Extracellular factors affecting the adhesion ofPseudomonas fluorescens cells to glass surfaces

Two factors affecting the adhesionof Pseudomonas fluorescens to glass surfaces were revealed in the culture liquid (CL) of this bacterium. One of these factors, adhesin, which is responsible for cell adhesion, was found to be a protein substance located both at the cell surface and in the CL. Bacterial cells grown in rich LB medium were less adhesive than cells grown in minimal M9 medium. The adhesive capacity of cells was independent of the growth phase. The other factor, antiadhesin (AA), which reduces cell adhesion, was found only in the CL. AA concentration in the CL increased with the culture age.     

A ground-based model to study the effects of weightlessness on lymphocytes

The mitogenic response of human lymphocytes was found to be markedly reduced in weightlessness conditions as compared to normal gravity. One possible explanation is that due to the non-existent sedimentation in space the lymphocytes could not adhere and spread on a substratum. Thus, we investigated the effect of substratum adhesiveness on lymphocyte responsiveness by reducing and blocking cell adhesion with poly-HEMA in a simple on-ground system. Lymphocyte adhesiveness was assessed by measuring the proportion of non-adhesive, slightly, and strongly adhesive 51Cr-radiolabelled cells on uncoated and poly-HEMA coated plastic. The amount of cell spreading on surfaces with varying adhesiveness was determined by measuring the area of cells. Cells grown on medium and thick poly-HEMA films were rounded in shape. By contrast, on tissue culture plastic, they showed clear signs of spreading. The mitogenic response of lymphocytes grown on thick poly-HEMA films was reduced by up to 68% of the control (tissue culture plastic). Interferon-gamma production was virtually nil when the cells were grown on the least adhesive substratum. These results show that activated lymphocytes need to anchor and spread prior to achieving an optimal proliferation response. We conclude that decreased lymphocyte adhesion could contribute to the depressed in vitro lymphocyte responsiveness found in the microgravity conditions of space flight.     

Growth and adhesion of Pseudomonas fluorescens in a batch culture: a kinetic analysis of the action of extracellular antiadhesins

The work varifies 6 hypothetical models simulating the growth, respiration, and adhesion of cells to the walls of the cultivation flask. All the models postulate the synthesis of antiadhesins (AAs), i.e., extracellular metabolites decreasing the degree of cell adhesion. The models have the following distinguishing features: (model 1) the blocking of sorption centers on the glass walls by antiadhesins (the competitive inhibition of adhesion); (model 2) the noncompetitive inhibition of adhesion; (model 3) the accelerated release of bound cells; (model 4) a combination of models 1 and 3; (model 5) a combination of models 1 and 3 with a delay; (model 6) a combined action of two AAs, one of which, AA1, inhibits cell adhesion, and the other, AA2 (its synthesis is induced when the concentration of AA1 reaches a threshold level), stimulates the detachment of bound cells. Model 6 fits the relevant experimental data best. The delay effect is relatively small. The sigmoid character of the curve showing cell adhesion as a function of the antiadhesin concentration implies the existence of a strong cooperative effect in the adhesion inhibition. The models proposed satisfactorily simulate the growth, respiration, and adhesion of cells and AA synthesis in a batch bacterial culture grown either in a fresh nutrient medium or in the medium supplemented with the filtrate of a mature culture of the same species.     

Protein-mediated adhesion of Lactobacillus fermentum strain 737 to mouse stomach squamous epithelium

