12_ANIMAL CELL TECHNOLOGY_707

723

M=Mitochondrion, S=smooth ER, G=glycogen rosettes, R=rough ER, N=Nucleus,
Nu= nucleolus, L= lipid droplet
2. HIGH DENSITY CULTURE IN STIRRED AND PACKED BED BIOREACTORS
HepG2 cells were inoculated into batches of 6 Porocell discs (Fig 3a) which were
subsequently transferred to surface-aerated, stirred cultures or into mini packed-bed
bioreactors. Two weeks of culture in stirred and packed-bed bioreactors resulted in
smooth, multi-layered rounded cells, growing deep into and over the porous structure of

Porocell (Fig. 3b). In both bioreactors, the cells were in such close proximity to each
other that cell-cell boundaries were difficult to identify by SEM. Extended culture for 28
days in the packed bed bioreactors showed that the cells had ‘fused’ to form tissue-like
sheets (Fig. 3c).

724

In high density, stirred and perfused cultures of HepG2 cells, the cells did not show any

significant biochemical and morphological changes when challenged with

dexamethasone at concentrations of (Fig. 4a). However, during repeated,

half-hourly exposures to concentrations of dexamethasone, the glucose
uptake rate by the cells was significantly reduced (Fig. 4a) compared with the control at
350 hr. During long-term exposure to dexamethasone

HepG2 cells no longer consumed glucose but were able to synthesize glucose by

gluconeogenesis and secrete it into the culture medium (Fig. 4b).

•

Conclusions
The origin of hepatic glucose production in vivo is either from glycogenolysis or
gluconeogenesis. Dexamethasone induces the regulatory enzymes of gluconeogenesis,

namely: pyruvate carboxylase, phophoenolpyruvate carboxykinase, fructose 1,6-

bisphosphatase, and glucose 6-phosphatase [6]. It is therefore most probable that the

dexamethasone stimulated production of glucose is due to enhanced gluconeogenesis

from substrates in the medium such as glutamine. The ATP needed for gluconeogenesis

is likely to be provided by oxidative phosphorylation coupled to of fatty

acids bound to the serum albumin included in the medium. The vigorous induction of

gluconeogenesis in response to dexamethasone is evidence that all the biochemical

processes are functioning in HepG2 cells cultured in Porocell.

References

1. Wolff, M., Fandrey, J. and Jelkman, W. (1993). Am J. Physiol. 265, p 1266-1270.
2. Knowles,B.B., Howe, C.C. and Aden, D.P. (1980). Science, 209, p 497-499.
3. Rang, H.P. and Dale, M.M. (1991). Pharmacology 2nd ed, Churchill Livingstone, N.Y.
4. Newsholme, E.A. and Leech, A.R. (1983). Biochemistry for the Medical

Sciences, J. Wiley and Sons, Chichester.
5. Handa-Corrigan, A., Hayavi, S., Ghebeh, H., Mussa, N.A. and Chadd, M.

(accepted). Novel porous matrix and bioreactors for high density cultures of
insulinoma cell lines: Insulin secretion and response to glucose. J. Chem. Technol. &
Biotechnol. (December 1997 issue).
6. Mayes, P.A. (1996) in Murray, R.K., Granner, D.K., Mayes, P.A, and Rodwell, V.W. (eds.)
Harper’s Biochemistry, 24th ed., Appleton & Lange.

APPLICATION OF PRIMARY CULTURES OF RAT FETAL NEURONS
TO THE STUDY OF NEUROTROPHIC ACTION OF PEPTIDES

O.V. DOLOTOV, I.A. GRIVENNIKOV
Institute of Molecular Genetics, Russian Academy of Sciencies,
Moscow, Russia, 123642 Kurchatov sq., fax: 7(095)196-0221

1. Introduction
Primary cultures of neurons are extensively used in neurobiological studies

and testing in vitro of compounds for neurotrophic/neurotoxic action. The effect
on neuronal survival may depend on conditions of isolations and cultivation of
neurons. However these factors are not as yet properly investigated.

The subject of our investigations is the study of neuroactive properties of
some new stable analogues of N-terminal fragment of ACTH(4-10) [1, 2]. One
of these was synthesized in the Institute of Molecular Genetics and named
“Semax” [2]. This peptide has the structure: Met-Glu-His-Phe-Pro-Gly-Pro.

Semax penetrates into the brain after intravenous injection [3] or intranasal
application [2]. As of now, no toxic effects of Semax have been reported. The
Russian Pharmacological Committee gave permission for Semax to be used as a
remedy for some mental disorders accompanying brain injury and to improve
the consolidation of memory and the adaptation to hypoxia and ischemia.

To test the hypothesis that Semax might exhibit neurotrophic effects on
nervous tissue, we tried to examine Semax for enhancement of fetal basal
forebrain neurons survival during the cultivation. Because of ambiguity of results
of using of primary neuronal cultures for testing of neurotrophic effects in
literature and from our experience, we tried to reveal factors which might
influence on magnitude of the effects. Part of this investigation is presented here.

2. Methods
1. Cell culture
Fetal (E16-18) rat brain primary cultures were established as described [4],

with some modifications. Cells were plated at 1000 - 2000 cells per mm2.

Composition of culture medium was standard [5]. The tested compounds were
added after cell plating. NGF was applied in concentration 100 ng/ml.

2. Estimation of cell survival

Semi-quantitative estimation of number of living cells was carried out on the
base of determination of cells with neurite length at least equal to the cell body

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726

diameter [6].Measurement of cellular MTT reduction (which reflects
number of living cells) was carried out as described [7], with minor
modifications. Cholinergic neurons were identified using cytochemical
visualization of actylcholinesterase (AChE) as described [4].

3. Results
Initial examination of Semax for neurotrophic activity was quite successful.

Injection of Semax into culture medium enhanced survival of neurons of the rat
basal forebrain by a factor of 1.5 by a week of cultivation. Injection of Semax at
day of cultivation other than first had no effect on neuronal survival.

More recent similar experiments have failed since effect of Semax and NGF
(as positive control) on neuronal survival was not revealed.

In attempt to tregger neuronal death we tried to prolong time period between
isolation of brains and cell plating. Isolated brains were incubated at 5°C (24 h)
and 37°C (30 min) in the solution of isolation and number of living cells were
estimated 7 and 3 days later, respectively, after cells plating. Both NGF and
Semax extremely increased number of living cells (Figure 1). In the case of

immediate isolation of basal forebrains and cell plating Semax and NGF did not
exhibit survival effect (Figure 1).

4. Discussion
Although primary neuronal cultures is widely applied for investigation of

neurotrophins, there is some ambiguity of results. In particular, a number of
investigators have described the survival effect of well-known classic
neurotrophic factor, NGF, on survival of rat fetal basal forebrain neurons in vitro
while others have not revealed this effect (for review see [8]). On the other hand,
there is the set of data describing neurotrophic effects of NGF in vivo [9].

Data presented indicate that cellular processes happening in all stages of
isolation and cultivation of neurons must be given proper weight in designing of
investigations of neurotrophic compounds. We believe that speed and
temperature of isolation are no less important than culture conditions and must
be controlled because they dictates the value of survival effect of the compound

727
of interest. Moreover, there is the risk of musking of action of a neurotrophic/
neuroprotective compound under good isolation conditions.

On the other hand we hope that our data may serve as base for development
of novel model of the pathological state of brain - hypoxia/ischemia which might
occupy place intermediate between recently used models of this state in vivo and
in vitro because it allows to investigate on the cell culture level processes initiated
on the level of the fetal brain.

It counts in favour of this speculations that there is correlation between our
data and clinical anti-insult results obtained for Semax [10].

We believe that there is common base for these effects. It is assumed that
when insult is occurred, a region of apoptotic neurons is formed. However, a
possibility of the reversing of these neurons to normal state with the help of
neurotrophic factor exists. When described here manipulations with fetal brain
are carried out, apoptosis is triggered and neurotrophic factors may prevent
cultivated neurons from activation and/or progression of this process.

We recognize that our data are not sufficient for a clear understanding of
described processes but hope that further study in this direction may be useful.

5. Acknowledgments

Supported by INTAS-RFBR N 95–1246. The presentation of poster was sponsored by ESACT.

6. References

1. Verhoef, J. and Witter, A.: In vivo fate of behaviorally active ACTH(4-9) analog in rats
after systemic administration, Pharmacol. Biochem. Behav. 4 (1976), 583-590.

2. Potaman, V.N., Alfeeva L.Y., Kamensky A.A., Levitzkaya N.G., and Nezavibatko V.N.:
N-terminal degradation of ACTH(4–10) and its synthetic analog semax by the rat blood
enzymes, Biochem. Biophys. Res. Commun. 176 (1991), 741-746.

3. Potaman V.N., Antonova L.V., Dubynin V.A., Zaitzev D.A., Kamensky A.A.,
Myasoedov N.F., and Nezavibatko V.N.: Entry of the synthetic ACTH(4–10) analogue
into the rat brain following intravenous injection, Neurosci. Lett. 127 (1991), 133–136.

4. Hefti F., Hartikka J., and Sanchez-Ramos J.: Dissociated cholinergic neurons of the
basal forebrain in culture, in A. Shahar, J. de Vellis, A. Vernadakis, and B. Haber (eds.),
A Dissection and Tissue Culture Manual of the Nervous System, Wiley-Liss, New York,
1989, pp. 172-182.

5. di-Porzio U., Daguet M.C., Glowinski J., and Prochiantz A.: Effect of striatal cells on in
vitro maturation of mesencephalic dopaminergic neurones grown in serum-free
conditions, Nature 288 (1980), 370-373.

6. Veprintsev B.N., Viktorov I.V., and Vilner B.J.: A Manual on Cultivation of the Neural
Tissue, Nauka, Moscow, 1988, pp. 172-174.

7. Hansen M.B. Nielsen S.E., and Berg K.: Re-examination and further development of a
precise and rapid dye method for measuring cell growth/cell kill, J. Immunol. Methods
119 (1989), 203-210.

8. Nonner D., Barrett E.F., and Barrett J.N.: Neurotrophin effects on survival and
expression of cholinergic properties in cultured rat septal neurons under normal and
stress conditions, J. Neurosci. 16 (1996), 6665-6675.

9. Hefti, F.: Nerve growth factor promotes survival of septal cholinergic neurons after
fimbrial transections, J. Neurosci. 6 (1986), 2155-2162.

10. Gusev E.I., Skvortsova V.I., Myasoedov N.F., Nezavibatko V.N., Zhuravleva E.Yu., and
Vanichkin A.V.: Semax efficiency in acute period of hemispheral ischemic stroke,
Korsakov Zhurn. Nevrol. Psikh. 6 (1997), 26.

