12_ANIMAL CELL TECHNOLOGY_707

663

5. Conclusion
Preliminary data suggests that factors such as pH, serum levels, cell to disc ratio and
incubation period do have an effect on the levels of cell attachment to the
microcarriers. As hepatocytes are essentially non-dividing cells it is important to
achieve high initial cell attachment if these microcarriers are to be utilised in a
bioartificial liver system where high numbers of cells are needed.

METABOLIC COMPETENCE AND HORMONAL REGULATION OF
PRIMARY PORCINE

HEPATOCYTES IN A 3-D SANDWICH CONFIGURAITON

1 A. Bader, 1D. Rocker, 1A. Acígköz, S. 1Schwintek, 1Jarosch von Schweder
1M. Maringka, 3V. Armstrong, 2R. Wagner, 1Steinhoff G, 1Haverich A
1Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Division
of Cardiovascular Surgery, MHH, 2GBF, Braunschweig, Klinische Chemie, University of
Göttingen, Germany

The necessary culture conditions for maintenance of metabolic competence
such as benzodiazepine metabolism or lidocaine biotransformation in primary
hepatocyte cultures are a matter of intense debate. We therefore investigated
and developed novel culture conditions with respect to tissue culture
requirements such as hormonal additives, medium exchange rates, matrix
geometry, collagen additives, nutrient and trace element composition. These
results finally enabled us to achieve a fully serum free long-term culture of
primary porcine hepatocytes maintaining oxidative biotransformation and
protein secretion for at least 8 weeks in culture.
This cell culture technology is crucial for a bioartificial liver based on primary
cells.

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Discussion

Noé: I anticipate your system to be used for liver transplant as an
intermediate step. How far are you from the application of your
system in patients?

Bader: Scale-up has been difficult and we have lost a lot of time. However,
we start in vivo experiments 2 weeks from now. We have to do
safety studies, then studies on a hepactectomised model we have
been using for the past year. Everything is ready for human trials
once we get permission from the ethical committee.

Noé: What type of animal studies have you done?

Bader: A hepactectomised model of pigs is used.

Handa-Corrigan: In a recent clinical trial in London of hollow fibre bioreactors all the
patients were successfully taken on to transplantation. However, the
cells were non-viable after a day or two, so it is a dying population.
What was the viability in your bioreactor?

Bader: A plateau of viability of over 90% is maintained based on cell
specific activity during in vivo use.

Keck: I think your data showing expansion of primary porcine hepatocytes
is a tremendous advance for the bioartificial liver field. What was the

medium used and have you done any work on primary human
hepatocytes?

Bader: We are in the process of developing human cells expanding from 10
and 20,000 cm2 to 150,00 cm2. The culture medium is important
and has well known growth factors such as EGF, but it is a very
complex picture and needs foetal calf serum.

Bernard: What are the advantages of your system compared with in vitro
P450’s, and if you use such a system what would be the through-put
in terms of numbers of compounds tested per unit time?

Bader: 667

It is a complementary picture rather than an advantage. We have
carried out in vitro and in vivo correlations comparing metabolic
patterns. Using an isolated P450 system you can identify individual
peaks on an HLPC chromatogram or mass spectrometry to correlate
it with the P450 enzyme family. We can identity metabolites
generated by a specific type of enzyme. In primary hepatocytes we
get a full pattern. We have compared various culture configurations
and find an inversion of metabolite patterns between rats, and
humans, and the same was found in vitro with rat and human primary
hepatocyte sandwich cultures. It has been used against a range of
compounds including cyclosporin, a range of Bayer antibiotics, and
for predictive evaluation before going to the clinical phase. The
main advantage of a primary hepatocyte system is to find metabolite
patterns. This can also be done using a liver slice. The advantage of
a culture system is you can study drug interaction to look for P450
induction. This is a useful model for this.

Miltenburger: How large has your device to be for use as a human liver, and how
many cells do you need?

Bader: 100 g of cells to replace a human liver will detoxify plasma in a

patient whilst regeneration occurs over 2 weeks,

surface area is needed.

Lehmann: If you harvest cells from the pig you may not get a culture of single
hepatocytes. What procedure do you use to separate Kupffer cells,
endothelial cells, etc, from the cell mixture obtained from the

disintegration of the liver?

Bader: We do not separate the cells but just co-culture the mixture of cells.
It is useful to keep them because of cellular interactions. Also pig
cells are isolated as aggregates, so separation would be difficult
compared with the rat.

Lehmann: So you do not get overgrowth from fibroblasts?

Bader: Overgrowth of fibroblasts has not been seen over 8 weeks.

NOVEL MINI-BIOREACTORS FOR ISLET CELL CULTURE

A. HANDA-CORRIGAN, I.C. GREEN*, J. MABLEY*, S. HAYAVI,
G.N. KASS, R.H. HINTON, L.M. MORGAN and J. WRIGHT
University of Surrey, UK. and * University of Sussex, UK

Abstract
A brief summary of whole islet transplantation and the use of implants or Bioartificial
Pancreases (BAPs) for the treatment of diabetes is presented. We have recently
developed an alternative BAP design using a macro-porous carrier called Porocell
(Porvair Sciences, UK) for the culture of islet cells. The three dimensional organisations
of islets in Porocell closely resemble whole islets in vivo. We have demonstrated that rat
islet cells are able to survive, secrete insulin and respond to glucose when cultured in
Porocell.

Introduction
Diabetes mellitus comprises a heterogeneous group of disorders in which the regulatory
mechanism of blood glucose control by insulin is impaired. Treatment with insulin
results in a reduction in glucose levels but cannot achieve the degree of regulation of
blood glucose seen in non-diabetic subjects. An ideal long-term treatment is to transplant
islets of Langerhans cells to restore insulin production for maintaining glucose within
normal limits. Since 1990 approximately 150 Type 1 diabetic subjects world-wide have
received islet transplants from cadavers. The islets in most patients were unable to
control blood glucose or had completely lost their activity after 3 years or less [1]. The
alternative to transplantation is to use an implant or Bioartificial Pancreas (BAP)
containing whole or dispersed islets . The salient objective in BAP design is to achieve
long-term blood glucose control with viable & functional islets that do not stimulate the
recipient's immune system.

Various types of BAP devices have been developed over the years. Diffusion chambers
in the shape of discs, hollow fibres and tubular membranes have been tested. Disc-
shaped chambers induce fibrosis and islet necrosis has been reported in hollow fibre
membranes [2]. A wide-bore, tubular membrane diffusion chamber has been shown to
restore normoglycaemia in strepozotocin induced diabetic rats for 150 days, without
immunosuppression [3]. Vascular shunts have direct access to arterial circulation and are
easier to remove after implantation. However implant failure due to vascular thrombosis
is a major disadvantage of a device of this kind [4]. The encapsulation of islet cells in
capsules or gels surrounded by semi-permeable membranes is one of the more recent
developments in BAP design. The semi-permeable membrane protects the cells from
immunorejection and prevents islet cell leakage in the recipient animal. Recent examples
of encapsulated BAPs include modified poly (L-lysine) capsules which have shown to
support islet al lografts in diabetic rodents for up to one year [5]. Jain et al (1996) have
demonstrated restoration of normoglycaemia for over 100 days by implanting

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agarose/collagen macrobeads in streptozotocin-induced diabetic mice [6]. Although
encapsulation offers immune protection to the cells, islet dysfunction and cell necrosis
caused by poor oxygen and nutrient supply remain problematic with this technology. In
addition, evidence is presented here that clearly demonstrates that islets require a
technology which enables them to be anchored in a spatial arrangement which can not be
offered by encapsulation technology.

Techniques For Investigating Rat Islet Survival In Porocell
The isolation and culture of islet cells is both difficult and labour intensive. Poor yields,
viability and purity of islets can be expected unless highly skilled and experienced staff
are dedicated for the isolation and preparative stages. In this study, islet cell viabilities
were in the range of 85-95%, as assessed by the trypan blue exclusion method. Each
Porocell disc (6.2mm x 2mm) was inoculated with whole islets, islet clusters or single
cells. The discs were left undisturbed in an incubator for 1-2 hours prior to addition of
fresh medium. The following techniques were adapted for Porocell - islet studies:

1. SCANNING ELECTRON MICROSCOPY (SEM)
This technique has proved to be invaluable for microscopic examination of islet
morphology and Porocell-islet interactions. Porocell inoculated discs were prepared for
SEM by fixation in glutaraldehyde and subsequent dehydration in ethanol solutions. The
final stage of dehydration in acetone was minimised to 5 minutes because prolonged
acetone exposure caused microscopic “bubbling and pitting” of the Porocell surface.
Using SEM, we were able to show that rat islet preparations of single cells, islet clusters
and whole islets all formed cytoplasmic adhesion extensions directly onto Porocell (Fig.
1). The islet populations in all cases appeared healthy and had securely adhered to
Porocell. Another consistent observation was that of islet adhesion to the underlying
monolayer of fibroblasts (Fig. 2).

671

2. FLOURESCENCE MICROSCOPY

Many combinations of fluorescence stains normally suitable for use with animal cells

could not be used on Porocell because of problems with auto and induced fluorescence.
We have successfully developed a rapid fluorescent staining assay for islet cells on

Porocell which uses a combination of Hoechst 33342 and propidium iodide stains. The
method was carefully optimised so that cut sections of Porocell were first rapidly stained
with propidium iodide to detect dead cells, then each section was fixed in formaldehyde
and stained with Hoechst 33342 to stain the total cell mass. This combination of stains
could rapidly differentiate between live and dead cells on Porocell.

3. CONFOCAL MICROSCOPY

This technique was invaluable for islet cell measurements and for 3-D reconstruction of
islets in Porocell (Fig. 3). It appears that the best islet population for inoculation into
Porocell are single cells containing a mixed population of A, B and D cells. These single
cells re-aggregate on Porocell to form islet-like structures similar to those in vivo.