The mechanism of adhesion of Lactobacillus fermentum strain 737 to mouse stomach squamous epithelium was investigated. Adhesion inhibition tests involving chelators, monosaccharides, periodate and concanavalin A and the use of bacteria grown in the presence of tunicamycin failed to clarify the adhesive mechanism. Washed bacterial cells had reduced adhesive capacity, except in the presence of spent broth culture supernatant fraction or cell washings. Spent culture supernatant fractions of erythrosine-supplemented broth did not enhance adhesion of washed cells. The adhesion-promoting factor(s) in the spent broth culture supernatant fractions and cell washings bound to both bacterial and epithelial cell surfaces, but did not promote adhesion of two other Lactobacillus strains which were not of mouse origin, thereby indicating host specificity for the adhesion-promoting activity. Chemical characteristics of the adhesion-promoting factor were determined by pretreatment of the dialysis retentate of spent broth culture supernatant fractions with proteolytic enzymes, concanavalin A-Sepharose or periodate before the adhesion assay. The adhesin was non-dialysable, pronase-sensitive, heat sensitive at 100 degrees C, had no affinity for concanavalin A-Sepharose and contained no carbohydrate groups active in the adhesion process. The protein profiles of dialysis retentates of spent broth culture supernatant fractions after bacterial growth in the absence and presence of erythrosine were determined by 2-dimensional SDS-PAGE. Gel filtration by HPLC was used for purification of an adhesion-promoting fraction. The host-specific adhesion of L. fermentum strain 737 was mediated by a protein, with an Mr of 12-13000, that was not detectable in cells grown in the presence of erythrosine. A model for the mode of binding of the adhesin to host epithelia and bacterial surfaces is proposed.     

Growth and Adhesion of Pseudomonas fluorescens in a Batch Culture: A Kinetic Analysis of the Action of Extracellular Antiadhesins

This work tests 6 hypothetical models simulating the growth, respiration, and adhesion of cells to the walls of the cultivation flask. All the models postulate the synthesis of antiadhesins (AAs), i.e., extracellular metabolites decreasing the degree of cell adhesion. The models have the following distinguishing features: (model 1) the blocking of sorption centers on the glass walls by antiadhesins (the competitive inhibition of adhesion); (model 2) the noncompetitive inhibition of adhesion; (model 3) the accelerated release of bound cells; (model 4) a combination of models 1 and 3; (model 5) a combination of models 1 and 3 with a delay; (model 6) a combined action of two AAs, one of which, AA₁, inhibits cell adhesion, and the other, AA₂ (its synthesis is induced when the concentration of AA₁ reaches a threshold level), stimulates the detachment of bound cells. Model 6 fits the relevant experimental data best. The delay effect is relatively small. The sigmoid character of the curve showing cell adhesion as a function of the antiadhesin concentration implies the existence of a strong cooperative effect in the adhesion inhibition. The models proposed satisfactorily simulate the growth, respiration, and adhesion of cells and AA synthesis in a batch bacterial culture grown either in a fresh nutrient medium or in the medium supplemented with the filtrate of a mature culture of the same species.     

Functional adhesiveness of the CX3CL1 chemokine requires its aggregation. Role of the transmembrane domain

In its native form, the chemokine CX3CL1 is a firmly adhesive molecule promoting leukocyte adhesion and migration and hence involved, along with its unique receptor CX3CR1, in various inflammatory processes. Here we investigated the role of molecular aggregation in the CX3CL1 adhesiveness. Assays of bioluminescence resonance energy transfer (BRET) and homogeneous time-resolved fluorescence (HTRF) in transfected cell lines and in primary cells showed specific signals indicative of CX3CL1 clustering. Truncation experiments showed that the transmembrane domain played a central role in this aggregation. A chimera with mutations of the 12 central transmembrane domain residues had significantly reduced BRET signals and characteristics of a non-clustering molecule. This mutant was weakly adhesive according to flow and dual pipette adhesion assays and was less glycosylated than CX3CL1, although, as we demonstrated, loss of glycosylation did not affect the CX3CL1 adhesive potency. We postulate that cell surfaces express CX3CL1 as a constitutive oligomer and that this oligomerization is essential for its adhesive potency. Inhibition of CX3CL1 self-assembly could limit the recruitment of CX3CR1-positive cells and may be a new pathway for anti-inflammatory therapies.     