DEVELOPMENT AND VALIDATION OF AN IMAGE ANALYSIS SYSTEM FOR
SINGLE CELL CHARACTERIZATION IN CELL MONOLAYERS

D. KAISER, M.A.FREYBERG, G. von Wichert, P. Marenbach, H. Tolle and

P. Friedl, Technical University of Darmstadt, Petersenstraße 22, D-64287
Darmstadt, tel: +49 (6151) 163655, fax: +49 (6151) 164759,
e-mail: dh 7y@pop. tu-darmstadt. de

1. Subject

The characterization of single cells in a monolayer is a necessary and helpful tool for
a variety of applications: determination of cell proliferation, apoptosis and vitality as
well as questions concerning the presence or absence of various cell surface
molecules. Most of these techniques are based on the use of fluorescent dyes or
labeled antibodies. The direct microscopic morphological determination of labeled
cells is a very inconvenient, elaborate, time consuming procedure and cumbersome
for quantitative analysis. Unfortunately currently availale image-analysing systems are
very expensive and so we developed an economically reasonable alternative able to
identify in an endothelial cell-monolayer proliferating and apoptotic cells. It could also
be used for counting living and dead cells after Trypanlue staining.
The presented image analysis system is based on a conventional fluorescence
microscope (Nikon Diaphot). The only additional equippment is a CCD-camera (about
$ 1000) that is interfaced to a Matrox framegrabber board (about $ 500) and a newly
developed image analysis software that offers fundamental data analysis modes and
works Windows 3.11 and Windows 95.

2. Methods

2.1 DETERMINATION OF APOPTOTIC CELLS

The analysis of the nuclear morphology is used to determine apoptotic endothelial

cells (1, 2): The supernatant of the culture dish is carefully aspirated and the cells are

immediately fixed with 0.1 ml 10% (v/v) formalin solution on ice for 15 min.

The dish is once washed with PBS, the DNA stained for 15 min by addition of

4',6' Diamidine-2-phenylindoledihydrochloride (DAPI) in methanol and after a final

wash with PBS the nuclear morphology is evaluated by fluorescence microscopy with

a 20x objective. Fragmented nuclei show fine granules, brightly stained with DAPI

throughout the entire nucleus and are counted as apoptotic.

2.2 DETERMINATION OF THE PROLIFERATIVE INDEX OF A CELL MONOLAYER

The cell monolaye, is incubated with a culture medium containing BrdU. Incorporated
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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

730

BrdU is detected with a specific first antibody (3, 4): All incubation steps have to be

done in the dark because BrdU is light-sensitive. The BrdU/Deoxycytidin stock solution

(15 mM/15 mM) is mixed with culture medium 1:1000. The cell monolayer is incubated

with the modified medium for 60 minutes and washed two times with PBS. The cells

are fixed with 70% (v/v) ethanol for 30 minutes at 4°C and washed again three times

with PBS. The following steps are performed on a shaker. For denaturation of DNA the

cells are incubated with 3 N HCI for 20 minutes. To neutralise the acid, the cells are

washed five times with PBS. The antibody against BrdU (0.25 mg/ml) is diluted 1:1000

in PBS with 0.5% (v/v) Tween 20 (PBT) and 0.5% (w/v) bovine serum albumine (BSA)

are added. After 30 minutes incubation the cells are washed five times for three

minutes with PBT. For negative control another cell layer is treated with unspecific

at the same concentration as the monoclonal antibody. The
secondary antibody, a Rabbit Anti-Mouse Anti-IG (H+L)

(1 mg/ml protein) is diluted 1:3000 in PBT with 0.5% BSA. After two hours incubation

the cells are washed five times with PBT followed by the addition of

conjugated streptavidin for 60 minutes. After a final wash with PBT the labeled cells

are counted.

3. Results

3.1 SHELL OF THE PROGRAMM

The picture shows the shell of the programm while analysing the nuclear morphology

of DAPI stained endothelial cells (20x objective). [A] Grabbed picture: The grabbed
picture may be stored and saved for later analysis or print. [B] Segmented picture: The
processing of the grabbed picture results in the segmented picture: positive cell counts
i. e. apoptotic cells in a cell monolayer are marked with a margin. [C] Analysis site: The
segmentation specifications are defined in this area: the different signal intensities are

731
separated by their characteristic grey values. A free choosable size limit discriminates
positive signals from debris. [D] Calculation Site: The counting result of each picture is
displayed in this area; it may be added to a data file until a given number of events has
been counted.

3.2 DETERMINATION OF APOPTOTIC CELLS
The counitng results are based on the evaluation of the one randomly choosen visual
field shown as the grabbed picture on the shell of the programm:

3.3 DETERMINATION OF THE PROLIFERATIVE INDEX OF A CELL MONOLAYER
The counting results are based on the evaluation of the one randomly choosen visual
field:

4. Conclusions
The results received either by counting via eye sight or by the use of the image
analysis system coincided very well as demonstrated with the determination of the
apoptotic and proliferative index of an endothelial cell monolayer.
The presented system offers the possibility of rapid and reliable quantitative analysis:
higher cell numbers are scanned in shorter times, counting results saved as Excel-files
and processed for further data analysis or presentation.
Outlook: The system will further be tested for the analysis of the cell size.

5. References
1) Göhde, W., Schumann, J. and Zante, J. (1978) in Pulse Cytophotometry (Lutz, D., Ed), pp. 229-232,

European Press, Ghent
2) Kaiser, D., Freyberg, M.A., Friedl, P. (1997) Biochem. Biophys. Res. Commun. 231, 586-590.
3) Klöppinger, M. (1987) Zeitschr. d. GUM 4, 10-12.
4) Gratzner, H. G., Leif, R.C., Ingram, D. J. and Castro, A. (1975) Exptl. Cell Res. 95, 88-94.
6. Acknowledgement
Presentation of the poster and participation of D. Kaiser at the meeting was supported by the European
Commission Directorat General XII.

DEVELOPMENT OF AN OPTICALLY ACCESSIBLE PERFUSION CHAMBER
FOR IN SITU ASSAYS AND FOR LONG-TERM CULTIVATION OF
MAMMALIAN CELLS

M. A. FREYBERG and P. Friedl, Technical University of Darmstadt,
Petersenstraße D-64287 Darmstadt, tel: +49 (6151) 163655,
fax: +49 (6151)164759, e-mail: [email protected]

1. Subject

In vitro cells are usually grown in artificial culture media, in tissue culture flasks and in a
batch mode and thus are exposed to an unphysiological situation. An improved culture
system should provide the cells with an environment of constant composition. We have
developped an alternative culture system based on a conventional tissue culture plate
(3.5 cm diameter) which is changed into a closed perfusion chamber. The system can
be scaled up from one to several chambers. The shape and the size of the area of cell
growth is defined by silicone sheets and can be designed to individual demands.
The culture chamber is optically accessible, so cell growth and morphology can be
evaluated by light and fluorescence microscopy. Furthermore the cellular physiology
can be characterised by any fluorimetric assay using a bottom type fluorescence
reader. A peristaltic pump sustains a constant medium flow through the chamber thus
creating true homeostasis. The use of HPLC-valves and connectors allows the
switching between different media or assay solutions. Thus it is possible to perform in
situ assays also measuring transient effects.

2. Methods

2.1 CELL CULTURE

HUVEC are isolated from umbilical cord veins by a modification of the previously
published method of Gimbrone et al. (4) and maintained in culture as described (3).

Ea.hy926 cells are kindly provided by Dr. Edgell et al. (2).
For use in the perfusion system the media are modified: 25 mM HEPES is substituted

for the usual NaCI is added for maintaining the osmotic balance.

3. Results
3.1 PERFUSION SYSTEM

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734

3.2 Perfusion Chamber

3.3 CELL NUMBER CORRELATION

735

3.4 LONG-TERM CULTIVATION

3.5 DETERMINATION OF CRITICAL OXYGEN CONCENTRATION

736

4. Conclusions
We have developed a perfusion system on the basis of normal cell culture plates. The
cultivation of various cell lines is possible under more physiological conditions than in
static culture. Futhermore adherent cells are cultured under defined shear stress
conditions. The working system is accessible for fluorescence measurements and
microscopical observations so the cells can be characterised by any fluorimetric assay.
The wide range of fluorescence indicators allows the examination of many parameters
of cell physiology under perfusion mode.

5. References
1) DePaola N, Gimbrone MA Jr, Davies PF, Dewey CF Jr (1992) Vascular endothelium responds to fluid
shear stress gradients. Arteriosclerosis and Thrombosis 12,11: 1254-1257
2) Edgell CJS, Mc Donald CC, Graham JB (1983) Permanent cell line expressing human factor VIII related
antigen established by hybridisation. Proc Nat Acad Sci USA 80: 3734-3737
3) Friedl P, Tatje D, Czapala R (1989) An optimized culture medium for human vascular endothelial cells
from umilical cord veins. Cytotechnology 2: 171-175
4) Gimbrone MA, Shefton EJ Jr, Cruise SA (1978) Isolation and primary culture of endothelial cells from
human umbilical vessels. Tissue Culture Association Manual 4: 813-817
5) Kjellström BT.Örtenwall P, Risberg B (1987) Comparison of oxidative metabolism in vitro in endothelial
cells from different species and vessels. J of Cellular Physiology 132: 578-580
6. Acknowledgement
Presentation of the poster and participation of M.A.Freyberg at the meeting was supported by the
European Commission Directorat General XII.

ANALYSIS OF MITOGENIC ACTIVITY OF PROTEINS AFTER SEPARATION
BY GEL ELECTROPHORESIS

O. HOHENWARTER, G. MARZBAN, E. JISA and H. KATINGER
Institute of Applied Microbiology, University of Agricultural Sciences
Vienna, Austria

1. Introduction

Complex mixtures of proteins may be separated rapidly by Phast system gel

electrophoresis and tested subsequently in cell culture. Using transwell inserts in

microwell plates, gel slices were eluted directly into the cell culture supernatant and the

mitogenic effect was evaluated by incorporation (Kuo et al., 1991). We
used a modification of this procedure in conventional slab gels to be able to apply
sample volumes up to 0,1 ml. In order to avoid the use of radioactivity enzymatic assays

for the mitogenic effect were applied.

Two examples we used to evaluate the feasibility of the method:

– a purified recombinant fusion protein of human superoxid dismutase and interleukin 2
(SOD-IL2)

– an extract from bovine pituitaries

The activity of the fusion protein SOD-IL2 was tested with cell line CTLL-2 which is
strictly dependent on the presence of IL2 in the culture medium (Gillis et al., 1978).

Human umbilical vein endothelial cells were used to evaluate the mitogenic response of
pituitary extract.

2. Materials and methods

Preparation and cultivation of human endothelial cells has been described (Hohenwarter

et al., 1992). CTLL-2 cells (ATCC No TIB 214) were cultivated as described (Vorauer-

Uhl, 1993).
Frozen bovine pituitaries were pulverized and 1 g was dissolved in 15 ml 0,1 M

solution by stirring for 1h at 4°C. Unsoluble material was separated

by centrifugation. The protein content of the extract was 30 mg/ml.