672

4. INTRACELLULAR INSULIN EXTRACTION FROM ISLET CELLS IN
POROCELL

In this study we have established the assay techniques for measuring intracellular insulin
from islet cells cultured in Porocell. Two methods were developed: (a) Extraction for 18
hours in acid/ethanol mixture and (b) Sonication of each disc for three 20 second bursts,
with a pause of 5 seconds in between each burst. The total insulin extraction method is an
excellent method for estimating the number of B cells retained in Porocell discs because:

The total extracted insulin from Porocell discs, 8 days after inoculation with a mixed
suspension of 1 x 105 single cells was 129.4 ±13.5 pmol insulin/disc. The calculated
number of B cells on the disc is therefore 6.9 ± 0.7 x 104 cells per disc. Therefore, using
this total extracted insulin method, we estimated that 70% of the initial inoculum was a B
cell type and that this cell type was retained in Porocell for 8 days after inoculation. Since
the B cell type usually accounts for 70-80% of the total islet cell population we conclude
that most of these cells were retained in Porocell.

5. INSULIN SECRETION AND RESPONSE TO GLUCOSE

The secretion of insulin in response to challenges with glucose and secretagogues is the
most important quantitative test that determined islet survival and function in Porocell:
Porocell discs float on the medium surface and can therefore be cultured in simple
designed vessels, equipped with filters for gas exchange and magnetic bars for mixing.
We have demonstrated that rat islet cells cultured in Porocell responded to 12 mM
challenges of glucose, by secreting more insulin than that in basal medium supplemented
with a glucose concentration of 2.5 mM. (Fig. 4)

Conclusions

We have demonstrated that the macro-porous material, Porocell can be used for in vitro
culture of rat islet cells. Islet cells adhere directly to Porocell and to fibroblast
monolayers growing on Porocell. Our preliminary investigations show that islet cells
cultured on Porocell in stirred, surface aerated flasks are able to respond to elevated
concentrations of glucose, by secreting more insulin into the culture medium. The future
development of Porocell as an implantable device will require a combination of cell and
material engineering and remodelling. In its present form, the Porocell-islet bioreactor
provides an invaluable tool for research and drug evaluation studies in vitro.

References
1. Lacy et al. Scientific American, July 1995, p 40
2. Altman et al. In Islet-pancreas transplantation and artificial Pancreas, Federlink K, Pfeiffer E,

Raptis, Eds. New York, Theme-Stratton, 1982
3. Lanza et al. Transplant proc 1992, 24, p669-71
4. Maki et al. Diabetes, March 1996, 45, p342-347
5. Jain et al. Transplantation 1996, 61 (4), p532-536
6. Tun et al. Cell Transplantation 1996, 5 (5S1), pS59-S63

We wish to thank BBSRC for funding this study (Grant ref. no. 90/TO6356)

CULTIVATION OF SKIN CELLS SUITABLE FOR RECOVERY OF BURN
WOUNDS

T.D. KOLOKOLTSOVA, N.D. YURCHENKO, N.G.KOLOSOV*,
O.V. SHUMAKOVA, E.A. NECHAEVA
Research Institute of cell culture State Research Centre of Virology
and Biotechnology Vector, Koltsovo, Novosibirsk region, RUSSIA
* Sibirian Military Hospital N 133, Novosibirsk , RUSSIA

1. Introduction
Thermal injuries are-still among urgent problems of surgery, due to the increase in
number of burn patients in the world, especially in industrially developed coun-
tries. At present, for treatment and healing of burn wounds a cleaved autoplast
perforation is most extensively employed, helping to cover the surface of granu-
lated wounds. But the problem of deficiency of donors skin always exists. That is
why researches aimed at the development of new, less traumatic methods of re-
covery of missing skin integument are of high importance.
The method of growing skin cells in vitro with their consequent transplantation
to the wound seems the most promising. Recently it has been shown that not
only auto- but also allokeratinocytes can be used to restore the skin on a burn
wound [1]. At the same time the Russian scientists Sarkisov et al. developed an
entirely new method of treating burns by transplantation of cultivated fibro-
blasts to the wound surface [2].
The present work seeks to produce, to cultivate and to use of scin cells suitable
for recovery of bum wounds.

2. Materials and methods
More than 50 human skin speciaments as material for researchs were obtained
from different sources (burn victims, foreskins, aborted material, cadavers).
Fibroblasts were obtained according to standard methods of fragmentation and
enzymatic treatment of tissue pieces with trypsin solutions. The cell cultivation
was carried out in Eagle MEM nutrient medium supplemented with 10% FBS.
Firbroblast cells were passaged in 3-5 days at 1:2-1:4 seeding coefficient.

Keratinocytes were selected and cultivated accoding to modified Rheinwald and
Green's method [4]; keratinocytes were cultivated in a mixture of DMEM and F-

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12 media supplemented with 5-10% of FBS, insulin, hydrocortison and epi-
dermal growth factor .

3. Results and discussion

Production and cultivation of keratinocytes. During the last two years of work 12
strains of human epidermal cells were produced and cultivated from 20 skin
samples taken from donors of different age and slate of disease. In two cases
when the skin was taken from strongly burnt patients the cell suspension ap-
peared to be low viable, althow the percentage of live cells being 80%; the cells at-
tached to the culture vessel surface badly and practically did not proliferate. In
8 cases the cells were evenly distributed on the glass and formed colonies. At
regular change of the medium in 3-4 days they formed either a mono- or a multi-
layer by clay 20 of in vitro cultivation. Further passage of the cells appeared to be
not a success. The cells grew badly and then died.
Production and cultivation of fibroblasts. In the course of work about 60 strains of
human diploid fibroblasts were produced. 5 cell strains were produced from
skin pieces taken from adults, and 25 strains of fibroblasts were produced from
embrional dermomuscular and lung tissues. 4 cell strains were rejected after bac-
terial or mycoplasma contamination was revealed. In spite of preliminary medical
control of patients after obtaining the primary cell culture fibroblasts, one case
of human hepatitis B virus infection and one case of chlamidial infection were re-
vealed. In the latter case the cells demonstrated good cultural potential and
preserved normal morphology. These data demonstrate the importance of testing
the obtained cell cultures for contaminantion.
The more suitable way of initiation of fibroblasts from adult's derma was the
method of tissue explants. This method allous us to obtain fibroblasts when
there is limited quantity of donor's materials. In this case the cells started to mi-
grate out of the skin's explants on day 7-10. As fibroblasts produced from derma
of adults did not show a high proliferative activity in all the cases and required
the nutrient medium to be enriched and, besides, had a limited life time (up to
20-25 passages in vitro), we used them only for autotransplantation and did not
study their possible use in the future.
Embryo fibroblasts possessed a better growth potentiality as compared with fi-
broblasts of adults’ derma, the proliferation index being 2.0-3.0; the cells
formed an even monolayer on day 2 . As the experience of our work showed,
diploid cells preserved good cultural properties up to passages 45-50 in vitro.
Embryonal fibroblasts cell cultures which had been preliminarily tested for
contaminants and possessed good cultural characteristics were deposited for stor-
age as initial cell culture banks.
The use of keratinocytes and fibroblasts for recovery of burn wound. Transplanta-
tion of autokeratinocytes was carried out in 5 cases. Unfortunately, the experi-
ments were unsuccessful. Cells badly attached on the wound surface and died af-
ter 5-10 days.

675

On the other hand , according to literary data, the use of fibroblasts as an
alternative source of transplanted cells for skin restoration gives a number of ad-
vantages. Fibroblasts are shown to stimutale by synthesis of components of extra-
cellular matrix both adhesion of keratinocytes and proliferation of the latter
followed by differentiation. In this connection transplants of cultivated fibroblasts
can be used in most cases when there is no total lesion of the skin and stimula-
tion of proliferation of the organism’s own epidermocytes in preserved foci
without transplantation of epidermal layers is possible [2]. On the other hand, it
was shown that fetal fibroblasts promote healing of wounds without scar [3].
Lately it has been shown that embryo fibroblasts are not only efficient with re-
spect to epidermocyte stimulation but also actively migrate into the zones of
mechanic lesion of the cell layer unlike analogous cells of adults [5].
In connection with the above it was interesting to study the possibility of use of
human embryo fibroblasts for recovery of burn wounds. For this purpose a
monolayer of fibroblasts grown on a sublayer was transplanted to the wound af-
ter a burn. In patient B., 55 years old, with a flame burn of 8% (3%) of the body
and extremities the wounds of donor sites after autodermoplastics did not close
up and suppurated for a long time. On day 3 after transplantation of allofi-
broblasts the wound clearance of the pus was registered, and complete epitheliza-
tion was observed on day 7. Histologic examination proved the wound close up
with epithelium.
The obtained data confirmed the supposition of D.S. Sarkisov et al. [2] concern-
ing the possibility of use of allofibroblasts in treatment of wounds. Besides,
they demonstrated the high efficiency of application of human embryo fibro-
blasts. As fibroblasts differ from keratinocytes by a higher proliferative activity
and a simpler composition of the nutrient media required for their growth
and support in vitro, we consider it expedient to create a bank of certified
human fibroblast cell cultures suitable for wide application in medicine. Creation
of a cell cultures bank will allow to provide medical studies with a cell material
standardized in biological and genetical properties.

