Proc. Nati. Acad. Sci. USA

Vol. 76, No. 4, pp. 1863-1867, April 1979 Cell Biology

Growth and morphology of colonies of Chinese hamster ovary cells growing on agar is affected by insulin (quantitative assay for hormones/automated cell biology)

BRUCE D. AIDELLS, MICHAEL W. KONRAD, AND DONALD A. GLASER Molecular Biology and Virus Laboratory, Stanley Hall, University of California, Berkeley, California 94720

Contributed by Donald A. Claser, January 18,1979

ABSTRACT As a model for the effect of hormones and growth factors on three-dimensional growth of mammalian cells, we have analyzed the effect of insulin on the three-dimensional growth and morphology of Chinese hamster ovary (CHO) colonies grown on the surface of agar. Sequential photographs in dark-field illumination of growing colonies have been analyzed with computer-assisted techniques. In this analysis the entire shape of each colony in a sizeable population (up to 105 colonies per experiment) can be measured and distributions of parameters derived from these measurements can be studied. In fetal calf serum (FCS), insulin has a dose-related stimulatory effect on cell growth that is most pronounced when growth has slowed down. In 10% FCS, insulin has a similar but diminished effect. When CHO cells are grown conventionally on plastic substrata or in suspension, insulin has little effect on cell growth at 4% serum concentration. Computer analysis of changes in the distribution of colony morphology proved to be a sensitive, dose-dependent, and reproducible assay of a hormonal effect. As little as 5 ng of insulin per ml added to 10% FCS causes a shift in the distribution of colony morphologies. In 4% FCS, 50 ng of insulin per ml is required to produce a detectable change in the colony morphology distribution. Computer analysis of cells grown three-dimensionally on agar provides a powerful approach to studying the effects of hormones and provides observations not available when cells are grown on plastic substrata.

Since the pioneering work of Puck and his coworkers (1, 2), it has been evident that both the clonal growth and the colony morphology of mammalian cells are sensitive to the conditions of culture as well as to the origin and state of the cells used to seed the clones. Under well-controlled conditions, measurements of the growth of clones and the morphology of the resulting colonies can be the basis for useful bioassays of hormones (3). While the scoring of growth or no growth by simply counting colonies has led to its extensive applications in nutritional, genetic, and other studies (4), quantitative study of colony growth or morphology has not been exploited similarly. The primary obstacle to the use of such data is the instrumentation necessary for objective, quantitative determination of colony size and shape for any appreciable number of colonies. We have constructed a facility for making such measurements that allows analysis of time-sequential photographs of more than 105 colonies per day. There are many documented effects of hormones on cells grown in vitro. The list includes effects on proliferation, differentiation, metabolism, and other biochemical changes (5, 6). One important in vivo effect of hormones is on morphogenesis. Alveolar development in the mammary gland during pregnancy (7), cyclic changes in the uterus and vagina during the estrus cycle (8), and embryologic effect of hormone on urogenital development (9) are all prominent examples of effects of hormones on morphogenesis. Few in vitro mammalian The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