Purified SOD-IL2 (Vorauer-Uhl, 1993) and pituitary extract were separated by
isoelectric focusing (IEF) or native electrophoresis (pH 5,5 or pH 8,9) on polyacrylamid
gels (Clean gel from Pharmacia) according to the instructions of Pharmacia.
The gels were washed thoroughly before use in destilled water. After separation one lane
was cut into equal pices. The slices were transferred into tissue culture inserts (8 well

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738

strip inserts from Nunc) which contained culture medium (Fig. 1). The inserts

were placed in 96-well microplates which contained the test cells (CTLL-2: 3000

cells/well, Endothelium: 1500 cells/well). For the bioassays culture medium with

antibiotics was used. In parallel wells 3 ng/ml SOD-IL2 or EGGS were added

as positve control. After 24 h the inserts were removed. After 72 h the the mitogenic

activity on CTLL-2 cells was evaluated by an MTT test procedure (details described by

Hohenwarter et al., 1996). The effect on human endothelial cell was evaluated after 96 h

using the acidic phosphatase assay (Connolly et al., 1986). The results correlated with

microscopic observation.

Parallel lanes of the gel were stained by silver staining.

3. Results

100 ng purified SOD-IL2 with a known isoelectric point of 5,85 were focused (pI 3-10).
The protein has serveral isoforms which are active in the bioassay (Fig 2).

pituitary extract were separated by isoelectric focusing (pI 5-8). In the bioassay of
the gel slices a broad region with mitogenic activity was found (Fig. 3). The most active
fraction lies between pI 5,3 and 6,2.

of pituitary extract were separated by native gel electrophoresis either under acidic
(pH 5,5) or basic (pH 8,9) conditions.
In both gels mitogenic fractions could be identified (data not shown).

739

4. Discussion

The combination of gel electrophoresis and a cell culture assay in microwell plates is a
rapid method to screen for mitogenic fractions. Only small amounts of material are
needed. Since most growth factors are active in very low concentrations, the cell culture
assay allows the detection of proteins in the gel which cannot be visualized by the most
sensitive staining techniques.
The use of transwell inserts is essential to separate the gel slices from the cells to avoid
inhibitory effects. Sometimes we observed toxic effects which could be minimized by
extensive washing of the gels before electrophoresis.
We have shown that the recombinant fusion protein SOD-IL2 remains biologically
active during the isoelectric focusing procedure. Furthermore the activity of single
isoforms could be determined.
The growth promoting activity of bovine pituitary extract on endothelial cells shows a
broad peak after isoelectric focusing. This could be due to different proteins or different
isoforms of one protein. The pituitary is a source of many mitogenic peptides for
example FGFs, EGF, TGFs and IGFs (Houben and Denef, 1994). The separation in
narrow pI ranges will allow a more detailed characterization of the mitogenic proteins.

6. References

Connolly, D.T., Knight, M.B., Harakas, N.K., Wittwer, A.J. and Feder, J. (1986)

Determination of the number of endothelial cells in culture using an acid phosphatase
assay. Anal. Biochem. 152, 136-140.
Gillis, S., Ferm, M.M., Ou, W. and Smith, K.A. (1978) T cell growth factor: parameters
for production and a quantitative microassay for activity. J. Immunol. 120, 2027-2032.
Hohenwarter, O., Schmatz, C. and Katinger, H. (1992) Stability of Von Willebrand
Factor production in different human endothelial hybrid cell lines. Cytotechnology 8, 31-
37.
Hohenwarter, O., Waltenberger, A. and Katinger, H. (1996) An in vitro test system for
thyroid hormone action. Anal. Biochem. 234, 56-59.
Houben, H. and Denef, C. (1994) Bioactive peptides in anterior pituitary cells. Peptides
15, 547-582.
Kou, K., Yeh, H., Chu, D.Z.J. and Yeh, Y. (1991) Separation and microanalysis of
growth factors by Phast system gel electrophoresis and by DNA synthesis in cell culture.
J. Chromatography 543, 463-470.
Vorauer, K. (1993) Expression, Reinigung und Charakterisierung eines SOD-IL2-
Fusionsproteins. Thesis, University of Agricultural Sciences, Vienna.

Acknowledgements

We thank Karola Vorauer-Uhl for generous gifts of purified SOD-IL2 protein.

WORKSHOP ON :

THE USE OF ANIMAL CELLS VERSUS THE USE OF TRANSGENIC
ANIMALS FOR THE PRODUCTION OF RECOMBINANT PROTEINS

The moderator of the workshop, Dr. Simon Barteling, expressed in his introduction the

back to the future feeling of ESACT members, who build their careers on animal

cells grown in fermenters or other sophisticated high-tech systems. Now, the

animal has to do the job.
Presentations were given by Dr. L. M. Houdebine, Dr. H. Meade, Dr. I. Garner, Dr. H.

Yoshida, and Dr. R. Werner. They discussed the and more general
aspects.
Dr. Houdebine (INRA, France) explained what enabled the production of proteins in the

milk of transgenic animals: knowledge of milk protein gene promoters, targeting of gene

constructs into the casein locus, in vitro fertilization and embryo development, and the
identification of transgenic embryos before implantation into the mother animals. The

quality of the proteins must be evaluated for glycosylation, carboxylation, and cleavage.

Dr. Meade (Genzyme Transgenics) described the production of proteins in the milk of

several animal species. Some of the proteins are very difficult to produce in animal cell

cultures. Yields of 1 mg of active protein per ml of milk are typical, which is at least 10

times as much as obtained from cell culture.
Dr. Garner (PPL, Roslin, Scotland) described how by prescreening of cells for the

expression of the milk promoter driven transgene, the highest expressing line could be

detected and used for the cloning of highly productive animals (e.g. Sheep Dolly).

Dr. Werner (Dr. Karl Thomae) discussed the pros and cons of the production of proteins

in transgenic animals versus the production in animal cell cultures. In general, the
production in animals seems to be more economical than in animal cells, however, the

animals are a less defined system, which might hamper the registration of the products.
Also, production of certain pharmaceuticals (e.g. insulin) might, by leakage into the
blood, impair the health of the animals. Production of such unhealthy transgenic animals

should be prohibited.
Technical, economical, and ethical aspects of the use of transgenic
animals were vividly discussed for almost an hour.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

SECRETION OF FUSION PROTEINS INTO MILK BY TRANSGENIC
MOUSE MAMMARY EPITHELIUM

D. POLLOK, L. CHEN, H. LIEM, B. W1LBURN, J. WILLIAMS,
M. HARRINGTON, Y. ECHELARD, H. MEADE
Genzyme Transgenics Corporation
1, Mountain Road
Framingham, MA 01701-9322, USA

Abstract: Mammary epithelial cell processing may be more efficient than that found in
traditional cell cultures. Certain fusion proteins were not efficiently secreted from traditional
cell culture, which includes COS and BHK cells. Yields of the proteins were very low and
sometimes undetectable. The same proteins were secreted into milk by animals transgenic for
the constructs at typical levels of 1 mg per ml active protein. The fusion proteins are
antibodies fused to enzymes and heterologous proteins, as well as protein fusions to
Transgenics has enabled the secretion of proteins not normally secreted and has permitted the
secretion of complex proteins that remain a significant challenge for cell culture.
The economic viability of these fusion proteins to be used therapeutically is thereby enabled,
whereas a cell culture process could either be technically impossible and/or economically not
feasible. The economics of the production of these proteins will be also discussed.

Discussion

Hauser: You suggest that the secretion system in the uterus is completely different from
other mammalian cells. Could you comment on this; for instance, why is the
protein stuck in the CHO cell and not in the others?

Meade: I have no explanation but can make a comment. It was in an experiment, that
totally surprised me, when the protein was able to get out of the memory cell.
When I think, back early on when we got monoclonals to try, one which was
humanised was put into the mouse system and only got 0.1 mg/ml. We
subsequently found that that cell did not secrete any antibody in tissue culture.
I do not know whether the memory gland is more efficient or it does not just
edit as well. As we test the proteins, the monoclonal binding and enzyme
activity are OK.

Hauser: You are saying that the memory gland has different properties in the secretion
system. Did you also test different mammalian cell lines because it could be
due to differentiation?

Meade: We have not tested different cell lines. We only used COS cells because they
are easy to handle.

743

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 743.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

THE PRODUCTION OF PROTEINS IN THE MILK OF TRANSGENIC
LIVESTOCK: A COMPARISON OF MICROINJECTION AND NUCLEAR
TRANSFER

I. GARNER
PPL Therapeutics,
Roslin,
Edinburgh,
EH 25 9PP, UK

1. Introduction

The milk of transgenic livestock offers a commercially viable source for the production

of pharmaceutical and nutraceutically valuable proteins and peptides. High-level

expression of the required protein in the mammary gland of transgenic animals can be

achieved and complex post-translational modification is accommodated. The technique

has been successful in producing human proteins in the milk of a variety of species

including mice, rabbits, pigs, goats, sheep and cattle. The high expression levels

achieved make the transgenic route an attractive alternative to mammalian cell culture

which can produce some complex proteins but at large scale can be costly and yields can

be low.

However, even in cases where large amounts of complex proteins are required,

the commercial viability of projects could be improved through developments in

transgenic technology, which has remained largely unchanged for over a decade.
This paper summarises the use of transgenic livestock in the production of two

medically important proteins, (AAT) and fibrinogen. It also focuses on

the potential of nuclear transfer technology to improve the efficiency and range of

transgenic technology.

2. The core technology - microinjection

The basic elements of transgenic protein production in livestock for commercial use
have remained largely unaltered since 1982 (James, 1993). However practical advances
have been made, such as the use of interference contrast microscopy and centrifugation
of the eggs to make pro-nuclei more easily visible. Techniques for obtaining supplies
of fertilised eggs at the right stage of development from supér-ovulated donors and for
the re-implantation of injected eggs into pseudopregnant recipients have also been
developed. While these have increased the ease with which the method can be
performed the fundamental strategy remains the same.

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O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 745-750.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

746

A few hundred molecules of the DNA construct are injected, via a microscopically small

glass needle, into one of the pro-nuclei of a fertilised egg prior to fusion. The embryos

are then implanted into recipient animals which act as surrogate mothers. The presence
of the transgene in the resultant offspring can be determined, and expression of the
protein detected, using standard techniques such as PCR, Southern blot analysis and

Western blot analysis. In most cases, it is desirable to have the protein present in the

milk of the animal, although for some applications, expression in the blood is employed.

In order to direct expression of the transgene to the mammary gland, consideration must

be given to the nature of the DNA construct. Milk-specific expression is effected by

fusing the target gene downstream of the regulatory sequence from any of a number of

milk-protein genes including murine whey acidic protein, ovine bovine

-lactalbumin, bovine and caprine With the exception of whey

acidic protein-driven genes in pigs, the regulatory sequences have been shown to be

approximately equivalent. However, the configuration of the target gene is of

importance. Experiments in mice have shown that genomic DNA is almost always

superior to cDNA. This trend has also been shown in the production of -antitrypsin

in sheep (Wright et al, 1991; Carver et al, 1993).
The choice of species for a commercial transgenic project is influenced by a

number of factors including generation time, disease status of the animals, number of

offspring and the volume of milk produced (Colman, 1996). Thus for pilot studies,

mice are routinely used. The time from birth to milk production for mice is

approximately 3.25 months. Where higher levels of expression are required, a larger

species would be necessary such as sheep, goats or cattle. However, in these species

generation time has traditionally presented a limitation for transgenic programmes as no

indication of expression level is possible until first lactation which, even when

hormonally induced, takes place after 9 months for sheep or goats and 12 months for

cattle. Only then can selection of suitable animals for flock or herd development take

place. Purification of proteins such as AAT from the milk of transgenic livestock has

not presented considerable hurdles (Colman, 1996). Of crucial importance is the

removal of lipid which is achieved by low-speed centrifugation, differential

precipitation and chromatography. Challenges in separating highly homologous proteins

remain but these are common to other production systems.