References

1. Wood, E.J. and Raxworthy, M.J. (1994) In vitro reconstruction of human skin, Biochem-
istry 1, 3 -7
2. Sarkisov, D.S., Fyodorov.V.D., Glushchenko, E.V., Alekseev, A.A. et al. (1995) Use of cul-
tured fibroblasts for reconstruction of scin’s cover in strongly burnt patients, Byulleten experimental-
noy biologii i medicini 6, 566-570 (rus.)
3. Rheinwald, J.G. and Green, II. (1975) Serial cultivation of human epidermal keratinocytes: the
formation of keratinizing colonies from single cells, Cell 6, 331-344
4. Sullivan, K.M., Meuli, M., MacGillivray, T.E., Adzick, N.S. (1995) An adult-fetal skin interface
heals without scar formation in sheep, Surgery 118(1), 82-86
5. Kondo Hiroshi, Matsuda Rei, Yonezawa Yumiko (1993) Autonomous migration of human fetal
skin fibroblasts into denuded area in a cell monolayer is mediated by basic fibroblast growth factor and
collagen, In Vitro Cell. and Dev. Biol. Anim. 12, 929-935

TISSUE THERAPY FOR TREATMENT OF PRIMARY MYODYSTROPHIES

Krokhina T.B., Raevskaja G.B., Shishkin S.S..
Research Center for Medical Genetics, Moscow, Russia

1. Introduction.

Myoblast transfer therapy (MTT) is an universal treatment for inhereted
muscular disorders with defect of dystrofin gene - Duchenne and Becker
dystrophies. The main ideas of MTT are: 1) the establishment of primary lines
of myoblasts derived from byopsies from healthy donors; 2) the large-scale
cultivation of donor's myoblasts (MB); 3) injecting of normal donor's MB into
patient's muscles. Donor's MB and patient's myofibers after injection fuse
together and form myofibers with normal dystrophin genes [3]. As a result of
MTT normal dystrophin appears in patient's muscles. This effect was
registrated by immunohistochemical assay of tangential muscle sections from
patient's biopsy.

Muscle is composed of a mixture of cell types present in variable ration.
When muscle is dissociated and the cells plated in culture, both myoblasts
and fibroblasts (FB) are obtained and the rate of proliferation of FB
frequently exceeds that of MB. After several passages of such culture quantity
of myoblasts is decreased [1]. Hight per cent of injected FB ( more than 10%)
leads to immunological regection reaction. That is why the obtaining of pure
noncontaminated by FB myogenic culture is one of the main problems of cell
transfer therapy of myodystrophies. One possibility to resolve this problem
is using of special selective conditions for preferential growth of MB and at
the same time inhibition of FB proliferation.

2. Materials and Methods

Muscle samples were obtained from volonteers during surgical treatment
for orthopedic nonmuscle problems. To isolate cells from biopsy (1g) muscle
was dissected to remove connective tissue and treated with trypsin (0.25%)
and 0.2% collagenase. Dissociated cells were plated on tissue culture dishes
or flasks ("Nunc") in growth medium. After 3-6 day in culture, the medium
containing the debris of dissociated myofibers was removed. The growth
medium contained Ham's nutrient mixture F-10 with 15% fetal calf serum,
MOPS, vitamins, sodium pyruvate and antibiotics. Cell were grown at 37°C and
5% For identification of myoblasts in the mixture we used a standart
cytochemical test for alkaline phosphotase activity (APA).

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3. Results and Discussion
During the cultivation morphology of human muscle cells changes. Three or
five days old culture consists of proliferating mononucleated cells. Then MB
begin to align in preparation for fusion; early MB fusion forms immature
multinucleated myotubes interspersed with mononuclead cells and aligning MB
and at the end multinucleated myotubes are formed. It is one of the best
evidenses of myogenic nature of our culture.
To improve selective growth of MB we used fibroblast growth factor (FGF)
and epidermal growth factor (EGF) - 5 ng/ml. The growth factors were in the
growth medium constantly. MB were cultivated on flasks. Cell number was
counted for some flasks and thier APA was tested every week. The results
of this experiment are presented on Fig. 1,2. As we can see, EGF stimulates
proliferation of our culture more effective then FGF. Moreover, EGF leads to
increasing of per cent of real MB upto 50% compared to control. That is why we
can use EGF in the combinative selective medium in our next investigations.

Then we tried other chemical selective agent - sodium butyrate (SB). SB
delays the expression of differentiation in L6 cell line culture. SB (3mM)
reversibly inhibits the formation of myotubes without affecting the normal
program the formation of differentiation. An almost complete careful arrest in
thymidine incorporation was observed in the presence of 2 mM SB for
fibroblasts and 5 mM for HeLa cells after 24 h exposure [2,4].

We used these data and treated MB and FB cultures from our cell bank by
SB (2,4,8 mM). Like in the experiment with growth factors we cultivated cells on

679

flasks with SB. After 4,9,14 days the amount of cells was counted and
tested for APA. SB depressed FB proliferation stronger then proliferation of
MB. That is why the amount of MB in the mixed culture increases in 1,5 -
2,0 times. We suppose that sodium butyrate would be very useful for selective
medium becouse of SB can decrease not only FB proliferation but can
inhibite MB differentiation. It is very important for large-scaile cultivation of
MB.
Acknowledgments. We thank European Society for Animal Cell Technology
and Russian Foundation for Fundamental Research for the financial support
of participation of Dr.T.Krokhina in The 15-th Meeting of ESACT.

4. References.

1. Blau, H.M.,Webster C., Pavlath G.K. (1991) Purification and proliferation of human
myoblasts isolated with fluorescence activated cell sorting, in R.C.Griggs and G.Karpati (eds.),
Myoblast transfer therapy, Plenum Press, New York and London, pp. 97-100.
2. Kruh, J. (1982) Effects of sodium butyrate, a new pharmacological agent, on cells in culture,
Molecular and Cellular Biochemistry, 42, 65-82.
3. Law, P.K. (1994) Myoblast Transfer: Gene Therapy for Muscular Dystrophy, R.G. Landes
Company Austin, Medical Intelligence Unit.
4. Prasar, K,N. (1980) Butiryc acid: a small fatty acid with livers biological functions, Life
Sciences, 27, 1351-1358.

POSSIBILITY OF APPLICATION OF THYROID ORGAN CULTURE
FOR THE TREATMENT OF PERSISTENT HYPOTHYROIDISM

I.P. PASTEUR, N.D. TRONKO, E.N. GORBAN, V.I. KRAVCHENKO
Institute of Endocrinology and Metabolism, Acad. Med. Sci. Ukraine
Vyshgorodska Str. 69, 254114 Kyiv, Ukraine

1. Introduction

Persistent hypothyroidism is rather a frequent after-effect of an excessive surgical
intervention on the thyroid gland, and it requires an additional treatment with L-
Thyroxine for a long period of time, and even for life [1]. Hormonal therapy of
persistent hypothyroidism using transplantation of thyroid tissue represents an
attractive alternative, because it may deliver such patients from a long-term
pharmacotherapy [1-3]. Reports already exist on clinical use of autotransplantation of
cryopreserved thyroid gland in patients with postoperative hypothyroidism [1-3].
However, all other forms of hypothyroidism (besides postoperative one) exclude the
possibility of autotransplantation of thyroid gland.

Moreover, transplantation as a method of treatment faces today an important and
increasing problem: that of shortage of donor organs and tissue [4]. A number of in-
vestigators try to resolve this problem, considering that xenotransplantation combined
with modern immunosuppressive therapy is a quite realistic way out of this situation
[4]. Porcine tissue is considered as one of the most suitable donor material. The anti-
genie affinity between human and porcine tissue proteins and blood proteins makes
these animals quite convenient donors, and a preliminary organ culturing allows to
significantly decrease the immunogenic properties of transplantation material [5].

2. Materials and Methods

Newly removed newborn pig thyroid gland were cut into pieces (less than
washed and cultured in medium 199 with a 10% calf serum (“Sigma”, USA) at 37 °C.
By the end of each term of culturing, aliquots of medium were selected for quantitative
determination of thyroxine and triiodothyronine in them by radioimmunoassay. After
this, 74 kBq of 131-I were introduced in each sample for 90 minutes in order to assess
the level of iodine uptake by thyroid tissue using radiometric method. Statistical pro-
cessing of experimental results was performed using methods of analysis of variance.

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3. Results

The investigations carried out by the authors showed a capacity of newborn pig
thyroid organ culture to actively uptake iodine from cultural medium during a long

period of time (Table). A high functional activity of thyroid tissue in vitro has also

been confirmed by the results of a quantitative determination of thyroid hormones

(thyroxine and triiodothyronine) in cultural medium. The levels of these hormones

were inversely proportional to the terms of culturing. Contrary to this, T4/T3 ratio

increased from (on day 3 of culturing) to (on day 20).

4. Discussion

Thyroid transplantation requires a functioning thyroid follicle, whose structure inclu-
des follicular cells, follicular colloid, basement membrane and capillaries, as a mini-
mum unity ensuring thyroid function [3]. For synthesis and secretion of hormones into
an appropriate medium, thyroid tissue of a maximum size of pieces up to 1 (such
a size prevents central necrosis in each tissue sample) is quite sufficient and it guaran-
tees good results in transplantation of thyroid tissue [6,7]. Moreover, thyroid tissue
transplantation instead of thyrocyte transplantation is a more simple process from
technical point of view [1,3].

Iodine uptake by thyrocytes represents the first phase of thyroid hormones´ biosyn-
thesis and has a diagnostic impact, since iodine accumulation at the transplantation
site confirms, in addition, the viability of thyroid tissue graft [1-3,8]. The radioisotopic
criteria of the viability of thyroid tissue graft are the following: scintigraphically -
radioisotopc accumulation at the transplantation site [1-3], radiometrically - a more
than 4-fold relationship between its accumulation by transplantation region and
control one (i.e. symmetric part of body) [5,8]. The amount of radioiodine accumu-
lation is considered as a qualitative index of graft viability [5]. Our results also
confirmed a report on the capacity of thyroid cells of pig in vitro to actively produce
thyroid hormones [9].

683

When using results of such investigations in experimental and clinical transplanto-
logy, it should be taken into account that the degree of hormonal compensation of
hypothyroidism and real amount of radioiodine absorbed by a thyroid graft depend on
its functional activity at the moment of study, on the amount of transplanted tissue and
level of thyrotropic hormone which circulates in the blood of the recipient [5].

Though functioning of an additional source of thyroid hormones is recognized as
the main mechanism of therapeutic effect of transplantation in case of hypothyroidism
[1-3], one may not exclude a possible stimulating effect on the recipient’s thyroid
gland [2]. So, a study of iodine-accumulating function of a thyroid gland stump in
patients with postoperative hypothyroidism showed that, after autoimplantation of
cryopreserved thyroid parenchyma, a progressive increase of the capacity for iodine
accumulation was observed [2].