Role of anionic lipid in bacterial membranes

The major phospholipids of Bacillus stearothermophilus are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). Under the growth conditions used in this study the concentration of anionic lipid (PG + CL) was determined by the pH of the culture medium. Cells grown in a complex medium at pH 5.8, 7.0, and 8.0 contained 17, 29 and 36 nmol of anionic (PG + CL) lipid/mg cell (dry weight). The concentration of the zwitterionic lipid phosphatidylethanolamine (PE) was 17-20 nmol/mg cell (dry weight) under all conditions. Analysis of isolated membrane preparations suggested that the amount of anionic lipid per unit area of membrane increased as the pH of the growth medium was increased. Membranes from cells grown at pH 5.8 and 8.0 contained 130 and 320 nmol anionic lipid/mg membrane protein, respectively. Phosphatidylethanolamine appeared to be localized on the inner membrane surface in cells grown under all conditions. Increasing the ionic strength of the culture medium by the addition of NaCl or KCl had little effect on growth at pH 5.8 but inhibited growth at pH 7 and 8. It was concluded that anionic phospholipid plays an important physiological role in maintaining an acidic pH at the outer membrane surface.     

Effect of iron on adherence and cytotoxicity of Entamoeba histolytica to CHO cell monolayers

Iron is an essential element for almost all living organisms. The possible role of iron for growth, adherence and cytotoxicity of Entamoeba histolytica was evaluated in this study. The absence of iron from TYI-S-33 medium stopped amebic growth in vitro. However, iron concentrations in the culture media of 21.4-285.6 microM did not affect the growth of the amebae. Although growth was not retarded at these concentrations, the adhesive abilities of E. histolytica and their cytotoxicities to CHO cell monolayer were correlated with iron concentration. Amebic adhesion to CHO cell monolayers was significantly reduced by low-iron (24.6 +/- 2.1%) compared with 62.7 +/- 2.8 and 63.1 +/- 1.4% of amebae grown in a normal-iron and high-iron media, respectively. E. histolytica cultured in the normal- and high-iron media destroyed 69.1 +/- 4.3% and 72.6 +/- 5.7% of cultured CHO cell monolayers, but amebae grown in the low-iron medium showed a significantly reduced level of cytotoxicity to CHO cells (2.8 +/- 0.2%). Addition of divalent cations other than iron to amebic trophozoites grown in the low-iron medium failed to restore levels of the cytotoxicity. However, when E. histolytica grown in low-iron medium were transferred to normal-iron medium, the amebae showed completely restored cytotoxicity within 7 days. The result suggests that iron is an important factor in the adherence and cytotoxicity of E. histolytica to CHO cell monolayer.     

Influence of collagen gel on the orientation of epithelial cell polarity: follicle formation from isolated thyroid cells and from preformed monolayers

The influence of collagen gels on the orientation of the polarity of epithelial thyroid cells in culture was studied under four different conditions. (a) Isolated cells cultured on the surface of a collagen gel formed a monolayer. The apical pole was in contact with the culture medium and the basal membrane was attached to the substratum. (b) Isolated cells embedded inside the gel organized within 8 into follicles. The basal pole was in contact with collagen and the apical pole was oriented towards the interior of the follicular lumen. (c) Cells were first organized into floating vesicles, structures in which the apical surface is in contact with the culture medium, and the vesicles were embedded inside the collagen gel. After 3 d, cell polarity was inverted, the apical pole being oriented towards the cavity encompassed by cells. Vesicles had been transformed into follicles. (d) Monolayers formed on collagen gels as in a were overlaid with a second layer of collagen, which was polymerized in contact with the apical cell surface. A disorganization of the continuous pavement occurred within 24 h; cells attached to the upper layer of collagen and reorganized into follicles in the collagen sandwich within 4-8 d. A similar process occurred when the monolayer was grown on plastic and overlaid with collagen, or grown on collagen and covered with small pieces of glass cover slips. No reorganization was observed between two glass surfaces. In conclusion, first, a basal pole was always formed in the area of contact between the cell membrane and an adhesive surface and, second, the interaction of a preformed apical pole with an adhesive surface was not compatible with the stability of this domain of the plasma membrane. The interaction of the cell membrane with extracellular components having adhesive properties appears to be a determinant factor in the orientation and stabilization of epithelial cell polarity.     

