Primary suspension culture of calf anterior pituitary cells on a microcarrier surface

In order to achieve a steady-state primary culture system for mammalian cells, with the potential to eventually correlate and control cell function and growth, a serious evaluation of various suspension systems was made. Calf anterior pituitary cells were employed as a differentiated cell type and successfully cultivated in a microcarrier suspension culture system. DEAE-Sephadex was demonstrated to be a satisfactory type of microcarrier. The cells readily attached to the bead and, after a short lag period, they actively proliferated on the bead surface to yield growth of a predominantly epithelial cell type. Under specific conditions the microcarrier supported primary cell growth up to levels of 2 × 10⁶ cells per ml. High bead concentrations inhibited cell growth. The inhibition could be overcome by using proportionately higher cell inoculum so that a concentrated culture with 5 × 10⁶ cells per ml was achieved. The inhibitory effect of high bead concentration was found to be due to the absorption of serum protein and certain growth enhancing factors. The fact that the growth enhancing factors were released from cells during the period of trypsinization and were both thermostable and nondialyzable, seems to suggest one approach to a dialysis culture system. In addition, relatively trauma-free primary cell cultures can be achieved by using explant culture without prior trypsinization. In microcarrier suspensions direct growth of primary rat mammary tumor cells was also demonstrated.     

Homogeneous cultivation of animal cells for the production of virus and virus products

Homogeneous technique facilitates the cultivation of large quantities of cells, reduces the risk of contamination by eliminating many manipulations, and makes practical the control of conditions such as pH and oxygen tension. Although most animal cells will not multiply in free suspension, certain cell lines have lost the requirement of being attached to a solid surface. These cells can be subcultured indefinitely but have some resemblance to cancer cells such as their abnormal karyotype. Certain cell linen developed from human embryonic tissue maintain their diploid character after repeated subculture and would seem to be ideal for the production of vaccines. However, strict regulations exist for viral products for human injection in that only cells taken from normal tissue and subcultured but once may be used. A microcarrier method in which cells adhere to DEAE-Sephadex beads permits a suspension culture which may be termed quasihomogeneous. The attached cells may be retained by sedimentation or by screening as the medium is replaced. Cell debirs from the original tissue is difficult to remove from microcarrier cultures; modifications of the trypsinization technique have alleviated but not solved this problem. Conditions for virus replication can be less critical than those for cell growth in that oxygen tension seems to have little influence on virus production. In cases where rate of virus production increases with specific growth rate of cells, homogeneous culture would have a advantage in maintaining a high cell mogeneous culture would have a valuble advantage in maintaining a high cell growth rate for a longer time. Some virus infections destroy cells, but others cause little change in cellular mteabolism except that virus is continually produced. The latter type can be conducted with a microcarrier in continuous culture with a virus titer exceeding 10⁷ plaque forming units per milliliter for over 50 days with Rubella-infected BHK cells.     

Serum-free cultivation of anchorage-dependent cells on microcarrier: Effective production of human macrophage colony-stimulating factor

For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalising the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 10⁷ cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.     

Serum-free cultivation of anchorage-dependent cells on microcarrier: Effective production of human macrophage colony-stimulating factor

For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalising the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 10⁷ cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.     

Microcarrier culture: Applications in biological production and cell biology

This report reviews recent progress in micro carrier technology at the Massachusetts Institute of Technology. This progress includes new understanding of the nutritional needs of high-cell density micro carrier cultures, the demonstration of cell growth on microcarriers in a hormone-supplemented, serum-free medium, and the continuous cell propagation of epithelial cell types in microcarrier culture by medium modification that permits efficient bead-to bead transfer. Also, a technique for obtaining large quantities of mitotic cells by selective detachment from microcarriers is reported. Finally, recent progress in interferon production from human foreskin fibroblasts grown on microcarriers is outlined: Our improvements in the interferon “super induction” process have increased the interferon yield per cell fivefold to tenfold.     