An increase in T4/T3 ratio in the process of culturing, in the presence of a
progressive decrease of indices of both hormones’ secretion, points out the capacity of
thyroid tissue, under unusual conditions for itself, to maintain on a higher level (in the
first place) thyroxine secretion as the main thyroid hormone, what is evidence of its
high adaptive properties.

Therefore, the newborn pig thyroid organ culture preserves its functional activity
for a long period of time and it may be used in transplantology.

5. References

1. Shimizu, K., Nagahama, M, Kitamura, Y., et al.: Improvement of thyroid function after autotransplantation
of cryopreserved thyroid tissues in rats: clinical application of the procedure to patients with persistent
hypothyroid Graves’ disease after thyroidectomy, Thyroidol Clm. Exp., 8 (1996), 55-62.

2. Puchkar, N.S., Makedonskaya, V.A., Utevsky, A.M., et al.: Autoimplantation of cryopreserved
(-196°C) thyroid parenchyma as a method of treatment of post-operative hypothyroidism, Problemy
Endokrinologii (Problems of Endocrinology) (Moscow), 30 (1984), 42-46.

3. Shimizu, K., Nagahama, M., Kitamura,Y., et al.: Autotransplantation of cryopreserved thyroid tissues for the
treatment of irreversible postoperative hypothyroid Graves’ disease. Report of the first case, Thyroidol. Clin.
Exp., 9 (1997), 23-26.

4. Kemp, E.: Xenotransplantation, J. Intern. Med. 239 (1996), 287-297.
5. Lafferty, K.J., Cooley, M.A., Woolnough, J., and Walker, K.Z.: Thyroid allograft immunogenicity is reduced

after a period in organ culture, Science, 188 (1975), 259-261.
6. Bauer, M.F., and Herzog, V.: Mini organ culture of thyroid tissue: a new technique for maintaining the

structural and functional integrity of thyroid tissue in vitro, Lab. Invest., 59 (1988), 281-291.
7. Kitamura, Y., Shimizu, K., Nagahama, M., and Shoji, T.: Cryopreservation of thyroid pieces - Optimal

freezing condition and recovery, J. Jpn. Surg. Soc., 95 (1994), 14-20.
8. Fischel, R.J., King, N.J., Boyle, E.M., et al.: Evaluation of xenogeneic thyroid transplants as a model of cell-

mediated response, Transplant. Proc., 24 (1992), 535-536.
9. Gruffat, D., Venot, N., Marriq, C., and Chabaud, O.: Thyroid hormone synthesis in thyroglobulin secreted by

porcine thyroid cells cultured on porous bottom chambers. Effect of iodide, Endocrinology, 131 (1992),
2921-2927

HUMAN CELLS AS THERAPEUTIC AGENTS

H. GREEN
Harvard Medical School
Boston, MA 02115, USA

Abstract: Cell culture in the modem sense is about fifty years old and has been
indispensable for the development of modern biology. In addition, cultured cells have
been used for the production of some of the proteins encoded by recombinant DNA
and in this way have acted as synthetic machinery for the manufacture of therapeutic
proteins. Of more recent history is the use of cultured cells themselves as therapeutic
agents. There is a wide gulf in thinking and in practice between these applications.
Examples will be described of the cultivation and grafting or implantation of cells to
treat disease.

Discussion

Aunins: I was surprised in a recent talk by Advanced Tissue Sciences on

their skin graft product, when they said that they freeze down their

product without cryopreservatives, and that most of the cells in
their first generation product are dead. Can you comment on those
grafts, and why they are successful?

Green: That product contains fibroblasts; there are no epidermal cells, and
it is mainly designed for use as a temporary replacement for the
surface before conventional grafts are added later. So, it is not a
similar product. The epidermal cells are also not capable of
multiplication after cryopreservation but the products they put out
are obviously stable enough to carry out the desired objective.

Partridge: When corneal grafts are done, do the cells repopulate the limbus?

Green: Yes, they must do so. After they have regenerated the corneal
epithelium by a graft, they are sometimes obliged to do a corneal
transplant because the corneal stroma has been damaged. So they
remove the whole cornea and put a new one down and that gets re-

epithelialised by normal corneal epithelial cells The only place that
they could come from would be the perimeter and that is a re-
establishment of the limbus.

Hatzfeld: Do you know anything about the stem cells of keratinocytes, and
about ageing during the many divisions which occur when you do
these grafts? If you have ageing, as seen in many other types of

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686

cells, does it have any influence on the quality of the skin? I have
heard that some of the burns patients with grafts cannot go into the
sun, and that the skin is very fragile. Can you improve this by
purifying stem cells and using early progenitors?

Green: It is not easy to answer those questions. The stem cells are present
in the basal layer and, if the cultures are properly done, you will
preserve them for a long period because the culture life is of the
order of 150 cell generations. If the cultures are not in a good
condition when they are applied, they may take but give rise to an
atrophic epidermis. That is avoidable by having the best possible
cultures. As far as sun exposure is concerned, if the cultures are

performed fresh, ie not cryopreserved, the melanocytes will re-

populate and so should not be sunlight sensitive. Unfortunately the
melanocytes do not survive cryopreservation, so there would not be
many in a preserved culture. Perhaps what you are referring to is
heat intolerance, which is a different matter. This is because they
lack sweat glands. In third degree burns, sweat glands are destroyed
and there is no way of restoring them, even by conventional
grafting.

Handa-Corrigan: We have recently started culturing keratinocytes in perfusion
culture. At the moment we are obtaining fibroblasts from cadavers.
Would you advise me to change this procedure?

Green: There are additional problems of cells from this source imposed by
safety. The human keratinocytes do better on 3T3 cells, than on
human fibroblasts, because they are not so firmly attached to the
dish. It is necessary when the keratinocyte colonies expand to
excavate the 3T3 cells from the surface - they are displaced, so at
confluency there are virtually no 3T3 cells left. Human fibroblasts
prevent colony expansion to a degree.

Grammatikos: How long can you keep a patient alive without grafting, and what
other treatments can a hospital use?

Green: The first thing that kills is dehydration and loss of electrolytes, but
this is now an exact science and patients are protected from this.
They may also die if the lungs are burnt. The other factor is
infection later on, which always occurs, but here antibiotics play an
important role. Cultured epidermal cells in large enough quantities
are usually not available for 3 weeks, but it takes a good deal of
that time to stabilise the patient sufficiently for surgery to be
undertaken. In good hospitals patients are easily kept alive for that
period but the longer it goes on, the more that infection becomes a
problem.

SESSION ON:

USE OF ANIMAL CELLS FOR IN VITRO TESTING

Increasing links between animal cell technology and biomolecular screening were
reflected by numerous contributions which have been submitted, finally leading to a
session dedicated to the use of cells for screening in Vilamoura, in 1996, and a
subsequent one in Tours, in 1997, which is featured in this chapter.
Biomolecular screening is, like animal cell technology, a highly interdisciplinary and
technology-oriented discipline. It integrates well in biological hazard assessment,
pharmacology and pharmaceutical lead finding. The use of animal cell cultures within
these screening assays is steadily increasing and, therefore, the obvious common interests
and problems suggest the exploration of synergies in both fields.
With related goals in mind, both disciplines are using organ resembling and other
functional celluler models to investigate and manipulate cellular transport as well as
membrane functions and cellular signalling/regulation. One group is focusing on using
cells for the production of biomolecules whilst the other is utilizing the cellular product
as a reporter signal. The techniques applied for process analytics by the cell technologists
and for analyzing biologically active substances by the screening people are also very
similar. Both apply related staining methods in combination with microoptical devices as
well as physical methods including electrical, optical and others for similar sensors
despite their different geometry.
Today, cell technologists are probably ahead when controlling the physiological status of
cultured cells in order to optimize their processes whereas screening people may be
advanced in observing cellular responses for examining physiological effects of their
substances. Each group can learn from the other and cross-fertilize the generation of
ideas, which again will hopefully promote the technology development within both fields.
This session, dedicated to the use of animal cells for in vitro testing, was designed to give
an wide overview on the field covering new cell lines for routine in vitro screening of
pharmacological functions, toxicology, and tissue-resembling culture systems. Several
examples of the above mentioned numerous technological improvements were presented
in this or in other sessions; this attribution was performed in essential where the contents
fitted better to the session.

G. Barlovatz-Meimon, W. Scheirer
Chairpersons

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EMBRYONIC STEM CELL DIFFERENTIATION MODELS: CARDIO-
VASCULAR, MYOGENIC AND NEUROGENIC DEVELOPMENT IN VITRO

A.M.WOBUS, K. GUAN, J. ROHWEDEL, C. STRÜBING* and M. DRAB#

In Vitro Differentiation Group, IPK Gatersleben, D-06466 Gatersleben,
*Institute of Pharmacology, FU Berlin, D-14195 Berlin,
#Franz-Volhard Clinic, MDC, D-13122 Berlin-Buch,
Germany

Abstract

Embryonic stem (ES) cells, totipotent cells of the early mouse embryo, established as

permanent cell lines, provide one of the most important developmental systems for the

introduction of preselected genes into mice by using the gene targeting technology. ES

cells when cultivated as embryo-like aggregates, so-called 'embryoid bodies', are able to

differentiate into derivatives of all primary germ layers, endoderm, ectoderm and

mesoderm.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

690
Differentiation protocols for the in vitro development of ES cells into cardiomyocytes,
skeletal muscle, neuronal and vascular smooth muscle cells were established, and a
developmentally controlled expression of genes, proteins, ion channels, receptors and
action potentials was observed. The in vitro differentiation system is now being used for
genetic analyses by 'gain of function' and 'loss of function' approaches and for studying
regulation of differentiation by exogenous compounds.