systems exist, however, for the study of the role of hormones in three-dimensional morphogenesis. Rtkently it was shown that both transformed (10-12) and sown nontransformed cells (13-15) could be grown on top of led medium at the gas interface. This allows growth in Environment where the supporting substratum does not dom te the interaction among the cells, and where the anticonvection properties of the gel enable each clone to develop in its own self-defined microenvironment. This allows three-dimensional forms of growth (12) to develop that may express more of the intrinsic attributes of the cells than growth in liquid suspension or on more adhesive solid substrate under liquid (16). Chinese hamster ovary (CHO) is a cell line highly adapted to the artificial environment of the tissue culture vessel. It has been cultured almost continuously for over 20 years (1), displays transformed properties such as growth in suspension and soft agar, and grows at a rapid rate (15-hr doubling time). Using this system, we have shown that cells grown on agar demonstrate a quantitative, proliferative, and morphogenic response to insulin that is dose dependent in the physiological range. Preliminary results with fibroblast growth factor show different changes and indicate that these changes in morphology and growth rate may be sensitive to a variety of growth factors, each producing a different set of effects. METHODS Cells. CHO-S cells were obtained from L. Thompson (Lawrence Livermore Laboratories) and were adapted to spinner culture. The two clones used in this study are called SCI and SC3. [SC1 has been studied in previous reports (12). In those publications SCI was called clone 2. We wish to use the new nomenclature in this and later publications. ] All clonal stocks were stored in liquid nitrogen. When an experiment was to be undertaken, frozen stocks were thawed, placed in T-flasks for 2-3 days, and then seeded into spinner flasks at 104 cells/ml in a minimal essential medium plus 10% fetal calf serum (FCS). The cells were then used for an experiment within 2 weeks. For experiments reported here cells were grown on the surface of 0.5% Noble agar containing a minimal essential medium and serum. Hormones. Porcine insulin was obtained from Sigma. Stocks were made up fresh for each experiment in 5 mM HCl and stored at 4VC. The final concentrations of insulin used in the study reported here were 5, 50, 500, and 5000 ng/ml. Facility for Automated Experiments in Cell Biology (FAECB). The facility centers on an instrument named "Cyclops," which is housed in its own environmentally controlled building. Cyclops is a semi-automated system for sorting and inoculating cells for clonal growth and for incubating and photographically recording the growth of the resultant colonies. It is a large-scale (about 9000 100-mm dishes can be incubated at one time) versatile system, in which some components are

Abbreviations: FCS, fetal calf serum; CHO, Chinese hamster ovary. 1863

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Proc. Natl. Acad. Sci. USA 76 (1979)

fully automated and computer controlled for ease and reproducibility of operation (e.g., photography). The general scheme for using Cyclops is as follows: A cell sorter/inoculator (modified Becton-Dickinson cell sorter) is used to inoculate cells. To do this, the sorter/inoculator mounted over the Cyclops movable x-y stage is used to drip uniformly spaced rows of selected single cells onto a tray of 32 dishes. The dishes contain culture medium solidified with a gel, so that growth of the resulting clones is at the gel/gas interface. This convection-limited mode of growth ensures that no daughter satellite clones will be seeded during the course of an experiment. Incubators can be set at any temperature from 15'C to 450C within ±0.100C at controlled humidity, and using any of a number of gas mixtures. In our experiments cells were maintained at 370C, 100% relative humidity, and 71/2% C02/air. The Cyclops clean room provides access to seven independently regulated incubators with a total storage space of 280 trays. As desired, trays of dishes can be removed from the incubators, mounted on the Cyclops stage, and photographed by an automated camera system. The system includes two light sources, an on-axis source for bright-field photography and an off-axis source, used in our experiments for dark-field photography. Photography uses black-and-white film only, but the spectrum of the light source can be varied by the use of a series of color filters mounted on a rotating wheel. Different types of colonies have different photographic requirements. In our experiments, animal cell colonies growing on a gel are photographed through a red filter to avoid interference by the phenol red absorption of the medium. In this case the image of a colony is formed by scattered light, and the intensity of the image is proportional to the mass of the colony. The camera's lens system is flexible and a number of magnifications from XO.24 to X4.8 are possible (100 X 100 and 5 X 5 mm field of views, respectively). The film is analyzed with a Digital Equipment Corporation (DEC) PDP-KL10 computer interfaced with a flying spot gray level scanner, specifically designed and built for the analysis of film from Cyclops photography experiments. The DEC System-10 facility is further equipped with numerous auxiliary input/output devices to facilitate the image analysis and pattern recognition phase of the work. The flying spot scanner acts as an optical microdensitometer with a resolution of 50,um and measures the amount of light scattered at each point of each colony. A condensed but essentially complete 3-dimensional description of each colony in an active experiment is then written on a high-speed magnetic disc. This information can be accessed by the computer to produce the statistical distribution of any of a number of desired colony characteristics (see Fig. 2C). These characteristics include diameter, total light scattered (optical mass), thickness of a colony at the center, and 3-dimensional shape. By using sequential photographs taken at various times, growth changes in the characteristics of each clone can be determined. In this way each clone can be observed in quantitative detail and in time lapse. This quantification can be done for a large population of clones, so that very small changes in behavior can be observed at a statistically meaningful level. Such differences could be unnoticed by the naked eye or if only a few clones were examined. Correlations among colony characteristics can be established and isolation of new variants can be carried out with the computer, because the physical location of each colony is also recorded.

