glucose and glutamine concentrations lead to overflow metabolism while
limiting concentrations provoke the use of more efficient energy pathways and reduce
formation of inhibitory by products [2].
3. Materials and Methods
Cell lines and cell growth systems
rBHK 21A cells were obtained from Dr. Hansjörg Hauser (GBF, Braunschweig,
Germany). Cells were grown in DMEM with 5% PCS. Studies were performed in
static flasks containing 10 ml of medium and as inoculum.
Metabolite analysis
Glucose- Sigma # G20, glutamine- Sigma # GLN-2, Lactate- Boehringer Mannheim #
139084, Ammonia- Boehringer Mannheim # 1112732.
219
O. - W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 219-221.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
220
4. Results and Discussion
4.1. GLUCOSE METABOLISM
The results obtained from the glucose uptake as a function of glucose concentration
show a hyperbolic behavior, with value of 1.64 ± 0.17 mM (similar to the reported 2
mM in chick embryo fibroblast cultivation [3]) and
(the range of values obtained includes those found in the literature [4]: 12 nmol
at 20 mM of glucose).
For the lactate production a similar behavior to that found for glucose was obtained,
with lactate production being very low under low glucose concentrations
The values obtained for are higher
than those found in the literature [4] but are consistent with the
ones obtained in the glucose uptake assay.
The glucose to lactate yield decreases dramatically at very low glucose levels. This
clearly indicates a metabolic shift from a high lactate to a low lactate producing
metabolism- more efficient pathways are involved. This shift is observed for glucose
below 1.64 mM or which is comparable with results obtained for hybridomas
[5].
4.2. GLUTAM1NE METABOLISM
The glutamine uptake has a hyperbolic behavior with and
mM for glucose 0.05 and respectively. These values are lower than those found
in the literature that range from 2 mM in normal rat kidney cells to 4.5 mM in extracts
of Ehrlich ascite cells [6]. The values obtained for were
for glucose and respectively. These values
are comparable to those in the literature: 3.0 to 4.9 nmol cells at 3 mM
glutamine [4] and 5.7 nmol cells for MDCK cells in medium with fructose
instead of glucose [7]. The glutamine consumption being higher at the lower glucose
concentration shows the interactive and complementary nature of both these main
nutrients for animal cells.
The ammonia production showed a similar behavior to that found for glutamine uptake,
with and at glucose 0.05 and respectively.
Reported results showed that ammonia production of MDCK cells can be reduced by
maintaining the glutamine concentration at less than 1.0 mM [7]. The values obtained
for were and at glucose 0.05 and 1.0
respectively.
The glutamine to ammonia yield increases at very low glutamine, meaning that a more
efficient glutamine metabolism is occurring [2]; although the yields are higher, the
overall ammonia production will be lower at low glutamine concentrations.
221
4.3. PRODUCTIVITY
A significant variation (approximately 2 fold) in the cell specific productivity is
observed with the glucose concentration.
With glutamine the same behavior is observed at glucose but at glucose
the trend is to increase productivity with decreasing glutamine.
5. Conclusions
Significant reductions in the glucose consumption were observed for low glucose
concentrations; the same occurred with lactate production. Lactate showed a significant
decrease in yield from glucose at very low glucose concentrations, showing that a shift
towards a more efficient glucose metabolism occurred.
Significant reductions in the glutamine consumption were observed for low glutamine
concentrations; the same occurred with ammonia production. Ammonia showed an
increase in yield from glutamine at very low glutamine concentrations which shows
higher efficiency in glutamine metabolism. Glutamine consumption and ammonia
production increased significantly with the decrease in glucose concentration.
The cell specific productivity can be significantly increased by manipulating nutrient
concentrations.
Acknowledgements
The authors acknowledge the financial support received from the European Commission (B1OTECH
PL933069) and Junta Nacional de Investigação Cientifica e Tecnológica- Portugal (PBICT/BIO/20333/95 and
PRAXIS XXI/BD/2764/94) and thank Dr. Sadettin Ozturk for the revision and discussion of this work.
References
1. McKeehan WL (1982) Mini-review: glycolysis, glutaminolysis and cell proliferation. Cell Biol Int Rep 6:
635-649.
2. Ljunggren J & Häggström L (1994) Catabolic control of hybridoma cells by glucose and glutamine fed
batch cultures, Biotechnol Bioeng 44: 808-818,
3. Fagon JB & Racker E (1978) Determinants of glycolytic rate in normal transformed chick embryo
fibroblasts. Cancer Res 38: 749-758
4. Neermann J, Wagner R (1996) Comparative analysis of glucose and glutamine metabolism in
transformed mammalian cell lines, insect and primary liver cells. J Cell Physiol 166: 152-169,
5. Zhou W, Rehm J & Hu WS (1995) High viable cell concentration fed-batch cultures of hybridoma cells
through on-line nutrient feeding. Biotechnol Bioeng 46: 579-587.
6. Xie L & Wang DIC (1994) Fed-batch cultivation of animal cells using different medium design concepts
and feeding strategies. Biotechnol Bioeng 43: 1175-1189.
7. Glacken MW, Fleischaker RJ & Sinskey AJ (1986) Reduction of waste product via nutrient control:
Possible strategies for maximizing product and cell yields on serum in cultures of mammalian cells.
Biotechnol Bioeng 28: 1376-1389.
EFFECT OF GROWTH ARREST IN BHK METABOLISM: ON LINE
MONITORING BY AND NMR SPECTROSCOPY
P.M. ALVES 1, A.V. CARVALHAL1, J.L. MOREIRA1, H. SANTOS1,
M.J.T. CARRONDO1,2
1Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia
Quimica e Biológica, Apt. 12,2780 Oeiras, Portugal.
2Faculdade de Ciências e Tecnologia/Universidade Nova de Lisboa, 2825
Monte da Caparica, Portugal.
1. Introduction
Controlling proliferation of cell growth has been successfully applied recently [1].
Nevertheless, the physiological and metabolic behaviour of the cells after growth arrest
is not well understood.
Nuclear magnetic resonance (NMR) techniques are potentially very attractive to
explore cell metabolism due to their unique non-invasive characteristics. With a suitable
perfusing system it is possible to monitor biochemical processes in real time under
physiological conditions and various metabolic manipulations can be simulated. The
low-sensitivity of the NMR method requires high cell densities in the detection zone;
basement membrane gel (BMG) threads are a good strategy for cell immobilization
because their matrix can support a high cell number and allow protection from shear
damage [2]. The aims of this work were: on line monitoring of cells energetic status by
NMR spectroscopy and on line monitoring of glucose uptake and lactate production
by NMR spectroscopy, after estradiol addition to a BHK cell line growth regulated
by the IRF-1 (interferon-regulated-factor-1) system.
2. Materials and Methods
2.1 Cell line and growth systems
BHK-21 cells genetically modified to depend on estradiol addition to regulate growth
were obtained from Dr Hansjörg Hauser (GBF, Braunschweig, Germany). Cells were
immobilized in BMG threads as described before [2]. An inocullum of cells was
used for 1.5 ml of BMG. After immobilization, the threads were placed in 14 cm culture
dishes containing 50 ml of Dulbecco’s modified Eagle’s medium (DMEM) with lg/1
glucose and supplemented with 10% fetal bovine serum (FBS). Cells were mantained for
2 days in a humidified atmosphere of 10% CO2/90% air at 37 °C.
2.2 On line NMR
For NMR measurements the immobilized cells were transferred to a 10 mm tube fitted
with a perfusion apparatus [2]. Cells were perfused at 1.5 ml/min with DMEM
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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
224
containing glucose (1 g/1) and estradiol (100 nM). The media was oxygenated with
and mantained at 37 °C.During the experiment, the medium was changed
or supplemented with glucose in order to avoid nutrient limitations and inhibition of cell
metabolism by waste products. NMR spectra and NMR spectra were obtained
using a 10 mm broad band probehead on a Brucker DMX500 NMR spectrometer.
3. Results and Discussion
3.1. Effect of Growth Inhibition on Cell Energetic Status
Growth inhibition was successfully achieved after estradiol addition as no increase on
NTP levels was observed during the following 100 hours (figure la).
During the first 24 hours after estradiol addition, NTP drops steadily. After that
period a slight decrease was observed and NTP levels almost reach a plateau (figure 1b).
This behaviour is consistent with the induction of growth arrest as an adaptation of cells
energetic state to the new metabolic conditions is expected. As can be seen in the spectra
(figure la), GPC/NTP, PC/NTP and PE/NTP ratios were affected with the time-course of
the experiment, which indicates that phospholipid content of the cells is changing. The
effect of estradiol on membrane fluidity is well reported [3] and further studies are
necessary to confirm the integrity of cell membrane after long periods in contact with
estradiol.
3.2. Effect of Growth Inhibition on Glucose and Lactate Metabolism
Using labelled substrates, as glucose, it is possible to monitor the distribution of
the label within various metabolites during the time-course of an in vivo NMR
experiment. As can be seen in the spectra (Figure 2), labelling from glucose was
225
incorporated mainly in lactate. Following time trajectories for labelling from
glucose into lactate it is possible to compare glucose uptake and lactate
production rates along time after estradiol addition.
For the first 12 hours, a higher rate for glucose consumption and lactate production
was observed as compared to the following 80 hours and to the rates obtained before
growth inhibition (data not shown). This can be due to the fact that cells are adapting
their metabolism immediately after growth arrest. This is in agreement with the decrease
in the NTP levels observed in the spectra. An interesting result is that the glucose
uptake rate slightly increases again after the re-feed done at time 60h, with no significant
increase in lactate production rate. Further experiments have to been performed in order
to evaluate the importance of this behaviour.