Growth of Fish Cell Lines on Microcarriers

Microcarrier beads were evaluated as substrates for the propagation of five anchorage-dependent fish cell lines. Growth of rainbow trout gonad (RTG-2) and Atlantic salmon cells was limited on microcarriers maintained in suspension. However, stationary microcarriers were suitable substrates for the growth of RTG-2, AS, Chinook salmon embryo (CHSE-214), and fathead minnow cells. Cell yields ranged from 2 × 10⁶ to 2.9 × 10⁶ cells per ml, representing 7- to 10-fold increases over the initial cell concentrations. The yield of new RTG-2 cells per unit volume of growth medium was 2.8 times greater in microcarrier cultures than in standard monolayer cultures. Northern pike cells failed to grow on microcarriers. Yields of infectious pancreatic necrosis virus propagated in microcarrier cultures of RTG-2 cells were more than twice the yields in standard monolayer cultures. The greater economy of microcarrier cultures in terms of growth vessel and medium requirements holds great promise for the large-scale production of anchorage-dependent fish cell cultures and fish viruses.     

Anchorage-dependent animal cell culture by using a porous microcarrier

CHO-K1 cells were cultured by using a porous microcarrier. The effects of microcarrier concentration and agitation rate on cell growth in porous microcarrier cultures were investigated. The specific growth rate of 0.041 h^–1 in porous microcarrier cultures was independent of both microcarrier concentration and agitation rate. By estimating the total surface area occupied by cells from the maximum cell number, it was found that not all the surface area of the porous microcarrier was utilizable for cell growth.The maximum cell number decreased with increasing the microcarrier concentration and the agitation rate. From this result, it was also found that not all the cells grown on the interior surface of the porous microcarrier were protected against mechanical damage due to agitation. The protection capacity of the porous microcarrier was estimated to be 300 cells/carrier. The direct gas sparging into the culture broth in porous microcarrier cultures improved the cell density without mechanical damage to animal cells.List of Symbols d m microcarrier diameter - d _i m impeller diameter - d _p m mean pore diameter - n _i s^–1 agitation rate - p Pa pressure difference - v m/s velocity of microcarrier - v _p m/s average velocity flowing through cyclinder - Pa · s viscosity of medium - angle measured from stagnant point - Pa average shear stress - Pa shear stress distribution     

Performance study of perfusion cultures for the production of single-chain urokinase-type plasminogen activator (scu-PA) in a 2.5 l spin-filter bioreactor

A perfusion-control strategy based on cellular consumption rates of oxygen and glucose was established for the production of single-chain urokinase-type plasminogen activator (scu-PA). Employing this strategy, the influences of microcarrier types and the culture media on culture performances were evaluated. In the control perfusion culture, which used a solid microcarrier and a 1% fetal bovine serum (FBS) medium, viable cell density reached 3.1?×?10⁷?cells?ml^?1. However, formation of large, heterogeneous aggregates (500–1,000?μm) resulted in a gradual decrease in viable cell density to less than 1.0?×?10⁷?cells?ml^?1. Accordingly, declines in the production of urokinase-type plasminogen activator (u-PA) and in the scu-PA portion of u-PA were observed. In the serum-free media, cell growth and u-PA production were suppressed 2–3?times, but were significantly enhanced when a porous microcarrier, Cultispheer G, was used. The cell-growth profile showed a continuous increase in cell density, reaching 5.1?×?10⁷?cells?ml^?1, and the production of u-PA remained stable throughout the culture (1586?±?247?IU?ml^?1). The values of all the parameters associated with cell growth and u-PA production were fairly comparable to or even higher than those in the control culture. Moreover, a 13% higher scu-PA portion of u-PA was observed in the serum-free culture, regardless of the microcarrier type, compared with scu-PA portion of u-PA in the control culture.     

Cell growth optimization in microcarrier culture

Summary Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephadex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm² of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per millitier of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for a scale up into large volume production units.     