pictorially the two effects of insulin: (i) with as little as 50 ng/ml, insulin stimulated growth in both clones on day 14 of culture; (ii) insulin caused distinct changes in morphology in clone SCI but not SC3. Effects of Insulin on Growth. The effect of insulin on the growth of colonies on agar was measured and was found to be most pronounced for clone SC3. Growth curves and dose-response curves for this clone are illustrated in Fig. 2. On the y axis each unit represents a doubling in optical mass. During the rapid phase of growth, insulin had little effect on growth in 4% FCS (Fig. 2A), but as growth slowed, the effect of insulin became significant; it was most pronounced on the 20th day of culture. When the effect of insulin is plotted as a dose-response curve (Fig. 2B), there is found a roughly linear relationship between log of colony mass and log of insulin dose in which a 12-fold increase in colony optical mass is observed over the range of insulin studied for growth in 4% FCS. In 10% FCS the stimulatory effect of insulin is not as pronounced, probably because the cells are growing nearly maximally in this serum concentration. Colony mass values in 4% FCS with higher doses of insulin are equivalent to those in 10% FCS without insulin. The significance of this observation will be discussed later. Using the PDP-10 computer, the flying spot scanner, and the data analysis system, we prepared population profiles of growth for the entire clonal population at each of the five doses of insulin (Fig. 2C). This type of analysis demonstrates a distinct advantage of the automated system because the entire distribution of the population can be examined and distinct subpopulations in each population can be detected. From Fig. 2C

RESULTS Description of CHO Clones. The two clones of CHO cells used for quantitative measurements in these studies are designated SCi and SC3 (Fig. 1). SC3 is flat, while SCI shows mounding in the center of the colony. In Fig. 1 are illustrated

FIG. 1. Photographs of the two clones of CHO cells used in our study. (A and C) Clone SC3; (B and D) clone SC1. All the colonies were photographed on the 14th day of culture. Colonies in A and B were grown in 10% FCS without insulin, while for colonies in C and D insulin was present in the medium at 50 ng/ml during the whole experiment. (X7.)

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Proc. Natl. Acad. Sci. USA 76 (1979)

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FIG. 3. Growth curve of clone SC3 cells grown on plastic or in suspension culture in 4% FCS (0) or on 4% FCS plus insulin at 500 ng/ml (0). (A) Six-centimeter tissue culture dishes were seeded with 1000 cells and observed for the next 10 days. Cells on the dish were removed with trypsin and those released into the medium were counted with a particle counter and the values were combined. The medium was not changed during the experiment. Each point is the average of duplicate plates. (B) Two thousand cells were seeded into 6-cm bacteriological dishes. Cells remained in suspension for the next 11 days and duplicate dishes were counted with a particle counter.

Effect of Insulin on Colony Morphology. Another and possibly more sensitive way to monitor the effect of insulin is by means of its effect on CHO colony morphology. For this 1 I Pi rCL 0 study, our clone SCI was employed. With as little insulin as 5 5 ng/ml in 10% FCS, there was a striking and reproducible F / change in colony morphology. There was no longer a large flat \ ledge surrounding a central mound and the mounded central A1'\I~ I,, -s,~~~~~~~~~~~~~~~~~~..K area became larger. When the insulin dose was increased, there 20 12 14 22 18 24 16 10 was a decrease in the flat peripheral ledge and an increase in Optical mass of colony the mounded central portion, as will become apparent in the next section on quantitation of the change in morphology. FIG. 2. Effect of insulin on growth of clone SC3 cells. (A) Growth The striking effect of insulin on SCI morphology is depicted curve for clone SC3 grown in 4% FCS plus insulin. Each unit on the y axis represents a doubling in mass. (B) Dose-response curve drawn in Fig. 4, which includes two computer-drawn topological maps from the 20th day of culture from A. The lower curve is for 4% FCS; representing light-scattering information. The left figure is a 0