A novel Dictyostelium gene encoding multiple repeats of adhesion inhibitor-like domains has effects on cell-cell and cell-substrate adhesion

The Dictyostelium protein AmpA (adhesion modulation protein A) is encoded by the gene originally identified by the D11 cDNA clone. AmpA contains repeated domains homologous to a variety of proteins that influence cell adhesion. The protein accumulates during development, reaching a maximal level at the finger stage. Much of the AmpA protein is found extracellularly during development, and in culminants, AmpA is found in association with anterior-like cells. Characterization of an ampA- strain generated by gene replacement reveals a significant increase in cell-cell clumping when cells are starved in nonnutrient buffer suspensions. Developing ampA- cells are also more adhesive to the underlying substrate and are delayed in developmental progression, with the severity of the delay increasing as cells are grown in the presence of bacteria or on tissue culture dishes rather than in suspension culture. Reintroduction of the ampA gene rescues the developmental defects of ampA- cells; however, expression of additional copies of the gene in wild-type cells results in more severe developmental delays and decreased clumping in suspension culture. We propose that the AmpA protein functions as an anti-adhesive to limit cell-cell and cell-substrate adhesion during development and thus facilitates cell migration during morphogenesis.     

The effect of a collagen tripeptide fragment (GER) on fibroblast adhesion and spreading depends on properties of an adhesive surface

The effect of collagen tripeptide fragment GER on the adhesion and spreading of mouse embryonic fibroblasts STO to different substrates (polystyrene plastic, poly-L-lysine, fibronectin, gelatin) has been studied. It was found that tripeptide GER was involved in fibroblast adhesion and spreading. The cell response depended both on the mode of tripeptide addition to culture medium and the substrate type. Coincubation of fibroblasts with tripeptide stimulated the cell attachment and spreading to untreated plastic and plastic coated with fibronectin or gelatin but did not change cell adhesion to immobilized poly-L-lysine. Preincubation of cells with tripeptide resulted in partial inhibition of fibroblast adhesion and spreading on fibronectin- and gelatin-coated substrata. It was shown that activation and inhibition of adhesive processes after tripeptide treating was higher on fibronectin than gelatin. The data obtained support the assumption about concerted action of tripeptide GER (activity was dependent both on the used concentration of the tripeptide and the mode of tripeptide addition to culture medium) and chemical characteristics of substrate (polymers of styrene and L-lysine, ECM proteins in native (fibronectin) or partly denatured (gelatin) form) on the cell adhesion and spreading. The main targets that GER peptide may affect during the formation of cell-substrate interactions are discussed.     

Effect of halothane on lung carcinoma cells A 549

The halogenated hydrocarbons, such as halothane, are widely used as anesthetics in clinical practice; however their application is often accompanied with metabolic, cardiovascular and respiratory complications. One of the possible factors for this negative outcome might be the severe toxicity of these agents. In this paper, we investigate in vitro effects of halothane on human lung carcinoma A 549 cells, namely on their cytotoxicity, adhesive properties and metabolic activity. The cytotoxicity response of lung carcinoma A 549 cells to halothane was determined by lactate dehydrogenase (LDH) assay (for cytotoxicity), by detachment assay after adhesion to type IV collagen (for cell adhesive properties) and by surface tension measurements of culture medium (for cell metabolic activity). Regarding the cytotoxicity, the determined maximal non-toxic concentration of halothane on A 549 cells, given here as volume percentages (vol.%) was 0.7 vol.% expressed as aqueous concentration in the culture medium. Direct measurement of the actual halothane concentration in the culture medium showed that 0.7 vol.% corresponds to 1.05 mM and 5.25 aqueous-phase minimum alveolar concentration (MAC). Concentrations equal or higher than 1.4 vol.% (2.1 mM; 10.5 MAC) of halothane provoked complete detachment (cell death), or reduction of initial adhesion to collagen IV in half of the cell population. Surfactant production of A 549 cells, registered up to 48 h after halothane treatment, was inhibited by halothane concentrations as low as 0.6 vol.% (0.9 mM; 4.5 MAC). Our results demonstrate that sub toxic halothane concentrations of 0.6 vol.% inhibits surfactant production; concentrations in the range 0.8-1.4 vol.% induce membrane damages and concentrations equal and higher than 1.4 vol.%--cell death of approximately 50% of the cells.     