3. Transgenic production of -antitrypsin

One of the first commercial targets for transgenic protein production was AAT.
antitrypsin is a 394 amino acid, single chain glycoprotein which is normally present at
2 grams per litre in plasma. It is a serine protease inhibitor secreted from hepatocytes
and mononuclear phagocytes and its major substrate is thought to be neutrophil elastase.
A deficiency in AAT is a comparatively common human genetic disorder. A certain
proportion of sufferers develop breathing problems leading to emphysema and resulting
in premature death. In addition, in other diseases of the lung such as cystic fibrosis (CF)
there is an imbalance between the concentrations of AAT and elastase in the lung. The

747

latter is an enzyme involved in the maintenance of the tissue lining of the lung. It acts
by cleaving bonds adjacent to neutral amino acids in the protein elastin which is present
in this lining. An overabundance of elastase is thought to cause lung damage in diseases

such as CF. In addition, elastase can act in the degradation of immunoglobulin which

may hinder the immune response to lung infections in CF patients. Thus, if the balance

between AAT and elastase could be restored, the progress of these lung disorders could

be slowed and the quality of patient life improved. A plasma-derived form of AAT is

currently available commercially but supplies are limited. The protein was thus a

suitable candidate for production in the milk of transgenic animals.

High level expression of active human AAT (hAAT) in the milk of transgenic

sheep was achieved using a hybrid ovine AAT gene (Wright,

1991). The transgene was shown to be present in several lambs by Southern blot

analysis. A comparison of band intensities suggested that the number of copies

incorporated varied between these lambs. Human AAT expression was demonstrated

using radial immunodiffusion assays and ELISA. The protein level from one of the

animals reached 63 grams per litre hAAT in week one of lactation and stabilised at 35

grams per litre. This remains the highest reported expression level for a protein in the

milk of a transgenic animal. Analysis of the milk from the founder animals by SDS-

PAGE indicated a novel band of apparent 54 kDa molecular weight. The mobility of

this band corresponded with that of native plasma-derived AAT. Cleavage with N-

glycosidase F indicated that the milk-derived protein was glycosylated. The amino

terminal sequence of AAT purified from the milk of these animals was shown to be

identical to that predicted for the human protein (Carver et al, 1992). Isoelectric

focussing showed some minor differences between plasma and milk derived hAAT.

However, the biological activity of the protein from the two sources was shown to be

indistinguishable (Wright et al, 1991). A production flock for human AAT has been
established and the transgenically-derived protein is currently in Phase II clinical trials
in the UK.

4. Fibrinogen

The work described above established the viability of transgenic protein production for
a single chain protein. The versatility of the technique has since been confirmed
through the production of human fibrinogen in the milk of transgenic mice and sheep.

Fibrinogen is a complex plasma protein required for blood clotting. The
protein is a heterodimer made up of 2 sets of 3 different polypeptide chains,

with molecular weights of 66.0, 54.0 and 48.5 respectively. The protein is
synthesised in liver cells where the six chains are assembled and linked by 29 disulfide
bonds. The assembled molecule is then secreted into the bloodstream. During the
coagulation process, thrombin acts enzymatically to remove amino-terminal peptides
from fibrinogen forming fibrin monomers which polymerise into insoluble fibrin clots.
Factor XIII acts to cross link adjacent fibrin strands and stabilise the clot. While various
mammalian cell culture systems have produced apparently functional human fibrinogen,
high level expression has not been achieved. Fibrinogen could have a widespread use

748

in the development of tissue sealants for surgical applications but only if larger amounts

can be economically produced than seems feasible with conventional expression

systems.

High-level expression of recombinant human fibrinogen has been achieved in

the milk of transgenic mice (Prunkard et al, 1996). Genomic sequences of the
were placed under the control of the sheep promoter. An

equimolar mix of the three constructs was introduced into mouse embryos by

microinjection. The presence of the transgenic sequences in founder animals was

detected by PCR. Southern blot analysis was used to identify animals carrying all three

fibrinogen genes. Fibrinogen protein was detected in the milk of transgenic animals by

Western blot analysis. Assembly of the protein subunits was analysed using a

monoclonal antibody (ZG177.4.1.2.3) which specifically recognises the assembled

fibrinogen hexamer. The percentage of total fibrinogen that existed as a fully assembled
hexamer was established by comparisons between reducing and non-reducing gels.

Figures from the highest producing animal suggested that up to 100% of the fibrinogen

was present as the fully polymerised form. The functional activity of transgenic

fibrinogen was investigated using clotting reactions. The addition of thrombin to

recombinant fibrinogen in mouse milk resulted in an insoluble clot which was collected

by centrifugation. Western blot analysis of this material indicated that both

fibrinopeptides had been removed by thrombin and cross-linking had occured. The

addition of Factor XIII enhanced the reaction to produce complete cross-linking.

Electron microscopy has shown that clots made from transgenically-derived fibrinogen

display the expected structure.

Having established that functional fibrinogen could be produced in transgenic

mice, work was initiated to produce the protein in the milk of sheep. Expression levels

of 5 grammes per litre have been achieved and several founder animals have been

produced with the aim of creating a production flock to generate material for clinical

trials.

5. Nuclear Transfer

The examples given above indicate the commercial potential of transgenic protein
production using microinjection. Complex proteins can be produced at high levels and
purification carried out to a degree sufficient to allow regulatory approval for clinical
trials. However, the technique has several important limitations. Using microinjection,
only a small number (aproximately 5%) of the animals born are transgenic. The site
of integration is random and this results in variations in levels of transgene expression.
To generate a production flock, conventional breeding is necessary, often taking several
years depending on the sex of the founder. A further limitation is that microinjection is
suitable only for the addition of genes. Gene deletion or replacement (gene targeting)
cannot be carried out.

Thus an alternative approach to generating transgenic animals would be
advantageous. Embryonic stem cells offer one avenue. Genetic manipulation of these
cells in culture, followed by reintroduction to a recipient embryo can give rise to

749

transgenic animals in which genes have been deleted, modified, replaced or added. This
has allowed the development of a number of disease models in mice. However, the use
of embryonic stem cells has yet to be successful in the generation of transgenic
livestock.

An alternative approach is nuclear transfer which has been shown to be
successful in sheep (Campbell et al, 1996; Wilmut et al, 1997). In these experiments,
cells were taken from a number of tissues and cultured (Campbell et al, 1996). The cell
cycle was arrested in G0 by the reduction of foetal calf scrum in the culture medium

from 10% to 0.5%. These quiescent cells were then fused with oocytes recovered from
ewes between 28 and 33 hours after injection of gonadotropin-relcasing hormone
(GnRH) and subsequently enucleated. Fusion of the donor cell and enucleated oocyte
and activation of the oocyte was induced using electrical pulses. Reconstructed
embyros were cultured in the ligated oviducts of sheep or in a chemically-defined
medium. Embryos which developed to morula or blastocyst after six days of culture
were transferred to recipient ewes and allowed to develop to term. The technique has
been successful in producing sheep from embryonic, foetal and adult tissues (Wilmut
et al, 1997) and led to the much-publicised birth of Dolly, produced from mammary
epithelial cells taken from a 6-year-old ewe.

Applied to the production of transgenic livestock, nuclear transfer offers
solutions to a number of the shortfalls inherent in microinjection. The cell culture step
provides an opportunity for gene targeting so that genes can not only be added but also
deleted or replaced. The use of suitable selectable markers can ensure that all nuclear

donor cells contain the desired construct and thus all transplanted embryos are

transgenic. It may also be possible to pre-select for high expression at the cell culture
stage. Furthermore, it would be possible to produce a production flock in a single
generation rather than through conventional breeding as is the case for microinjection.

The technique has already shown the potential to be more efficient than
microinjection in terms of the numbers of transgenic lambs born per recipient ewe used.
Between 1993-1996 our microinjection programme resulted in 49 live births of
transgenic lambs from a total of 2875 sheep used. The 1996 nuclear transfer programme

resulted in 5 live births from 207 sheep. Expressed as a ratio of sheep/lamb, these
figures are 58.6 for microinjection and 41.4 for nuclear transfer. Assuming that all
lambs born following nuclear transfer would be transgenic, this represents a promising
increase in efficiency.

For commercial use, a protocol for the addition of a transgene using nuclear
transfer would involve the transfection of a suitable cell type with a construct containing
the gene for the protein of interest. Of the cell types so far used successfully for nuclear

transfer in livestock species, foetal fibroblasts are the most promising, possessing a
number of advantages. Large numbers of cells can be derived from a single foetus and

primary cultures can be readily established and maintained under standard culture
conditions. This cell type is also amenable to the introduction of exogenous DNA via
transfection.

The production of transgenic livestock (sheep) through nuclear transfer of

transfected foetal fibroblasts has now been achieved with an efficiency of 1 transgenic
lamb per 27 sheep used. (Schnieke, unpublished results). Thus nuclear transfer has
distinct advantages in terms of both time scale and efficiencies.

750

6. Conclusions

The potential of transgenic protein production in the milk of livestock has been clearly
demonstrated. Complex therapeutic proteins, such as fibrinogen, can be obtained at
higher yields than would be possible through mammalian cell culture. Purification has
been shown to be reasonably simple and cost effective for a number of proteins. The
issue of regulatory compliance has also been addressed and products derived from the
milk of transgenic livestock, such as AAT, have entered clinical trials.

However, despite these successes, for microinjection, nuclear transfer
promises, and has begun to deliver, marked improvements over the technique. Instant
flocks or herds could be generated from a transfected cell line greatly reducing the time
for development of a product. Cell culture prior to nuclear transfer offers the
opportunity for more sophisticated genetic manipulation than the simple addition of
genes, including gene removal, modification or replacement. There is also the potential
to exploit the technique to increase the yields of proteins obtained from livestock.
Nuclear transfer thus offers a significant advance in the application of transgenic
technology to the production of proteins and peptides.

7. References

James, R. (1993) Human therapeutic proteins generated in animals, The Genetic Engineer and
Biotechnologist 13, 189-197.

Wright, G., Carver. A., Cottom, D., Reeves, D., Scott, A., Simmons, P., Wilmut, 1., Garner, I., and Colman,
A. (1991) High level expression of active human alpha-1-antitrypsin in the milk of transgenic sheep,
Bio/Technology 9, 830-834.

Carver, A., Dalrymple, M., Wright, G., Cottom, D., Reeves, D., Gibson, Y., Keenan, J., Barrass, J., Scott, A.,
Colman, A., and Garner, I. (1993) Transgenic livestock as bioreactors: stable expression of human alpha-1 -
antitrypsin by a flock of sheep, Bio/Technology, 11, 1263-1270.

Colman, A. (1996) Production of proteins in the milk of transgenic livestock: problems, solutions and
successes, Am. J. Clin. Nutr., 63, 639S-645S.