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we can see that at each dose of insulin there were only a few colonies that failed to respond (those at the left of the graph). Insulin did not cause a significant change in the shape of the distribution curve, but it did cause a shift in the median colony size to larger sizes. When SC3 cells were grown clonally on plastic tissue culture dishes under liquid media, insulin at 500 ng/ml had little or no effect on growth even after growth had slowed down (Fig. 3A). By growing cells as 3-dimensional colonies on agar, we were able to measure a growth effect not observed with traditional plastic tissue culture technology. Cells from clone SC3 were also grown in suspension culture in plastic bacteriological dishes. Very few cells stick to these dishes and most remain in suspension. Cells were grown in either 4% FCS or 4% FCS plus insulin at 500 ng/ml. Fig. 3B shows the growth curve for this experiment. During the exponential phase insulin had no effect on growth. After the growth had slowed down, insulin had its maximal effect on the 10th day of culture, causing only a 2-fold increase in cell number.

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FIG. 4. Determination of mounding index. By dividing the radius of each colony into thirds, we can quantitate morphological differences, using a parameter we call the mounding index, defined as loglo I(B/(C)/(A/B)J, in which A, B, and C are the amounts of light scattered by the corresponding zones of the colony. On the bottom are computer-drawn topological contours of a typical colony grown in 10% FCS on day 14 (mounding index 0) (Right). The vertical axis is exaggerated.

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Proc. Nati. Acad. Sci. USA 76 (1979)

typical colony of SCi grown in 10% FCS without insulin; the right figure shows a colony grown in 10% FCS plus insulin at 50 ng/ml. In order to quantitate the effects of insulin on morphology we have divided the radius of each colony into three equal parts defining regions A, B, and C as shown in Fig. 4. From the amounts of light scattered by the three regions, we can compute a quantity we call the "mounding index." The miounding index is defined as logjo[(B/C)/(A/B)]. This definition of the mounding index was chosen because it distinguishes well between flat and mounded colonies, corresponds with intuition by having a large value for mounded colonies and a small value for flat ones, and is easily computed and displayed by using the colony measurements (Fig. 5). Using the mounding index, we have examined the effect of insulin on a population of clone SCi on day 14 of culture in 10% FCS. Insulin added to 10% FCS has a dose-dependent effect on the median mounding index. This dose-response curve illustrated in Fig. 5B shows that insulin at 5 ng/ml has increased the mounding index significantly. Comparison of Growth and Effect of Insulin on the Two Clones. Growth comparison of clones grown in 10% FCS without insulin and those grown with 4% FCS plus insulin at 500 ng/ml (Fig. 6) showed the importance of quantitative analysis of colony morphology. In both clones the growth curves on agar under the two conditions were virtually identical. The addition of insulin at 500 ng/ml to 4% FCS has the equivalent effect on growth as an increase of 6% FCS. By simply counting the number of cells per colony one cannot distinguish between the effects of insulin and of increasing serum, but quantitative analysis of changing morphology clearly distinguishes between the effects of serum and of insulin.

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DISCUSSION Effect of Insulin on Growth. Our results show that insulin in physiological doses stimulates growth and causes distinct changes in colony morphology of CHO cells grown on the surface of agar. Insulin stimulates colonial growth of the same CHO cells only slightly when they are grown on plastic or in suspension. Similarly, Hamilton and Ham (3) were unable to detect a growth-stimulating effect of insulin on CHO clonal growth in serum-free media on plastic. Because insulin changes both the morphology of the colony and the shape of the total growth curve (colony mass versus time of incubation), it is possible that the two effects are related. It seems unlikely, however, that the changes in colony shape and colony growth rate have a simple causal relationship because insulin stimulates growth less in SC1 than in SC3, while it induces greater changes in colony shape in SCI than in SC3. Furthermore, insulin induces increased mounding in the center of the colony together with a reduction in overall colony diameter while it also stimulates overall growth. It seems plausible, then, that different mechanisms account for the changes in morphology and the change in growth rate, although they may share receptors and other parts of the molecular machinery responsible for sensitivity to insulin. On agar, in liquid suspension over a nonadhesive substrate, and on an adhesive substrate, growth does not seem to be stimulated appreciably by insulin during the phase of most rapid growth. In all three environments, insulin increases growth during the period between the stage of rapid growth and the stage at which net growth stops altogether. This is consistent with the fact that the largest total effect of insulin on growth is seen when cells are grown on agar, where the gradually decelerating part of the growth curve occupies the largest fraction of the total period of net growth. The difference in shape of the growth curve of colonies growing on agar contrasted to cells growing on a solid substrate under liquid probably results from the compact configuration of the colonies on agar and the fact that nutrients must get to the cells and waste products must be removed only by diffusion. In contrast, cells growing on a solid substrate tend to spread out to a greater extent, and thermal convection plus mechanical vibration in the incubator results in closer equilibrium between the cells and the liquid medium. Thus growth continues until the entire volume of liquid medium becomes unfavorable to growth, at

