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Spatial Distribution of Mammalian Cells Grown on Macroporous Microcarriers with Improved Attachment Kinetics Hyun-Soo Lim, Bong-Kwan Han, and Jung-Hoe Kim Department of Biotechnology and Bioprocess Engineering Research Center, Korea Advanced Institute of Science and Technology, Taejon, Korea
Madhusudan V. Peshwa and Wei-Shou Hu* Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455-1103
Vero and HepG2 cells were cultivated on macroporous gelatin microcarriers prepared by the calcium carbonate inclusion method. Cell attachment to these microcarriers was slow. For HepG2 cells the subsequent growth was poor. Modification of the microcarriers by incorporation of (diethy1amino)ethyl-HC1 improved HepG2 attachment and subsequent growth. Optical sectioning with confocal microscopy allowed visualization of the distribution of cells within microcarriers. In most microcarriers, cells were found to preferentially populate regions close to the external surface and some cavities in the interior. Despite the incomplete occupancy of the interior of the microcarriers, high cell concentrations were achieved.
Introduction One of the major areas of animal cell biotechnology research in the last decade has been the development of bioreactors for cell culture (1, 2). Great emphasis was given to increasing cell concentration. Microcarriers are frequentlyused for the cultivationof anchorage-dependent cells. Cells adhere to the external surface and grow to confluence or even to form multilayers. In these microcarrier cultures the cell concentration can be increased by increasingthe microcarrier concentration. However,using this culture method, damage to cells caused by agitation is often a concern (3). Macroporous microcarriers were developed to allow cells to grow to high densities in the interconnected pores of the interior and to protect them from fluid damage caused by flow (4). Macroporous microcarriers were originally made of gelatin (5, 6) and collagen (7). Cellulose porous microcarriers have since become commercially available (8). Recently, porous polystyrene (9) and polyethylene (10) beads have been employed to cultivate Vero, hybridoma, and CHO cells. Fluidized bed or stirred tank bioreactors are used for porous microcarrier culture. Porous microcarrierssupport a higher cell concentration than conventional solid microcarriers at comparable bead concentrations. The presence of cells in the pores was confirmed by microscopic observation after thin sectioning (11). The fraction of internal volume occupied by cells in gelatin- and cellulosebased porous microcarriers was estimated for a number of cell lines and varied from 5 to over 40% (8,111. The initial attachment of cells to gelatin-based macroporous microcarriers is relatively slow compared to their attachment to conventional dextran-based microcarriers (11). Many investigators have studied how various surface modifications affect cell attachment to microcarriers. Among the factors that were considered important were charge density (12)and the length of the carbon backbone
* Author to whom all correspondence should be addressed. Phone: (612)625-0546. Fax: (612)626-7246. 8756-7938/92/3008-0486$03.00/0
which carriesthe charged molecules (13,14). In comparing cell attachment rate to gelatin-coated and to controlledcharge microcarriers,it was observed that the attachment rate was faster to controlled-charge microcarriers (15). However,charge density alone may not be the determining factor for the rate of cell attachment as microcarriers of similar charge density at physiologicalpH supported very differentattachment rates (16). In this study, weexamined the effect of incorporating a charged molecule, 2-(diethy1amino)ethyl chloride-hydrochloride (DEAECl-HCl), which has been shown to increase the cell attachment rate in conventional dextran-based microcarriers, to gelatin macroporous microcarriers for improving cell attachment and subsequent growth. Because of the opaque nature of gelatin macroporous microcarriers, it is difficult to visualize the attachment and spreading of cells using conventional microscopic techniques. In this study we demonstrate the use of optical sectioning employing confocal microscopy for visualizing the distribution of cells at different stagesof growth within the macroporous microcarriers.