Carver, A., Wright, G., Cottom, D., Cooper, J., Dalrymple, M., Temperley, S., Udell, M., Reeves, D., Percy,
J., Scott, A., Barrass, D., Gibson, Y., Jeffrey, Y., Samuel, C., Colman, A. and Garner, I. (1992) Expression
of human antitrypsin in transgenic sheep, Cytotechnology, 9, 77-84.

Prunkard, D., Cottingham, I., Garner, I., Bruce, S., Dalrymple, M., Lasser, G., Bishop, P., and Foster, D.
(1996) High-level expression of recombinant human fibrinogen in the milk of transgenic mice, Nature
Biotechnology, 14, 867-871.

Campbell, K., McWhir, J., Ritchie, W., and Wilmut, I. (1996) Sheep cloned by nuclear transfer from a
cultured cell line. Nature, 380, 24-25.

Wilmut, I., Schnieke, A., McWhir, J., Kind, A., and Campbell, K. (1997) Viable offspring derived from fetal
and adult mammalian cells, Nature, 385, 810-813.

CREATION OF MICE EXPRESSING HUMAN ANTIBODY BY INTRODUCTION

OF A HUMAN CHROMOSOME

H. YOSHIDA1, K. TOMIZUKA1, H. UEJIMA2, H. KUGOH2, K. SATOH1,
A. OHGUMA1, M. HAYASAKA3, K. HANAOKA3, M. OSHIMURA2 and
I. ISHIDA1
1 Central Laboratories for Key Technology, Kirin Brewery Co., LTD., 1-13-5,
Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236, Japan
2 Department of Molecular and Cell Genetics, School of Life Sciences, Tottori
University, Nishimachi 86, Yonago, Tottori 683, Japan
3Laboratory of Molecular embryology, Department of Bioscience, Kitasato
University School of Science, 1-15-1, Kitasato, Sagamihara, Kanagawa 228,
Japan

ABSTRACT. A human chromosome or its subchromosomal fragment (SCF) derived from

normal fibroblasts was introduced into mouse embryonic stem (ES) cells via microcell-

mediated chromosome transfer (MMCT) and viable chimaeric mice were produced from

them. Serum antibodies with each human Ig were detected and various V segments were

identified in human Ig H, and transcripts. Upon immunization of the chimaeras with

HSA, HSA-specific antibodies with were detected in the sera. A HSA-

specific human antibody with producing hybridoma was obtained from fusion of murine

myelomas with spleen cells from the chimaeric mouse .

1. Introduction
Monoclonal antibodies are not only used in human diagnostics, but also in human therapy.
People cannot be immunized in vivo with any kind of antigen, and many challenges have
been done to obtain human monoclonal antibodies for therapeutic use. Transgenic
approach is useful to generate high affinity monoclonal antibodies. But only a limited
amount of DNA can be transferred using standard techniques. We have developed a novel
procedure to introduce foreign genetic material into mice by using a chromosome itself as a
"vector". Human chromosome(hChr.) 14,22,or hChr.2-derived fragment including Ig
heavy, lambda or kappa genes was transferred into mouse ES cells via microcell-mediated

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O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 751-756.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

752

chromosome transfer(MMCT) [1]. In this study we immunized the chimaeric mice by
human serum albumin(HSA) and obtained a hybridoma expressing specific antibody to
HSA comprised human chain from chimaeric mouse containing hChr. 14-fragment.

2. Material and Methods

2.1. CONSTRUCTION OF MH(ES) CELLS AND CHIMERA PRODUCTION

Our strategy to introduce human chromosomes into mice is outlined in Fig. 1. MMCT was

utilized to introduce a human chromosome tagged with G418-resistance gene into mouse

ES cells. First, we constructed the libraries of mouse A9 cells containing a human

chromosome tagged with pSTneoB suitable for conferring G418-resistance to mouse ES
cells. Human primary embryonic fibroblasts were used as a source of human chromosomes.

About 3,000 independent G418-resistant colonies of HFL-1 transformed with pSTneoB

were divided into 30 pools and then each pool was fused with mouse A9 cells. G418- and

ouabine- double resistant human-mouse hybrids were used to prepare microcells for the

next fusion experiments with mouse A9 cells. Finally, we cloned about 700 independent

G418-resistant human-mouse microcell-hybrids. Mouse ES cells (TT2: 40,XY) were fused

with the microcells prepared from these donor hybrid A9 clones and selected with G418 or

puromycin. The drug-resistant MH(ES) clones were analyzed by PCR. Representative

microcell hybrids, MH(ES)2-1 (containing a hChr.2-SCF), MH(ES)22-1 (containing an

intact hChr.22), MH(ES)14-4 and MH(ES)14-5 (containing an intact hChr.14), MH(ES)14-

and MH(ES) (containing a gamma-ray irradiated hChr. 14) were used to produce

chimeras. The SCF(2-W23) was further tagged with puromycin resistant marker was used
as a microcell donor. One of the resultant puromycin-resistant MH(ES) clone, MH(ES)2-

21, was confirmed to retain SCF(2-W23) by the PCR and the FISH analysis (data not

shown) and used to produce chimeras.

From ten to twenty MH(ES) cells from each cell line or wild type TT2 were

injected into a 8-cell stage embryo derived from Jcl:MCH(ICR) mice. Injected embryos

were then transplanted to the uteri or oviducts of pseudo pregnant recipients and allowed to

proceed to term. Chimerism in a resulting offspring was determined by extent of coat

pigmentation. The TT2 line, derived from C57BL/6xCBA-F1 embryo, gives an agouti coat

color in an albino MCH(ICR) background.

753

2.2. HYBRIDOMAS
The spleen was removed from the chimaeric mouse bearing human chromosome #14
immunized human serum albumin. The spleen cells were fused with a P3X63-Ag8.653
myelomas using PEG. After a fusion, the cells were seeded in 96-well plates in HAT
supplemented medium. After HAT selection, G418 was supplemented to select human
chromosome containing clones. The number of wells positive for hybridoma growth was
determined visually and the human antibody-secreting hybridomas were screened by
ELISA.
2.3. ASSAYS
Human immunogloblin were assayed using anti-human antibodies immobilized on
ELISAplates and detected with peroxidase-conjugated anti-human immunogloblin
antibodies as described before [1]. Anti-HSA human immunogloblin were assayed using
HSA immobilized on ELISAplates and detected with peroxidase-conjugated anti-human
immunogloblin antibodies using human IgM for the negative control. The samples,
standard and antibody conjugates were diluted with mouse serum supplemented PBS.
Absorbance was measured using a spectrophotometer .

754

3. Results and Discussion

3.1. ANTIBODIES WITH HUMAN IMMUNOGLOBULINS IN CHIMAERIC MICE

We assessed human immunogloblin expression in the sera of chimaeric mice. Human Igs

in the sera from non-immunized chimaeric mice were identified by ELISA assays using

anti-human Ig antibodies (Table 1). All the tested chimaeric mice (14/14) derived from

MH(ES)14-4, 14-5, produced polypeptides in their

sera. Further analysis revealed the production of all four in

chimera C14-15. Chimaeric mice derived from MH(ES)2-1 (15/19) and from MH(ES)22-1

(5/5) also produced hk and h1 polypeptides, respectively.

Upon immunization of these chimeras with human serum albumin (HSA), HSA-

specific antibodies with each human were readily detected in their sera

(data not shown).

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3.2. CREATION OF HYBRIDOMAS FROM THE CHIMERAS

The spleen was removed from chimera C14-1 and were fused with a P3X63-Ag8.653
myelomas. The number of growth positive wells in HAT medium was 670. The
number of growth positive wells in G418 medium, it was supposed to contain human
chromosome, was 140. The frequency of G418 resistant clones was similar to that
estimated from coat color of mouse. Six human antibody positive clones was obtained.
The anti-HSA human positive wells were cloned by limiting dilution culture. The
clone was named H4B7 (Table 2).

756

The amino acid sequences were deduced from variable region ofhuman antibody

cDNA derived from clone H4B7. It was revealed that the H4B7 hybridomas contained a

combination of genes for VH4 family and JH2. These results show that the chimaeric

mouse retaining human #14 partial fragment produced a functional human antibody heavy

chain protein (data not shown).

The clone H4B7 was cultured and the culture supernatant was diluted, followed

by ELISA using HSA as an antigen with peroxydase-labeled anti-human IgM goat

antibody. As a result, the absorbance decreased with the increase in the dilution of the

culture solution. Two of human showed low absorbance regardless of dilution

ratios. This suggests that the antibody produced by hybridoma H4B7 had a specificity to

HSA(Fig.2).

4. Conclusions

1. Human chromosome or its fragment into mouse ES cells by MMCT and viable chimaeric

mice were produced from them. In the case of #2-fg., the fragment was transmitted to
the offspring.
2. Serum antibodies with each human Ig were detected.
3. A HSA-specific human antibody with hm producing hybridoma was obtained from
fusion of murine myelomas with spleen cells from the chimaeric mouse.

5. Acknowledgements
We thank M. Kato, A. Kurimasa and M. Shimizu for technical advice and valuable

discussions; T. Kato and N. Inoue for giving facility to use Gamma Cell 40 in Yokohama
City University; A. Fujiyama and J. Hourov for efforts in early stages of the project; H.
Kondoh for giving pSTneoB; S. Watanabe, T. Yagi and P.W. Laird for giving pPGKpuro.

6. References
1. TOMIZUKA, K., YOSHIDA, H., UEJIMA,H., KUGOH, H., SATOH, K.,
OHGUMA, A., HAYASAKA, M., HANAOKA, .K. , OSHIMURA, M. and ISHIDA, I.
(1997) Functional expression and germline transmission of a human chromosome
fragment in chimaeric mice, Nature Genet., 16, 133-143

TRANSGENIC TECHNOLOGY-A CHALLENGE FOR
MAMMALIAN CELL CULTURE PRODUCTION SYSTEMS

ROLF G. WERNER
Boehringer Ingelheim Pharma Germany,

88397 Biberach an der Riss

Due to price restrictions ofthe health care system, innovative biopharmaceutical
products have to compete with low price products on the market or have to
demonstrate higher efficiency and safety, which add value to the
biopharmaceutical product.

Thus, as well as product safety, optimizing the economy of biopharmaceutical
manufacturing processes is the main goal of process development. This can be
achieved by more efficient expression systems, shorter generation time of the
host cell, media optimization such as use of serum free or protein free media
with corresponding feeding strategies as well as improved automatization and
sterile technology to increase the overall success rate of the manufacturing
process. An optimized combination of these factors will improve the
productivity during the fermentation process.

