Growth of three established cell lines on glass microcarriers

Three established cell lines were examined for growth on a newly developed microcarrier which consists of glass beads. The cells were simultaneously exmined for growth on commercially available microcarriers made from DEAE-dextran and from plastic. Cell yields on the glass microcarriers were comparble to the cell yields on the commercially available products. Cells grown on the glass microcarriers were easily separated from the substratum by trypsinization (as were the cells grown on the plastic substratum) while the cells grown on the DEAE-dextran particles were much more trypsin resistant. After removal of cells from the glass microcarriers, the cells reattached and spread out in plastic flasks as readily as cells harvested from monolayer. Scanning electron microscopy revealed dramatic differences in the appearence of the cell grown on the glass microcarriers and cells grown on the DEAE-dextran microcarriers. On the glass microcarriers, cells attached to the substratum through lond, slender filopodia while on the DEAE-dextran microcarriers, the entire edge of the cell appeared to be in contact with the substratum. This dissimilarity in attachment could underly the difference in sensitivity to trypsin-mediated detachment. Finally, the glass microcarriers were washed after being used once and retested for their ability to support cell growth a second time. Nearly identical results were obtained with the reprocessed beads as with previously unused ones.     

Cell growth on microcarriers: comparison of proliferation on and recovery from various substrates

Three commercially-important types of cell were grown on four different microcarrier substrates. The cells, which included normal human diploid fibroblasts (MRC-5), primary chick embryo cells and Madin-Darby bovine kidney cells (MDBK), were compared with regard to proliferation on the substrates and with regard to recovery of viable cells from the same substrates. The substrates used included glass-coated microcarriers (Biosil), collagen microcarriers (Ventregel), DEAE-dextran microcarriers (Cytodex I) and collagen-linked DEAE-dextran microcarriers (Cytodex III). The established cell line (MDBK) grew well on all of the substrates and a high percentage of viable cells could be harvested from each substrate. The MRC-5 cells also grew well on all four substrates but high recovery rates were achieved only with cells grown on the glass-coated microcarriers or collagen microcarriers. In contrast, the primary chick embryo cells grew well only on the glass microcarriers and the recovery rate of cells harvested from this substrate was high. In some industrial operations, the re-utilization of cells after removal from the substrate is necessary. In these situations the appropriate choice of microcarriers for the cultivation of the cells may be critical.     

Proteolytic enzymes and arachidonic acid metabolites produced by MRC-5 cells on various microcarrier substrates

Summary Human diploid fibroblasts were cultured on microcarriers made from DEAE-dextran, denatured collagen, DEAE-dextran linked to denatured collagen, and glass. Cells grown on these four substrates were examined for the production of proteolytic enzymes and arachidonic acid metabolites. Culture fluids from cells grown on the DEAE-dextran microcarriers contained the highest amounts of proteolytic enzyme activity. Both plasminogen-independent and plasminogen-dependent fibrinolytic activities were present and the plasminogen-dependent activity seemed to result from the presence of both urokinase and tissue plasminogen activator. Culture fluid from the cells grown on the glass microcarriers contained the least amount of protease activity, and nearly all of the plasminogen-activator activity seemed to be of the urokinase type. Protease activity in the culture fluids of cells grown on the other two substrates were intermediate. With regard to arachidonic acid metabolites, cells grown on the DEAE-dextran microcarriers produced the highest amounts of cyclooxygenase products but very low levels of lipoxygenase metabolites. Cells grown on the other three substrates produced comparable amounts of various cyclooxygenase products (lower than that produced by cells on the DEAE-dextrans substrate). Cells grown on the glass microcarriers also produced detectable amounts of two lipoxygenase metabolites—leukotriene B₄ and leukotriene C₄. Inasmuch as both proteolytic enzymes and arachidonic acid metabolites regulate basic cell properties, the differential amount of these metabolites observed in the culture fluids on the various substrates may contribute to the biological differences that exist on these substrates. This study was supported in part by grants R44 CA 36656 and IK08HL01332-01 from the Public Health Service, U. S. Department of Health and Human Services and by grant BC-512 from the American Cancer Society. JDH is a research fellow of the American Lung Association.     