Materials and Methods Cells and Cell Maintenance. African green monkey kidney (Vero) cells and HepG2 cells were maintained in T-150 flasks (Corning Glass Works, Corning, NY) at 37 "C in a humidified 5 % COz environmental incubator. The growth medium for both cells was Dulbecco's modified Eagle's (DME)medium (GibcoLaboratories,Grand Island, NY) supplemented with 10% and 5% (v/v) fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT) for Vero and HepG2 cells, respectively. Preparation of Microcarriers. The macroporous microcarriers were prepared by a proceduremodified from the method of Nilsson (17). One gram of gelatin (Sigma Chemical Co., St. Louis, MO) was dissolved in 10 mL of distilled water at 40 "C. Calcium carbonate (Sigma) particles, in a size range of 5-30 pm, were added to a concentration of 10-25% (w/v). This suspension was
0 1992 Amerlcan Chemlcal Society and Amerlcan Institute of Chemlcal Engineers
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transferred to a 100-mL mixture of toluenechloroform (73/27,v/v) containing 1% (v/v)Tween-80, and the mixture was stirred vigorously to form small droplets. At room temperature the droplets that formed solidified, entrapping calcium carbonate particles within them. They were sieved, and the size fraction between 125 and 500 pm was collected. The beads were then washed extensively with water and suspended in dilute HC1 solution to dissolve the entrapped calcium carbonate particles. The beads, now porous, were further washed with distilled water and then cross-linked with 2-8 % glutaraldehyde for 30 min to increase their mechanical strength. The macroporous microcarriers thus formed were extensively washed with distilled water and freeze dried. Modification of Microcarriers with DEAECl-HCl. DEAEC1-HC1 (Sigma) (5.0 mL) containing 0.3 g of macroporous gelatin microcarriers was heated to 60 "C in a water bath. Subsequently, 5.0 mL of 3.0 M sodium hydroxide was added, and the reaction was allowed to proceed for 12 h. The reacted microcarriers were washed sequentially with 200 mL of distilled water, 150 mL of 0.1 M HC1,250 mL of 0.1 mM HC1,500 mL of distilled water, and 1.0L of Ca2+/Mg2+-free phosphatebuffer saline (PBS). The microcarriers were resuspended in PBS, autoclaved, and stored at room temperature until use. Titration of DEAE-Derivatized Microcarriers. The macroporous microcarriers were washed extensively with 1.0 M NaOH followed by 0.15 M NaC1. They were subsequently titrated against 0.3 M HC1. Titrations were also carried out with the same amount of 0.15 M NaCl without microcarriers as controls. At pH values above 2.5, the amount of HC1 added to the controls was insignificant. The amount of HC1 added at various pH values was recorded. From this data, the amount of HC1 added per mass amount of microcarriers was calculated and reported as the charge density at the corresponding PH. Microcarrier Culture. Microcarrier cultures were performed at concentrations of 3.0 g/L microcarriers in 250-mL spinner flasks with a 100-mL working volume (Wilbur Scientific, Boston, MA) and 60 rpm agitation rate. The spinners were equipped with two Teflon-coated 45" pitched blades attached to the impeller, suspended approximately 1.5 cm from the bottom of the vessel. These spinners were filled with washed beads and media to a volume of 90 mL and placed in the COz incubator for 1 h to allow for equilibration of pH and temperature. Cells growing in T-flasks were trypsinized with 0.25 % trypsin in PBS containing 0.02 % ethylenediaminetetraacetic acid (EDTA). Ten milliliters of cell suspension was used to inoculate each spinner. Cell Enumeration. For cell enumeration, a 0.5-mL sample was transferred to a centrifuge tube, and the microcarriers were allowed to settle by gravity. The supernatant was withdrawn, and 0.5 mL of 0.1% (w/v) crystalviolet in PBS containing 0.1 M citric acid was added. The sample was incubated at 37 "C for 60 min. The suspension was then sheared with a Pasteur pipet to detach the nuclei from the microcarriers. The nuclei were then counted on a hemocytometer. Cell Attachment to Microcarriers. The procedure for measuringcell attachment kinetics has been described previously (16).Spinner cultures were inoculated with approximately3.5 X lo5 cells/mL while the spinners were stirring. Periodically the agitation of the spinners was briefly stopped to allow microcarriers to settle partially and a microcarrier-free zone to appear near the top of the culture fluid. The microcarrier-free zone (0.5 mL) was