For improvement of the yield in the downstream processing, the efficiency of

the purification steps and the number of the purification steps are decisive. In

addition, loss during sterile filling and lyophilisation contribute to the overall

yield of the finished product.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

758

One of the most efficient expression vectors in mammalian cell culture is
pEE 14 in NSO cells, regulated by CMV promoter and SV 40 termination
region. The gene of interest is expressed up to 1 gram protein per liter. In
addition the glutamine synthesis mini-gene provides a detoxification of
ammonium during the mammalian cell culture process. Although the
productivity can be increased significantly with this expression vector in
comparison to the expression of the desired gene in the pPA DHFR vector
system, which is controlled by a SV 40 promoter and the hepatitis surface
antigen termination region, glycosilation of the glycoprotein is different in the
N-acetyl neuraminic acid content. Whereas the CHO cell glycosilates the
proteins with a N-acetyl neuraminic acid content of about 20 mole N-acetyl
neuraminic acid per mole protein in the NSO expression system, the N-acetyl

neuraminic acid content is almost zero. However, the N-glycolyl neuraminic
acid content in the NSO expression system is increased to the level of 10 mol
N-glycolyl neuraminic acid per mole protein, whereas in the CHO expression
system N-glycolyl neuraminic acid is not present. Since normal human tissue
cells do not glycosilate proteins with N-glycolyl neuraminic acid it can be
expected that glycoproteins expressed in the NSO expression system might be
antigenic.

An alternative expression system is the Neospla-vector where the gene of
interest is spliced into an impaired neomycin resistance marker. With this weak
selection marker, clones are selected which are inserted into highly active
regions of the chromosome. Titers which are obtained in this expression system

for monoclonal antibodies, are in the range of 1 to 2 g/l.

Although these expression titers are enormous, expression systems in transgenic
animals are more productive by one order of magnitude. Transgenic expression
systems make use of the bovine lactoglobulin promoter or the

759

promoter. Both are proteins which are highly expressed in the mammary gland.

The gene of interest is the genomic DNA which provides higher titer than the

c-DNA. In the expression vector the gene of interest is inserted

between the exon 2 and exon 7. Proteins which have been expressed by the

expression vector in mice are summarized in the following table.

Proteins which are further developed and are expressed by the

expression system in goat or by the expression system in sheep

are presented in the following table.

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These data demonstrate that recombinant DNA derived proteins can be

expressed in sheep in the case of antitrypsin with a titer of up to 30 g/1 and

monoclonal antibodies like BR 96 expressed by the vector system in

goat with a titer up to 14 g/1. These high titer in the milk of transgenic animals

implicate low manufacturing cost. However, economic manufacturing in

transgenic animals require a high annual output of the purified protein. The

transgenic approach becomes superior to the mammalian cell culture

fermentation systems at a range of 20 and 50 kg of purified protein per year.

With an annual output of 100 kg protein in CHO cells or in goat milk, cost of

goods per gram protein are estimated for the CHO cell fermentation systems in

the range of US $ 300 to 3.000 depending on the growth conditions and the

economy of scale and are estimated to be US $ 105 per gram protein in goat

milk. At these quantities, the production of recombinant DNA derived proteins

certainly is superior using transgenic animal technology.

The downstream processing has to be designed in a way that it can handle
variations in total protein, in casein, whey protein, fat and lactose during the
lactation period or during different feeding of the animals in order to obtain
consistent product quality from animal to animal and from batch to batch.

761

As far as glycosilation of the protein is concerned, there are only very few
examples where mammalian cell culture systems have been compared to
transgenic animals. One example is interferon gamma which is glycosilated at
two sites, asparagin 25 and asparagin 97. In Chinese hamster ovary cells,
interferon gamma is glycosilated as a complex biantennary type core focusylated
at asparagin 25 and complex triantennary type non-core focusylated at
asparagin 97. However, in transgenic mice, interferon gamma at asparagin 25 is
a complex biantinnary type core focusylated and at asparagin 97, oligomannose
hybride and complex N-glycans are present and the molecule is non-core
glycosilated. This implicates that it is not very likely to change from a
mammalian cell culture system to a transgenic animal expression system,
without changing the glycosilation of the protein.

If product safety aspects are considered there are a number of issues that have to
be addressed. Raw materials in transgenic technology are less defined and more
heterogeneous than in biotechnology. In biotechnology, the basis for the
fermentation process is a well characterized Master Cell Bank (MCB).

Transgenic technology usually uses genomic DNA for the expression of the
desired gene in order to obtain higher expression levels. The definition of the
MCB in transgenic technology is not uniform.

Whereas biotechnology uses a single well characterized cell clone and validated
fermentation processes, transgenic technology uses multiple individual animals.

In the case of mammalian cell culture, the absence of viral contaminants in the
MCB as well as in biological raw materials can be analyzed. In the case of
transgenic technology this is not realistic for infectious agents such as scrapies
or bovine spongiform encephalitis. The trust in a reliable source of animals has

762

to replace the proof of absence of such prions. Testing for the absence of prions
in the transgenic animals would take more than one year for the final product.

A non issue for biotechnology is the sickness of animals. What happens if one
or more of the individuals of a flock become sick? Does this have any influence
on product quality or on the impurity profiles? Are the analytical tools
appropriate to address changes? Can the batch be released?

The Commission of the European Communities has some answers in their
guideline: "Use of transgenic animals in the manufacture of biological
pharmaceutical products for human use (1993)."

For transgenic technology these guidelines require the characterization of the
genomic DNA vector construct, expression of the gene in the appropriate tissue,
description of the measure for creation of transgenic animals, determination of
the gene stability during breading and production and demonstration of similar
productivity in different individuals. In comparison to the authorization of a
MCB, in the transgenic approach market authorization for a single individual
can be given or market authorization for a number of identified individuals can
be provided. Compared to sterile fermentation or closed systems, specific
pathogen-free conditions have to be provided and no antibiotics or hormones
have to be given in order to prevent infections. The health status of the animals
have to be evaluated and infected animals can not be used for production. The
purification process has to be validated to handle a wide range of compound
variations. The specification for product activity has to be given per unit of non
fat dry solid. Removal of unknown adventitious agents have to be demonstrated
by validation procedures. For product purity the same requirements are valid as
for biopharmaceutical derived from mammalian cell cultures. However special
attention has to be given to product processing, homologous proteins,

763

glycosilation of the protein and lipid association to the protein. Immunogeneity
aspects of trace impurities might have an higher impact in the manufacturing of
proteins derived from transgenic animals since these proteins usually are used
in high dosage forms or for chronic diseases.

In conclusion, transgenic technology is an economic challenge for
biopharmaceutical manufacturing of proteins or glycoproteins, especially if high
dosages of the active ingredients are required or a high annual capacity for the
proteins in the upper kilogram range is needed. Drawbacks for the expression of
proteins can be the tolerance of the synthesized biopharmaceuticals to the
animals. In respect to product quality and safety, issues have to be addressed
such as genetic stability in different individuals, variation in productivity and
impurity profile during lactation period, product processing in milk and variable
biological active contaminants which are usually a non issue in biotechnology.
If these safety issues can be addressed properly and if high volume of the
biopharmaceutical have to be produced on a constant annual basis, transgenic
technology will be the choice for such products.

Workshop Discussion

Hoppie: We hear about mad cow disease and scrapie in sheep but is there a
Panel: similar disease in goats?
Massie:
Goats can get scrapie and they get sick, and you can see that they
Panel: are sick. There have been 4 cases in the USA since 1947.
Massie:
Panel: With respect to economics, there was a factor of 3 or 4 in the cost
of monoclonal antibodies per gram. When you look at production
Berthold: in transgenic animals the yield is much higher, so what other
factors contribute to the cost?
Panel:
The problem is that we have not been able to make the advances in
Berthold: purification that we have in production. A great deal of the cost is
purification.

Do you take into account the time that it takes to get the animals
ready for production?

I cannot compare the time that it takes to derive a cell line, to

optimise the bioreactor and to produce the monoclonal antibodies,
with preparing an animal. The slow part is getting your processes
ready.

Can we have more information on the new transfection technology
which we have heard about? There is the possibility to have
plasmids that define single genes, then you take chromosomes;
now you take nuclei. What is the next extension?

The use of cloning techniques to transfer genes is just at the
beginning. It will be possible to replace the gene, but we are just at
the beginning, and it will take us more than 5 years before it
becomes routine.

My question concerns the fact that the elegance of molecular
biology is using a single gene. With a chromosome, or nucleus,
you have a lot of non-translated material, ie it is complex with a lot
of unknowns.

765

766 Whether we are using a CHO cell line or an embryo, we are
Panel: introducing a single gene so the system is basically the same.
Naveh:
Panel: Did you look at sero-conversion, antigenicity and antibody
formation for AT3?
Naveh:
Panel: Yes, but these were for only 1 injection and there are no antibodies
after just 1 injection. Whether there will be sero-conversion after
Naveh: repeated injections, will be known after the next trial.
Aunins:
Especially in light of the differences in glycosylation you see?
Panel:
Aunins: Yes, we will be focusing on these differences but then we all realise
Panel: there are differences with the human plasma proteins, including
proteins from CHO cells.
Builder:
By serendipity, it just so happens that proteins made by CHO cells

show very little sero-conversion.

Some of the issues between transgenics and cell culture were
discussed by Dr Werner. Could you tell us what you have done as
process validation on the goats or sheep?

We have spiked at various points with specific viruses.

I was referring to the animals themselves.

We have a closed flock of sheep from New Zealand that are scrapie
free. We have a monitoring system and four vets to examine their
health status. Sick animals are removed from the flock until they
are better and batches are held from such cases until they are
screened. We have an array of ELISA’s for product consistency
against common milk contaminants, eg caseins and beta lacto
globulins. We look at the product the same way as for CHO cells -
MWt, amino acid composition, etc.

The time taken to get herds ready is a major barrier to the use of
transgenics. Can someone comment on the use of transomatics?

Panel: 767
Panel:
Infection by retrovirus, or adenovirus, is not very high or efficient.
Anon: Remember, the gene is submitted to a formidable number of events
Panel: from the first cell stage to the differentiated stage. If you introduce
Panel: the gene into the differentiated mammary gland, it is just like a
Hauser: transfection of a cell in a culture system.

Panel: If you want a quick answer to how would your protein look, you
Szperalski: can use retrovirus packaged in a cell line which is put into the
mammary gland. The problem is that the yields are very low.
Another system uses a cell line which mimics the goat or cow
mammary gland. Having a transient system in the mammary gland
gives you a quick look but sometimes it takes as long to make the
test as to get the animal ready.

If you test the construct in a mouse, can you predict how it would
be in a goat?

With AT3 the mouse product is more heavily sialyated, but it is
similar to monoclonal antibodies.

The mouse does not process in the same way as a sheep does. The
mouse has trouble in gamma carboxylating some proteins.

We have heard that certain proteins, which are not secreted by
CHO cells, are secreted by the mammary gland. A thought about
why they are not secreted is that there is some sort of control
system. Mis-folded and incompleted proteins are held back. Is the
mammary gland a more relaxed system as this sort of control is not
so important? Did you look for the amount of proteins that are
mis-folded and compare it to an alternative system, such as
mammalian cells?

No, we have not tried this although it would be an interesting
experiment. I should imagine that the mammary glad is much more
efficient in secretion. We have found that protein sitting in COS
cells is not degraded but just remained inside. We are also more
selective in using proteins that we know can be secreted.

As every milk drinker knows, taste and quality is dependent upon
the season and kind of food which the animal gets. What is your
experience of these points for the quality of the product?