4. Conclusions and Perspectives
• BHK cells can be immobilized in gel threads (BMG) and perfused inside a NMR tube
for long periods of time (up to 100 hours) without significant losses in cell viability.
• Estradiol addition is efficient to induce growth arrest (NTP does not increase with
time) and no significant changes in glucose metabolism were observed. Differences in
phospholipid content ofthe cells were detected after growth inhibition.
• Work using different labelling -glucose and other labelled precursors is in progress
in order to evaluate metabolic fluxes in BHK before and after growth arrest. Further
studies are necessary to elucidate the effect of estradiol on phospholipid metabolism.
References:
1 Kirchhoff, S., Kröger, A., Cruz, H., Tümmler, M., Schaper, F., Köster, M. and Hauser, H. (1996)
Regulation ofcell crowth by IRF-1 in BHK-21 cells, Cytotechnology 22,147-156.
2 Alves, P.M., Flögel, U., Brand, A., Leibfritz, D., Carrondo, M.J.T., Santos, H. and U. Sonnewald (1996)
Immobilization of primary astrocytes and neurons for on-Line monitoring of biochemical processes by
NMR, Dev. Neurosci. 18, 478-483.
3 Schwarz, S.M. (1988) Estrogen modulates ileal basolateral membrane lipid dynamics and Na+-K+-
ATPase activity. Am. J. Physiol. 254(5), G687-G694
Ackowledgments: The authors acknowledge the financial support received from European Comission BIO4-
CT95-0291, Fundação para a Ciência e Tecnologia PBICT/BIO/20333/95 and PRAXIS XXI/BD/2721/94.
CELL SURVIVAL IN CHO CELL CULTURES GROWN IN A DEFINED PROTEIN FREE MEDIUM
ABSTRACT
Animal cells, primarily hybridomas, important for diagnostic and pharmaceutical production have been shown
to die almost exclusively via apoptosis when entering the stationary phase. Recently similar reports have been
presented for CHO (Chinese Hamster Ovary) cells grown in batch cultures under optimal conditions for high
product yield 1. Experiments performed in small scale using a defined protein free medium, with the sole
addition of recombinant insulin, enabled us to investigate the importance of physiological parameters and their
effect on cell survival. We show here that a CHO cell line engineered to produce a recombinant therapeutic
protein is capable of inducing the apoptotic programme under batch culture conditions in a 2 L reactor, as
measured by the TUNEL assay (DNA end-labeling). Cells are arrested in the Gl phase of the cell cycle though
insulin is present in excess and no apparent depletion of nutrients is at hand. Proliferation ceases and viability
decreases although the concentration of inhibiting metabolites is low.
INTRODUCTION
Animal cells in culture are constantly exposed to environmental changes. There are several factors determining
the health status of the cells in a bioreactor: the availability of growth and survival factors, the availability of
nutrients, the accumulation of both toxic and beneficial metabolites, variations in pH and DO levels and the
physical shear forces encountered by the cells 2. If exposed to sudden toxic insult animal cells die by necrosis,
morphologically characterised by swelling and the flocculation of nuclear chromatin, with subsequent cell lysis.
Cells which experience mild environmental perturbations in culture conditions respond by undergoing an active,
energy dependent programmed cell death called apoptosis. Apoptosis is characterised morphologically by cell
shrinkage, condensation and fragmentation of the nucleus and biochemically by cleavage of DNA into
nucleosomal fragments as a result of induction of transglutaminases, proteases and nucleases 3. A number of
agents are reported to induce apoptosis in vivo and in vitro. Hormone and growth factor deprivation and loss of
matrix attachment triggers apoptosis in different cell systems. Damage related inducers such as heat shock, viral
infections, activation of oncogenes, or the tumour suppressor as well as exposure of cells to nutrient
deprivation, free radicals and high concentrations of antimetabolites results in cell death4 .
METHODS
Batch growth studies were performed both in spinners and in a controlled 2 L stirred tank bioreactor (Belach
bioteknink AB, Sweden). pH and dissolved oxygen (DO) were monitored and controlled at optimal levels. pH
was controlled on line through additions of on demand and oxygenation by bubble free
aeration using air and pure oxygen. The reactor culture was characterised in respect to consumption of nutrients
(glucose and amino acids), the growth factor (insulin RIA) and formation of metabolites (lactate and ammonia).
Glucose and lactate was determined with a YSI 2700 analyser; amino acids with Waters PicoTag method and
ammonia using an Orion electrode. Temperature was maintained at 37° C. Cells were grown in a proprietary
defined serum free medium supplemented with 10 mg/L recombinant insulin, (nucellin-Zn, Eli Lilly Inc.).
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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
228
Cell count and viability were assesed using a particle counter (CASY, Schärfe system, Germany) and by
Erythrosin B dye exclusion with a Bürker chamber. Cell cycle distribution analysis were performed using the
method of Vindeløv. Apoptosis was measured by several independent methods: cells were harvested twice daily
and fixed according to the protocol for the TUNEL reaction, (Boehringer Mannheim), and analysed with an
Epics Elite flow cytometer. Different modified protocols for DNA fragmentation were employed according to
”Techniques in Apoptosis”, T. G Cotter and S. J. Martin, 1996. Additional experiments were performed in 100
ml stirred spinner cultures where the effect of decreased concentrations and consecutive supplementations of the
growth factor were investigated, as well as different inoculum densities during batch growth.
RESULTS
The proliferation of CHO cells in batch culture stops after 100 h of culture at a cell density of approximately
2,5X106 cells/ml (Fig. 1). Practically all cells are viable throughout the growth phase. The cessation of growth is
immediately followed by rapid cell death which obviously occurs almost exclusively by apoptosis as measured
by TUNEL whereas no DNA fragmentation could be detected in the viability ranges examined. At this time (100
h) neither the level of glucose were limiting nor were the accumulated levels of lactate and ammonium inhibiting
(Fig. 2). The consumption and accumulation patterns of the amino acids show that only a few of them were used
to any great extent (Fig. 3). The consumption of aspartate and asparagine was most significant and it seems
likely that the cells are using these amino acids for their energy metabolism. Almost no uptake of the dipeptide
glycyl-glutamine (gly-gln) occured and the concentration in the medium did not change until the viability
dropped. At this point both glutamine and glycine were accumulated in the culture medium which indicates an
extracellular cleavage of the dipeptide probably due to the release of proteases from the dying cells. The uptake
of serine may also have contributed to the accumulation of glycine. Alanine was the sole additional amino acid
which accumulated extracellularly. The concentrations of the remaining amino acids, (ser, glu, met, cys, val, leu,
ile, pro, lys, arg, thr, his, tyr, phe and trp) were not affected significantly during the growth phase.
Analysis of insulin concentrations in the medium showed that nucellin-Zn levels were high during almost the
entire culture period and dropped to lower levels only during the last day, i.e. long time after cell death was
initiated (Fig 1).
Cell cycle distribution analysis from cells grown in spinner cultures (Fig. 4) revealed that the cells arrest in G1
phase of the cell cycle long before the viability is negatively affected and while the insulin still is plentiful.
Insulin concentrations below 2,5 mg/L resulted in a reduced growth rate and decreased viability whereas total
omission of the growth factor caused a rapid cell death. To investigate whether the insulin present was bioactive,
additional nucellin-Zn (10 mg/L) was added intermittently to the spinner cultures at 4 time points after the
commencement of the cultures. No improvement of the survival, nor any augmentation of the proliferation could
be observed in this test indicating that a sufficient amount of nucellin-Zn is present (Fig. 5). Using different
inoculum densities with the medium containing 10 mg/L nucellin-Zn, we could obtain an increasing amount of
maximal total cell concentration, which would indicate an autocrine growth factor effect.
References
1 ”Apoptosis in CHO cell cultures, examination by flow cytometry”, A Moore et al, Cytotechnology 17: 1-17, 1995.
2 ”Cell death (apoptosis) in cell culture sytems”. T G. Colter and M Al-Rubcai. TIBTECH vol 3 pp 150-155, April 1995
3 ”The genetic regulation of apoptosis”, A. H. Wyllie, Current Opinion in Genetics and Development, 5: 97-104, 1905.
4 ”Apoptosis in the pathogcnesis and treatment of disease”, C. B. Thompson, Science vol 267, pp 1456-1462, March 1995.
229
ENHANCED APOPTOSIS IN INSECT CELLS CULTIVATED IN SIMULATED
MICROGRAVITY
N. COWGER and K. O’CONNOR
Tulane University, Chemical Engineering Department, and
Molecular & Cellular Biology Program, New Orleans, LA 70118
Abstract
There is a shift from necrosis to apoptosis when batch cultures of Spodoptera frugiperda
are grown in the low turbulence environment of NASA’s High-Aspect Rotating-Wall
Vessel (HARV) versus a shaker flask. The NASA vessel was developed for the
cultivation of animal cells in an environment which simulates microgravity on earth.
Fluorescence microscopy with the DNA stains acridine orange and ethidium bromide
showed 33% of S. frugiperda cells were apoptotic in the HARV when the viability was
50%, while cells grown in shaker flask had no more than 11% apoptotic cells at the same
viability. We believe that the shift to apoptosis in the HARV is primarily the result of
reduced hydrodynamic forces.
Background
The HARV bioreactor used in our research was developed by NASA in 1990 for the
cultivation of animal cells in an environment which simulates the effects of microgravity
here on earth. As described in our earlier publications [1,2], the combination of end-
over-end mixing, bubble-free aeration and solid-body rotation greatly reduces the
magnitude of hydrodynamic forces within the HARV relative to conventional
bioreactors. Since its introduction, the HARV has mostly been applied as a tool for
tissue engineering studies [1]. In addition, our laboratory chose to study the usefulness
of the HARV for insect-cell suspension culture. Our work with insect cells was the first
application of simulated microgravity to a non-mammalian and a freely suspended cell
line. Insect cells were chosen primarily because their reported sensitivity to
hydrodynamic forces made them amenable to the quiescent environment of the HARV.