1. Introduction

Embryonic stem (ES) cells, totipotent cells of the early mouse embryo were established
as permanent lines which retain their developmental capacity in vitro (Fig. 1; Martin,
1991; Evans and Kaufman, 1981; Wobus et al., 1984; Doetschman et al., 1985; Wobus
et al., 1991). After retransfer of ES cells into the mouse blastocyst, ES cells in vivo are
able to generate cells of all lineages including the germ line (Bradley et al., 1984).
Therefore, ES cells provide an important cellular system for introducing preselected
genes into mice by the gene targeting technology: Mutations introduced into ES cells by
homologous recombination are transferred into the germ line and result in the
generation of mice strains containing specific genetic defects (Thomas and Capecchi,
1987; see Fig. 1).
In addition, ES cells differentiating in vitro via embryo-like aggregates, the so-called
'embryoid bodies', develop into cellular derivatives of all three primary germ layers of
endodermal, ectodermal and mesodermal origin. Undifferentiated ES cells develop
from early stages into terminally differentiated and specialized cells of the cardiogenic,
myogenic and neurogenic lineage. During this differentiation we found a

691
developmentally controlled expression of genes, ion channels and receptors (Maltsev et
al., 1993; 1994; Rohwedel et al., 1994; Strübing et al., 1995). Recently, we also
described epithelial differentiation and the in vitro formation of vascular smooth muscle
cells (Bagutti et al., 1996; Drab et al., 1997). Other laboratories established the
differentiation of ES cells into haematopoietic and endothelial cells (Risau et al., 1988;
Wiles and Keller, 1991; Weiss et al., 1994).
This controlled in vitro developmental pattern may be modulated by exogenous factors,
for example, retinoic acid (Wobus et al., 1994; 1997b), or by genetic means: the
targeted inactivation of genes (= "loss of function", Fässler et al., 1996) or the
overexpression of genes (= "gain of function", Rohwedel et al., 1995).

2. In vitro differentiation of ES cells

2.1. DIFFERENTIATION PROTOCOLS

The ES cell differentiation protocol is shown in Fig. 2 and the details have been
described in detail elsewhere (Wobus et al., 1991; Maltsev et al., 1993; Wobus et al.,

1997a). In principal, ES cells are cultivated in hanging drops for two days, and after
transfer into bacteriological plates, cultivated in suspension for additional two to five

days. During this process the ES cells differentiate in the embryoid body (EB) and build
up differentiated cellular structures of endodermal, ectodermal and mesodermal origin.
The EBs are plated at day 4 (neuronal cells), 5 (cardiac or myogenic cells) or 7 (cardiac
or vascular smooth muscle cells) and the differentiated cells are growing out (for details

692
see Wobus et al., 1991; Rohwedel et al., 1994; Maltsev et al., 1994; Strübing et al.,
1995; Drab et al., 1997).
During in vitro development of EBs the expression of cardiac-, muscle-, neuron-,
epithelial and vascular smooth muscle-specific genes and ion channels was analysed by
RT-PCR and by patch clamp analysis, respectively (reviewed by Wobus et al., 1997a).
The formation of proteins was estimated by immunofluorescence on differentiated cells
of embryoid body outgrowths.

2.2. FACTORS THAT INFLUENCE IN VITRO DEVELOPMENT

The differentiation efficiency of ES cells is determined by several parameters; (i) the
number of cells differentiating in the embryoid body, (ii) culture conditions, media and
additives, the quality of fetal calf serum, and (iii) the time of plating the EBs. In
addition, different ES cell lines show a different developmental pattern in vitro. To
obtain maximal differentiation of ES cells into defined cell types specific differentiation
conditions and cell lines were used (see Wobus et al., 1997a).

3. ES cells differentiate into cardiogenic, myogenic, neurogenic, epithelial and
vascular smooth muscle cells in vitro

In the following chapter a short overview about the characterization of ES cell-derived
phenotypes is given. The specific genes, proteins and ion channels are explained in the
legend to Figure 3.

693
Cardiogenesis: ES cells of several lines (D3, Doetschman et al., 1985, R1, Nagy et al.,
1991, and CCE, Wiles and Keller, 1991) differentiate via EBs into clusters of
spontaneously beating cardiomyocytes. During differentiation, ES cells develop into
mesodermal progenitor cells and early cardiomyocytes which further specialize into
cardiac cells representing atrium-, ventricle- and pacemaker-like cells (Maltsev et al.,
1993). They are characterized by developmentally controlled gene expression,
formation of proteins and receptors as well as cardiac-specific ion channels (Fig. 3;
Miller-Hance et al., 1993; Maltsev et al., 1993; 1994; Wobus et al., 1997a; Hescheler et
al., 1997). Furthermore, we found that the differentiated cardiomyocytes respond with
characteristic chronotropic responses to cardiotropic drugs (Wobus et al., 1991).
Therefore, in vitro differentiated cardiomyocytes were used to establish a semi-
automatic computer-assisted imaging system for a routine screening of chronotropic
cardioactive drugs (Pich et al., submitted).
Myogenesis: Myogenic differentiation of ES cells into skeletal muscle cells is shown by
the formation of myoblasts which during terminal differentiation fuse into
multinucleated myotubes. During myogenic development, muscle-specific genes,
proteins and ion channels are time-dependently expressed. The data obtained from ES
cell line BLC6 are summarized in Fig. 3 (Rohwedel et al., 1994; Rohwedel et al., 1995;
K. Guan, unpublished data).
Neurogenesis: ES cells of line BLC6 differentiate after induction with retinoic acid
(RA) into neuronal cells which express neuron-specific genes and possess the complex
electrophysiological and immunocytochemical properties of postmitotic nerve cells
(Strübing et al., 1995; Bain et al., 1995). Data about gene expression, formation of
neurofilament proteins and synaptic vesicle proteins (synaptophysin), voltage-

694
dependent ion currents and neuron-specific receptor-operated ion channels are

presented in Fig. 3. In addition to the expression of specific neuronal receptors neuronal
cells generate Na+-driven action potentials and are functionally coupled by inhibitory

(GABAergic) and excitatory (glutamatergic) synapses as revealed by measurements of

postsynaptic currents (Strübing et al., 1995; Wobus et al., 1997a).

Epithelial cell differentiation: One of the most prominent cell types in the ES cell-

derived embryoid body outgrowths are epithelial cells. Genes and proteins

characteristic for early (K8, K18, K19), intermediate (K14) and terminal (involucrin)

differentiation of epithelial cells are expressed (Fig. 3; Bagutti et al., 1995).

Vascular smooth muscle cell differentiation: A complex cell type which differentiate

from ES cells, are spontaneously contracting vascular smooth muscle (VSM). We

established a specific differentiation protocol by using RA and db-cAMP for the

induction of VSM cells (Drab et al., 1997). RT-PCR and immunostaining confirmed the

expression of VSM cell-specific transcripts and VSM-specific MHC proteins. In

addition, smooth muscle cells expressed typical ion channels and responded to specific

agonists with an increased intracellular release (Fig. 3; Drab et al., 1997).

4. Summary and conclusions

The establishment of ES cell differentiation models allowed to study cellular
differentiation processes during early embryonic development in vitro. The
developmental systems permit the analysis of differentiation of early embryonic cells

695

via progenitor cells into highly differentiated and specialized cells of the

cardiovascular, myogenic and neurogenic lineages.

With respect to cardiogenic development, the ES cell differentiation system is suitable

for a routine screening of pharmacological functions on differentiated cardiomyocytes

(Wobus et al., 1991; Wobus et al., 1997b; Pich et al., submitted). Furthermore, the

effects of growth and differentiation factors or extracellular matrix proteins may be

investigated (Wobus et al., 1994; 1997b; Johansson and Wiles, 1995).

In addition, the differentiation of genetically modified cells by "gain of function"

(Rohwedel et al., 1995) and "loss of function" (Fässler et al., 1996; Wobus and Guan,

submitted) of totipotent ES cells in vitro is an excellent alternative and substitute to in

vivo studies with transgenic animals to analyse the phenotypes of mutant cells during

early embryogenesis.

Acknowledgements
We wish to thank the Deutsche Forschungsgemeinschaft (Wo 503/1-3, SFB 366/YE1)
and Fonds der Chemischen Industrie for financial support of our research projects.
Jürgen Rohwedel present adress: Institute of Medical Molecular Biology, University of
Lübeck.

696

Permanent ES cell lines were cultivated from the inner cell mass (ICM) of mouse
blastocysts. These pluripotent ES cells are able to differentiate via embryoid bodies into
derivatives of the endodermal, ectodermal and mesodermal lineage. In addition, ES
cells were used to inactivate genes by homologous recombination (Thomas and
Capecchi, 1987). After retransfer of these mutant ES cells into the blastocyst, the cells
can colonize the ICM and, after transplantation into pseudopregnant foster mothers
result in chimaeric animals showing different phenotypes. If the mutation results in
early embryonic death (i.e., integrin-deficient embryos die around day 5 of
pregnancy, see Fässler et al., 1996), the in vitro "loss of function" approach is an
alternative to analyze effects of the specific mutation on cellular differentiation.

697

ES cells (ESC) were differentiated as embryoid bodies (EBs, schematical picture) in
hanging drops for two days, and after further cultivation in suspension, plated between
day 5 or 7 (depending on the differentiation into the specific cell lineage). EBs attach to
tissue culture plates and differentiated cells develop in the EB outgrowths. The cells are
characterized by a controlled expression of genes and proteins as well as functional
properties.