Growth of endothelial and HeLa cells on a new multipurpose microcarrier that is positive,negative or collagen coated

A new cell culture microcarrier that can be covalently bonded by cell attachment proteins and can be thin-sectioned for electron microscopy was synthesized. It was easily made by sulfonating cross-linked polystyrene beads for a negative surface charge followed by covalent attachment of polyethylenimine for a positive charge. Cell attachment proteins, e.g. collagen, was covalently bonded directly to the microcarrier using a carbodiimide or after activating the microcarrier surface with glutaraldehyde. HeLa-S3 cells attached, spread and grew to confluence more efficiently on the positive microcarriers and those coated with collagen than on the negative ones. Endothelial cells grew best on those with a negative surface charge. The nature of the microcarrier surface was not the only aspect involved in cell adhesion but also the type of serum proteins adsorbed. Qualitatively different proteins coated the microcarriers depending upon whether the carrier was negative, positive or coated with collagen. Comparison of various types of available microcarriers indicated that the modified cross-linked polystyrene beads used here were best for transmission and scanning electron microscopy. Endothelial cells grown on the microcarriers had the same ultrastructure as cells grown in monolayers in culture dishes. Of a variety of microcarriers tested the modified cross-linked polystyrene beads were the only ones that could be used for both ultrastructural and biochemical techniques.     

Sustained high-yield production of recombinant proteins in transiently transfected COS-7 cells grown on trimethylamine-coated (hillex) microcarrier beads

The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.     

Serial propagation of mammalian cells on microcarriers

For the large-scale operation of microcarrier culture to be successful, a technically feasible method for sequential inoculation is essential. Using human foreskin fibroblasts, FS-4, we have achieved this by detaching cells viably from microcarriers employing a selection pH trypsinization technique. Cells thus detached are able to reattach to microcarriers and grow normally after subsequent reinoculation into new cultures. However, after reinoculation cells attach to new microcarriers at a higher rate than to used microcarriers on which cells have previously grown. The effect of this differential cell attachment was analyzed and overcome by employing a low inoculum concentration. FS-4 cells could thus be serially propagated on microcarriers and subsequently used for beta-interferon production. This technique has also been applied to the cultivation of a monkey kidney cell line, Vero. We have also shown that Vero cells directly inoculated from a seed microcarrier culture could be used for virus production.     

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.     

Expression of plasminogen activator and plasminogen activator inhibitor mRNA in human fibroblasts grown on different substrates

mRNA levels for urokinase type plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) were examined in human diploid (neonatal foreskin) fibroblasts grown in 200-ml microcarrier suspension culture. Four different substrates were used. These included gelatin-coated polystyrene plastic, DEAE-dextran, glass-coated polystyrene plastic and uncoated polystyrene plastic. Our previous studies have shown that culture fluids from diploid fibroblasts grown on DEAE-dextran contained higher levels of plasminogen-dependent fibrinolytic activity than culture fluids from the same cells grown on other substrates. The increased plasminogen activator activity was due largely to elevated amounts of tPA (In Vitro Cell. Develop. Biol. 22: 575–582, 1986). The present study shows that there is a corresponding elevation of tPA mRNA in diploid fibroblasts cultured on DEAE-dextran relative to the other substrates. There does not appear to be any difference in uPA mRNA or in mRNA for PAI-1 or PAI-2 produced by the same cells on the four substrates. These data suggest that the influence of the substrate on plasminogen activator production is mediated at the genetic level.     

Formation of bridges and large cellular clumps in CHO-cell microcarrier cultures: Effects of agitation dimethyl sulfoxide and calf serum

We have investigated conditions that inhibit the tendency of CHO K1 cells to form cellular bridges between microcarriers and dense clumps of cellular overgrowth in microcarrier cultures. Microcarrier aggregation by cellular bridge formation was found to occur only during periods of rapid cell growth. The level of microcarrier aggregation decreased with increasing agitation intensity. Dense masses of cellular overgrowth formed inside bridges connecting the microcarriers and in clumps that protruded off the microcarrier surface. To replace cells that were continuously sheared from the microcarriers, cell growth occurred preferentially in areas of overgrowth after confluent microcarriers were maintained in a serum-free medium. This ultimately led to poor surface coverage as bare spots developed on the microcarrier away from the areas of dense cellular overgrowth. The development of bare spots was inhibited when confluent microcarriers were maintained in medium supplemented with 1% serum. The development of cellular overgrowth was inhibited by dimethyl sulfoxide. Thus, maintaining confluent microcarriers in medium supplemented with 1% dimethyl sulfoxide and 1% calf serum resulted in microcarriers that appeared similar to monolayer cultures. There was also a decrease in bridging in cultures supplemented with either 1% calf serum or 1% dimethyl sulfoxide/1% calf serum compared to serum-free cultures.     