Our previous studies [2] have revealed profound differences in the growth profile and
metabolism between S. frugiperda cells grown in the HARV compared to those in shaker
flask. The shaker was selected as a control vessel since it is one of the most gentle forms
of conventional cultivation. Our studies showed less nutrients consumed and less wastes
produced in HARV insect-cell cultures. The metabolic differences are evidence for the
use of alternate metabolic pathways in the HARV culture. In the quiescent HARV, the
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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
232
energy required for cell repairs may be redirected to other metabolic processes. Earlier
studies [3] also showed changes in morphology and DNA fragmentation indicative of
increased apoptosis in the HARV culture. It is only recently that apoptosis has been
documented [4] during routine subculturing of S. frugiperda cells with no viral infection
present.
Methods
The cultivation conditions for IE1FB2 cells, a subclone of Sf9 S. frugiperda, were as
presented previously [3]. In the fluorescence microscopic protocol [5], cell samples were
stained with acridine orange and ethidium bromide and viewed on an Olympus BX60
microscope with UPlanFl objective at 40x magnification.
Results and Discussion
Figure 1A shows representative growth curves for S. frugiperda cells. Though maximum
cell densities were virtually identical in the two vessels, stationary phase in the HARV
was much extended and the death rate constant in the HARV was a factor of 20 to 90
times less than for the control culture [3]. The increased longevity of insect cells in the
HARV is not an artifact from experimental conditions, such as feeding interval and cell
density, and most likely results from the HARV’s combination of reduced shear and
lower accumulation of waste.
Fluorescence microscopy was performed to quantify the subpopulations appearing in the
two cultures during death phase. The protocol used relies on the differential uptake of
the DNA-binding dyes acridine orange and ethidium bromide. Cells are classified as
viable, early apoptotic, late apoptotic, or necrotic based on their staining characteristics
and morphology. From Figures 1B and 1C, it can be seen that apoptosis is dominant for
the HARV culture, while necrosis is the controlling mechanism for shaker cells. When
the viability is 50%, up to 33% of total cells are apoptotic in the HARV. In comparison,
cells grown in shaker flask had no more than 11% apoptotic cells at the same viability.
Thus, necrotic cells at this stage represented approximately 17% of the population in the
HARV and 40% in the shaker.
233
In a previous publication [6], we discussed some of the factors which may influence cell
death. One pertinent example is that the intensity of a mechanical stress may affect the
relative occurrence of apoptosis and necrosis, as shown in a study [7] where animal cells
were subjected to different agitation rates in a stirred-tank bioreactor. In our studies,
since there was less turbulence in the HARV than in the shaker flask, a higher percentage
of apoptosis relative to necrosis may have been favored in the microgravity vessel. Other
cultivation conditions have been shown to enhance apoptosis, for example, glutamine,
serum, or glucose deprivation, and ammonia toxicity. These do not appear relevant to
the HARV culture, particularly since it is characterized by reduced glucose utilization
and reduced ammonia accumulation relative to the control culture.
Conclusion
Knowledge of death mechanisms in insect-cell cultures could potentially impact
commercial processes by offering a means of control over the culture to improve
productivity. During long-term cultivation of Spodoptera frugiperda insect cells in
simulated microgravity, there is an apparent shift to apoptosis relative to a shaker control.
Our work with HARV cultivation has further shown significant effects on the
metabolism, morphology, and growth profiles of insect cells. We believe, first, that these
phenomena were primarily the result of reduced hydrodynamic forces in the HARV, and
second, that these results would not have been readily achieved with conventional means.
Acknowledgments
This work was funded by a grant from NASA (NAG 9-826). We are also grateful to Dr.
Carol Burdsal and Dr. Kyriakos Popadopoulos for the use of their microscopes.
References
1. Clejan. S., O’Connor, K.C., Cowger, N.L., Cheles. M.K.. Haque, S., and Primavera, A. (1996) Effects of
simulated microgravity on DU 145 human prostate carcinoma cells. Biotechnol. Bioeng 50. 587-597
2. Francis, K.M., O’Connor. K.C., and Spaulding, G.F. (1997) Cultivation of fall armyworm ovary cells in
simulated microgravity. In Vitro Cell Dev. Biol. 33, 332-336.
3. Cowger, N.I., O’Connor. K.C., and Bivins. J.E. (1997) Influence of simulated microgravity on the
longevity of insect-cell culture. Enzyme Microb. Technol. 20, 326-332.
4 Palli, S.R., Sohi. S.S., Cook, B.J., Primavera. M., and Retnakaran. A. (1997) Screening of 12 continuous
cell lines for apoptosis, in K. Maramorosch and J. Mitsuhashi (eds.), Invertebrate Cell Culture: Novel
Directions and Biotechnology Applications, Science Publishers, Inc., Enfield, New Hampshire, pp. 53-
60.
5. Duke. R.C. and Cohen. J.J. (1992) Quantitation of apoptotic index and cell viability using fluorescent
dyes, in J.E. Coligan, A.M. Kruisbeek, D.H. Margulies. E.M. Shevach, and W. Strober (eds.), Current
Protocols in Immunology, Greene Publishing Associates and Wiley-lnterscience, New York. pp. 3.17.1-
3.17.3.
6. Cowger, N.L. and O’Connor. K.C. (1997) Application of simulated microgravity to insect-cell culture, in
K. Maramorosch and J. Mitsuhashi (eds.), Invertebrate Cell Culture: Novel Directions and
Biotechnology Applications, Science Publishers, Inc., Enfield, New Hampshire, pp. 131-138.
7. Al-Rubeai, M., Singh. R.P., Goldman. M.H., and Emery, A.N. (1995) Death mechanisms of animal cells
in conditions of intensive agitation. Biotechnol Bioeng. 45, 463-472.
MODULATION OF APOPTOSIS BY BCL-2 EXPRESSION FOLLOWING
AMINO ACID DEPRIVATION AND IN HIGH CELL DENSITY PERFUSION
CULTURES
R. P. Singh*, D. Fassnacht**, A. Perani*, N. H. Simpson*, C. Goldenzon*,
R. Pörtner** and M. Al-Rubeai*
*School of Chemical Engineering, University of Birmingham, Edgbaston,
Birmingham, B15 2TT, U.K. and **Technical University of Hamburg-
Harburg, Bioprozess und Bioverfahrenstechnik, Dnickestr. 15, D-21071,
Hamburg, Germany.
Key words: Apoptosis, Hybridoma, Bcl-2, Amino Acids, Perfusion Culture
1. Introduction
A number of studies have now demonstrated that cell death during commercial cell
cultures proceeds by the active, genetically determined mechanism called apoptosis,
rather than the passive accidental form of cell death known as necrosis (Al-Rubeai et
al., 1990; Franek and Dolnekova, 1991; Mercille and Massie, 1994; Singh et al., 1994;
Perreault et al., 1994). Suppression of apoptosis significantly improves the robustness
of the cells, thus raising culture productivity. However, there are still major gaps in our
knowledge in terms of the role of specific components of the culture environment in
terms of the induction and suppression of apoptosis.
Studies conducted thus far have demonstrated that the deprivation of glutamine,
threonine and cysteine results in the induction of high levels of apoptosis (Mercille and
Massie, 1994; Perreault and Lemieux, 1994; Singh et al., 1994). However, it is not
clear whether this is a feature specific to these particular nutrients, or an effect which
is seen following the deprivation of any of the amino acids found in culture medium. In
order to answer this question, we have identified the mechanism of cell death following
deprivation, individually, of all amino acids. We then went on to investigate the effect
of over-expression of the anti-apoptosis gene bcl-2 under these conditions. Finally, the
impact of bcl-2 overexpression on antibody productivity in the extremely stressful
environment of high cell density perfusion culture systems was studied.
2. Materials and Methods
2.1 CELL LINES AND CELL MAINTENANCE
The cell lines used in this study were TB/C3.bcl-2 and TB/C3.pEF derived from the
murine hybridoma TB/C3 which was transfected with the expression vector pEF bcl-2-
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236
MC1neopA and the control vector pEF-MC1neopA respectively (see Simpson et al.,
1997). Cells were maintained at 37°C in RPMI 1640 medium (Gibco, UK.)
supplemented with 5% (v/v) Foetal Calf Serum (FCS) (Sigma, UK.). Bcl-2 expression
levels were monitored by immunostaining of the Bcl-2 protein followed by flow
cytometric analysis as previously described ( Simpson et al., 1997). The medium used
for the fixed bed culture was a 1:1 mixture of Iscove's MDM and Ham's F12
supplemented with 2 mmol 1-1 L-glutamine, 2 g 1-1 NaHCO3, 0.01% PEG and 50 mmol
1-1 ethanolamine supplemented with 1% (v/w) of a protein-free iron-rich supplement
(IR) containing 20 mmol 1-1 ferric citrate.