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sampled. The number of unattached cells in the supernatant was determined by counting on a hemocytometer. Cell Staining. A combined procedure of fixation and staining was used for the visualization of cell distribution within microcarriers. A 0.5-mL sample was transferred to a centrifuge tube. After the beads settled by gravity, the supernatant was withdrawn and 1.0 mL of 40% methanol in PBS containing 20 pg/mL ethidium bromide was added. Methanol fixes the cells within the macroporous microcarriers, and ethidium bromide stains the DNA of the cells. The nuclei of the cells appear orange when viewed under the confocal microscope using a 568 nm long pass filter. Dual staining with fluorescein diacetate (FDA) and ethidium bromide was used for visualization of cell viability (18). Staining solution was prepared by mixing 10 pL of FDA (5mg/mL in acetone) and 10mL of ethidiumbromide (20 pg/mL in PBS). The sample was incubated at room temperature with the staining solution for 3-5 min and was then washed with PBS. The stained samples were viewed using the FITC filter set on the confocal microscope. Confocal Microscopy. The confocal system used consists of an MRC-500 confocal imaging system (BioRad, Boston, MA) linked with an Olympus BH-2 microscope equipped for epifluorescence. The main optical component of the setup consists of a pair of computercontrolled galvanic mirrors. These mirrors are used to scan light originating from a pinhole illuminated by an argon ion (488 nm) laser in a raster pattern across the specimen. The fluorescence signal emitted from within the specimen is descanned by the same pair of galvanic mirrors and focused onto a pinhole aperture in front of a photomultipliertube. The system allows illuminationand imaging of one picture element (pixel) at a time. The digitized output from the photomultiplier at each point in the raster scan is assembled into an image by means of framestore in the computer. The frame buffer is also used to integrate successive frames to improve the signal-tonoise ratio by filtering out the noise. The plane of focus can be adjusted by a computer-controlled stepper motor and allows for serial optical sectioning at varying depths within the specimen, without distortion by signals generated from out-of-focusplanes. These stored images can be projected to form a three-dimensional reconstruction of the specimen.
Results Charge Density of Microcarriers. The charge density of microcarriers is affected by the pH and the ionic strength of the solution in which they are suspended. The charge density of unmodified and DEAE-derivatized microcarriers was determined by titration against HCl. The resulting titration curve is shown in Figure 1. At a pH of 7.2 (which corresponds to the pH of the culture medium), the unmodified gelatin mcroporous microcarriers have a charge density of 0.5 mequiv/g, compared to a charge density of 0.8mequiv/g for microcarrier modified with DEAECl-HC1. Thus, DEAE modification yields approximately a 60% increase in the positive charge density of the macroporous gelatin microcarriers. Cell Attachment Kinetics. The attachment of HepG2 and Vero cells to unmodified and DEAE-derivatized macroporous gelatin microcarriers was examined under identical conditions. The rate of cell attachment was estimated by measuring the unattached cell concentration as a function of time after inoculation. Under conditions where the surface area is not limiting, the kinetics of attachment of cells to the microcarriers has been shown
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Figure 1. Charge density as a function of pH for unmodified (0) and modified ( 0 )macroporous microcarriers.
