Serum-free cell culture on insulin-immobilized porous collagen beads

Insulin or albumin was immobilized on collagen beads using water-soluble carbodiimide. Adhesion of STO mouse fibroblast cells onto the beads decreased with increasing the amount of immobilized proteins. Growth of the cells was remarkably accelerated on the insulinimmobilized collagen beads, which can be used for serum-free cell culture. The growth acceleration became larger with increasing the amount of immobilized insulin, while it became smaller with increasing the amount of immobilized albumin. In addition, the immobilized insulin more strongly accelerated the cell growth than free insulin plus collagen beads. (c) 1995 John Wiley & Sons, Inc.     

Cell growth on immobilized cell growth factor. 8. Protein-free cell culture on insulin-immobilized microcarriers

In order to develop a new protein-free cell culture system, microcarriers immobilized with insulin were synthesized. For the synthesis, glass and polyacrylamide beads were treated for the introduction of amino groups on the surface, and insulin was immobilized on the surface by using several method. Anchorage-dependent cells. mouse fibroblast cells STO and fibroic sarcoma cells HSDM(1)C(1), and the anchorage-independent cells, mouse hybridoma cells SJK132-20 and RDP 45/20 were cultivated on the microcarriers immobilized with insulin. The insulin-immobilized microcarriers did not have any effect on the proliferation of the anchorage independent cells but promoted the growth of anchorage-dependent cells remarkably. The activity of immobilized insulin was larger than that of free or adsorbed insulin. The repeated use of the insulin-immobilized microcarrier was possible, and the promotion activity in the the repeated use was greater than that in the use. (c) 1992 John Wiley & Sons, Inc.     

Influence of phenols on growth and membrane permeability of free and immobilized Escherichia coli

The inhibition of the exponential growth of Escherichia coli K-12 by different phenolic compounds was examined. Cells entrapped in calcium alginate showed a greater tolerance than cells grown in suspension. The extent of inhibition of growth of the immobilized cells depended on the period of growth in the gel matrix. After the addition of bacteriostatic concentrations of phenol or 4-chlorophenol, a dose-dependent efflux of metabolites such as ATP and of K+ ions was elicited. Provided that glucose was supplied as an energy substrate, a reaccumulation of K+ ions at low phenol concentrations was observed. The restoration of the membrane gradient for K+ always preceded the continuation of growth in the presence of the toxic compounds. Compared with free cells, those cells immobilized and grown in alginate suffered a smaller loss of cations after the addition of 4-chlorophenol. The reestablishment of gradients was observed at higher concentrations of the pollutants with entrapped cells than with free cells. Corresponding to the increase in tolerance, the membrane damage was reduced in cells grown in immobilized form for longer times. These data offer a mechanistic explanation of the protection of immobilized microorganisms from phenolic solvents. The data point to the membrane as an important cell component in the toxicity of these pollutants.     

Influence of phenols on growth and membrane permeability of free and immobilized Escherichia coli.

The inhibition of the exponential growth of Escherichia coli K-12 by different phenolic compounds was examined. Cells entrapped in calcium alginate showed a greater tolerance than cells grown in suspension. The extent of inhibition of growth of the immobilized cells depended on the period of growth in the gel matrix. After the addition of bacteriostatic concentrations of phenol or 4-chlorophenol, a dose-dependent efflux of metabolites such as ATP and of K+ ions was elicited. Provided that glucose was supplied as an energy substrate, a reaccumulation of K+ ions at low phenol concentrations was observed. The restoration of the membrane gradient for K+ always preceded the continuation of growth in the presence of the toxic compounds. Compared with free cells, those cells immobilized and grown in alginate suffered a smaller loss of cations after the addition of 4-chlorophenol. The reestablishment of gradients was observed at higher concentrations of the pollutants with entrapped cells than with free cells. Corresponding to the increase in tolerance, the membrane damage was reduced in cells grown in immobilized form for longer times. These data offer a mechanistic explanation of the protection of immobilized microorganisms from phenolic solvents. The data point to the membrane as an important cell component in the toxicity of these pollutants.     

Signaling pathways of immobilized FGF-2 on silicon-substituted hydroxyapatite

Therapeutic strategies for bone regeneration involve the selection of suitable biomaterials, growth factors, and cell types to mimic the cellular microenvironment where molecular and mechanical signals control the reconstruction of bone tissue. The immobilization of basic fibroblast growth factor (FGF-2) on powdered silicon-substituted hydroxyapatite (Si-HA) allows to prepare a biofunctional biomaterial able to interact with bone cells in a very specific way. The biological activity of FGF-2/Si-HA, evaluated in Saos-2 osteoblasts and MC3T3-E1 preosteoblasts through the PLCγ and MAPK/ERK signal transduction pathways, shows that FGF-2 immobilized on Si-HA provides the right signals to cells stimulating crucial intracellular mechanisms of osteoblast proliferation and differentiation.     