Effect of glucocorticoid hormones on growth of human fibroblast cells and interferon production in a microcarrier culture system

Glucocorticoid hormones promoted the growth of fibroblast cells derived from human neonatal foreskins and prolonged their life span in a microcarrier culture system that used Eagle's minimum essential medium (MEM) supplemented with fetal calf serum (FCS). But, these hormones suppressed cell growth in conventional monolayer cultures. Precolostrum newborn calf serum (PNCS) was the only species that supported the serial propagation of fibroblast cells on microcarriers, possibly because of its high content of hydrocortisone (HC). Fibroblast cells grown on microcarriers in the presence of glucocorticoid hormones maintained their ability to produce interferon (IFN)-beta in a superinduction method with poly I: poly C and antimetabolites. These cells had more than 93% diploidy and no chromosomal aberration or translocation. Use of PNCS for the cultivation of human fibroblast cells has high potential for providing a microcarrier culture system for the mass production of human IFN-beta.     

Effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of human mesenchymal stem cells

With the aim to utilize human mesenchymal stem cells (hMSCs) grown in large scale for regenerative medicine, effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of hMSCs were studied. hMSCs could attach and grew on surface-type microcarriers of Cytodex 1, whereas almost no cell elongation and growth were observed on porous type microcarriers of Cytopores. The percentages of aggregated Cytodex 1 microcarriers at an agitation rate of 60 and 90 rpm were lower than that at 30 rpm, which was the lowest agitation rate necessary for the suspension of Cytodex 1 microcarriers, and the cells grew fastest at 60 rpm. hMSC could be subcultivated on Cytodex 1 by the beads-to-beads method at both 30 and 60 rpm without trypsinization. However, agitation at 60 rpm resulted in a markedly lower percentage of aggregated microcarriers not only before but also after subcultivation. The percentages of CD90- and CD166-positive cells among cells grown on Cytodex 1 at 60 rpm (91.5 and 87.6 %) were comparable to those of cells grown in the pre-culture on dishes. In conclusion, hMSCs could be subcultivated on Cytodex 1 by beads-to-beads method maintaining the expressions of the cell surface antigens CD90 and CD166, while adjusting agitation rate could decrease the microcarrier aggregation.     

Attachment characteristics of normal human cells and virus-infected cells on microcarriers

The attachment kinetics of normal and virus-infected LuMA cells were studied to improve the production of live attenuated varicella viruses in human embryonic lung (LuMA) cells. Normal LuMA cells and LuMA cells infected by varicella virus at various cytopathic effects (CPE) were grown on microcarriers. Ninety-three percent of suspended LuMA cells attached to the solid surface microcarriers within fifteen minutes and cell viability was greater than 95% when the cell suspension was stirred. Low serum levels did not affect the attachment rate of virus-infected cells in the microcarrier culture system. Kinetic studies showed that varicella infected cells had a lower attachment rate than normal LuMA cells. Virus inoculum (= infected cells) at low CPE showed a relatively better attachment rate on cell-laden microcarriers than virus inoculum at a higher CPE. Maximum titers were obtained at 2 days post-infection. Based on cell densities, the use of viral inoculum showing a 40% CPE led to an approximately 2- and 1.2-fold increase in the cell associated and in cell free viruses, respectively, than a virus inoculum with a CPE of 10%.However, the ratio of cell-free to cell-associated virus in a microcarrier culture was very low, approximately0.04–0.06. These studies demonstrate that the virus inoculum resulting in a high CPE yielded a high production of cell-associated and cell-free virus in microcarrier cultures because of the high cellular affinity of the varicella virus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Virus production with a newly developed microcarrier system.