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to be firsf. order with respect to the unattached cell concentration (16). Spinner cultures were inoculated at approximately 3.5 X lo5 cells/mL. The changes in unattached cell concentration for HepG2 and Vero cells are shown in Figure 2a,b, respectively. HepG2 cells attached to unmodified microcarriers at a very slow rate. The unattached cell concentration was 2.0 X lo5cells/mL at 6 h after inoculation. Even after a cultivation period of 9 h, only 50% of the inoculated cells had attached. DEAE derivatization markedly improved both the rate and efficiency of attachment of HepG2 cells with more than 90% of the inoculated cells attached after 6 h of cultivation. The attachment of Vero cells to unmodified microcarriers was substantially faster than that of HeptG2 cells, with more than 80% of the cells attached within 6 h of inoculation. DEAE derivatization increased the attachment rate slightly, but the improvement was less than that for HepG2 cells. The unattached cell concentrations 6 h after inoculation for unmodified and DEAE-modified beads were 8.0 X lo4and 6.0 X lo4cells/mL, respectively. The attachment rate constants (and correlation coefficients) as determined by linear regression of the initial slope were 3.45 X h-l (R2 = 0.94) and 0.16 h-l (R2= 0.99) for HepG2 cells and 9.71 X h-l (R2 = 0.97) and 0.13 h-l (R2= 0.95) for Vero cells on unmodified and DEAE-derivatized macroporous microcarriers, respectively. However, this attachment rate for both Vero and HepG2 cells to modified macroporous microcarriers is still relatively slow compared to their attachment to conventional DEAE-dextran microcarriers (11, 19). Cell Growth Kinetics. The growth kinetics of HepG2 cells on unmodified and modified macroporous microcarriers is shown in Figure 3a. The low efficiency of attachment to unmodified microcarriers is reflected in the decrease of cell concentration after inoculation. In contrast, in the culture with modified microcarriers the cell concentration increased in the same time period. Although cells grew exponentially after the initial lag phase in unmodified microcarrier cultures, the maximum cell concentration achieved was 3.5 X 106 cells/mL. This was substantially lower than the maximum cell concentration of 1.0 X lo7 cells/mL achieved in modified microcarrier cultures. Microscopicobservations revealed that multiple cell layers grew on the exterior surface of the modified microcarriers, which partially accounts for the higher cell concentration achieved. In contrast to HepG2 cells, Vero cells do not exhibit any lag phase on either the unmodified or the modified microcarriers (Figure 3b). Although the final cell concentration achieved for both unmodified and modified microcarrier cultures was in the range of 5.0 X 106 to 6.0 X lo6 cells/mL, differences in growth kinetics were seen, and the cell concentration achieved was slightly
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Figure 2. Kinetics of cell attachment on macroporous microcarriers: (a) HepG2 attachment to unmodified (0) and modified ( 0 )microcarriers; (b) Vero attachment to unmodified (0) and modified ( 0 )microcarriers. l.?e+7
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Figure 3. Kinetics of cell growth on macroporous microcarriers at a microcarrier concentration of 3.0 g/L: (a) HepG2 growth on unmodified ( 0 )and modified (0)microcarriers; (b) Vero cell microcarriers. growth on unmodified ( 0 )and modified (0)
higher in the culture with modified microcarriersthroughout the entire culture period. Cell Distribution by Confocal Microscopy. The gelatin macroporous microcarriers are optically opaque.
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Figure 4. Confocalmicrographsof Vero cells cultivated on macroporousmicrocarriers(a) after initial cell attachment. Image represents a composite of nine optical sections from the top to a depth of 40.0 pm within the microcarrier. (b) Reconstructed three-dimensional image during exponential growth phase. Image represents a composite of 15 optical sections from the top to a depth of 100.8 pm within the microcarrier. (c) Reconstructed three-dimensional confocal image of the top 100.8 pm of the bead during the stationary phase. (d) Reconstructed three-dimensionalconfocal image as in c but with the top 37.8 pm optically removed to visualize the distribution of cells inside the bead from a depth of 37.8 to 100.8 pm.