Microcarrier culture of vascular endothelial cells on solid plastic beads

The culture of vascular endothelial cells on solid plastic beads is described. A greater than 30-fold increase in cell numbers was achieved in stationary culture medium. The inclusion of fibroblast growth factor slightly improved the rate of growth from low densities. Addition of fresh beads to colonized beads resulted in colonization of the newly introduced microcarrier. In common with the behaviour of endothelium in conventional culture, the cells cultured on beads changed from a fusiform to a polygonal shape after reaching confluence. Cell proliferation was also observed by [³H]thymidine autoradiography of DNA. The fraction of radiolabelled nuclei declined at confluence on each bead, indicating density-inhibition of growth. By electron microscopy, the cells displayed the typical ultrastructural appearance of endothelium. Following transfer of colonized beads to a chromatography column with slow perfusion of the bead bed, cell viability was maintained over a 24 h period and proportional synthesis of prostaglandin I₂ upon stimulation by ionophore A23187 was demonstrated. This simple microcarrier technique allows the generation of large numbers of vascular endothelial cells for subcellular fractionation with economical use of space and medium. When set up as a perfused bead bed, it offers possibilities for the short-term collection of concentrated endothelial metabolites.     

Selection of microcarrier diameter for the cultivation of mammalian cells on microcarriers

The kinetics of mammalian cell growth in a microcarrier culture are affected by the distribution of cells on microcarriers. It has been shown previously that a critical cell number per microcarrier is required for the growth of FS-4 cells on microcarriers. It is advantageous to alter the cell distribution on microcarriers to allow for a larger fraction of microcarriers to acquire enough cells to initiate normal growth. This can be achieved by selecting the diameter of the microcarriers employed. It has also been shown previously that the critical cell number could be reduced by choosing a better culture medium to support low density growth. However, even if all cells inoculated into a culture are capable of growing to confluence, it is still necessary to select the microcarrier diameter ration ally to improve the growth kinetics. The method of selecting the microcarrier diameter is discussed. By employing a improved medium as well as using microcarriers of selected diameter, the multiplication ratio was in creased to 15- to 16-fold for FS-4 cells, as opposed to 3- to 4-fold typically obtained in a batch culture.     

Human bone marrow colony growth in agar-gel

A technique for growing human bone marrow cell colonies in agar-gel medium is described. “Feeder layers” containing 1 × 10⁶ normal human peripheral white blood cells are used as the stimulus for colony growth. Human bone marrow aspirates are collected in heparinized syringes and plated as 2 × 10⁵ cells on “feeder layers.” Normal human bone marrow yields 32–102 colonies per 2 × 10⁵ cells plated. Colonies are almost exclusively granulocytic. Growth rate of colonies is slower than with mouse bone marrow but colonies reach a comparable size (500–1500 cells) at days 12–16.     

A mechanistic analysis of the inoculum requirement for the cultivation of mammalian cells on microcarriers

For the cultivation of mammalian cells on microcarriers a minimum inoculum concentration is required to initiate cell attachment and subsequent cell growth. A critical cell number model has been proposed to elucidate the mechanism of the inoculum requirement. In this model it was hypothesized that after inoculation a critical number of cells per microcarrier is required for normal growth to occur; failure to acquire enough cells will impede cell growth. This critical cell number model was expressed mathematically and used to simulate cell distribution and growth on microcarriers under different cultivation conditions. By comparing the simulated growth kinetics with the experimental results, the actual critical cell number per microcarrier was identified. The critical number could be reduced by employing an improved medium for the cultivation.     

Expansion of human mesenchymal stem cells on microcarriers

The effects on human mesenchymal stem cell growth of choosing either of two spinner flask impeller geometries, two microcarrier concentrations and two cell concentrations (seeding densities) were investigated. Cytodex 3 microcarriers were not damaged when held at the minimum speed, N_JS, for their suspension, using either impeller, nor was there any observable damage to the cells. The maximum cell density was achieved after 8–10 days of culture with up to a 20-fold expansion in terms of cells per microcarrier. An increase in microcarrier concentration or seeding density generally had a deleterious or neutral effect, as previously observed for human fibroblast cultures. The choice of impeller was significant, as was incorporation of a 1 day delay before agitation to allow initial attachment of cells. The best conditions for cell expansion on the microcarriers in the flasks were 3,000 microcarriers ml⁻¹ (ca. 1 g dry weight l⁻¹), a seeding density of 5 cells per microcarrier with a 1 day delay before agitation began at N_JS (30 rpm), using a horizontally suspended flea impeller with an added vertical paddle. These findings were interpreted using Kolmogorov’s theory of isotropic turbulence.     