Primary cell cultures as well as established lines have been grown on a recently developed microcarrier configuration that overcomes the problem of toxicity attendant on earlier developments in this technology. Virus yields from these cells propagated on the new microcarriers have been measured. Microcarrier-grown cells, when compared to roller-bottle-grown cells, gave virus yields on a per-cell basis that varied from slightly greater with the Sindbis virus-Chinese hamster ovary cells and polio-WI-38 combinations to approximately one-third with Moloney murine leukemia virus-Cl-1 mouse cells and vesicular stomatitis virus-chicken embryo fibroblasts. Yields ranged from 8.0 X 10(7) to 3.6 X 10(8) cells per 100-ml microcarrier culture and from 3.7 X 10(7) to 4.1 X 20(8) cells per roller-bottle culture. Secondary chicken embryo fibroblast yields were approximately four times as great in microcarrier cultures as in standard roller-bottle cultures, per unit volume of medium consumed. In spite of the reduced virus yields per cell seen in some instances, the greater cellular productivity of microcarrier cultures appears to hold great promise for large-scale virus production. Optimizing microcarrier conditions for specific cell-virus systems should result in improved yields.     

Serial propagation of mammalian cells on gelatin-coated microcarriers

The ability to serially propagate mammalian cells in microcarrier cultures is essential for large-scale operation. The success of such serial propagation depends on viable dissociation of cells from microcarriers and the normal growth and product formation after subsequent reinoculation. The high pH treatment developed for dissociating cells from DEAE-derivatized microcarriers was not as effective for a number of cell strains cultivated on gelatin-coated microcarriers. By prewashing the cell-laden microcarriers with buffer containing a chelating agent, bovine kidney cells, BK, human embryonic foreskin fibroblasts, FS-4, and continuous human kidney cells, TCL-598 which produces prourokinase, were viably dissociated from commercially available gelatin-coated microcarriers, Cytodex-3. Cells dissociated from microcarriers reattached and grew on micro-carriers subsequent to inoculation into subcultures. However, after subculturing, cells may attach at different rates to newly added beads and to conditioned microcarriers which cells had previously grown. It resulted in an uneven cell distribution on microcarriers and inferior growth kinetics. This effect was more profound for BK and FS-4 cells which are propagated with a low multiplication ratio. Specifically, BK cells attach to conditioned beads at a faster rate than to new beads, while FS-4 cells attach to new beads faster than to conditioned beads. Thus, for these two cell strains, a separator was used to separate the microcarriers from the suspension of dissociated cells before subsequent inoculation. For TCL-598 cells, which are propagated at a high multiplication ratio, this dissociation technique can be applied directly without the separation of dissociated cells and conditioned microcarriers. All the three cell lines tested exhibit normal growth kinetics in serial propagation on microcarriers. Furthermore, the production of prourokinase by TCL598 cells serially propagated on microcarriers was comparable to that inoculated from roller bottles.     

人皮肤成纤维细胞在不同培养系统中的生长代谢特性

大面积烧伤病人及多种皮肤溃疡病人很难用自体皮肤移植来进行治疗.早期治疗方法采用尸体来源的皮肤移植,但由于来源有限、且有传播疾病的危险,因此应用组织工程技术构建生物活性人工皮肤已成为近十几年来在组织工程和创伤治疗领域的研究热点,目前已有几种人工皮肤成功地走向临床[1].然而,在构建大面积皮肤组织过程中,如何大量制备皮肤种子细胞仍然是一大棘手的难题,成为人体皮肤组织工程迫切需要解决的技术关键.获得大量扩增的皮肤细胞,解决种子细胞的供应问题,是构建人工皮肤的一个关键.     

Virus production with a newly developed microcarrier system.