698

699
Fig. 3: Developmentally controlled expression of tissue-specific genes, proteins and ion

channels after differentiation of embryonic stem cells into cardiac, skeletal muscle,

neuronal, epithelial and vascular smooth muscle cells in vitro. ,
Cardiogenesis:

ANF (atrial natriuretic factor), MLC-2v (ventricular

isoform 2 of myosin light chain), MHC (myosin heavy chain); cardiac-specific ion
currents:

Myogenesis: Myogenic determination genes Myf5, Myogenin, MyoD and Myf6,

nicotinic acetylcholine receptors skeletal muscle-specific L-type and

channels

Neurogenesis: NFL (68 kDa), NFM (160 kDa) and NFM (200 kDa) neurofilament
proteins; neuron-specific voltage-dependent ion currents:

neuron-specific receptors:

Gly (Glycin), Kai (Kainate), NMDA (N-methyl-D-aspartate),

Epithelial differentiation: K8, K10, K14, K18, K19 (keratins 8, 10, 14, 18, 19), inv

(involucrin),

Vascular smooth muscle cell differentiation: V-SM-MHC-A (vascular smooth muscle

myosin heavy chain A), I-SM-MHC-B (intestinal smooth muscle myosin heavy chain

B), (smooth muscle ), A II-R (angiotensin II-receptor), PDGF AB

(platelet-derived growth factor AB),

700

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Discussion How do you get the smooth muscle cells to contract spontaneously?
Bader:
Wobus: The embryonic bodies were differentiated from a totipotent stem cell
line and specifically induced by retinoic acid and dibutyrl c AMP in a
Singhvi: very defined window of embryonic development. You can induce
Wobus: different cell types with retinoic acid but it depends upon the
concentration and the time during development on how the retinoic
acid acts. We use very defined protocols of medium, number of cells,
etc. which are specific for cardiac, or neuronal, cells.

What sub-stratum was used for cell out-growth, and was it different
for the different types of cells?

It is not different for the different lineages. We use gelatin (0.01%)
coated tissue culture plates just to get an efficient adhesion of cells.
The main characteristic of the embryonic stem cell is totipotency, the
capacity to differentiate into cells of different lineages.

RESPONSES OF HUMAN LUNG EPITHELIAL CELLS (A549) TO
PATHOGENIC INFECTION BY MYCOPLASMA PNEUMONIAE

A549 cells and Mycoplasma pneumoniae.

GOODMAN J., MORLEY K., PACKER P., BATTLE T.
CAMR, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK.

1. Abstract

Mycoplasma pneumonia have been shown to be infectious to the human respiratory
system. The cells were grown in 'Transwells' to investigate whether they could be used
in a new in vitro model of mycoplasmal infection. Trans-Epithelial Electrical Resistance
(TEER) of M. pneumoniae-infected A549 cells, laser-beam counting (FACS) and PCR
were used for monitoring infection. Mycoplasma orale served as a non-pathogenic
control. Three hours after inoculation with M. pneumoniae, TEER Values showed a
marked disruption of the A549 monolayers combined with an increase in the number of

mycoplasma. In comparison M. orale caused no significant change to the monolayer.
The number of mycoplasma was calculated from standard curves (FACS). Elimination
of external M. pneumoniae and PCR of the infected A549 cells, produced specific bands

of amplified DNA. Thus suggesting that the cells become penetrated.

2. Introduction

Mycoplasma are the smallest free living, self-replicating type of bacteria known. An

individual cell can range from in diameter and a colony varies between 10-

in diameter (Kenny,G. 1985). M. pneumoniae is pathogenic to humans. It
causes primary atypical pneumonia, accounting for 10% of all X-ray proven pneumonia.

It is also responsible for other minor respiratory illnesses such as pharyngitis. As a

consequence it carries much scientific interest. The main aim of this project was to

investigate an in vitro test for mycoplasma infection using M. pneumoniae and human
lung epithelial cells (A549) with M. orale as a non-pathogenic control. The cells were
grown on Transwell systems (Costar). The cells grew on a collagen-coated, porous
membrane that was suspended 1mm above the base of the well, creating compartments

above and below the cell monolayer. The responses of individual cell monolayers to
infection were measured by Trans-Epithelial Electrical Resistance (TEER). The

untreated confluent cell monolayer has a specific resistance if this monolayer is altered

in any way then the resistance will change. Counting mycoplasma is difficult and time
consuming as they can only be seen through a light microscope when they are grown on

agar plates. The use of Fluorescent Activated Cell Sorter (FACS) was therefore
investigated. An invasion assay aimed to determine the location of M. pneumoniae
during infection.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

706

3. Materials and Methods

A549 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) purchased
from Sigma chemical co., which was supplemented with 10% foetal calf serum and 1%
L-Glutamine. Mycoplasma were grown in mycoplasma broth prepared in the Culture
Collection.

3.1 TRANSWELLS

The bottom compartment was filled with 0.5 ml of DMEM. A volume of 0.5ml of cell
suspension was grown on each membrane until reaching confluency, in 5%
atmosphere at 37°C. Mycoplasma were added to the cells at a concentration of

in their exponential growth phase.

3.2 TEER

Using a Millicell-ERS system, three Transwells were measured between 30-330 minutes
after infection and each individual Transwell was measured in triplicate to ascertain the
accuracy of the measurements. The changes in resistance were compared to the original
resistance of the same cells before infection.

3.3 FACS

Standard curves were set up from a serial dilution of mycoplasma of known
concentration (standard curves). The number of counts in 60 seconds against the Log of
concentration was plotted. M. pneumoniae was inactivated by 0.6% v/v formaldehyde,
before use in the FACS machine. Samples were taken, in duplicate, from above and
below the cell monolayer at each time interval and counted in the FACS machine.

3.4 INVASION ASSAY

The external mycoplasma were eliminated by kanamycin in DMEM. The
solution was removed after two hours and the cells washed three times with phosphate

buffered saline (PBS). The cells were then lysed by 0.1% TritonX-100. The DNA was

extracted using Qiagen QIAamp blood kit. A polymerase chain reaction was carried out
on the extracted DNA sample using M. pneumoniae specific primers.

4. Results

The stability of uninfected A549 cells over time was ascertained. The Ohms. were

calculated using the surface area of the Transwells The values for the

untreated A549 cells were stable between 30 and 330 minutes; the resistance did not

increase above (Graph 1). The mean TEER profile of three experiments

of M. pneumoniae infected A549 cells showed a change in resistance. The resistance

707

decreased gradually after infection and reached the lowest value of at

210 minutes (Graph 1). This can be compared to the control profile of A549 cells which

displays around at 180 minutes.

The experiment was carried out on M. orale, a non-invasive mycoplasma, in order to
confirm that the TEER profile of M. pneumoniae was due to infection. The profile of

M. orale was similar to that of untreated A549 cells. The values were stable and the
resistance did not fall more than -30 Ohms.cm2 the profile being very close to zero

at 180 minutes (Graph 2).

i

708

4.1 FACS COUNTS

The concentrations of the mycoplasma samples were calculated from the standard
curves. Two separate counts experiments showed a similar pattern for M. pneumoniae
over time. The numbers in the top compartment in the first experiment began to

increase at 90 minutes, reaching a peak of at 150 minutes. The numbers

decreased and levelled out at 180 minutes. The amount in the lower compartment
showed no large change over the infection time apart from a small rise at 210 minutes

(Graph 3).

709

4.2 INVASION ASSAY
The primers used in the PCR experiments were tested for their specificity. A control
PCR carried out on different strains of M. pneumoniae proved that the primers were
specific. M. orale, M. genitalium and A549 cell DNA were also tested on the primers
and none of their DNA was amplified. The invasion assay experiment allowed for a
long infection time, in order to maximise the chance of invasion. The cells were left to
infect the cells overnight for 16.5 hours. The primers amplified DNA that formed bands
at 345bp suggesting that M. pneumoniae DNA was detected inside of the cells.
5. Discussion

TEER has been shown to be a sensitive and reliable method for measuring small
changes to the confluent A549 cell monolayers over a period of time. The profiles of
pattern of change over time produced are important rather than the absolute values. The
TEER experiment on untreated A549 cells demonstrated the stability of the TEER
device. The TEER of M. pneumoniae infected cells portrayed a definite decrease in
resistance, indicating an alteration in the cell monolayer. The subsequent increase in
resistance suggests that the cells recovered and the monolayer is restored. It is not clear
whether the disruption is due to the M. pneumoniae penetrating, attaching to, or passing
through the gap-junctions of the cells. The recovery of the monolayer implies that M
pneumoniae cause only temporary alteration to the cells. The profile of M. orale was
similar in shape to that of the untreated A549 cells, indicating that the addition of M
orale does not physically damage or disrupt the A549 cell monolayer between 30 and
330 minutes. The large difference between the profiles of the two types of mycoplasma
implies that they are behaving differently.
The two FACS counts experiments on M. pneumoniae showed no significant change in

the numbers below the cell monolayer between 30 and 330 minutes. It therefore seems

710
unlikely that they were passing through the gap junctions into the lower compartment.
The peak above the monolayer that occurs in both of the separate experiments has no
obvious explanation. The increase in numbers may simply be due to the growth of M.
pneumoniae. The subsequent decrease in numbers could be due to either the attachment
to or penetration of the cells or to the death of M. pneumoniae. The M. orale FACS
counts experiment showed no peak in the numbers above the cell monolayer. The lower
and upper compartments contained similar amounts that remained stable throughout the
time period (30 and 330 minutes). Since M. orale were also added to the cells in the
same growth phase, it seems unlikely that the rise in numbers of M. pneumoniae were
due to rapid growth. The invasion assays aimed to determine the location of the M.
pneumoniae during infection. The results obtained suggest that the M. pneumoniae do
penetrate the cells. Without photographic evidence such as electron microscopy it is
hard to determine their exact location.

Further studies in this area are needed before any definite conclusions can be drawn
about the process of M. pneumoniae infection. The results obtained from this study
show that M. pneumoniae, a pathogen, temporarily disrupts A549 cell monolayers
between 30 and 330 minutes after infection. It behaves differently to M. orale, a non-
pathogen and comes into contact with the A549 cells.

6. Conclusion

The Transwell systems provided an effective method for studying mycoplasmal
infection of cell monolayers in vitro. The TEER is a sensitive method for monitoring
changes in cell monolayers. M. pneumoniae temporarily disrupts the cell monolayer,
whereas M. orale has no apparent effect. The FACS analysis is an effective way of
monitoring the pattern of infection of the mycoplasma. M. pneumoniae acts differently
to M. orale. The invasion assay experiments suggested that M. pneumoniae either enter
the cells or become firmly attached to the cell membrane.

7. References

Kenny, G. (1985) The manual of clinical microbiology, 4th edition, American Society
of Microbiology, Washington DC,USA.