768 The sheep’s rations do affect the quality of the milk but we have
Panel: not seen any major differences in consistency.
Panel:
Panel: Throughout lactation, glycosylation is very consistent between
Sasaki: animals. In fact, it is probably more consistent than in cell cultures
Panel: where glycosylation is often very variable.
Sasaki:
Panel: The only major difference we have found was when the sheep were
Panel: given salt licks as this changed the electrolyte composition of the
Miller: milk.

Panel: A comment on proteins secreted from mammary epithelial cells but
not CHO cells. Casein would be secreted as a micelle, not as a
single protein, and other proteins would be in the micelle.

There are proteins in milk which are not bound to micelles, such as
beta lacto globulin. So it is not certain that a micelle would help

these whey proteins to be secreted.

If the protein has an affinity for the casein, that would be included
in the micelle form.

Most of the proteins we work with are in the whey fraction but this
fraction seemed to be associated with the caseins, so this is an
interesting observation.

BHK and HepG2 cells are very poor at secreting fibrinogen which
is not associated with micelles when it comes out in the milk.

Importing your sheep from New Zealand does not protect you
from prions suddenly appearing - no-one knows how these appear
and it maybe that some part of the genome escapes control. My
response is that the patient should be able to choose whether he
receives the protein from transgenics or from biotech. The figures
you gave should be evaluated independently of the costs. It does
not mean that biotech is dead as there is still much basic science
that can be done to improve yields. The cost figures were
impressive but I would not like to be led by financial considerations
alone.

If you take industry as independent, we recalculated all these
figures. Manufacturing costs are cheaper but there is a need to
produce a large amount of material - above 20 to 30 Kg per year.

Panel: 769

Panel: A comment on prions. In Scotland we operate a closed flock on
Lupker: arable land which has never had sheep on it. We have not had a
Panel: case of scrapie in the years we have used this site. If it was a
Merten: spontaneous event, as suggested in the question, we would have
Panel: had a case by now. Anyway, our spiking experiments show that
Rhyll: we have 10 to 20 logs of clearance over and above what we would
Panel: need to clear anything found in milk. Prion particles have never
been found in milk. WHO categorises milk as a category 4
Panel: substance which is as safe as you can get.

The patient may not get a choice because some proteins can only
be produced by transgenic technology.

There are not that many therapeutic proteins that have to be
produced in ton quantities. Has anyone looked at producing other
than therapeutic proteins in transgenic animals, and how does the
production compare to that in micro-organisms?

We think that we can make a protein for the same price as in
E. coli. The advantage is that it is properly folded.

When you compared costs, was it only the production costs or was
it validation and regulatory costs as well?

The $105 included everything.

I would be interested in more details of your prion validation
studies, and what contingency plans have you for a herd getting
sick?

Prion validation is done by outside contractors so I cannot tell you
what the strain was. We have monitored it for 18 months and the
clearance rates have been better than expected. Contingency plans
for infected herds include having multiple production sites, and
back-up animals.

We also have a semen bank.

770 In the presentations some differences were mentioned between cell
Julien: culture processes and transgenic processes in terms of downstream
processing. I noticed that there was a one log reduction factor of
Panel: viral safety. Do you pay special attention to viral safety in the
Panel: downstream processing? Are there any technological differences
Aunins: between the two processes?

Panel: The short answer is no, there are no differences. We have viral
inactivation and removal in transgenic processes using the same
Panel: technology with the same result. The actual viral load in milk is
Grammatikos: very low.
Panel:
In the AT3 process we have cross-flow filtration which is a great
viral removal step and which is not usually used in cell culture
processes.

Transgenics is a great technological achievement but, as far as a
production vehicle goes, what exactly is the big deal and what are
your concerns? If you look at licensed products, you have
influenza vaccines made in embryonic eggs, anti-venoms from
snakes and horses, and OKT3 from mice. What is fundamentally
different about transgenics?

As long as you require low-dosage proteins there is no economical
advantage in transgenics, and because of the well characterised
status of proteins from cell cultures this is the preferred process.
However, if you need 50 Kg or more of proteins, then transgenics
is a good choice.

Another example is plasma proteins from humans but the fact is
that none of these examples are cloned. There is a new set of
regulations because you have cloned genes, as in cell culture.

Can you tell us about the conditions that the animals are kept in?

We have 3,000 sheep in Scotland which are well looked after with
4 full-time vets - in fact a higher vet to sheep ratio than GPs to
humans! Animals are kept until they are 7-9 years old. Sheep in
Scotland are kept outside in winter but we have housing for 75%
of ours. They get out into the fields and graze normally. They
have a specific diet.

Miller: 771
Panel:
Panel: If there is an alternative, we should not use animals to satisfy
human needs. Pressure has stopped animal testing of cosmetics. I
am strongly against this example of animal eugenics which is purely
for financial reasons.

The animals, as I have described, are well looked after and some of
the products cannot be made by other means in the necessary
quantities. We are making over a metric ton of AAP a year, and
many tons of human serum albumin. If you can show me another
way of producing this, I would be happy to try it.

Society has to decide whether it is worth spending the money on
medicines. If it decided to pay 100 times more to produce it in
tissue culture, then many people will not receive the medicine.

The trade-fair-sponsors of the 15th ESACT-Meeting:

Adi Biotech Sarl (Applikon) JRH Biosciences

Aber Instruments Ltd. Life Technologies

B. Braun Biotech International Lonza Biologies plc

Bayer Diagnostics France LSL-Biolafitte SA

Bibby Sterilin Ltd. Medi-Cult A/S

Biolnvent Production AB Microbiological Associates

Bioprocessing MicroSafe BV

Boehringer Ingelheim Bioproducts Molecular Devices GmbH

Partnership New Brunswick Scientific Sa

CanSera International Inc. Nunc A/S

Cellex Biosciences Inc. PAA Laboratories GmbH

Cellon Sarl Pall Europe Ltd.

Compex B.V. Pharmacia Biotech AB

Corning Costar Corp. PPL Therapeutics

Covance Laboratories Ltd. Q-One Biotech Ltd.

Dr. Karl Thomae GmbH Quest International B.V.

ECACC-ESACT Secretary CAMR Sarstedt

Genespan Corp. Schärfe System GmbH

Genetic Engineering News Selborne

Greiner Labortechnik GmbH Sigma-Aldriche-Chimie

Heraeus Instruments GmbH Sorvall Ltd.

Hyclone Europe S.A. Spectrum Europe

Inceltech France SA Stedim SA

Institut Pasteur Relations TC Tech Corporation

Industrielles TCS Biologicals Ltd.

Integra Biosciences The Automation Partnership

Inveresk Research Unisyn Technologies

773

INDEX

A549 cells, 705 322
A549 lung cells, 713 437
acoustic perfusion, 379 153
action potentials, 690
activators, 158 BHK, 215
active hydrogen, 94 BHK cell, 219
active oxygen, 93 BHK metabolism, 223
Adeno-associated virus, 493 BHK-21, 157, 209
adenovirus, 513 BHK-21/BRS cells, 561
agitation, 201, 285, 399 BHK-21A, 185
AIDS, 588 bioartificial liver, 661
biodistribution analysis, 545
2,6-sialyltransferase, 131, 185 biologicals, 561
1 -antitrypsin, 745 biopharmaceuticals, 433, 481
alphaviruses, 584 bioprocess effects, 5
amino acid consumption, 277 bioreactor, 117, 399, 409, 513
amino acids, 235 biosafety testing, 469
ammonia, 135 biosensor, 357
ammonium, 157, 281 bone marrow, 613
animal protein free medium (MDSS2N), BrdU-incorporation, 729
buffered salt solutions, 445
561 buffers, 445
annexin-V, 259 burn wounds, 673
antigen expression, 285
antigenicity, 163 293 cells, 121, 127, 293, 513
antigens, 583 4647 cells, 577
antisense, 247 calcium phosphate, 122, 125
antisense glutamine synthetase, 168 calorimetry, 355
antisense RNA, 191 capacitance, 322
antisense RNA expression, 157 carbon dioxide, 135
apoptosis, 227, 231–233, 235, 243, 247, cardiogenesis, 693
cardiomyocytes, 690
255, 259, 643, 729 CD4 modulation, 601
apoptosis-resistant, 247 cell, 637
applicator, 613 cell counter, 329
arrested in the G1 phase, 227 cell counting, 729
arrhenins, 363 cell culture, 399
artificial organs, 657 cell cycle, 77, 247, 643
artificial skin, 673 cell cycle distribution analysis, 228
ATP content, 629 cell density, 324, 630
ATPase activity, 39 cell distribution, 627
atrium-like cells, 693 cell factories, 101
bacitracin, 59 cell growth, 94, 627
baculovirus, 35 cell metabolism, 277, 458
baculovirus infection, 153, 277, 329 cell physiology, 457
baculovirus-insect cell expression, 303 cells, 321
baculovirus-insect cell system, 597 cell-settler, 395
baculovirus-Sf9 insect cell system, 39 cell shuttles, 531
bag, 399 cell size, 324, 329
Bcl-2, 235 775

776 DNA, 333
dog skeletal muscle, 541
cell substrate, 577 downstream processing, 513, 757
cell technology, 463 Drosophila melanogaster, 29
cell volume, 627
cellular immunity, 583 economy, 757
centrifugal elutriation, 77 electrolyzed reduced water, 93
characterize animal cells, 317 embryoid bodies, 689
Chinese Hamster Ovary (CHO), 59, 77, embryonic stem (ES) cells, 689, 751
emphysema, 746
81, 141, 205, 227, 293, 321, 459 endoplasmic reticulum, 101
CHO cells, 131, 181, 310, 359 endoprotease, 69
cholera toxin B subunit, 617 endothelial cells, 593
chromatography, 441, 481 endothelium, 733
c-jun, 247 energetic status, 223
clonal variability, 81 enolase, 168
cloning, 175 environmental stress, 243
epithelial differentiation, 691
clumped cells, 395 erythropoietin, 463
CNAH, 191 estradiol, 215
concentrates, 445 EX-CELL ™293-S, 293, 302, 420
conductive electronical cell count, 333 EX-CELL ™ Vero SF, 293
excitotoxicity, 255
conductivity, 324 expression hosts, 5
continuous culture, 351 expression of genes, 690
core 181 expression systems, 757
co-stimulatory factors, 583 expressions, 209
count, 317 extracellular matrix, 637
cryopreservation, 510
CTP, 458 Factor X, 69
culture, 541 fatty acids, 198, 200
culture conditions, 460 fed-batch, 347
culture systems, 595 fed-batch and continuous cultivations,
cultures, 542
cystic fibrosis, 746 141
cytochrome P450 isoenzymes, 97 fed batch cultures, 267
Cytodex, 570 fed-batch process, 460
cytokines, 583, 637 FIA, 343
cytomegalovirus (CMV) immediate early fibrinogen, 745
fixed-bed, 243, 657
promoter, 585 fixed bed bioreactor, 627, 635
flow chamber, 733
2D microcarriers, 378 flow cytometry, 77, 259
3-D, 665 fluidized bed, 381, 385, 437
3-D culture, 717 fluidized bed adsorption, 429
dead cells, 333 fluidized-bed bioreactor, 281
derivatives, 601 fluorescence, 231–233, 733
development of safer vectors, 527 flux, 351
dexamethasone, 721 freeze drying, 417
dhfr, 81 fructose-6-phosphate, 157
dialysis, 347 fully automated PC-based image analysis
dielectric spectroscopy, 321, 355
dielectrophoresis, 369 system, 317
differential gene expression, 97 Furin, 69
differentiation, 690
diploid cell culture, 577 ganglioside GM3, 601
direct capture, 429
DISC HSV-2, 569
displacement chromatography, 441
disposable, 399
DMSO, 29