It is difficult to visualize the morphology and viability of cells growing within the microcarriers. Thus, it is not possible to distinguish whether the cells attach and grow only on the outside of these macroporous microcarriers or whether they populate cavities inside the microcarriers as well. Optical confocal microscopy allows serial optical sectioningof a three-dimensional opaque object. Each of the optically sectioned focal planes is mapped onto its corresponding image plane in a pixel by pixel manner. This technique thus allows one to observe the distribution of cells on the inside of these macroporous microcarriers without having to section the beads. The macroporous microcarriers used in this study fluoresced in the green wavelength region of the spectrum and interfered with the detection of the fluorescein emission signal obtained from FDA-stained viable cells. To avoid interferencefrom the microcarriers, cells on microcarriers were fixed with methanol and stained with ethidium bromide only. The ethidium bromide signal (orange spectrum) was collected to locate the position of the cells within the macroporous microcarriers. Vero Cells. Figure 4a shows a confocal micrograph of Vero cells after inoculation,with about 8-10 cells attached to the microcarrier. The cells are primarilyattached within the cavities on the external surface of the macroporous microcarrier, and the cavities or pores on the inside of the microcarriers are essentially empty. Figure 4b depicts a three-dimensional composite projection of the top 70 pm
through a Vero cell-ladenmicrocarrier in midexponential phase. The cells appear to be growing and filling up available pores both on the outside and the inside of the macroporous microcarriers. However, it is difficult to concludewhether all available surface area is occupied by cells. Figure 4c shows a similar three-dimensional reconstruction of Vero cells from the late exponential phase of culture. It consists of the superimposition of 25 image planes separated by a distance of 4.2 pm, corresponding to 25 optical sections taken from the top of the bead to a distance of 100.8 pm within the bead. The cell density is high, and the cells appear to be confluent and covering the entire bead. To examinewhether the cells in the center of the image are cells which actually populate the internal cavities of the macroporous microcarrier or whether it is a projection of cells occupyingthe external cavities on the top of the bead, we sectioned optically and removed the upper section of the bead corresponding to the thickness of the external cavities. Figure 4d shows the threedimensional reconstruction of the same bead without the upper 10image planes. Thus, it is essentiallya projection of a slice of the microcarrier, 63.0 pm thick, comprising optical sections from between 37.8 and 100.8pm from the top of the bead. Figure 4d indicates that there are no cells occupying internal cavities in the macroporous microcarrier. Similar conclusions can be drawn by analyzing a series of optical sections at increasing depths within the micro-
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Figure 5. Serial optical sections through a Vero cell-laden macroporous microcarrier. Each optical section is separated by a distance of 7.2 pm. Images were acquired from the top of the microcarrier to a depth of 79.2 pm inside the microcarrier.
carrier. Figure 5 shows such an image of a series of 12 optical sections separated by a distance of 7.2 pm. The top sections have cells distributed throughout the bead surface, whereas as one progresses inward the cells are located only on the peripheral cavities and some cavities within the inside of the microcarrier. From images shown
in Figures 4c,d and 5, one observes that the cells preferentiallypopulateonly the cavities near the externalsurface of the macroporous microcarrier, and only a few of the internal cavities are occupied by the cells. Similar observations were made by examination of histopathological sections of macroporous microcarriers. No signif-
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Figure 6. Confocal micrographs of HepG2 cells cultivated on macroporous microcarriers. (a) Cell distribution on unmodified beads after initial cell attachment. Image represents a projection of the upper 15 optical sections, each separated by 6.8 pm. (b) Cell distribution on DEAE-modified beads after initial cell attachment. Image represents a projection of the upper 13 optical sections, each separated by 5.0 pm. (c) Reconstructedthree-dimensionalprojection of HepG2 cells on unmodified beads in the late exponential phase. Image represents a compositeof 10 optical sectionsfrom the top to a depthof 75.6 pm within the microcarrier. (d) Reconstructed three-dimensional projection of HepG2 cells on DEAE-modified beads in the late exponential phase. Image represents a composite of 10 optical sections from the top to a depth of 46.8 pm within the microcarrier.