2.2 AMINO ACID DEPRIVATION: EXPERIMENTAL PROCEDURE
48 Hours Deprivation. Cells taken from the mid exponential phase of batch cultures
were centrifuged for 5 minutes at 1000 rpm. They were washed once in phosphate
buffered saline (PBS) and then resuspended in the Select-Amine basal medium with
5% dialysed FCS containing all amino acids except the one under investigation at a
target viable cell number of 2xl05 /ml in a 15 ml volume. The cells were then
dispensed in 5 ml aliquots into 3x5 ml plastic wells on 12 well plates, giving triplicate
cultures for each experiment. The Plates were incubated for 48 hours at 37°C in a
humidified 5% CO2/air incubator. Prior to analysis, the cells were trypsinized using a
trypsin EDTA solution (Sigma UK), centrifuged at 1000 rpm and resuspended in the
original 5 ml of culture medium (to ensure that any floating dead or viable cells in the
original culture medium were included in the analysis). Levels of apoptosis, necrosis
and viability were assessed by fluorescence microscopy as previously described in
Simpson et al., 1997.
Time Course Study Following Deprivation of Individual Essential Amino Acids. Cells
were prepared as described above at a target cell number of 2xl05 /ml in 90 ml of
selectamine medium (containing all amino acids except for the one under study). The
cells were dispensed in 5 ml aliquots into 18x5 ml wells. At regular intervals, 2 wells
were “sacrificed” and cells removed by trypsinization and assessed for viable cell
number, viability and levels of apoptosis and necrosis as described above. This
allowed for the analysis of the cultures at 9 time points during the course of the culture.
2.3 PERFUSION CULTURES
Fixed-bed Reactor. The fixed bed reactor (Meredos, Germany) used in this study
consists of a 100 ml fixed-bed in which the cells are immobilised on porous carriers
and a 1 litre conditioning vessel. During operation, the medium was constantly
pumped from the conditioning vessel through the fixed-bed and back. Bubble aeration
took place in the conditioning vessel to supply the cells with oxygen. The maximum
superficial flow velocity in the fixed-bed was kept below 1.1 mm s-1 in order to prevent
cells from being washed out of the fixed-bed. The pH was measured and controlled
237
automatically by adding CO2 to the air. The medium in the conditioning vessel was
agitated using magnetic stirrer and the temperature maintained at 37°C.
Porous glass bead carrier with a diameter of 3 to 5 mm (Siran, Schott, Germany) were
used. The fixed bed was inoculated with 1xl06 cells (ml fixed-bed)-1 which corresponds
to a cell density of 1xl05 cells (ml suspension)-1. Monoclonal antibody titre was
measured by an IgG ELISA as described in Simpson et al., 1997.
Hollow Fibre System (Tecnomouse). Hollow fibre studies were carried out using a
Tecnomouse bioreactor (Integra Biosciences, UK) with two OxyCell hollow fibre
cassettes, one for each cell line. Temperature was controlled at 37°C and the cultures
were aerated with 5%CO2/air. Medium was recirculated through the cassette at a flow
rate of l00ml/hr from a 2 L reservoir bottle. Samples were harvested from the extra-
capillary space at weekly intervals and assayed for levels of mouse IgG using a
sandwich enzyme-linked immunosorbent test as described previously (Simpson et al.,
1997). Glucose levels were determined using a Reflolux II® system (Boeringher,
Germany) The medium bottle was replaced at regular intervals such that the glucose
concentration remained above 3 mM.
3. Results and Discussion
3.1 INDUCTION OF APOPTOSIS BY AMINO ACID DEPRIVATION: INFLUENCE
OF BCL-2 EXPRESSION
The percentage of viable, necrotic and apoptotic cells in the control cell line cultures
was determined following 48 hours of deprivation of each amino acid. The majority of
cell deaths in each case occurred by apoptosis. However, there were clearly two distinct
groups of amino acids. Deprivation of essential amino acids exhibited the greatest loss
of viability, with over 70% of all cells being apoptotic. Deprivation of the non-essential
amino acids had the least effect on viability , although apoptosis still accounted for
most of the dead cells.
The effect of bcl-2 expression on the viability of the culture was also assessed. For
most amino acids, the viability of the bcl-2 cultures was greater than 70 % after 48
hours, representing a substantial improvement in viability over control cultures.
Time course studies were then conducted to examine the death profile following
deprivation of each essential amino acid. As illustrated by the 3 examples given in
Figure 1, the cultures could be divided into two phases: an initial high viability phase,
followed by a rapid death phase. Both of these phases were significantly extended
during the bcl-2 cultures. However, the specific level of protection offered varied from
one amino acid to the next, which is clearly illustrated by comparing threonine to
238
239
isoleucine. In the former, the viability fell constantly between 40 and 80 hours,
whereas in the latter, there was a significantly lower fall in viability.
Development of fed batch culture processes is an iterative process which relies on
feeding of exhausted nutrients and is therefore dependent on intensive monitoring of
nutrient levels. However, in many cases the exhaustion of a particular amino acid
willresult in the induction of apoptosis before feeding has commenced. The use of bcl-2
transfected cell lines should prevent this from happening, thus considerably speeding -
up the process of feeding strategy development by removing the need to constantly
having to re-initiate the culture from the beginning.
3.2 INFLUENCE OF BCL-2 ON ANTIBODY PRODUCTIVITY IN HIGH CELL
DENSITY PERFUSION CULTURES
The high cell numbers achieved in perfusion culture systems normally lead to an
exacerbation of nutrient and oxygen limitations. Therefore, it is expected that bcl-2
transfected cells would have a significant survival advantage in the relatively stressful
240
environment encountered in such systems. In order to investigate this possibility,
growth of bcl-2 transfected and control cell lines was studied in fixed bed and hollow
fibre perfusion systems. Direct estimation of viability is impossible, although a
flourometric technique was applied to the estimation of the free DNA concentration in
the culture medium from the fixed bed reactor. This revealed a higher concentration of
free DNA in the control cell line culture, indicating a significant level of cell death
when compared to the control cell line. The difference in the antibody productivity of
the two cell lines in each culture system is shown in Figures 2 and 3. In both systems,
the antibody titre in cultures using the bcl-2 transfected cell line was approximately
100% higher than that of the control cell line cultures. This increase must reflect the
reduction,, in the bcl-2 transfected cultures, of the amount of perfused medium
required to generate replacement cellular biomass.
4. References
Al-Rubeai, M., Mills, D., Emery, A. N. (1990) Electron Microscopy of hybridoma cells with special regard to
monoclonal antibody production. Cytotechnology. 4:13-28.
Franek, F. and Dolnikova, J. (1991) Nucleosomes occurring in protein-free hybridoma cell cultures. Evidence for
programmed cell death. FEBS Letters 248:285-287.
Mercille, S. and Massie, B. (1994a). Induction of apoptosis in nutrient-limited cultures of hybridoma cells.
Biotechnology and Bioengineering: 44, 1140-1154.
Perreault, J. and Lemieux, R. (1994). Essential role of optimal protein-synthesis in preventing the apoptotic death
of cultured b-cell hybridomas. Cytotechnology. 13, 99-105.
Simpson, N., Milner, A. N. and Al-Rubeai, M. (1997) Prevention of hybridoma cell death by bcl-2 during sub-
optimal culture conditions. Biotechnology and Bioengineering 54, 1-16.
Singh, R. P., Al-Rubeai, M., Gregory, C. D., and Emery, A. N. (1994). Cell death in bioreactors: a role for
apoptosis. Biotechnology and Bioengineering 44, 720-726.
5. Acknowledgements
This work is supported by the EC Framework IV Programme
Discussion 241
Massie:
Singh: What is the stability of the clones expressing bcl-2, not only in
Massie: terms of the expression of the genes but also the phenotype
Singh: following passaging?
Massie: We have passaged the cells for 6 months and found very high levels
of stability in bcl-2 expression, even in the absence of geneticin.
Singh: The same is true for antibody productivity.
I assume that there is no selection pressure in the perfusion system?
Massie: No.
Singh: A question concerning the relative amounts of apoptotic cells in
Ryll: batch culture in the bcl-2 transfectant: do you still see cells dying
by apoptosis in a typical batch culture?
Singh: Yes, we do see the classical apoptosis morphology but at a lower
level. This may reflect the rate of apoptotic death and progression
of cells into a secondary necrotic state, so it becomes more difficult
to clearly identify them morphologically as apoptotic.
Do you think that there is a relationship between the level of bcl-2
and protection? Can you saturate the dominant effect of protection
which bcl-2 confers on hybridoma cells?
I think that you probably can but we have not reached that point
with these cells. Clearly, in the amino acid deprivation
experiments, the cells are still dying of apoptosis.
You found that cells over-expressing bcl-2 have reduced amino
acid uptake rates. Is that something one would expect? Did you
do this with just on clone that you selected, or is it a population? If
it was a clone, you could have selected for reduced amino acid
uptake by chance.
There are mixed populations, not clones, for that precise reason.
The cells are under starvation conditions, so one would expect the
cells to adapt by down-regulating metabolic activities. This is
evidenced by the utilisation rates.
EFFECT OF BCL-2 EXPRESSION ON HYBRIDOMA CELL
GROWTH DURING STRESSFUL CONDITIONS
D. FASSNACHT1, S. ROSSING1, F.
M. AL-RUBEAI3, R. PORTNER1
1 Technische Universitat Hamburg-Harburg, und
Bioverfahrenstechnik, D-21071 Hamburg, Germany
2 Institute of Molecular Genetics, Academy of Sciences of the Czech
Republic, CZ-14220 Praha 4, Czech Republic
3 Centrefor Biochemical Engineering, School of Chemical Engineering,
University ofBirmingham, Edgbaston, Birmingham, B15 2TT, UK
1. Abstract
Two transfected hybridoma cell lines TB/C3-bcl2, (overexpressing the Bcl-2 protein)
and TB/C3-pEF (control cell line), were compared to assess the potential of Bcl-2 in
preventing apoptosis. The conditions examined were batch cultures, growth in diluted
media, and long-term fermentations in a high cell density fixed-bed reactor.