Specific capture of mammalian cells by cell surface receptor binding to ligand immobilized on gold thin films

Aldehyde-terminated self-assembled monolayers (SAMs) on gold surfaces were modified with proteins and employed to capture intact living cells through specific ligand-cell surface receptor interactions. In our model system, the basic fibroblast growth factor (bFGF) binding receptor was targeted on baby hamster kidney (BHK-21) cells. Negative control and target proteins were immobilized on a gold surface by coupling protein primary amines to surface aldehyde groups. Cell-binding was monitored by phase contrast microscopy or surface plasmon resonance (SPR) imaging. The specificity of the receptor-ligand interaction was confirmed by the lack of cell binding to the negative control proteins, cytochrome c and insulin, and by the disruption of cell binding by treatment with heparitinase to destroy heparan sulfate which plays an essential role in the binding of bFGF to FGF receptors. This approach can simultaneously probe a large number of receptor-ligand interactions in cell populations and has potential for targeting and isolating cells from mixtures according to the receptors expressed on their surface.     

Growth factors are released by mechanically wounded endothelial cells

Growth factors may be required at sites of mechanical injury and normal wear and tear in vivo, suggesting that the direct action of mechanical forces on cells could lead to growth factor release. Scraping of cells from the tissue culture substratum at 37 degrees C was used to test this possibility. We show that scraping closely mimics in vitro both the transient plasma membrane wounds observed in cells subject to mechanical forces in vivo (McNeil, P. L., and S. Ito. 1989. Gastroenterology. 96:1238-1248) and the transient plasma membrane wounds shown here to occur in endothelial cells under normal culturing conditions. Scraping of endothelial cells from the culturing substratum released into the culture medium a potent growth-promoting activity for Swiss 3T3 fibroblasts. Growth-promoting activity was released rapidly (within 5 min) after scraping but was not subsequently degraded by the endothelial cells for at least 24 h thereafter. A greater quantity of growth-promoting activity was released by cells scraped 4 h after plating than by those scraped 4 or 7 d afterwards. Thus release is not due to scraping-induced disruption of extracellular matrix. Release was only partially cold inhibitable, was poorly correlated with the level of cell death induced by scraping, and did not occur when cells were killed with metabolic poisons. These results suggest that mechanical disruption of plasma membrane, either transient or permanent, is the essential event leading to release. A basic fibroblast growth factor-like molecule and not platelet-derived growth factor appears to be partially responsible for the growth-promoting activity. We conclude that one biologically relevant route of release of basic fibroblast growth factor, a molecule which lacks the signal peptide sequence for transport into the endoplasmic reticulum, could be directly through mechanically induced membrane disruptions of endothelial cells growing in vivo and in vitro.     

Product inhibition of immobilized Escherichia coli arising from mass transfer limitation

Mass transfer-limited removal of metabolic products led to product-inhibited growth of Escherichia coli that was immobilized in a model system. Comparison of the growth kinetics of immobilized and free-living cells revealed no further physiological differences between cells in these two modes of existence beyond those manifested in the local concentrations of substrate and product. Bacteria were retained on a microporous membrane in a dense, planar aggregate and were grown anaerobically on a glucose-based minimal medium. Radioisotope labeling of the immobilized cell mass with 35S was used to determine growth kinetic parameters. Growth rates in the immobilized cell layer were measured by an autoradiographic technique which allowed comparison of the size of the growing region with the rate of cell convection caused by growth. Immobilized cell growth rates and growth yields ranged from near maximal (0.56 h-1 and 39 g of dry cell weight/mol of glucose, respectively) to substantially reduced (0.15 h-1 and 15 g/mol). The depression of these kinetic parameters was attributed to product inhibition arising from mass transfer-limited removal of acidic waste products from the cell mass. A simple one-dimensional reaction-diffusion model, which incorporated data on the product-inhibited growth kinetics of free-living cells collected in a product-limited chemostat, satisfactorily predicted product inhibition of immobilized cell growth.     

Product inhibition of immobilized Escherichia coli arising from mass transfer limitation.