icant differencesin cell distributionwere observed for Vero cells grown on unmodifiedor surfacemodifiedmacroporous microcarriers. HepG2 Cells. Figure 6a represents a composite image depicting the distribution of HepG2 cells after initial attachment on an unmodified macroporous microcarrier. Figure 6b shows a composite image for HepG2 cells on a modified macroporousmicrocarrier. The latter image does exhibit an increased number of attached cells, indicating improved attachment due to increased charge density. However, in both cases the cells appear to be attached only to the external surface and external cavities of the macroporous microcarrier. Also, in some instances, the attached cells appear to be clumped together. Figure 6c,d represents composite images for HepG2 cells on unmodified and modified macroporous microcarriers in the late exponential phase of culture. A higher cell density on modified microcarriers can be seen, as would be expected from the growth kinetics data. HepG2 cells, when cultured in suspension,tend to clump and form aggregates. After inoculation, a fraction of the cells may form aggregates instead of attaching to the microcarriers. This phenomenon is more profound when the attachment rate is slow as in the case of unmodified microcarrier culture. The absence of any macroporous
microcarrierin the interior of the aggregatewas confirmed by optical sectioning (data not shown). Figure 7 depicts a reconstructed image of such a self-aggregated clump of HepG2 cells, obtained from the unmodified microcarrier culture. The image representsa compositeof seven optical sectionsthrough an aggregatedual stained with a viability stain consisting of fluorescein diacetate (FDA) and ethidium bromide. FDA stains viable cells green,and ethidium bromide stains the nuclei of dead cells orange. Fluorescence signals from both FDA and ethidium bromide emissions were collected. There was no signal detectable above the noise level in the ethidium bromide emission spectra. Thus, the image also indicates good cell viability in the spinner cultureswith essentiallyall cells being viable.
Discussion Our confocal microscopic examination demonstrates that the cells initially attach only on the external surface or within the external cavities of the macroporous microcarriers. Subsequent migration toward the interior of the beads is relatively slow. Macroporous microcarriers provide a larger surface area for cell growth compared to conventionalmicrocarriers. Our results alsodemonstrate that not all of the available surface is utilized. However, it should be noted that, if the pores are uniformly
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Figure 7. Confocal micrograph of an aggregate of HepG2 cells. Image depicts projection of the top seven optical sections, each separated by 10.0 pm.
distributed, penetration and growth of cells to within the outer half of the radius of the microcarriers accounts for 87% surface utilization efficiency. The central core comprising the inner half radius of the spherical particles contributesonly 12.5% of the total availablevoid fraction. Our results also indicate that promotion of initial cell attachment leads to better utilization of available surface area. Charge density has been shown to be partly responsible for cell adhesion to surfaces. Plastics, such as polypropylene and polystyrene, in their native state are incompatiblefor cell attachment. Surfacetreatment, such as electric corona discharge, leads to an increase in their charge density, and the resulting surfaces are more compatible for cell adhesion (20). However, the attachment and subsequent cell growth are not merely affected by charge density. DEAE-Sephadex, which is similar to a widely used dextran-based microcarrier, Cytodex 1, except that it has a high charge density, suffered from some toxicity to cells. Subsequent optimizationof charge density led to improved growth kinetics (12). The beneficial effectof DEAE derivatizationon cell attachment observed in our study thus may not solely be due to the increased charge density. To elucidate the mechanism, further studies using other molecules carrying different charged groups are necessary. Developmentof the confocal imaging technique can be extended from visualizing the distribution of cells to studying the distribution of various cellular properties, such as cell proliferation using stains for incorporated bromodeoxyuridine,within the macroporousmicrocarriers. It is envisagedthat such property distributions would help elucidatethe effectsof local microenvironmentson cellular activity and function as related to their spatial location within the microcarriers.
Acknowledgment This work was supported in part by grants from the (BCS8552670) National Science Foundation to W.-S.H. and from the Korea Science and Engineering Foundation to J.H.K.
Literature Cited (1) Hu, W.-S.; Peshwa, M. V. Animal cell bioreactor-Recent advances and challenges to scale-up. Can.J. Chem.Eng. 1991, 69,409-420. (2) Hu, W.-S.; Piret, J. M. Large scale mammalian cell culture:
methods, applications and products. Curr. Opin. Biotechnol. 1992,3,110-114.