Cell Biology: Aidells et al. which time a more abrupt halt is seen than for growth on agar. It is also possible that the substrate itself may be responsible for the difference in response to hormones. Androgen-dependent mammary tumor cells show enhanced growth response to androgens when grown in suspension culture or in agar as compared to growth on plastic (17). Corneal endothelial cells failed to respond to epidermal growth factor when grown on plastic, but the cells were stimulated when grown on collagen (18). The effect of the various substrates may be to change the geometry of the cells, in turn affecting their responses. This may be the case with the corneal endothelial cells (18). Folkman (19) has clearly shown that one effect of substrate on growth is related to the cell shape. Similarly, the shape of a CHO colony on agar is correlated with the growth of that colony. Clone SC3, which is a flatter colony than SC1, was able to grow for a longer period, and for other colony types we have also found that, the more mounded the clone, the shorter the growth phase. This diminished growth is related to the cell thickness of the colony, the availability of nutrients, and the extent of necrotic regions. Studying growth of spheroids in agar, Sutherland et al. (20) have shown that there is a finite limit to the size a spheroid can reach that is determined in part by necrosis of cells in the center of the spheroid. Effect on Colony Morphology. The effect of insulin at a concentration of 50 ng/ml on the morphology of CHO SC1 colonies growing on agar is obvious to the eye. To be precise, what the eye sees, and what the computer-controlled densitometer also measures on the photographs, is the thickness of the colony perpendicular to the plane of the agar surface. Because colonies grow down into the agar as well as up from the agar surface, these data do not specify completely their threedimensional shape. Computer-assisted analysis allows quantitative measurements to be made and statistics to be obtained on the shapes of hundreds or thousands of colonies. From these quantitative measurements a dose-response curve can be constructed, and the measured responses can be averaged over many colonies to allow detection of subtle effects by diminishing the influence of heterogeneity in colony shape. Analysis and use of the shapes of hundreds or thousands of colonies as a measure of the biological action of a substance such as a hormone can be a complex problem. The information needed to specify the-shape of even one colony at one stage of growth is large, being approximately proportional to the square of the linear resolution of the system. In the system used here, the resolution is better than 1/30th of the diameter of a typical colony, so that about 1000 numbers are required. The fact that most colonies are approximately cylindrically symmetric and, more importantly, that we consider the information of biological interest to be preserved after the shape is averaged over all angles around the center of the colony, reduces the number to about 30. Visual observation of the shape changes caused by insulin suggests to us that the ratio of colony thicknesses over a few zones of the colony can be used to quantitate this effect, and led to the partially arbitrary definition of the "mounding index" used in this report. It should be emphasized, however, that the reduction of the shape to one parameter results in a great loss of information in general. Thus, while "mounding index" is clearly of use in these experiments, it may not be in others, in which the shape of colonies may be altered, but not in a way to produce a monotomic change in the "mounding index," or even necessarily in a way to produce any dramatic change in this derived parameter. One of the advantages of our system is that essentially all the data characterizing colony shape, for up to 100,000 colonies at several stages of growth, can be stored on high-speed magnetic discs. This allows the appli-

Proc. Natl. Acad. Sci. USA 76 (1979)

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cation of the quantitative techniques of pattern recognition, as well as more intuitive searches for meaning, to be conducted repeatedly, if necessary, in a few minutes per pass.