Mass transfer-limited removal of metabolic products led to product-inhibited growth of Escherichia coli that was immobilized in a model system. Comparison of the growth kinetics of immobilized and free-living cells revealed no further physiological differences between cells in these two modes of existence beyond those manifested in the local concentrations of substrate and product. Bacteria were retained on a microporous membrane in a dense, planar aggregate and were grown anaerobically on a glucose-based minimal medium. Radioisotope labeling of the immobilized cell mass with 35S was used to determine growth kinetic parameters. Growth rates in the immobilized cell layer were measured by an autoradiographic technique which allowed comparison of the size of the growing region with the rate of cell convection caused by growth. Immobilized cell growth rates and growth yields ranged from near maximal (0.56 h-1 and 39 g of dry cell weight/mol of glucose, respectively) to substantially reduced (0.15 h-1 and 15 g/mol). The depression of these kinetic parameters was attributed to product inhibition arising from mass transfer-limited removal of acidic waste products from the cell mass. A simple one-dimensional reaction-diffusion model, which incorporated data on the product-inhibited growth kinetics of free-living cells collected in a product-limited chemostat, satisfactorily predicted product inhibition of immobilized cell growth.     

Oxygen uptake rate of immobilized growing hybridoma cells

Summary The specific oxygen uptake rate of hybridoma cells immobilized in calcium alginate gel particles was measured, and the observed data was compared with those of non-immobilized cells. The uptake rate of the immobilized cells coincided with that of the non-immobilized hybridoma cells just after immobilization, but increased with cell growth. On the other hand, the cellular glucose consumption rate decreased slightly during the experiments. The increased oxygen uptake rate by immobilized cells was closely related to the formation of cell colonies in the gel particles.     

Contact inhibition of growth of human diploid fibroblasts by immobilized plasma membrane glycoproteins

The human embryonic fibroblasts used in this study show pronounced inhibition of growth when reaching a critical cell density. High cell density and growth inhibition has previously been mimicked by the addition of glutaraldehyde-fixed cells or of isolated plasma membranes to sparsely seeded proliferating fibroblasts (Wieser, R. J., R. Heck, and F. Oesch, 1985, Exp. Cell Res., 158:493-499). In this report, we describe the successful solubilization of the growth-inhibiting glycoproteins and their covalent coupling to silicabeads (10 microns), which had been derivatized with 3-isothiocyanatopropyltriethoxysilane. The beads, bearing the plasma membrane proteins, were added to sparsely seeded, actively proliferating fibroblasts, and growth was measured by the determination of cell number or of incorporation of [3H]thymidine into DNA. The growth was inhibited in a concentration-dependent manner, whereby 50% inhibition was achieved with 0.3 micrograms of immobilized protein added to 5 X 10(3) cells. Terminal galactose residues of plasma membrane glycoproteins with N-glycosydically bound carbohydrates were responsible for the inhibition of growth. Dense cultures of human fibroblasts are characterized by an accelerated synthesis of procollagen type III. We have found that this cellular response can also be induced by the addition of immobilized plasma membrane glycoproteins to sparsely seeded cells. These observations support the conclusion that the addition of immobilized plasma membrane glycoproteins to sparsely seeded fibroblasts mimics the situation occurring at high cell density. These results show that cell-cell contacts via plasma membrane glycoproteins carrying terminal galactose residues are important for the regulation of the proliferation of cultured human fibroblasts and presumably of the accelerated synthesis of collagen type III.     

Glucose regulates insulin mitogenic effect by modulating SHP-2 activation and localization in JAr cells

The glucose effect on cell growth has been investigated in the JAr human choriocarcinoma cells. When JAr cells were cultured in the presence of 6 mm glucose (LG), proliferation and thymidine incorporation were induced by serum, epidermal growth factor, and insulin-like growth factor 1 but not by insulin. In contrast, at 25 mm glucose (HG), proliferation and thymidine incorporation were stimulated by insulin, serum, epidermal growth factor, and insulin-like growth factor 1 to a comparable extent, whereas basal levels were 25% lower than those in LG. HG culturing also enhanced insulin-stimulated insulin receptor and insulin receptor substrate 1 (IRS1) tyrosine phosphorylations while decreasing basal phosphorylations. These actions of glucose were accompanied by an increase in cellular tyrosine phosphatase activity. The activity of SHP-2 in HG-treated JAr cells was 400% of that measured in LG-treated cells. SHP-2 co-precipitation with IRS1 was also increased in HG-treated cells. SHP-2 was mainly cytosolic in LG-treated cells. However, HG culturing largely redistributed SHP-2 to the internal membrane compartment, where tyrosine-phosphorylated IRS1 predominantly localizes. Further exposure to insulin rescued SHP-2 cytosolic localization, thereby preventing its interaction with IRS1. Antisense inhibition of SHP-2 reverted the effect of HG on basal and insulin-stimulated insulin receptor and IRS1 phosphorylation as well as that on thymidine incorporation. Thus, in JAr cells, glucose modulates insulin mitogenic action by modulating SHP-2 activity and intracellular localization.     