8. Acknowledgements
This work was in part sponsored by the Department of Health.

Discussion 711
Onadipe:
Did you have any visual confirmation of the disruption of the
Goodman: monolayer?
No, it is very difficult to see them breaking apart. That is why we
Noé: use TEER as it is very sensitive.
Can this be used as a very quick test for detection of mycoplasma
Goodman: infection?
The TEER? Yes, although with M. orali there was no difference
Noé: with TEER, so only pathogenic mycoplasma can be used.
Goodman: Can you state the time-span to perform the test?
Shirahata: 330 min.
Why did you choose the A549 cell line? Can any other cell line
Goodman: derived from lung be used?
We chose a human cell line because it is a human respiratory
Shirahata: pathogen and the A549 was convenient to use at ECACC.
Goodman: Have you tried other cell lines?
Bernard: No.
Did you check whether the amplitude of your TEER response was
Goodman: dependant on the number of mycoplasma particles?
We added mycoplasma to each transwell. We did not try other
concentrations.

POLAR LIPID PROFILING OF MYCOPLASMA PNEUMONIAE-INFECTED
HUMAN LUNG EPITHELIAL CELLS.

Polar lipids and Mycoplasma pneumoniae infection.

GOODMAN J., WAIT R., BATTLE T.
CAMR, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK.

1. Abstract

Some mycoplasma are known to contain novel glycolipids which have no counterparts
in eukaryotic cells. Profiling of polar lipids is thus a potential strategy for the detection
of mycoplasmal contamination of cell lines. Polar lipids were isolated by Bligh-Dyer
extraction, and were analysed by positive and negative ion fast atom bombardment
mass spectrometry (FAB-MS). Individual phospholipid molecular species were
identified by helium collisional activation and linked scanning techniques. Polar lipid
profiles were determined from A549 lung cells, Mycoplasma pneumoniae-infected
A549 cells and pure cultures of M. pneumoniae. Uninfected A549 cell extracts
contained hexadecanoate- and octadecenoate substituted phosphatidylcholines, whereas
the major phospholipids of M. pneumoniae were sphingomyelin, phosphatidylglycerol
and phosphatidylcholines, the latter apparently derived from the culture medium.

2. Introduction

Mycoplasmal contamination is a common problem in experimental cell culture [1], M.
orale, M. arginini, M. hyorhinis, M. fermentans, M. salivarum and Acholeplasma
laidlawii accounting for the majority of instances. Infection of eukaryotic cells by
mycoplasma may have subtle and unpredictable effects, including growth inhibition and
altered physiology and metabolism [1]. Moreover, since several mycoplasma are
pathogenic to humans, contamination of cell lines or cell-derived therapeutic products
poses a potential health hazard. Mycoplasma species have been shown to contain
unusual phospholipids [2], some of which have no counterparts in eukaryotic cells.
Profiling of polar lipids might thus provide a potential strategy for the detection of
mycoplasmal contamination of cultured cell lines, and could possibly also aid diagnosis
of human infections, as current methods of clinical diagnosis are relatively
unsatisfactory. Since fast atom bombardment mass spectrometry (FAB MS) is one of
the most sensitive and powerful methods available for the characterization of polar
lipids, we have used it to investigate the influence M. pneumoniae infection on the
phospholipid composition of a human bronchial epithelial cell line, A549.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

714
3. Materials and Methods

A549 cells were grown in Dulbecco’s modified Eagle medium (DMEM) until

confluent, trypsinized, harvested by centrifugation and freeze-dried. M. peumoniae was
cultured in Mycoplasma broth at for 3-4 days until exponential growth phase,

recovered by centrifugation and lyophilized. For infection experiments A549 cells were

incubated with M. pneumoniae, and harvested as described after 2 or 5

days growth.

Polar lipids were extracted from the freeze-dried biomass by the procedure of Bligh and

Dyer [3]. Fast atom bombardment mass spectra were recorded with a Kratos MS80

RFA spectrometer. Spectra of phospholipids were recorded in both the positive and

negative ion modes using 3-nitrobenzyl alcohol (3-NBA) as liquid matrix.

4. Results

In the positive ion FAB mass spectrum of a Bligh-Dyer extract of A549 cells,
protonated molecules are observed at m/z 732, 760, 786 and 788. An abundant ion at
m/z 184 was consistent with the presence of phosphocholine. Collisional activation
experiments indicated that this ion was a daughter fragment of the protonated molecules
at m/z 732, 760 and 786/788, suggesting that the major species present were
phosphatidylcholines with differing fatty acid substitution patterns. In the
corresponding negative ion spectra carboxylate anions were observed at m/z 253, 255,
281 and 283, attributable to the presence of hexadecenoate (16:1), hexadecanoate
(16:0), octadecanoate (18:0) and octadecenoate (18:1) substituents. Thus the major
phospholipids are phosphatidylcholines substituted with 16:0/16:1 (m/z 732), 16:0/18:1
(m/z 760) and 18:1/18:1 (m/z 786). Minor signals at m/z 718 and 746 were identified as
phosphatidyl ethanolamines by means of constant neutral loss scan for 141, the
characteristic mass of the ethanolamine phosphate head group. Confirmation of these
assignments was provided by analysis of an authentic standard of 1-hexadecenoyl-2-
octadecenoyl-sn-glycero-3-phosphocholine.
The predominant high mass signal in the positive ion FAB spectrum of the lipids
extracted from a pure culture of M. peumoniae was observed at m/z 703, with less
abundant ions at m/z 731, 758 and 786. A fragment at m/z 184 indicated the presence of
phosphocholine containing components; however the protonated molecules at m/z 758,
786 and 808, although consistent with the protonated molecules of phosphatidyl
cholines, are too weak to account for its high abundance. Moreover, in the
corresponding negative ion spectrum the major carboxylate anions were observed at m/z
255 and 283, suggesting that the predominant fatty acyl groups were hexadecanoate and
octadecanoate, rather than octadecenoate substituents originating from phosphatidyl
choline. The major deprotonated molecules in the negative ion spectrum were observed
at m/z 747 and 775, corresponding to phosphatidly glycerols substituted 16:0/18:1 and
18:0/18:1.

715

The unidentified component at m/z 703 was identified by collisional activation and B/E
linked scans. The major daughter ion was observed at m/z 183.9, consistent with a facile
loss of phosphocholine, which suggested that the ion at m/z 703 is the protonated
molecule of a hexadecanoate-containing sphingomyelin. The species at m/z 731 is
presumably an octadecanoate-containing homologue. This observation is consistent
with previous reports of sphingomyelins in the membranes of mycoplasma, including
M. pneumoniae [5]. The phosphatidylcholine composition of mycoplasma broth was
very similar to that of the M. pneumoniae cells, suggesting that exogenous PC was
being directly incorporated rather than synthesised de novo. We did not observe the
modification of endogenous PC to a disaturated species which has been reported in M.
gallisepticum, M. pulmonis and M. pneumoniae [6]. Only trace amounts of
sphingomyelin were detected in the broth, suggesting either that this material is
synthesised de novo, or, alternatively that M. pneumoniae is able to scavenge it with
high efficiency.
After two days infection of A549 cells with M. pneumoniae there was little change in
polar lipid profile compared to uninfected cells. There appears to be no significant
difference in the proportion of sphingomyelin, phosphatidylglycerol, or of fully
unsaturated phosphatidylcholines, suggesting that the population of mycoplasma
remains relatively low, and causes no significant disruption of cellular phospholipid
metabolism.

5. Conclusions.

The polar lipid profiles of A549 cells as determined by FAB MS are stable and
reproducible, and are predominantly comprised of phosphatidylcholines. M.
pneumoniae, when grown in mycoplasma broth, exhibits a polar lipid profile
characterized by phosphatidyl glycerol, sphingomyelins and di-unsaturated
phosphatidylcholines. Co-culture of A459 cells with M. pneumoniae for two days does
not result in any significant change in cellular phospholipid profiles.

6. References.

[1] Hay, R. J., M. L. Macy, and T. R. Chen. 1989. Mycoplasma infection of cultured cells. Nature 339:487-
488.
(2] Matsuda, K., T Kasama, I. Ishizuka, S. Handa, N. Yamamoto, and T. Taki. 1994. Structure of a novel
phosphocholine-containing glycoglycerolipid from Mycoplasma fermentans. J. Biol. Chem. 269:33123-
33128.
[3] Bligh, E. G., and W. J. Dyer. 1959. A rapid method for total lipid extraction and purification. Can. J.
Biochem Physiol. 37:911-917
[4] Jensen, N. J., and M. L. Gross. 1988. A comparison of mass spectrometry methods for structural
determination and analysis of phospholipids. Mass Spectrom. Rev. 7:41-69.
[5] Razin, S., S. Kutner, H. Efrati, and S. Rottem. 1980. Phospholipid and cholesterol uptake by mycoplasma
cells and membranes. Biochim. Biophys. Acta. 598:628-640.
[6] Rottem, S., L. Adar, Z. Gross, Z. Ne'Eman, and P. J. Davis. 1986. Incorporation and modification of
exogenous phosphatidylcholines by mycoplasmas. J. Bacteriol. 167:299-304

COMPARISON OF THREE DIMENSIONAL (3-D) RAT HEPATOCYTE
CULTURES IN SIMULATED MICROGRAVITY CONDITIONS

MAGUIRE T., MOULSDALE H.J., STACEY G., BATTLE T.
CAMR, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK.

1. Abstract

Amongst the existing 3-D culture models, the Rotary Cell Culture System (RCCS)
enables 3-D growth in a unique environment (increased mass transfer and minimal shear
forces). We investigated whether these conditions could generate improved hepatocyte
3-D models as potential tools for in vitro toxicological assays. Cells were seeded into
three types of culture vessel which rotate on a horizontal axis and differ in their
respective chamber dimensions.
Primary and immortalised cells were both observed to respond differently in the
alternative culture vessels. Non-uniform large lobular aggregates (0.5cm diameter) of
primary cells were observed in the High Aspect Ratio Vessel (HARV), whereas the
Disposable Cell Culture Vessel and the Cylindrical Cell Culture Vessel (CCCV) tended
to yield homogenous suspensions of numerous smaller spheroid-like aggregates
provided the appropriate rotational speed was used. Preliminary studies with
immortalised hepatocytes showed variation in the degree of vacuolation in cellular
aggregates between the HARV and the larger CCCV.