The membrane intact index (percentage of cells with intact membranes determined
by trypan blue staining) of the TB/C3-bcl2 cell line decreased much slower than that of
the control cell line during the dying phase of the batch cultures. No significant
difference in the consumption (glucose, glutamine) and production rates (lactate,
ammonia, antibodies) was notable in the exponential phase of the experiments.
When comparing the cell lines in diluted media the advantage of the Bcl-2
overexpressing cell line was again a higher membrane intact index at increasing dilution
steps.
Comparing both cell lines in a fixed-bed reactor at various dilution rates revealed a
significant difference in antibody production. TB/C3-bcl2 produced twice as many
antibodies as the control cell line for all steady-states. But no difference in the
consumption (glucose, glutamine) and production (lactate, ammonia) rates was
observable.
2. Cell Line and Culture Conditions
Both mouse hybridoma cell lines produce an IgG antibody against an antigenic
determinant in the hapten domain in the Fc region of human IgG. These cell lines
were transfected with the Bcl-2 (pEF bcl2-MClneopA) and control (pEF-MClneopA)
plasmids by Simson et al. (1997).
243
O.-W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 243-245.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
244
For growth, a 1:1 mixture of Iscove’s MDM and Ham’s F12 supplemented with
L-glutamine, 0.01% PEG and ethanolamine
was used as basal medium. This basal medium was either supplemented with 1% (v/w)
of a protein-free iron-rich supplement (IR) containing ferric citrate
and Dolníková, 1991) or 3% (v/w) horse serum (HS). The total amount of glutamine in
both media was and for glucose.
For the batch experiments, cultivation took place in stationary flasks. The
flasks were inoculated with exponentially growing cells at an initial cell density of
Growth curves were obtained by removing a doublet of flasks each day
for counting and analyses.
A fixed-bed reactor (meredos, Germany) was used for continuous fermentation. The
reactor consists of a 100 ml fixed-bed in which the cells are immobilised on porous
carriers and a 1 litre conditioning vessel. The carrier used was Siran (Schott, Germany)
which proved especially suitable for hybridoma cells (Pörtner et al., 1997). pH was
measured and controlled automatically by aandedliencgtrCicOal2hteoattehre. air. The medium of the
conditioning vessel was heated to 37 °C by No separate heating was
required for the fixed-bed because it was integrated in the conditioning vessel. This also
assured a convenient handling of the whole system.
3. Results
3.1. BATCH EXPERIMENTS
The main aspect of comparing the two
cell lines in batch cultures was to verify
the effect of Bcl-2 during ideal growth
conditions (exponential phase) as well as
in sub-optimal conditions (death phase).
These experiments were performed with
two different medium supplements: 3%
HS and 1% IR to clarify if the medium
supplement has an effect on the
behaviour of the cell lines.
The two cell lines grew in both media
to the same maximal cell density of
approx. (Fig. 1). In the
HS medium, Bcl-2 seemed to prolong the
exponential phase and to delay the dying
of the cells for 48 hours compared to the
TB/C3-pEF cell line. The growth of both
cell lines in the IR medium did not only
show the delay of the death phase but
also a significant reduction in the specific
death rate for the TB/C3-bcl2 cell line.
Fifty percent of the TB/C3-bcl2 cells in
245
the IR medium still had intact membranes after 9 days of cultivation whereas no
viability was detected in all other experiments after this time.
There was no significant difference in glucose and glutamine consumption as well
as lactate and ammonia production for the exponential phases of the experiments. This
was also true for the antibody titre, which reached a concentration of approx. 50 mg l-1
in all experiments.
3.2. GROWTH IN DILUTED MEDIA
These experiments were carried out in diluted media in order to compare the two cell
lines under nutrient limitation. Both hybridoma cell lines reached a viable cell density
of 6.105 to 7.105 cells ml-1 in the undiluted IR medium after 72 hours. The membrane
intact index was close to 90%. The reduction of the content of nutrients, achieved by
medium dilution, resulted both in a lower relative viable cell density and in a lower
membrane intact index. The TB/C3-bcl2 cell line displayed higher resistance to the
starvation stress. At the verge of starvation-induced death, i.e. in 40% to 50% medium,
the membrane intact index clearly documented the superiority of the TB/C3-bcl2 cell
line. Here the membrane intact index was approx. 15% higher than that of the control
line.
3.3 FIXED-BED REACTOR
Both cell lines were also compared in the fixed-bed bioreactor at various dilution rates.
Comparing the results of the cell lines revealed a significant difference in antibody
production. TB/C3-bcl2 produced twice as many antibodies as the control cell line for
all steady-states. But no difference in the consumption (glucose, glutamine) and
production (lactate, ammonia) rates was observable.
For details of the results, please refer to R. P. Singh et. al ‘Manipulation of
apoptosis by Bcl-2: Enhancement in survivability after amino acid deprivation and in
intensive culture systems’.
4. Conclusions
The overexpression of Bcl-2 did not have an influence on the metabolism of cells
growing under ideal conditions (exponential phase of batch cultures). But the Bcl-2
overexpressing cell line survived much better when induced to stressful conditions such
as the late exponential phase in batch experiments, growth in saline diluted media or
during continuous fermentations in fixed-bed reactors at low dilution rates.
References
F., and Dolníková, J, 1991. Hybridoma growth and monoclonal antibody production in
iron-rich protein-free medium: Effect of nutrient concentration. Cytotechnology 7: 33-38
Pörtner, R., Rössing, S., Koop, M., Lüdemann, I. 1997. Kinetic studies on hybridoma cells
immobilized in fixed bed reactors. Biotechnology and Bioengineering 55: 535-541
Simpson, N. H., Milner, A. E., Al-Rubeai, M. 1997. Prevention of hybridoma cell death by Bcl-2
during sub-optimal culture conditions. Biotechnology and Bioengineering 54: 1-16
INHIBITION OF c-jun EXPRESSION IN F-MEL CELLS CAUSES CELL
CYCLE ARREST AND PREVENTION OF APOPTOSIS
YON HUI KIM1, TAKEHIKO IIDA1, EDWARD V. PROCHOWNIK2,
EIJI SUZUKI1
1. Department of Chemistry and Biotechnology, Graduate School of
Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
2. Division of Pediatric Hematology/Oncology, Children's Hospital of
Pittsburgh, Pittsburg, PA 15213-2583, USA
ABSTRACT
We are able to provide an evidence that will support the suppression of cell proliferation to
withdrawal from the cell cycle and suppression of onset apoptosis with conditional
expression of c-jun antisense transcript in F-MEL cells. F-MEL cells were transfected
with c-jun antisense gene located downstream of a glucocorticoid-inducible MMTV
promoter, and named as c-jun AS cells. By treating the c-jun AS cells with dexamethasone
(DEX) in DMEM supplemented with 10% serum, the growth could be suppressed for
duration of 16 days with high cell viability of 92%. When DEX-treated c-jun AS cells
were serum deprived, the cell viability remained at high of 86% for upto 10 days, the onset
of apoptosis was suppressed, and the internucleosomal cleavage of DNA was not detected
upto 8 days. In contrast, when wild type F-MEL cells were serum deprived, the cell
viability is low (50%.) and the onset of apoptosis is induced within 2 days, and
internucleosomal cleavage of DNA was detectable.
1. INTRODUCTION
Biologically active proteins such as cytokines, vaccines, and antibodies are produced
by mammalian cell culture. The protein production of such cultures usually increases with
increase in cell integration, the viable cell number integrated with respect to culture time.
Viable cells in the late logarithmic growth phase and the following non-growth viable
phase are major contributors to cell integration. Cells often produce protein at a rate higher
in the non-growth viable phase than in the logarithmic growth phase (Suzuki and Ollis,
1990). However, cells tend to die quickly after reaching the maximum cell density due to
adverse conditions caused by over-growth. Hence the viable non-growth culture period may
be as short as only two to three days (Duval et al., 1990; Vomastek and Franek, 1993).
Therefore, preventing cell death which starts in the late logarithmic growth phase
(Vomastek and Franek, 1993) and maintaining them viable in batch culture for longer time
period should increase protein production of the culture. The cells die due to depletion of
nutrients such as amino acids (Franek and Chladkova-Sramkova, 1995) and glucose
(Marcille and Massie, 1994; Singh et al., 1994), limitation of growth factors such as
serum components(Singh et al., 1994), or accumulation of toxic metabolites (Glacken et
al., 1988; Tohyama et al., 1990). The over-growth inevitably leads to some of these cell
death inducing conditions.
Cell death may follow two distinct patterns: necrosis and apoptosis. Apoptotic cell
death follows a well-defined sequence of events characterized by cell shrinkage, nuclear
247
O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 247-254.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
248
condensation, and fragmentation of the cell into discrete membrane enclosed bodies. In the
early apoptotic death process, an endogenous endonuclease is activated and cleaves DNA
within the internucleosomal spacer regions. Hence, a characteristic ladder pattern consisting
of multiplies of 180 bp fragments is seen when the DNA from apoptotic cells is
electrophoresed on an agarose gel. Reportedly, the cell death in the late logarithmic growth
and stationary phases of batch culture is mostly apoptosis (Mercille and Massie, 1994).