Expansion of mouse sertoli cells on microcarriers

Background: Sertoli cells (SCs) have been described as the ‘nurse cells’ of the testis whose primary function is to provide essential growth factors and create an appropriate environment for development of other cells [for example, germinal and nerve stem cells (NSCs), used here]. However, the greatest challenge at present is that it is difficult to obtain sufficient SCs of normal physiological function for cell transplantation and biological medicine, largely due to traditional static culture parameter difficult to be monitored and scaled up. Objective: Operational stirred culture conditions for in vitro expansion and differentiation of SCs need to be optimized for large‐scale culture. Materials and methods: In this study, the culturing process for primary SC expansion and maintaining lack of differentiation was optimized for the first time, by using microcarrier bead technology in spinner flask culture. Effects of various feeding/refreshing regimes, stirring speeds, seed inoculum levels of SCs, and concentrations of microcarrier used for expansion of mouse SCs were also explored. In addition, pH, osmotic pressure and metabolic variables including consumption rates of glucose, glutamine, amino acids, and formation rates of lactic acid and ammonia, were investigated in culture. Results: After 6 days, maximal cell densities achieved were 4.6 × 10⁶ cells/ml for Cytodex‐1 in DMEM/FBS compared to 4.8 × 10⁵ cells/ml in static culture. Improved expansion was achieved using an inoculum of 1 × 10⁵ cells/ml and microcarrier concentration of 3 mg/ml at stirring speed of 30 rpm. Results indicated that medium replacement (50% changed everyday) resulted in supply of nutrients and removal of waste products inhibiting cell growth, that lead to maintenance of cultures in steady state for several days. These conditions favoured preservation of SCs in the undifferentiated state and significantly increased their physiological activity and trophic function, which were assessed by co‐culturing with NSCs and immunostaining. Conclusion: Data obtained in this study demonstrate the vast potential of this stirred culture system for efficient, reproducible and cost‐effective expansion of SCs in vitro. The system has advantages over static culture, which has major obstacles such as lower cell density, is time‐consuming and susceptible to contamination.     

Rapid electroelution of nucleic acids from agarose and acrylamide gels

The alkaline/filter DNA elution technique measures single-strand DNA breaks in mammalian cells based on the DNA molecular weight dependent retention of the macromolecule on 2-μm-pore-size filters. Described here is a modification of the technique which uses [³H]thymidine-labeled DNA of γ-irradiated cells as an internal reference. Thus, an increased precision is obtained in the assessment of this type of DNA damage at biologically significant radiation doses (i.e., where cell survival occurs). The measure of DNA damage is based on the actual initial DNA elution rate, i.e., arithmetic ratio of the elution of “test” DNA (i.e., ¹⁴C-labeled DNA) relative to the elution of “reference” DNA (i.e., ³H-labeled DNA). The repair of this damage on postirradiation incubation of the cells is detected as a decrease in the rate of “test” DNA cluted relative to “reference” DNA from unincubated cells. For Chinese hamster V79–171 cells irradiated with 5 Gy (500 rads), repair can be resolved into two first-order processes having rate constants (at 24°C) of ～0.190 and ～0.017 min^?1.     

Plate-Induced tumors of BALB/3T3 cells exhibiting foci of differentiation into pericytes,chondrocytes, and fibroblasts

The BALB/3T3 clone A31 mouse embryo cell line has been used by many investigators as a model “normal” “fibroblast” line for a variety of in vitro studies. It has been shown, however, that these cells are not “normal” because they will produce tumors within 2–4 months if 3 × 10⁴ cells are implanted subcutaneously in BALB/c mice attached to 0.2 × 5 × 10-mm plastic plates. Previous studies also suggested that these cells were not fibroblasts because they gave rise to tumors with the characteristics of vascular endothelium not fibroblasts. We now report that BALB/3T3 (clone A31), BALB/3T3-T, a proadipocyte subclone of clone A31 cells, and six recent subclones of BALB/3T3-T cells show additional differentiation patterns when tumors derived by implantation of these cells attached to plastic plates are examined. Differentiation into pericytes, chondrocytes, and fibroblasts was observed. We conclude that the BALB/3T3 clone A31 cell line and related lines are multipotent mesenchymal cells which are capable of differentiation into a variety of cell types.     