(3) Papoutsakis, E. Fluid Mechanical damage of animal cells in bioreactors. Trends Biotechnol. 1991,9,427-437. (4) Looby, D.; Griffiths, B. Immobilization of animal cells in porous carrier culture. Trends Biotechnol. 1990,8,204-208. (5) Reiter, M.; Hohenwarter, 0.;Gaida, T.; Zach, N.; Schmatz, C.; Cluml, G.; Weigan, F.; Nilsson, K.; Katinger, H. The use of macroporousgelatin carrirsfor the cultivation of mammalian cells in fluidized bed reactors. Cytotechnology 1990,3,271277. (6) Lim, H. S.; Choi, J. H.; Kim, J.-H. Development of macro-
porous gelatin microcarrier and its characterization for anchorage dependent animal cell culture. Proc. APBioChEC, Kyunglu, Korea, April 22-26, 1990, pp 161-164. (7) Dean, R. C., Jr.; Karkare, S. B.; Phillips, P. G.; Ray, N. G.; Runstadler, P. W., Jr. Continuous cell culture with fluidized sponge beds. In Large Scale Mammalian Cell Culture Technology;Lyderson, B. K., Ed.; Hanser Publications: New York, 1987; pp 145-167. (8) Shiragami, N.; Ohira, Y.; Unno, H. Anchorage-dependent animal cell growth in porous microcarrier culture. In Animal Cell Culture and Production of Biologicals, Proceedings of the Third Annual Meeting of the Japanese Association for Animal Cell Technology, Kyoto, Japan, December 11-13,1990; Sasaki, R., Ikura, K., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1991; pp 121-126. (9) Lee, D. W.; Piret, J. M.; Gregory, D.; Haddow, D. J.; Kilburn, D. G. Polystyrene macroporous bead support for mammalian cell culture. In Biochemical Engineering VI& Dibiasio, D., Mutharason,R., Eds.; New York Academy of Sciences, in press. (10) Reiter, M.; Bliiml, G.; Caida, T.; Zach, N.; Unicluggauer, F.; Doblhoff-Dier, 0.; Noe, M.; Plail, R.; HUSS,S.; Katinger, H. Modular Integrated Fluidized Bed Bioreactor Technology. BiolTechnology 1991,9,1110-1102. (11) Nikolai, T. J.; Hu, W.-S. Cultivation of mammalian cells on macroporous microcarriers. Enzyme Microb. Technol. 1992, 14,203-208.
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(12) Levine, D. W.; Wang, D. I. C.; Thilly, W. G. Optimization of Growth Surface Parameters in Microcarrier Cell Culture. Biotechnol. Bioeng. 1979,21, 821-845. (13) Reuveny, S.; Mizrahi, A.; Kotler, M.; Freeman, A. Factors Affecting Cell Attachment Spreading Growth on Derivatized Microcarriers I Establishment of Working System and Effect of Types of Amino-charged Groups. Biotechnol. Bioeng. 1983, 25,469-480. (14) Reuveny, S.; Mizrahi, A.; Kotler, M.; Freeman, A. Factors Affecting Cell Attachment Spreading Growth on Derivatized Microcarriers 11: Introduction of Hydrophobic Elements. Biotechnol. Bioeng. 1983,25, 2964-2980. (15) Tao, T.-Y.; Ji, G.-Y.; Hu, W.4. Human fibroblastic cells attach to controlled-charge and gelatin-coated microcarriers at different rates. J . Biotechnol. 1987, 6 , 9-12. (16) Himes, V. B.; Hu, W.-S. Attachment and growth of mammalian cells on microcarriers with different ion exchange capacities. Biotechnol. Bioeng. 1987,29, 1155-1163.
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(17) Nilsson, K.; Buzsaky, F.; Mosbach, K. Growth of anchoragedependent cellsonmacroporousmicrocarriers. Biol Technology 1986,4,989-990. (18) Nikolai, T. J.; Peshwa, M. V.; Goetghebeur, S.; Hu, W.-S. Improved microscopic observation of mammalian cells on microcarriers by fluorescent staining. Cytotechnology 1991, 5, 141-146. (19)Kim, J.-H.; Hu, W.-S. Initial culture conditions affect the sensitivity of HepG2 cells to excessive mechanical agitation. Cytotechnology 1989,2, 135-140. (20) Ramsey, W. S.; Hertl, W.; Nowlan, E. D.; Binkowski, N. J. Surface treatments and cell attachment. In Vitro 1984, 20, 802-808.
Accepted August 5, 1992. Registry No. DEAEC1-HCl, 869-24-9.