gel electrophoresis, 737 777
gene expression, 94, 101, 105 high fire cells, 277
gene therapy, 442, 503, 531 high five cells, 153, 277, 329
gene therapy vector, 545 high-spin condition, 613
genetic manipulation, 1 high titre virus, 569
gene transfer, 495, 541 hippocampus, 255
histidine tagged, 73
gluconeogenesis, 267 HIV-1, 588
glucosamine-6-phosphate, 157 hollow fibre, 417
glucosamine-6-phosphate isomerase, 157 hollow fibre cultures, 43
glucosamine-6-phosphate synthase, 158 hollow-fibre bioreactors, 627
homing, 531
glucose, 669 homologous recombination, 690
glucose and glutamine levels, 219 hormonal regulation, 665
glucose transporter, 168 host system, 163
glucose uptake and lactate production, human agamma serum, 607
human cell lines, 273
223 human cells, 685
glucose-6-phosphate, 158 human chromosomes, 751
glutamate, 255 human EPO, 185
glutamine, 158 human GM-CSF, 163
glutamine synthetase, 43
glyceraldehyde-3-phosphate human hematopoietic progenitor cells,
635
dehydrogenase (G3PDH), 597
glycoforms, 5, 411, 433 humanized MAb, 417
glycolipids, 158 human monoclonal antibodies, 617, 751
glycoprotein production, 359 human multidrug transporter (MDR1), 39
glycoproteins, 158, 175, 191 humoral, 583
glycoprotein structure-function hybridoma, 197, 235, 243, 281, 351, 459,

relationships, 5 469
glycosylation, 35, 135, 149, 157, 181, hybridoma cells, 167, 347
hydroxyapatit, 443
395 IDE, 59
glycosylation engineering, 5 IEF, 310
glycosylation pattern, 141 IEF analysis, 409
green fluorescent protein (GFP), 125 image analysis, 729
growth-controllable cell line, 247 immortalised hepatocyte, 97
growth curves, 511 immortalised mouse hepatocyte, 657
growth deprivation, 247 immortalization, 643
growth equation, 355 immune glycoprotein, 566
growth factors, 273 immune system, 613
growth inhibition, 215 immunofluorescence, 85
growth potential, 457 immunoglobulin A, 149
growth regulation, 209 immunomodulator, 613
GST-sialidase fusion protein, 178 in vitro development, 690
5-HT3 receptor production, 449 in vitro immunization, 617
heat flow rate, 355 inactivation of hepatitis A virus, 485
heat flux, 355 influenza, 551, 555, 587
HEK293, 117 inhibitor, 158
HEK 293 cells, 105, 381 insect cell culture, 153, 277, 329
hematopoietic, 637 insect cells, 29
hematopoietic progenitor cells, 503 insulin, 669
hemicellulose, 274 insulin-degrading enzymes, 59
hepatitis C, 35 Interferon Regulatory Factor 1, 209
hepatocytes, 643, 661, 665, 717 interferons, 613
hepatoma, 721 interleukin, 617
HIA, 345
high cell density, 377

778 membrane-bound polysomes, 102
metabolic activity, 355
intracellular assay, 85 metabolic engineering, 157, 168
intracellular physiologic data, 458 metabolic flox, 357
intracellular proteins, 101 metabolic network analysis, 351
intralipid, 205 metabolic rates, 243, 356, 657
ion channels, 690 metabolic shift, 219
ion exchanger, 429 metabolism, 219
IRF-1, 209, 215 metal suspension, 613
ischemia, 725 metallothionein, 29
islet, 669 method of Vindeløv, 228
JQEF cells, 573, 577 mHep-R l cell line, 657
keratinocytes, 673 microcalorimeter, 355
L-68 cells, 577 microcarriers, 385, 513, 561
lactate, 281 microcell-mediated chromosome transfer
lactate and ammonia, 219
LDH, 333 (MMCT), 751
Leningrad-Ib-vaccine strain, 573, 577 microgravity, 231–233
leukocyte alpha interferon, 607 microinjection, 745
linoleic, 197, 199 migration, 637
linoleic acid, 197, 198, 200, 201 milk, 743
lipid, 197, 200
lipid supplements, 205 Milk-specific expression, 746
lipopolysaccharides (LPS), 441 mitogens, 737
long-term culture, 81
loss of function, 690 mixing device, 445
Louping iLL, 588 monitoring, 457
lyophilisation, 757 monkey, 573
lysophosphatidic acid, 205 monoclonal antibodies, 55, 85, 197, 389,
Mab, 197
macrophages, 531 409, 429
macroporous, 385 monolayer cultures, 593
macroporous carriers, 322, 627 morphogenesis, 577
macroporous microcarriers, 51, 381 mRNA retargeting, 101
magnetic resonance imaging, 627 multinucleated myotubes, 693
magnetic resonance spectroscopy, 627 murine leukemia virus, 469
major histocompatibility complex murine retrovirus, 473
mus dunni cells, 473
(MHC) class I, 583 mycoplasma, 705, 713
major histocompatibility complex Mycoplasma pneumoniae, 705, 713
mycoplasma testing, 523
(MHC) class II, 583 myoblasts, 677
mammalian cell, 43 myoblast transfer therapy, 677
mammalian cell culture, 757 myogenesis, 693
mammalian genome, 1 6-N-acetyl-D-glycosaminyltransferase,
mannose, 158
mannose-6-phosphate, 158 181
mass, 149 Na-butyrate, 131
mass spectrometry, 713 N-acetyl-glucosamine-6-phosphate, 158
mathematical model, 359 N-glycans, 157
MDCK, 551, 555 naked DNA, 584
MDR l-ATPase, 40 natural killer (NK), 583
measles vaccine, 573, 577 necrosis, 231-233
media optimization, 757 neurogenesis, 693
neuromuscular disorders, 531
neuronal, 690, 725
neuronal culture, 255
neurotrophic, 725
new drug form, 463

new form, 573 779
newborn pig thyroid organ culture, 682 process control, 347
NGF, 726 process development, 459
NMR spectroscopy, 223 processing, 69
NSO myeloma, 51 process monitoring, 329
NTP/U ratio, 458
nucellin-Zn, 227 product monitoring, 343
nuclear transfer, 745 product recovery, 437
nucleoprotein, 587 product safety, 757
nucleotides, 458 production medium, 55
nutrient medium, 445 production processes, 457
nutrient supply, 385 production schedules, 511
oleic, 197-199 productivity, 77, 363
oleic acid, 197, 200 progenitor, 637
oligosaccharides, 5, 175 proliferation control, 209
on line monitoring, 223 propeptide, 70, 73
on-line substrate, 343 protein expression, 29
optical density using laser light, 321 protein glycosylation, 153
optimisation, 509
oral administration, 573 protein processing, 153
oxygen demand, 277 protein production, 745
oxygen uptake rate (OUR), 347 protein secretion, 153
P-glycoprotein, 39 proteinase, 309
P53, 643 proteinase inhibitors, 304
pacemaker-like cells, 693 proteolytic activity, 306
packed bed reactor, 661 prourokinase, 389
Pasteurisation, 485 purification, 417, 433, 442
patient monitoring for RCR, 527
peptide, 725 rabies virus, 561
perfused bioreactors, 721 RAP-PCR, 97
perfusion, 733 rCHO, 85, 463
perfusion cultures, 56, 235, 369, 459 reactor pH, 629
perfusion system, 395 receptors, 690
persistent hypothyroidism, 681 recombinant, 309
pH, 285 recombinant cells, 357
pHi, 259 recombinant CHO cells, 389
phosphorylation mutants, 39 recombinant insulin, 227
pilot scale, 429 recombinant protein, 29
pituitary extract, 737 recombinant RNA, 584
plasmid DNA, 441 recombinant soluble human PSGL-1, 181
plasmid production, 113 reconstitution, 445
plasmid purification, 113 reduced conditions, 51
polar lipids, 713 regulation-friendly media, 293
polymerase chain reaction (PCR) replication-competent adenovirus (RCA)

technique, 545 testing, 526
polysaccharides, 274 replication-competent retrovirus (RCR)
porous carrier, 669
porous microcarriers, 281, 389 testing, 526
post-transcriptional control, 101 reproducible, 317
postmitotic nerve cells, 693 retinoblastoma, 643
primary cultures, 725 retroviral packaging cell, 503
primary porcine, 665 rFurin, 70, 71
rFX, 73
RNA, 168
Rotary Cell Culture System, 717
routine, 317
routine screening of pharmacological

functions, 695
RTPCR, 97

780 transgenic mouse mammary epithelium,
743
cells, 473
transgenic technology, 750
S2 cells, 29 transient expression, 121, 125
scale-up, 509, 595 transient protein expression, 36
scale-up schedules, 460 transient transfection, 105, 113, 117, 442
schistosomiasis, 597 TUNEL assay, 227
secretion, 101, 121 3'untranslated region, 101
semicontinuous process, 55 UDP-activated N-acetyl hexosamines,
Semliki Forest Virus (SFV), 449, 584
serum concentration, 285 157
UDP-activated sugars, 458
serum-free, 105, 197, 199 UDP-N-acetylhexosamines, 458
serum-free culture, 117, 273 DTP, 458
serum-free media, 513, 555 vaccination, 583
SF-9, 35, 293 vaccine adjuvants, 581
SF21, 293 vaccines, 561, 583
variable valency, 613
Sf21 cells, 153, 329 vascular smooth muscle cells, 690
sialic acids, 175, 191 vectors, 493, 584
sialidase, 175 ventricle-like cells, 693
sialylation, 131, 135 Vero cell line CR2C9, 569
signal peptide, 101 Vero cells, 561, 577
simulated microgravity, 717 viability, 333
Viable Cell Monitor, 321
SIV envelope gp 160, 588 viral disease, 583
viral removal, 481
skeletal muscle, 690 viral vectors, 583
skin fibroblasts, 673 volume fraction of viable cells, 324
SP6 RNA polymerase, 585 von Willebrand Factor, 69
specific antibody productivity, 81 vWF, 70
specific productivity, 141 wave, 399
spectrometry, 149 xenotransplantation, 681
spleen, 613 zymography, 313
Spodoptera frugiperda, 231–233
stability, 81,512
stirred tank, 594
stoichiometric coefficients, 356
streamline, 429
substrates, 36, 351
suicidal DNA/RNA, 583
suspension growth, 562
SV40 early promoter, 159
synapses, 694

T-cell receptor, 121
TB/C3-pEF, 243
TBK3-bcl2, 243
temperature, 363
therapeutic agents, 685
THOMAE, 459
thymus, 613
tissue engineering, 661
total virus input spike, 482
toxicology, 721
trans-epithelial electrical resistance, 705
transfectants, 43
transfection, 255
transgenic animals, 745
transgenic livestock, 745