Primary cell cultures as well as established lines have been grown on a recently developed microcarrier configuration that overcomes the problem of toxicity attendant on earlier developments in this technology. Virus yields from these cells propagated on the new microcarriers have been measured. Microcarrier-grown cells, when compared to roller-bottle-grown cells, gave virus yields on a per-cell basis that varied from slightly greater with the Sindbis virus-Chinese hamster ovary cells and polio-WI-38 combinations to approximately one-third with Moloney murine leukemia virus-Cl-1 mouse cells and vesicular stomatitis virus-chicken embryo fibroblasts. Yields ranged from 8.0 X 10(7) to 3.6 X 10(8) cells per 100-ml microcarrier culture and from 3.7 X 10(7) to 4.1 X 20(8) cells per roller-bottle culture. Secondary chicken embryo fibroblast yields were approximately four times as great in microcarrier cultures as in standard roller-bottle cultures, per unit volume of medium consumed. In spite of the reduced virus yields per cell seen in some instances, the greater cellular productivity of microcarrier cultures appears to hold great promise for large-scale virus production. Optimizing microcarrier conditions for specific cell-virus systems should result in improved yields.     

Microcarrier culture: A different approach to granulosa cell cultivation

Granulosa cells (GC) are steroid secreting and hormone responsive cells synthesizing increased amounts of progesterone (P₄) under the influence of gonadotropins. Up to now these cells have been cultivated as monolayers. However, in this system, cells dedifferentiate early and cease to respond to stimulation. GC are a good model for studying hormonal regulation of ovarian cell function. In this study, microcarrier culture was tested for the first time to see whether it would be a good system in which GC would grow and give a stronger responsivity to gonadotropins. Cells were grown in stationary culture on microcarriers Cytodex 3 (C3) coated with collagen and on pure gelatin beads. Only C3 cultures were successful. Cells proliferated better on C3 microcarriers and on day 4 of culture secreted more P₄ under the influence of FSH and LH than comparative monolayer cultures.     

Three-dimensional (3-D) structures formed by immortalized human fibroblast cells in simulated microgravity

3-D structures were obtained at rotatory cultivation of CH immortalyzed human fibroblasts attached to glass microcarrier beads. The morphology of cells from these cultures was studied by scanning electron microscopy. A number of structural alterations in fibrillar filopodia of CH cells were revealed as compared with cells grown in stationary monolayer cultures, namely, smaller length, uneven caliber, the presence of curvatures, and disturbed branching pattern. Filopodia displayed unusual formations: protuberance-like and "mammoth's tusk"-like off-shoots, foamy spreadings in distal segments, and spiral windings of filopodia. The susceptibility of CH cells morphology to mechanical environment makes them a promising model for gravitational biology studies.     

Production of reovirus type-1 and type-3 from Vero cells grown on solid and macroporous microcarriers

Two strains of reovirus were propagated in Vero cells grown in stationary or microcarriers cultures. Vero cells grown as monolayers on T-flasks or in spinner cultures of Cytodex-1 or Cultispher-G microcarriers could be infected with reovirus serotype 1, strain Lang (T1L), and serotype 3, strain Dearing (T3D). A regime of intermittent low speed stirring at reduced culture volume was critical to ensure viral infection of cells in microcarrier cultures. The virus titre increased by 3 to 4 orders of magnitude over a culture period of 150 h. Titres of the T3D reovirus strain were higher (43%) compared to those of the T1L strain in all cultures. Titres were significantly higher in T-flask and Cytodex-1 microcarrier cultures compared to Cultispher-G cultures with respect to either reovirus type. The viral productivity in the microcarrier cultures was dependent upon the multiplicity of infection (MOI) and the cell/bead ratio at the point of infection. A combination of high MOI (5 pfu/cell) and high cell/bead loading (>400 for Cytodex-1 and >1,000 for Cultispher-G) resulted in a low virus productivity per cell. However, at low MOI (0.5 pfu/cell) the virus productivity per cell was significantly higher at high cell/bead loading in cultures of either microcarrier type. The maximum virus titre (8.5 x 10(9) pfu/mL) was obtained in Cytodex-1 cultures with a low MOI (0.5 pfu/cell) and a cell/bead loading of 1,000. The virus productivity per cell in these cultures was 4,000 pfu/cell. The lower viral yield in the Cultispher-G microcarrier cultures is attributed to a decreased accessibility of the entrapped cells to viral infection. The high viral productivity from the Vero cells in Cytodex-1 cultures suggests that this is a suitable system for the development of a vaccine production system for the Reoviridae viruses.