William J. Hillegas

Use of recombinant and synthetic peptides as attachment factors for cells on microcarriers.

Polystyrene culture dishes and polystyrene microcarriers were coated with Pronectin-F and poly-l-lysine (polylysine), either alone or in combination. Pronectin-F is a recombinant peptide containing repeats of the RGD cell-attachment sequence from fibronectin. Polylysine is a polymer ofl-lysine. Pronectin-F supported attachment of Madin-Darby Canine Kidney (MDCK) cells at concentrations as low as 0.025 μg/cm2 of surface area. The cells rapidly spread after attachment. Polylysine at concentrations of 0.05–0.5 μg/cm2 also supported cell attachment but cells did not rapidly spread after attachment to this substrate. Higher concentrations of polylysine could not be used because of toxicity. When the two peptides were used in conjunction, MDCK cells attached and spread at lower peptide concentrations than they did when either substrate was used alone. These findings suggest that recombinant Pronectin-F alone or in conjunction with a cationic polymer could be a useful replacement for materials such as gelatin or collagen which are currently used as microcarrier surfaces.

Substrate-dependent differences in growth and biological properties of fibroblasts and epithelial cells grown in microcarrier culture

Normal diploid human fibroblasts and first passage monkey kidney epithelial cells were examined for growth and metabolic activity on microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells cm2 of surface area) on the glass microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells/cm2 of surface area) on the glass microcarriers than they did on the DEAE-dextran microcarriers and morphological differences were observed between the cells growing on the two substrates. On the DEAE-dextran microcarriers, the cells were much more resistant to protease-mediated detachment than were the cells on the glass microcarriers. In these respects, the cells grown on the glass microcarriers were similar to cells grown in conventional monolayer culture. Interestingly, the cells grown on the DEAE-dextran microcarriers expressed higher levels of proteolytic enzyme activity than the cells grown on the glass microcarriers. Substrate-dependent differences in prostaglandin production also occurred--both in unstimulated cells and in cells stimulated with 12-0-tetradecanoyl phorbol acetate. The unstimulated cells on the glass microcarriers produced slightly higher levels of three different prostaglandins than did the cells on the DEAE-dextran microcarriers. However, after stimulation the levels were much higher in the DEAE-dextran microcarrier cultures than in the glass microcarrier cultures. In contrast to these results, there was no significant, substrate-dependent difference in the production of infectious herpes simplex virus. Taken together, these findings suggest that when commercially-useful cells such as normal fibroblasts and epithelial cells are grown in large quantities on microcarriers, the nature of the substrate may have a profound effect on the growth and physiology of the cells. They also suggest that when microcarriers are used, unexpected results based on preliminary work in conventional monolayer culture may be obtained.

Attachment and growth of anchorage-dependent cells on a novel, charged-surface microcarrier under serum-free conditions

The present study describes a novel microcarrier substrate consisting of a swellable, copolymer of styrene and divinylbenzene, derivatized with trimethylamine. The co-polymer trimethylamine microcarriers support the growth of a number of different cell lines – Madin Darby Bovine Kidney, Madin-Darby Canine Kidney, Vero and Cos-7 – under serum-free conditions, and human diploid fibroblasts in serum-containing medium. Cells attach to the co- polymer trimethylamine microcarriers as rapidly as they attach to other charged-surface microcarriers (faster than they attach to collagen-coated polystyrene microcarriers) and spread rapidly after attachment. All of the cells examined grow to high density on the co- polymer trimethylamine microcarriers. Furthermore, cells are readily released from the surface after exposure to a solution of trypsin/EDTA. In this respect, the co-polymer trimethylamine microcarriers are different from other charged-surface microcarriers. Madin-Darby Bovine Kidney cells grown on this substrate support production of vaccine strain infectious bovine rhinotracheitis virus as readily as on other charged-surface or collagen-coated microcarriers. Thus, the co-polymer trimethylamine microcarriers combine the positive characteristics of the currently available charged-surface and adhesion-peptide coated microcarriers in a single product. The viral vaccine production industry is undergoing considerable change as manufacturers move toward complete, animal product-free culture systems. This novel substrate should find application in the industry, especially in processes which depend on viable cell recovery.

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.

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

AbstractmRNA 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.

Human diploid fibroblast growth on polystyrene microcarriers in aggregates

Polystyrene microcarriers were prepared in four size ranges (53–63 μm, 90–125 μm, 150–180 μm and 300–355 μm) 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 μm) (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.

The effect of substrate on the production of infectious virus by cells in culture

Herpes simplex virus type I (HSV-1), infectious bovine rhinotracheitis virus (IBR) and turkey herpesvirus were examined for growth in cells cultured on three different substrates. The substrates were glass, DEAE-dextran and collagen gel. With two of the viruses, HSV-1 and IBR, there were no apparent differences in production as a function of substrate. In contrast, the amount of the turkey herpesvirus which was recovered varied greatly with the substrate. Titers were highest on glass, followed by DEAE-dextran and then collagen gel. Our previous studies have indicated that the substrate on which anchorage-dependent cells are grown in vitro has an affect on a number of biological and biochemical properties. The present study indicates that the production of commercially important biologicals can be affected by the substrate.

Small Microcarrier Aggregates Yield High Cell Density

A commonly held belief has been that microcarrier-cell aggregates (clumps) leads to necrosis of the cells in the core of the aggregate. We, and others, have results that contradict that belief (1-4). Another commonly held belief is that microcarriers below a given size (approximately 75 microns) deter or disallow cell attachment. In our studies (a) we demonstrated that cell yields can be greatly increased by utilizing very small spheres (38-63pm), (b) we showed further that the high cell densities were achieved by virtue of rapid cell attachment to the microcarriers with concomitant microcarrier aggregation and high cell growth and (c) we demonstrated that cells growing in high cell density remained metabolically active and produced high levels of at least one commercially-relevant biological. On a per cell basis, cells growing in high-density, small-microcarrier cultures synthesized protein and produced infectious bovine rhinotracheitis virus as efficiently as cells in monolayer culture.