The general usefulness of observations of colony shape in cell biology will be greatly increased when it can be interpreted in terms of other more fundamental cellular parameters of direct biological interest. Although we do not yet have a comprehensive, quantitative model of colony morphogenesis, we propose that colony mounding is driven by the force of cell-cell adhesion and opposed by the force of cell-agar adhesion, the mechanical rigidity of the agar, and the surface tension of the air interface of the medium that wets the surface of the agar and the cells. At least in some cases, the observed colony shape does not seemn to be in equilibrium with respect to these forces, cell movement being retarded by effective cell-cell viscosity. This viscosity, that is the inhibition of the movement of cells over each other, is in turn likely to be the result of a balance among still more fundamental processes, including cell mobility, cell surface movement, and cell-cell attachment formation and breakage. At present then, we can only suggest that insulin might be modifying colony shape by increasing cell-cell adhesion, decreasing cell-agar adhesion, or decreasing cell-cell viscosity. Direct measurements of these parameters and the effects of insulin on them in this system should give further insight into the action of insulin and the morphogenesis of animal cell colonies. We feel that this is a promising system to study the effect of hormones on colony morphology and growth. By observing the effects of a number of hormones and growth factors on colony morphology and by analyzing them in terms of internal and external molecular responses of sensitive cells, we hope to develop a list of mechanisms that may play a role in morphogenesis in vivo as well as in cultured cells. This work was partly supported by National Institutes of Health Grant GM 22021 and LBL 7109500. 1.

Puck, T. T., Marcus, P. E. & Creciura, S. J. (1955) J. Exp. Med.

2.

Puck, T. T.

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Hamilton, W. G. & Ham, R. G. (1977) In Vitro 13,537-548. Puck, T. T. (1972) The Mammalian Cell as a Microorganism: Genetic and Biochemical Studies In Vitro (Holden-Day, San Francisco). 5. Gospodarowicz, D. & Moran, J. S. (1976) Annu. Rev. Biochem. 45,531-558. 6. Wigley, C. B. (1975) Differentiation 4,25-55. 7. Nandi, S. (1959) Hormonal Control of Mammogenesis and Lactogenesis in the C3H/He Crgl Mouse, University of California Publication 2001 (Univ. of California, Berkeley, CA), Vol.

3. 4.

65, pp. 1-128. 8. Martin, L., Finn, C. & Trindu, G. (1973) J. Endocrinol. 56, 133-144. 9. Cunha, G. R. (1978) In Vitro 14, 333 (abstr.). 10. Kuroki, T. (1973) Exp. Cell Res. 80, 55-62. 11. Kuroki, T. (1975) in Methods in Cell Biology, ed. Prescott, D. (Academic, New York), Vol. 9, pp. 157-178. 12. Konfad, M. W., Storrie, B., Glaser, D. A. & Thompson, L. H. 13. 14. 15.

(1977) Cell 10,305-312. Elsdale, T. & Bard, J. (1972) J. Cell Biol. 54, 626-637. Elsdale, T. & Bard, J. (1972) Nature (London) 236, 152-155. Michalopoulos, G. & Pitot, H. C. (1975) Exp. Cell Res. 94, 7078.

Aidells, B. D., Konrad, M. W. & Glaser, D. A. (1978) In Vitro 14, (abstr.). 17. Stanley, E. R., Palmer, R. E. & Sohn, U. (1977) Cell 10, 35-44. 18. Gospodarowicz, D., Greenberg, G. & Birdwell, C. R. (1978)

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19. Folkman, J. (1978) Nature (London) 273,345-349. 20. Sutherland, R. M., McCredie, J. A. & Inch, W. R. (1971) J. Natl. Cancer Inst. 46, 113-120.

Growth and morphology of colonies of Chinese hamster ovary cells growing on agar is affected by insulin.

Proc. Nati. Acad. Sci. USA Vol. 76, No. 4, pp. 1863-1867, April 1979 Cell Biology Growth and morphology of colonies of Chinese hamster ovary cells g...
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