Basic fibroblast growth factor-syndecan complex at cell surface or immobilized to matrix promotes cell growth.

Promotion of cell growth and differentiation by growth factors during early development and organ formation are both temporally and spatially very precise. Syndecan is a well characterized integral membrane proteoglycan that binds several extracellular matrix components via its heparan sulfate chains and is therefore suggested to participate in cell regulation. Syndecan-like molecules, as low affinity receptors for heparin-binding growth factors, have been recently suggested to also regulate growth factor activity. Heparin/heparan sulfate interaction is required before, e.g. basic fibroblast growth factor (bFGF) can associate with its high affinity cell surface receptors and trigger signal transduction. In this paper we show that syndecan, but not free heparan sulfate chains, can simultaneously bind both bFGF and extracellular matrix molecules. Moreover, increased DNA synthesis of 3T3 cells was observed when the 3T3 cells were exposed to beads coated with the fibronectin-syndecan-bFGF complex, indicating that bFGF remains biologically active even when immobilized to matrix via the heparan sulfate chains of syndecan. Finally, when bFGF was bound to the surface of another cell type (epithelial), co-culture with 3T3 cells stimulated 3T3 cell growth. Therefore, we suggest that syndecan-like molecules may determine sites of growth factor action at cell-matrix and cell-cell interfaces.     

Effective diffusivity of galactose in calcium alginate gels containing immobilized Zymomonas mobilis.

The effective diffusivity of galactose was measured for calcium alginate gel membranes containing immobilized live Zymomonas mobilis cells at concentrations ranging from 0 to 150 g dry wt/L of gel. Since galactose is not taken up by living Z. mobilis organisms, the diffusion of this representative six-carbon sugar could be studied independently of sugar consumption. Various immobilized biomass loadings were achieved by two different techniques: addition of biomass at known concentrations to the sodium alginate solution before membrane formation and growth of cells in the gel to various biomass concentrations. The highest immobilized cell concentration, attained by in situ growth, corresponds to the maximum of this system, as growth beyond this maximum concentration led to disintegration of the gel membrane. The galactose effective diffusivity measurements for both methods of immobilized cell loading overlap within experimental error and follow the same general monotonic decline with entrapped biomass concentration. Most of the data fall below the upper bound predicted by Hashin and Shtrikman (1962) and show good agreement with the random pore model of Wakao and Smith (1962, 1964). Available effective diffusivity data from the literature provide evidence that the random pore model is an excellent predictor of sugar effective diffusivity in gel immobilized cell systems in general.     

Methane production from wastewaters by immobilized methanogenic bacteria

A methanogenic population was immobilized onto agar gel, polyacrylamide gel, and collagen membrane. Agar-gel-entrapped methanogenic microorganisms gave the highest activity. The optimum agar concentration was between 1.5 and 3% (w/v), and the optimum microbial content was 20 mg wet cells/g gel. The optimum conditions for methane production by immobilized whole cells were pH 7.0–7.5 and 37–45°C. The rate of methane production was initially 1.8 μmol/g gel/hr. Methane productivity was gradually increased and reached a steady state (4.5μmol/g gel/hr) after 25 days of incubation. The immobilized methanogenic microbial population continuously evolved methane over a 90 day period. No difference in methane productivity was observed after three months of storage at 5°C. Methane was also produced by immobilized whole cells under aerobic conditions. Furthermore, carbohydrates, such as glucose, in wastewater completely decomposed by immobilized whole cells.     

Studies on immobilized Saccharomyces cerevisiae. I. Analysis of continuous rapid ethanol fermentation in immobilized cell reactor

Rapid fermentation of cane molasses into ethanol has been studied in batch, continuous (free-cell and cell-immobilized systems) by a strain of Saccharomyces cerevisiae at temperature 30 degrees C and pH 5.0. The maximum productivity of ethanol obtained in immobilized system was 28.6 g L(-1) h(-1). The cells were immobilized by natural mode on a carrier of natural origin and retention of 0.132 g cells/g carrier was achieved. The immobilized-cell column was operated continuously at steady state over a period of 35 days. Based on the parameter data monitored from the system, mathematical analysis has been made and rate equations proposed, and the values of specific productivity of ethanol and specific growth rate for immobilized cells computed. It has been established that immobilized cells exhibit higher specific rate of ethanol formation compared to free cells but the specific growth rate appears to be comparatively low. The yield of ethanol in the immobilized-cell system is also higher than in the free-cell system.     