Proto-oncogene c-jun known as the immediate early response' gene is rapidly induced
in response to mitogenic stimuli, and it plays a critical regulatory role in the commitment
of the cell to proliferate (Ryseck et al., 1988, Smith and Prochownik, 1992). A nuclear
phosphoprotein c-Jun is a major component of the AP-1 trans-acting transcriptional
activator complex that promotes transcription of various genes required for progression of
the cell cycle (Turner and Tjlan, 1989). The selective inhibition of c-jun expression caused
exit from the cell cycle, in other words, it blocked cell growth (Smith and Prochownik,
1992; Soprano et al., 1992). The possibility of c-Jun being the inducer for natural cell
death, apoptosis, was reported (Colotta et al., 1992; Estus et al., 1994; Rampalli and
Zelenka, 1995; Anderson et al., 1995). Interleukin (IL)-6- and lL-2-dependent mouse cell
lines underwent programmed cell death alter the growth factor deprivation; in this death
process, c-jun gene was rapidly induced; and the antisense oligonucleotides directed against
c-jun reduced this c-jun induction, while improving survival of the cells after the growth
factor deprivation (Colotta et al., 1992). The antisense oligonucleotides for c-jun prevented
HL-60 cells from apoptosis induced by ceramide (Sawai et al., 1995).
Using this understanding that the c-jun gene plays an important role in the progression
of the cell cycle and induction of apoptosis or programmed cell death. The objective of
the present work is to establish a cell line in which growth can be switched on and off
without induction of apoptosis. And we were successful in creating a cell line c-jun AS
that can be induced by glucocorticoid hormone to block cell proliferation and at the same
time suppress apoptosis even under growth factor deprivation. This growth and apoptosis
controllable cell line can be utilized to keep high viability in batch, fed-batch, or perfusion
culture. The cell line will be also suitable to culture for which use of any serum
components is prohibited.
2. MATERIALS AND METHODS
Cell Line and Cell Culture Conditions
Friend murine erythrolcukemia (F-MEL) cells were supplied by Riken Cell Bank. F-
MEL cells and all the cells derived from F-MEL cells in the present work were cultured in
Dulbecco's modified Eagle's medium (DMEM) (Nissui, Tokyo), supplemented with 10%
fetal bovine serum (v/v) (JRH, Biosciences), 4 mM glutamine, 100 mg/ml kanamycin,
0.2% NaHCO3 (complete medium), unless otherwise specified. The cells were maintained
in the logarithmic phase of growth in 25-cm2 plastic T-culture flasks (Sumitomo Bakelite.
Tokyo) at 37°C in a 5% CO2-95% air humidified incubator. For the induction of the
transfected sense or antisense c-jun transcripts, cells were cultured in the complete or serum
deprived medium supplemented with dexamethasone (Sigma) at a concentration of 1x10-6
M. Viable cell count was performed by the trypan blue dye exclusion method employing a
hemocytometer.
249
Transfection, Selection, and Gene A m p l i f i c a t i o n
pMS-G c-jun AS dhfr vector contains an antisense fragment of c-jun gene downstream
of a glucocorticoid-inducible murine mammary tumor virus (MMTV) promoter and a dhfr
(dihydrofolate rcductase) gene. The antisense c-jun gene is the inversely inserted sequence
that consists of 287 bp of 48 bp of coding sequence derived from the
extreme end of the coding region, and 929 bp of c-jun cDNA.
pMS-G c-jun dhfr vector contains a complete sense c-jun cDNA instead of the antisense c-
jun gene (Smith and Prochownik, 1992). For transfections, 2 x 1 0 6 F-MEL cells were
pelleted at 500 g for 10 min, washed twice in phosphate-buffered saline (PBS), and
resuspended in 200 ml of PBS. 20 mg of BamHI-linearized pMS-G c-jun AS dhfr DNA or
pMS-G c-jun dhfr DNA plus 2 mg of NdeI-linearized pSV2neo plasmid DNA in a total
volume of 50 ml of sterile water were added to the cell suspension. Transfection was
accomplished by electroporation with a homemade apparatus at settings of 650 voltage
with 10 pulses at resultant time of 250 msec. The cells were grown for 24 h in the
complete medium, and then G-418 was added to a final concentration of 0.4 mg/ml. G-418
resistant clones were detected within 4 to 8 days. Pooled G-418 resistant clones were then
cultured in the complete medium containing methotrexate (MTX) at a final concentration
of 50 nM for amplifying the sense or antisense c-jun gene together with the dhfr gene for
several months. For cloning the cell , the dilution plating techniques were applied using
96-weIl plates.
Detection of c-Jun Protein by Western B l o t t i n g
To assess the effect of c-jun antisense transcript induction on c-jun protein levels, the
c-jun AS cells were cultured in the complete medium containing 10-6 M dexamethasone
for 48 h. The 5x105 cells were harvested, pelleted at 2500 g for 10 min, washed twice in
phosphate-buffered saline (PBS) and resuspended in lysis buffer (1% Triton x-100, 0.15
mM NaCl, 10 mM Tris, pH 7.8) at 4°C for 30 min. The cell lysates were boiled in
sodium dodecyl sulfate (SDS) sample buffer for 5 min, and electrophoresed on a 10% SDS-
polyacrylamide gel, and then electroblotted onto nitrocellulose filter overnight. The blotted
nitrocellulose filter was blocked with 0.5% skim milk in TBST for 3 h at room
temperature. At size of 39-kDa, c-jun protein was detected by rabbit polyclonal antibodies
to c-jun /AP-1 protein (Medac, Diagnostika) to which peroxidase-conjugated goat anti-
rabbit Ig polyclonal antibody (Bio Source International, Inc. Tago Products) bound. The c-
Jun-specific band was then visualized by use of the ECL Western blotting system
(Amersham, Life Science). F-MEL cells and the c-jun AS cells were cultured free from
DEX, and treated in the same manner for Western blotting.
DNA Fragmentation Assay by Electrophoresis
1x106 cells were pelleted and lysed by vortexing vigorously in 0.5 ml of TE buffer (10
mM Tris-HCl, pH 7.4 and 1 mM EDTA) containing 0.2% Triton X-100, and centrifuged
for 10 min at 13,000g at 4°C. DNA was extracted after addition of 0.1 ml of 5 M NaCl
and 0.7 ml of isopropanol. The supernatant was vortexed, placed at -20°C overnight, and
then centrifuged for 10 min at 13,000 g at 4 °C. The DNA pellet was dissolved in TE
buffer and treated with RNase prior to electrophoresis on a 2% agarose gel. To visualize the
fragmented DNA bands, the gel was stained with 1 mg of ethidium bromide per ml.
250
3. RESULTS and DISCUSSION
Blocking Cell-Growth by Antisense c-Jun Expression
F-MEL cells were transfected with the linearized pMS-G c-jun AS dhfr vector carrying
the antisense c-jun gene as described in Materials and Methods. The vector contains a
glucocorticoid-inducible MMTV promoter and dhfr gene. The dhfr gene was applied for
amplifying the vector by culturing cells with methotrexate (MTX), an inhibitor of
dihydrofolate reductase. MMTV promoter was used for turning on or off the transcription
of the antisense gene depending on the presence or absence of a synthetic glucocorticoid,
dexamethasone (DEX). The transfected cells were selected with G418, and then cultured in
MTX for amplifying the antisense gene (generally 50 to 200 copies/cell) (Prochownik and
Kubowska,1986). The obtained cell line was named c-jun AS.
First we assessed the effect of c-jun antisense transcript induction on c-jun protein
levels. We exposed the c-jun AS cells to 10-6 M DEX for 48 h. The cells were harvested
and subjected to SDS-polyacrylamide gel electrophoresis and electroblotted onto
nitrocellulose. Control lysates were prepared from the wild type F-MEL cells. Rabbit
polyclonal antibodies to c-Jun and AP-1 proteins was used to detect c-Jun. Next we
examined whether we can block the growth of the c-jun AS cells and maintain the cells in
viable state. The c-jun AS cells in logarithmic growth phase in the complete medium
were collected and resuspended in 10 ml of the fresh complete medium at day 0 in each of
25 cm2 T-flasks. For inducing the antisense c-jun to block the cell growth, DEX was added
to the medium at 10-6 M on day 0. The viable cell density and viability are shown in
Fig. 1-a and -b, respectively. For comparison, we cultured in the same manner the wild type
F-MEL cells and those transfected with the complete sense c-jun gene. The growth of the
c-jun AS cells was almost blocked two days after DEX addition. After that the viable cell
density was kept almost unchanged for 14 days. A viability greater than 86% was
maintained for 16 days. In the absence of DEX, the c-jun AS cells grew to the over-growth
condition and then started dying. The wild type F-MEL cells also grew to over-growth ,
and then started dying either in the presence or absence of DEX. Jointly these results
indicated that the c-jun AS cell line was a desired cell line, that is, a viably growth-
arrestable cell line.
Suppressing Apoptosis by Antisense c-Jun Expression
At least two groups reported that antisense oligonucleotides directed for c-jun gene
suppressed apoptosis (Colotta et al., 1992; Sawai et al., 1995). This suggested that the c-
jun AS cell line is possibly free from apoptosis when cultured in the presence of DEX.
Use of oligonucleotides is limited to research purpose, because they are expensive and not
stable in culture. Contrarily, the antisense c-jun transcripts in the c-jun AS cells can be
induced with relatively inexpensive DEX when necessary. Therefore, we examined the
apoptosis resistance of the c-jun AS cells by applying serum-deprivation, one of typical
apoptosis inducing conditions. The logarithmically growing c-jun AS cells in the complete
medium were collected, and washed three time in PBS. Then the cells were placed in the
serum-deprived medium, and cultured in the presence or absence of DEX. The wild type F-
MEL cells were also subjected to the same procedure. The viable cell density and viability
are shown in Fig.2-a and -b, respectively. Under serum-deprivation, the viability of the
251
wild type F-MEL cells decreased to 50% in 3 days, while that of the c - j u n AS cells was
maintained above 80% for 8 days.