2. Introduction

The Rotary Cell Culture System was originally designed by NASA to create a
microgravity environment for the transport of cellular samples during space flight.
However the RCCS is also unique in that it allows for large scale tissue culture with
extremely low fluid shear stress and turbulence, spatial freedom and high mass transfer.
The combination of these factors has subsequently allowed for the culture of many cell
lines in 3-D, including cells which previously did not culture in large scale (Chantret et
al 1988). Hepatocytes cultured as tightly packed freely suspended 3-D spheroids have
previously proved their usefulness for in vitro toxicology.
We investigated whether primary rat hepatocytes could be cultured in the RCCS in order
to create reproducible 3-D models for toxicological assays. We also attempted to
evaluate the factors which govern tissue morphogenesis in the RCCS. Pilot studies with
immortalised cells were also performed.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

718

3. Materials and Methods

Three different types of culture vessel were used with the RCCS. Primary rat

hepatocytes were obtained form a two step collagenase perfusion of Wistar rats (200-

300g) for culture in; The Cylindrical Cell Culture Vessel (CCCV), The High Aspect

Ratio Vessel (HARV) and the Disposable Cell Culture Vessel. The effect of different

seeding densities, between cells per ml and rotational speed on

aggregate formation were also examined. Media samples were taken twice daily to

monitor cell growth using glucose/lactate analysis. The cell culture media used was
William’s E supplemented with albumin 1g/1, insulin 5mg/L and 10% FCS.

An immortalised rat hepatocyte cell line was established by transfection of primary rat
hepatocytes with SV40 large T antigen using strontium phosphate. Its culture within the

RCCS at a seeding density of cells per ml, was also examined.

4. Results

4.1 PRIMARY RAT HEPATOCYTES

In all circumstances aggregate formation was seen within eight hours after seeding. The
type of vessel used and the rotational speed proved to have a profound effect on 3-D

aggregate formation within the RCCS. There was no correlation between seeding density
and aggregate formation. Primary rat hepatocytes, when cultured in the HARV at the
lowest rotational speed (8 rpm) for 24 hours, formed a single giant lobular shaped
aggregate (0.5 cm2 diameter) Khaoustov et al 1995). Increasing the rotational speed did

not prevent the lobular aggregate from forming. Primary rat hepatocytes cultured in the

CCCV and Disposable Cell Culture Vessel formed loosely assembled aggregates after

24 hours. After 36 hours, smooth surfaced spheroids of approximately were

present However a high rotational speed was
essential to prevent spheroids from fusing together. Glucose/Lactate growth curves for
spheroids and giant lobular structure were similar. Histological examination of the

spheroids after four days of culture, revealed a small necrotic zone at the centre.

4.2 IMMORTALISED RAT HEPATOCYTES

Immortalised hepatocytes cultured well within the HARV and CCCV although
aggregates were not visible for 48 hours and did not form very smooth aggregates as
seen with primary cells. The aggregates (0.05-0.1 mm in diameter) were maintained in
culture for five days. Variation occurred in the degree of vacuolation in the cellular
aggregates cultured in the HARV (large vacuoles) and the CCCV (tightly packed).

719

5. Discussion

A microgravity environment is created within the RCCS irrespective of the vessel used.
However, it is clear that due to the particular dimensions of each vessel, different results
may be seen. Interestingly the HARV (55ml volume) was originally designed for the
culture of suspension cell lines. It would appear that in our circumstances the CCCV
(110ml volume) and the Disposable Cell Culture Vessel (50ml volume) are better suited
for the culture of attachment dependant cells. Primary rat hepatoctyes created in the
RCCS are better suited for toxicological model than the giant aggregate as they provide
a greater exposed surfaced area. Necrotic zones forming at the centre of spheroids may
reflect the poorer availability of oxygen and/or nutrients to deeper cells. It is hoped that
future experiments will elucidate the time when spheroids are at an optimum i.e. tightly
packed with a smooth surface and no necrotic zone. Glucose/Lactate analysis suggests
this time may be before 30 hours of culture, the point at which glucose consumption and
lactate secretion stabilise. Previously the culture of spheroids in William's E medium
has proved troublesome due to the fusion of many spheroids together. Our study has
shown that a homogenous culture of hepatocyte spheroids is possible in William’s E
medium in conjunction with the RCCS. Nevertheless it must be determined if spheroids
cultured in the RCCS have enhanced liver specific function notably the increased
secretion of albumin and expression of cytochrome P450 enzymes.
The use of an immortalised cell line for in vitro toxicology has many obvious
advantages such as increased feasibility and reproducibility and avoiding recurrent
sacrifice of animals. Consequently must work is required to characterise the cell line
established.

6. Conclusion

The culture of individual hepatocyte spheroids is possible within the RCCS. The type of
vessel and the rotational speed are fundamental in determining the subsequent tissue
morphology. The RCCS will no doubt provide a useful tool for the generation of

spheroids and for in vitro toxicology.

7. References

Chantret, I., Barbat, A., Dussaulx, E., Brattain, M.G., and Zweibaum, A. (1988) Epitheial polarity, villin
expression, and entererocytic differentiation of cultured human colon carcinoma cells: Asurvey of twenty cell
lines. Cancer. 48:1936- 1942.
Khaoustov, V.I., Darlington, G.J., Soriano, H.E., Krishnan, B.,Risin, D., Pellis, N.R., and Yoffe, B. (1995)
Establishment of 3-dimensiomal primary hepatocyte cultures in microgravity environment .Hepathology Vol
22 No.4 Pt.2

HIGH DENSITY CULTURE OF THE HUMAN HEPATOMA CELL LINE
HepG2: LONG-TERM CULTURE FOR IN VITRO TOXICOLOGY

A. HANDA-CORRIGAN, R.M TRAYNOR, I. ADAMOPOULOS AND
J. SALWAY, University of Surrey, UK.

Abstract
One of the major limitations to the use of cultured mammalian cells for in vitro
toxicological applications is the lack of robust, long term test systems that represent in
vivo responses. Recent advances in maintaining differentiated hepatocyte cultures in
fed-batch and perfused bioreactors offers an important means of extended toxicological
testing in vitro. However, the lack of sufficient animal and human material limits the
possibilities of using hepatocyte culture on an extensive scale for such applications. The
human hepatoma cell line, HepG2 retains many of the differentiated properties seen in
hepatocytes and has been used for many years as an alternative to primary hepatocytes
for in vitro toxicology studies. Despite its increasing use, the growth, functional status
and response of this cell line in high density cell cultures have not been well
characterised in the literature. In this paper we describe the morphological, growth and
biochemical responses of HepG2 cells to the glucocorticoid dexamethasone.
Transmission and scanning electron microscopy were used to define the morphological
and utrastructural status of the cells. Finally, batch and perfused mini-bioreactors were
used for culture of cells at high densities on a novel porous matrix, Porocell. In HepG2
cells cultured at high densities for periods of up to 29 days in Porocell, the
glucocortidosteroid dexamethasone was capable of inducing the production and
secretion of glucose. This response is comparable to the in-vivo effect of the drug.

Introduction
The HepG2 (human hepatoma) cell line maintains some differentiated, morphological
and biochemical characteristics of normal hepatocytes [1,2]. In this study, HepG2 cells
were challenged with the glucocorticosteroid, dexamethasone. Dexamethasone is
prescribed as an anti-emetic to help relieve nausea and vomiting caused by
chemotherapeutic and cytotoxic agents such as cisplatin and digoxin. Other clinical uses
include replacement therapy, anti-inflammatory therapy and immunosupression [3].
Dexamethasone has a relatively high affinity for the glucocorticoid receptor and is
clinically thirty times more potent as an anti-inflammatory agent than hydrocortisone.
The duration of action of the drug is between 36 to 72 hour post oral administration [3].
The long-term effects of dexamethasone dosing in cell cultures have not previously been
investigated. In this paper, HepG2 cells cultured on a novel porous matrix (Porocell,
Porvair Sciences, UK) were challenged with dexamethasone in static, stirred and packed

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722

bed bioreactors. The morphological, growth and biochemical responses of cells
challenged with dexamethasone were investigated.

Materials and Methods

Cell cultures: HepG2 cells were cultured in medium supplemented with 5% foetal calf

serum. Cells were adapted for one month in medium supplemented with dexamethasone

at concentrations of Where stated cells were also challenged with

dexamethasone at concentrations ranging from

Porocell disc culture: Monolayer cultures of HepG2 cells were trypsinized, washed and

concentrated to a density of Batches of six Porocell discs were transferred
to 6 well plates and 0.025ml of the cell suspension was carefully pipetted into each disc.

The discs were incubated at 36.5°C and subsequently transferred to stirred and mini-

packed bed bioreactors. The stirred cultures (50ml working volume) were surface-

aerated and fresh medium was periodically added to the culture. The packed bed

bioreactors were micro-bubble aerated and perfused continuously with fresh medium.

Further details of Porocell culture and microscopical analyses are described by Handa-

Corrigan et al, 1997 [5].

Results and Discussion

1. EFFECT OF DEXAMETHASONE ON HepG2 CELL MORPHOLOGY

HepG2 cells cultured on plastic showed two distinct morphologies, as observed by light

microscopy (Figures 1a and 1b). During the exponential growth phase, the cell

population consisted of (a) flattened cells which formed close contact with each other

and (b) small clumps of rounded cells which were attached to each other and to the

underlying flat cell sheet. During the death phase, the majority of cells had adopted a

rounded morphology and large clumps of cells were observed in culture. Adaptation of

cells to dexamethasone at concentrations of did not appear to affect the

morphology of this cell line. Transmission electron microscopy showed that

dexamethasone adapted HepG2 cells had a similar utlrastructure to cells cultured in

medium without dexamethasone (Figures 2a - 2d).