Growth requirements and characterization of rat cervical epithelial cells in culture

The extended culture of rat cervical epithelial cells can be achieved in the absence of a fibroblast feeder layer by utilizing collagen gels and a complex growth medium. The medium contains a 1:1 mixture of RPMI-1640 and Ham's F12 supplemented with 7.5% porcine serum and epidermal growth factor, cholera toxin, transferrin, insulin, and hydrocortisone. Under these culture conditions the cells show rapid log-phase growth and high saturation densities while retaining the ultrastructural characteristics of immature squamous metaplastic cells of the rat uterine cervix even after extended passage. In a manner similar to epithelial cells from a variety of sources, rat cervical epithelial cells form hemicysts at confluence in vitro when cultured on impermeable substrates. The development of these methods for culturing cervical epithelial cells provides an experimental system for the study of factors important in regulating the growth and differentiation of metaplastic squamous epithelial cells.     

Effects of Hydroxyurea on immobilized and suspended yeast fermentation rates and cell cycle operation

Hydroxyurea, an inhibitor of DNA synthesis in Saccharomyces cerevisiae, has been applied in order to restrict growth of immobilized cells. For comparison, the influence of hydroxyurea on suspended S. cerevisiae has also been investigated. Recovery from DNA synthesis inhibition, indicated by measurements of cell growth rate, DNA content, and light scatter properties, occurred faster in immobilized cells than in the suspended yeast. Morphogenesis in both populations was arrested by hydroxyurea, and there was an accumulation of single immobilized and suspended cells with large buds. Synthesis of protein and RNA was not adversely affected in either cell type. The specific rate of ethanol production by immobilized cells increased by an average of 24%, while, for the suspended cells, specific ethanol productivity was up to three times higher. Glucose consumption rates for both cell types also increased under the influence of hydroxyurea. Immobilized cell ethanol yields were reduced by ca. 16% in the presence of hydroxyurea; suspended cell yields were lower by an average of 50%. Total polysaccharide content was reduced by 65% for suspended cells and increased 30% for immobilized cells after hydroxyurea treatment. The data evidence disturbance of the yeast cell cycle due to immobilization.     

Culture and nitrite uptake in immobilized Scenedesmus obliquus

Summary The green alga Scenedesmus obliquus was immobilized in Ca-alginate beads. The cell growth after immobilization was studied by cell counting. The nitrite uptake was not affected by immobilization, except that a longer lag phase was observed in immobilized cells than in free ones. That result could be due to a barrier effect of the matrix against nitrite diffusion inside the beads. The treatment of cells by glycerol prior to their immobilization in a batch reactor induced an increase of nitrite uptake by the cells. This effect disappeared after a few runs. The glycerol effect on specific rates seemed also to decrease when the number of immobilized cells increased. This decrease can be related to the decrease of light efficiency as well as substrate accessibility when a high cell concentration was used. Several alternating runs of Tris-HCl buffer containing nitrite growth medium depleted in combined nitrogen were tested. Cellular growth occurred inside the beads up to a maximum followed by a decrease of cell number in the beads.     

Bioactivity of immobilized hyaluronic acid derivatives regarding protein adsorption and cell adhesion

Hyaluronic acid (HA) was chemically modified either by oxidation to obtain aldehyde-HA (aHA) or 3,3'-dithiobis(propanoic hydrazide) to obtain thiol-HA (tHA) that was covalently immobilized on model substrata such as amino-terminated surfaces or gold. Knowledge about the effect of modification with HA on physicochemical surface properties of these substrata and estimates of the quantities of immobilized HA were obtained by different physical methods such as contact angle measurements, ellipsometry, and atomic force microscopy. The bioactivity of aHA and tHA toward their natural binding partner aggrecan was studied by comparing surface plasmon resonance to native HA; this shows that binding of aggrecan was achieved in a similar way. Dermal human fibroblasts were used as a model cell to study how chemical modification and immobilization of HA impact adhesion and spreading of cells, which also affects cell growth and differentiation. A lower number and spreading of cells were observed on HA-modified surfaces compared to amino- and vinyl-terminated glass and silicon surfaces. Immunofluorescence microscopy also revealed that adhesion of fibroblast plated on HA-modified surfaces was mediated primarily by HA receptor CD44, indicating that bioactivity of HA was not significantly reduced by chemical modification.