In the absence of DEX, the c-jun AS cells, of which c-jun expression was partially
suppressed by the antisense c-jun expression due to the leakiness of MMTV promoter,
survived and grew well under serum-deprivation (Fig.2-a and -b). This meant that we could
unexpectedly establish a F-MEL cell line that can grow without any serum component. We
interpreted this result as c-jun expression in the c-jun AS cells cultured in the absence of
DEX was high enough to drive cell cycling and too low to induce apoptosis.
We examined emergence of the ladder pattern, a land mark of apoptotic cells, on
electrophoreses of DNA extracted from the cells of the above experiments. The c-jun AS
cells cultured with DEX under serum-deprivation were collected on day 4, 6, 8, 10, and 12.
DNA samples extracted from the cells were treated as described in Materials and Methods
for detecting DNA fragmentation. The wild type F-MEL cells cultured under the serum-
deprivation were similarly examined. The clear DNA ladder was detected for the wild type
F-MEL cells on day 4, but not for the c-jun AS cells treated with DEX (Fig.3). Jointly
with the result of Western blotting for detection of c-jun protein, this result indicated that
suppression of c-jun expression inhibits apoptosis.
A cell line c-jun AS that can be reversibly and viably growth-arrested was established
by transfecting F-MEL cells with inducible antisense c-jun gene. The cell line is resistant
to apoptosis induced by scrum-deprivation , or by cell cycle arrest. The c-jun AS cells can
grow even under serum-deprivation when cultured free from dexamethasone, while the wild
type F-MEL cells die quick. Jointly with several other reports on the relation between
apoptosis and c-jun expression, our result strongly suggested that c-jun expression is
required for onset of at least some types of apoptosis.
References
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gene proteins in cultured neurons by b-amyloid (Ab): association of c-Jun with Ab - induced
apoptosis. J. Neurochem. 65: 1487-1498.
Colotta, F., Polentarutti, N., Sironi, M. and Mantovani, A. 1992. Expression and involvement
of c-fos and c-jun protooncogenes in programmed cell death induced by growth factor
deprivation in lymphoid cell lines. J. Biol. Chem. 267: I8278-18283.
Duval, D., Demangel, C., Geahel, I., Blondeau, K. and Marcadcd, A. 1990. Comparison of
various methods for monitoring hybridoma cell proliferation. J. Immunol. Meth.
134: 177-185.
Estus, S., Zaks, W. J., Freeman, R. S., Gruda, M., Bravo, R., and Johnson, E. M. Jr. 1994.
Altered gene expression in neurons during programmed cell death: identification of c-jun as
necessary for neuronal apoptosis. J. Cell Biol. 127: 1717-1727.
Franek, F, and Chladkova-Sramkova, K. (1995) Apoptosis and nutrition: Involvement of amino
acid transport system in repression of hybridoma cell death. Cytotechnology 18: 113-117.
Glacken, M. W., Adema, E, and Sinskey, A. J. 1988. Mathematical descriptions of hybridoma
culture kinetics: I. initial metabolic rates. Biotechnol. Bioeng. 32:491-506.
Mercille, S. and Massie, B. 1994. Induction of apoptosis in nutrient-deprived cultures of
hybridoma and myeloma ells. Biotechnol. Bioeng. 44: 1140-1154.
Rampalli, A. M. and Zelenka, P. S. 1995. Insulin regulates expression of c-fos and c-jun and
suppresses apoptosis of lens epithelial cells. Cell Growth Differ. 6: 945-953.
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253
254
Discussion
Handa-Corrigan: Dexamethasone is a gluco-corticoid and most clinicians look at this
molecule as giving cells the ‘kiss of life’. The change in the
biochemistry of the cell is quite acute as the cell will start
producing glucose, rather than utilising it, and revert to using
amino acids and fatty acids instead of glucose. So when we add
dexamethasone to cells we find that there is a slowing of the
growth rate and they live slightly longer. In your study, what
happened to the parent cell line in the presence of dexamethasone?
Kim: The F-MEL cells were not affected at all by dexamethasone in any
respect.
Hauser: Can you use the system with cells that have endogenous
dexamethasone receptors? This would be a problem for changing
to other cells. You would probably have to change to another
inducible system. Also, have you tried to get a stable expression of
your growth anti-sense regulator, and can your cells sustain
stability with this type of regulator?
Kim: It is a problem in that sometimes dexamethasone does induce
apoptosis in some cell lines. So there is a limit to this model. A
year ago I did have a stability problem but, as I selected more and
more, I now have very strong expression even without
dexamethasone. So I believe that it is very strong, once selected.
Hatzfeld: Did you try adding for short cultures oligonucleotide anti-sense?
Kim: No, I have not tried oligonucleotides.
HIPPOCAMPAL CELLS IN CULTURE AS A MODEL TO STUDY
NEURONAL APOPTOSIS
I. FIGIEL, J. JAWORSKI and L. KACZMAREK
Nencki Institute of Experimental Biology
Pasteura 3, Warsaw 02-093, Poland
1. Abstract
Glutamate toxicity (excitotoxicity) has been well documented and proposed to play a
role in a broad spectrum of neurological diseases. There is a growing interest in
defining glutamate-mediated neurotoxic mechanisms, but the high degree of
complexity of the intact nervous system hampers a detailed analysis. Thus, cell cultures
may provide a useful alternative. We have applied a culture of hippocampal granule
cells of postnatal rats to study glutamate-evoked toxicity. These cells are known to be
particularly resistant to excitotoxic insult in the brain in situ. However, we have found
that granule neurons grown in culture die when they are exposed to high
concentrations of glutamate. Moreover, the dying cells display morphological and
biochemical features characteristic of apoptosis, a mode of cell death typical of
physiological neuronal elimination during development. To further investigate
molecular correlates of this phenomenon we have developed an improved calcium
phoshate coprecipitation procedure to transiently introduce DNA into cultured
neurons. Using this technique we have been able to investigate direct involvement of
particular transcription factors in the regulation of glutamate excitotoxicity. The results
of our study may be helpful to understand the molecular mechanisms of neuronal
apoptosis.
2. Introduction
Glutamate is the major excitatory neurotransmitter, that, in addition to interneuronal
communication, can also be neurotoxic. In the past several years there have been many
studies on isolated neuronal populations treated with glutamate, which have lead to the
hypothesis that toxicity is caused by an influx of calcium through receptor-controlled
ion channels, which in turn triggers a cascade of events leading to neuronal cell death
(Choi and Rothman, 1990; Randall and Thayer, 1992). However, details of the
complex process are still uknown. Recently it has been suggested that excitotoxicity
may have an apoptotic character, i.e., may belong to programmed cell death
phenomena (for review see: Dragunow and Preston, 1995).
255
O.-W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 255-258.
©1998 Kluwer Academic Publishers. Printed in the Netherlands.
256
Since apoptosis is supposed to be an active process, it is believed that it should
involve gene expression. Regulation of gene expression is brought about by
transcription factors. Activation of glutamate receptors causes an increase in
expression of AP-1 (activator protein-1) transcription factor (Morgan and Curran,
1991; Kaczmarek 1994; Kaminska et al., 1994). Major components of AP-1 are Fos (c-
Fos, Fos B, Fra-1, Fra-2) and Jun (c-Jun, Jun B, Jun D) proteins. Possible role of AP-1,
and more specifically c-Fos and c-Jun proteins, in active cell death has been initially
supported by results of experiments carried out on non-neuronal cells (Colotta et aI.,
1992). In the studies on the central nervous system, Smeyne et al. (1993) using c-fos-
lacZ transgenic mice expressing β-galactosidase under control of c-fos regulatory
element, observed that c-Fos expression is associated with apoptotic cell death
following kainate injection. Dragunow et al. (1993), on the other hand, reported a
prolonged increased in c-Jun immunoreactivity in a model of status epilepticus coupled
to neurodegeneration. Recently, Estus et al. (1994) and Ham et al. (1995) have
demonstrated that c-Jun is induced in sympathetic neurons undergoing programmed
cell death after NGF withdrawal, and moreover, blocking of AP-1 activity with either
microinjection of specific antibodies or introduction into the cells of dominant negative
mutant of c-Jun prevented neuronal death, whereas overexpression of c-Jun promoted
the neuronal death. Hence, it is expected that identification of genes as well as their
regulatory mechanisms involved in control of apoptosis should be of great value for
both understanding the process and development of therapeutic strategies.
3. Experimental approach and results
In our studies we have employed cultures of hippocampal dentate gyrus, a brain area
known to be rather resistant to excitotoxic insult. Dentate granule neurons were
obtained from 5-day-old rat pups and cultured as previously described (Figiel and
Kaczmarek, 1997). Cells grown for 6 days in vitro were exposed to glutamate.
Unexpectedly, in our culture conditions, we have observed an extensive neuronal
loss after treating the cultures with 0.5 mM glutamate. Generally, we noted somal
rounding, thining and fragmentation of dendrites. In some cases cell body became
enlarged, the nucleus vacuolated, and eventually the cell body detached from the
substrate.
We have used the specific DNA stain, Hoechst 33258, in order to assess changes of
nuclear structure following exposure to glutamate. Several features indicative of
apoptosis, such as the condensation of the nuclear chromatin and fragmentation of the
nucleus, were observed (Fig. 1)
These apoptotic-like morphological changes were acompanied by DNA
fragmentation, as revealed by the method of TdT-mediated dUTP-digoxigenin 3’ end
labeling (TUNEL) (Gavrieli et al., 1992).