A paradoxical effect of hydrated C60-fullerene at an ultralow concentration on the viability and aging of cultured Chinese hamster cells

The effect of an aqueous solution of hydrated C₆₀-fullerene (HyFn) on the growth and “stationary phase aging” (accumulation of “age-related” changes in cultured cells during the slowing down of their proliferation within a single passage and the subsequent “aging” in the stationary phase of growth) of transformed B11-dii FAF28 Chinese hamster cells was studied. The final calculated concentration of HyFn in the growth medium was 10^?19 M. A paradoxical result contrasting the available data on the absence of HyFn cytotoxicity at higher concentrations was obtained in our experiments: namely, HyFn decelerated cell proliferation (estimated by the growth of mass culture, as well as by the efficiency of colony formation) and accelerated the “stationary phase aging” of the cell culture. Moreover, repeated addition of an aqueous solution of HyFn (to the final calculated concentration of 10^?19 M) to the cells that had already reached the stationary phase of growth caused a rapid (within no more than 24 h) death of a significant part of the cell population. The observed effect of HyFn at ultralow concentration is supposed to arise from the alterations in the properties of the water surrounding the fullerene molecule: namely, water becomes a donor and acceptor of electrons and regulates redox processes (especially those involving oxygen) in aqueous systems. This effect of HyFn at an ultralow concentration may be specific for transformed cells, and, therefore, experiments on normal fibroblasts with limited mitotic potential are planned as a continuation of the present study. It is also possible that the reported antiaging effect of HyFn in experimental animals is due to its anticancer, immunostimulatory, antiviral, and antibacterial properties manifested only at the whole-organism level.     

Human diploid fibroblast growth on polystyrene microcarriers in aggregates

Polystyrene microcarriers were prepared in four size ranges (53–63 m, 90–125 m, 150–180 m and 300–355 m) and examined for ability to support attachment and growth of human diploid fibroblasts. Cells attached rapidly to the microcarriers and there was a direct relationship between cell attachment and microcarrier aggregation. Phasecontrast and scanning electron microscopic studies revealed that while aggregation was extensive, most of the aggregate consisted of void volume. Cell growth studies demonstrated that human diploid fibroblasts proliferated well in microcarrier aggregates, reaching densities of 2.5–3×10⁶ cells per 2 ml dish after 6 days from an inoculum of 0.5×10⁶ cells per dish. When cells were added to the microcarriers at higher density (up to 5×10⁶ cells per 2-ml culture), there was little net growth but the cells remained viable over a 7-day period. In contrast, cells died when plated under the same conditions in monolayer culture. When the microcarriers were used in suspension culture, rapid cell attachment and rapid microcarrier aggregation also occurred. In 100-ml suspension culture, a cell density of 0.7×10⁶ cells per ml was reached after 7 days from an inoculum of 0.1×10⁶ cells. Based on these data, we conclude that microcarrier aggregation is not detrimental to fibroblast growth. These data also indicate that small microcarriers (53–63 m) (previously thought to be too small to support the growth of diploid fibroblasts) can support fibroblast growth and this occurs primarily because microcarriers in this size range efficiently form aggregates with the cells.     

Long-term stable production of monocyte-colony inhibition factor (M-CIF) from CHO microcarrier perfusion cultures

Monocyte-colony inhibition factor (M-CIF) was produced in microcarrier perfusion cultures from engineered Chinese hamster ovary (CHO) cells. Three and fifteen liter microcarrier perfusion bioreactors equipped with internal spin filters were operated for over two months. Approximately 60 L and 300 L of culture filtrate were harvested from the 3L and 15L microcarrier perfusion bioreactors respectively. During the perfusion operation, cell density reached 2–6 × 10⁶ cells/ml. Importantly, stable expression of M-CIF from the CHO cells under non-selection condition was maintained at a level of 4–10 mg/L. Specific productivity was maintained at 1.8–3.4 mg/billion cells/day. The ability of the recombinant CHO cells to migrate from microcarrier to microcarrier under our proprietary HGS-CHO-3 medium greatly facilitated microcarrier culture scale-up and microcarrier replenishment. Future directions for microcarrier perfusion system scale-up and process development are highlighted. This revised version was published online in August 2006 with corrections to the Cover Date.