We have also employed immunocytochemistry to investigate expression of c-Fos
and c-Jun, two AP-1 transcription factor components, in the control and glutamate-
treated cells. We found that following glutamate treatment the level of c-Fos protein
was significantly increased in comparison to control (untreated) cultures. However the
pattern of this immunoreactivity was almost identical, regadless of the concentration of
glutamate. Moreover, there was much higher number of neurons expressing c-Fos,
than the number of dying cells identified with an aid of the aforementioned
approaches. On the contrary, c-Jun expression was found only in dying cells.
257
To test the hypothesis that c-Jun contributes to the induction of neuronal apoptosis,
we employed calcium phosphate transfection developed by M.E. Greenberg (Xia et al.,
1995) with modifications. PS65T-C1 plasmid (Clontech, GenBank accession -
#U36202) encoding red shifed variant of GFP under control of CMV promoter was
used for optimalization of transfection. Preliminary results are shown in Fig.2.
4. Conclusions
In conclusion, we have demonstrated that excitotoxicity produced by glutamate in
cultured hippocampal dentate neurons induces cell death by an apoptotic mechanism.
Results of our studies based on transient expression of c-jun dominant negative mutant
may show the correlation between cell death and c-Jun induction.
5. References
Choi, D. and Rothman, S.M. (1990) The role of glutamate neurotoxicity in hypoxic-ischaemic neuronal death
Annu. Rev. Neurosci. 13, 171-182.
Colotta, F., Polentarutti, N., Sironi, M., and Mantovani, A. (1992) Expression and involvement of c-fos and c-jun
protooncogenes in programmed cell death induced by growth factor deprivation in lymphoid cell
lines, J. Biol. Chem. 267, 18278-18283.
Dragunow, M. and Preston, K. (1995) The role of inducible transcription factors in apoptotic nerve cell death,
Brain Res. Rev. 21, 1-28.
Dragunow, M., Young, D., Hughes, D., Mac Gibbon, G., Lawlor, P., Singleton, K., Sirimanne, E., Beilharz, E.,
and Gluckan, P. (1993) Is c-Jun involved in nerve cell death following status epilepticus and
258
hypoxic-ischaemia brain injury? Mol. Brain Res. 18, 347-352.
Figiel, I. and Kaczmarek, L. (1997) Cellular and molecular correlates of glutamate-evoked neuronal programmed
cell death in the in vitro cultures of rat hippocampal dentate gyrus, Neurochem. Int. 31, 229-240.
Gavrieli, Y., Sherman, Y., and Ben-Sasson, S.A. (1992) Identification of programmed cell death in situ via
specific labeling of nuclear DNA fragmentation, J. CellBiol. 119, 493-501.
Ham, J., Babij, C., Whitfield, J., Pfarr, C.M., Lallemand, D., Yaniv, M., and Rubin, L.L. (1995) A c-Jun
dominant negative mutant protects sympathetic neurons against programmed cell death, Neuron 14,
927-939.
Kaczmarek, L. (1994) Glutamate-evoked gene expression in brain cells - Focus on transcription factors,
Amino Acids 7, 245-254.
Kaminska, B., Filipkowski, R.K., Zurkowska, G., Lason, W., Przewlocki, R., and Kaczmarek, L. (1994)
Dynamic changes in composition of the AP-1 transcription factor DNA dinding activity in rat brain
following kainate induced seizures and cell death, Eur. J. Neurosci. 6, 1558-1566.
Morgan, J.I. and Curran, T. (1991) Stimulus-transcription coupling in the nervous system: involvement of the
inducible protooncogenes fos and jun, Annu. Rev., Neurosci. 14, 421-451.
Randall, R.D. and Thayer, S.A. (1992) Glutamate-induced calcium transient triggers delayed calcium overload
and neurotoxicity in rat hippocampal neurons, J. Neurosci. 12, 1882-1895.
Smeyne, R.S., Vendrell, M., Hayward, M., Baker, S.J., Miao, G.G., Schilling, K., Robertson, L.M., Curran, T.,
and Morgan, J.I. (1993) Continuous c-fos expression precedes programmed cell death in vivo, Nature 363,
13-23.
Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J., and Greenberg, M.E. (1995) Opposing effects of ERK and JNK-
p38 MAP kinases on apoptosis, Science 270, 1326-1331.
DEVELOPMENT OF CARBOXY SNARF-1-AM AND ANNEXIN V ASSAYS FOR THE
DETERMINATION OF APOPTOSIS IN HETEROGENEOUS CULTURES
A. Ishaque & M. Al-Rubeai
Centre for Bioprocess Engineering, School of Chemical Engineering,
University ofBirmingham, Edgbaston, Birmingham, U.K.
1. Abstract
Accurate identification and quantitation of apoptosis is essential for developing efficient
strategies for optimisation of culture survivability and productivity. Flow cytometry in
conjunction with several fluoroprobes is increasingly used to identify apoptotic cells. We have
examined the possibility of using carboxy SNARF-1-AM, a pH sensitive fluoroprobe and FITC-
labelled annexin V, a probe specific to phosphatidylserine exposed on the outer surface of
apoptotic cells. Intracellular acidification was shown to precede the occurrence of apoptosis
thereby proving to be an early indicator of cellular deterioration and cell death. Annexin V in
combination with propidium iodide enabled identification of viable, transient apoptotic and
necrotic cells in heterogeneous cultures. Metabolic activity (pHi), and cell death population
dynamics (viable/ apoptotic/ necrotic fraction) were therefore effectively and reliably determined
using flow cytometry.
key words: flow cytometry, apoptosis, pHi and annexin-V.
2. Introduction
The presence of dead cells significantly limits the productivity of hybridoma cells in culture. Cell
death via apoptosis represents the predominant mode of cell death in culture prosesses, especially
in the batch culture of hybridoma cells (Singh et al., 1996). Therefore, monitoring the
development of early apoptotic cell state will significantly facilitate the control and optimisation
of bioreaction processes. Flow cytometry is the best method for detecting and analyzing apoptosis
in heterogeneous culture, revealing information on the state of cells in a near on-line mode and
time scale (Al-Rubeai et al., 1991). In the present study Annexin-V (AV) (a naturally occurring
phospholipid which has a high affinity for phosphatidylserine exposed on the surface of early
membrane intact apoptotic cells) conjugated to FITC was used in a simple flow cytometric assay
for the identification of apoptosis. FITC-AV in combination with the DNA binding dye
propidium iodide (PI) entered plasma membrane damaged cells to identify necrotic cells. Cell
death population dynamics (viable/apoptotic/necrotic cells) were therefore assessed in batch
culture of hybridoma cells using the AV-assay.
Intracellular acidification may represent a signal for the integration of a number primary signals
to enhance the development of apoptotic cell death. This hypothesis was tested by determining
the intracellular pH of hybridoma cells, using a pH sensitive fluoroprobe carboxy-SNARF-1-AM,
undergoing apoptotsis either spontaneously in batch culture or by induction after exposure to
campothecin.
259
O. - W. Merten et al (eds.), New Developments and New Applications in Animal Cell Technology, 259-261.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
260
3. Materials and methods
Transfected control hybridoma cell line (Pef), and hybridoma cells transfected with the anti-
apoptotic protein bcl-2 were cultured at 37 °C in RPMI 1640 basal medium supplemented with
5% (v/v) foetal calf serum. Cultures were maintained in 50 ml or 250 ml Duran-bottles stirred by
magnetic followers.
Pef and bcl-2 cells undergoing spontaneous apoptosis in batch culture were double stained with
FITC-AV and PI. Cells were washed in PBS (1%) and then resuspended in binding buffer (10
mM Hepes/NaOH, pH 7.4, 150 mM KCl, ImM MgCl2and 1.8 mM CaCl2). FITC-AV was added
to give a final concentration of 2.5 The mixture was incubated in the dark at room
temperature for 10 mins. PI (1mg/ml) was added 5 mins before flow cytometric analysis.
Pef and bcl-2 hybridoma cells induced to undergo apoptosis by campotheicn treatment or from
the death phase of batch culture were loaded with carboxy-SNARF-1-AM (1 mM in DMSO), to
give a final concentration of 10 in a HEPES-buffered medium. Cells were incubated for 30
min at 37 °C and then analysed by a Coulter Epic Elite flow cytometer, with excitation at 488 nm
to produce an acidic emission signal at 575 nm and a basic emission signal at 635 nm. The ratio
of basic and acidic forms gave an indication of the pHi. All cells were correspondingly assessed
for cell viability by fluorescent microscopy using a mixture of acridine orange and PI.
4. Results
4.1. FITC-AV staining of hybridoma cells in batch culture
AV/PI staining of control (Pef) hybridoma cells allowed the transition from viable to necrosis via
apoptosis to be followed in batch culture (fig.l). At 72 hrs the viable population [AV(-ve)/PI(-
ve)] moved into an early apoptotic compartment [AV(+)/PI(-)]. The emergence of early apoptotic
cells coincided with fluorescent microscopy results. At 84 hrs of culture the entire apoptotic
population migrated to a necrotic cell gate [AV(+)/PI(+)]. Bcl-2 transfected cells migrated
directly from [AV(-ve)/PI(-ve)] to [AV(+)/PI(+)] compartments during batch culture, therefore
by-passing apoptotic cell death demonstrated by phosphatidylserine externalization on cells with
intact plasma membranes.
4.2. Carboxy-SNARF-1-AM staining of hybridoma cells exposed to campothecin
Exposure of hybridoma cells to the DNA topisomerase I inhibitor campothecin (CAM) led to the
appearance a subpopulation of cells with a lower fluorescent ratio than the untreated cells. The
apoptotic population had a reduced forward scatter which was due to cellular shrinkage. In the
CAM treated cells a population with lower ratio (acidic and apoptotic ) appeared 3 hrs before the
maximum proportion of apoptotic cells could be detected by fluorescent microscopy.
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