Extracellular protein of propionic acid bacteria inhibits induced mutation in strains of Salmonella typhimurium

A culture of propionic acid bacteria grown in a glucose-containing minimal medium, as well as the culture liquid and logarithmic-phase cells obtained from this culture, were found to inhibit the base pair substitution mutations induced by 4-nitroquinoline N-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, and sodium azide and the frameshift mutations induced by 9-aminoacridine. The antimutagenic activity of the culture liquid (CL) was presumably due to the presence of an extracellular thermolabile protein with a molecular mass of no more than 12 kDa based on the facts that this activity considerably decreased after the treatment of the CL with pronase, its heating at 92 degrees C, and its dialysis in a cellulose sack, which retains substances with molecular masses greater than 12 kDa. The residual antimutagenic activity of the dialyzed culture liquid was probably related to the interaction of the mutagen with thiols, rather than to the presence of organic acids (acetic or propionic). Thiols may also contribute to the antimutagenic activity of the P. shermanii cells.     

Atherogenic lipids induce adhesion of human coronary artery smooth muscle cells to macrophages by up-regulating chemokine CX3CL1 on smooth muscle cells in a TNFalpha-NFkappaB-dependent manner

Recent genetic evidence has implicated the adhesive chemokine CX3CL1 and its leukocyte receptor CX3CR1 in atherosclerosis. We previously proposed a mechanism involving foam cell anchorage to vascular smooth muscle cells because: 1) CX3CL1 and CX3CR1 are expressed by both cell types in mouse and human atherosclerotic lesions; 2) foam cells are reduced in lesions in cx3cr1(-/-)apoE(-/-) mice; and 3) proatherogenic lipids (oxidized low density lipoprotein [oxLDL] and oxidized linoleic acid derivatives) induce adhesion of primary human macrophages to primary human coronary artery smooth muscle cells (CASMCs) in vitro in a macrophage CX3CR1-dependent manner. Here we analyze this concept further by testing whether atherogenic lipids regulate expression and function of CX3CL1 and CX3CR1 on CASMCs. We found that both oxLDL and oxidized linoleic acid derivatives indirectly up-regulated CASMC CX3CL1 at both the protein and mRNA levels through an autocrine feedback loop involving tumor necrosis factor alpha production and NF-kappaB signaling. Oxidized lipids also up-regulated CASMC CX3CR1 but through a different mechanism. Oxidized lipid stimulation also increased adhesion of macrophages to CASMCs when CASMCs were stimulated prior to assay, and a synergistic pro-adhesive effect was observed when both cell types were prestimulated. Selective inhibition with a CX3CL1-specific blocking antibody indicated that adhesion was strongly CASMC CX3CL1-dependent. These findings support the hypothesis that CX3CR1 and CX3CL1 mediate heterotypic anchorage of foam cells to CASMCs in the context of atherosclerosis and suggest that this chemokine/chemokine receptor pair may be considered as a pro-inflammatory target for therapeutic intervention in atherosclerotic cardiovascular disease.     

Differentiation of a clone isolated from the HT29 cell line: polarized distribution of histocompatibility antigens (HLA) and of transferrin receptors

The HT29 cell line, derived from a human colon adenocarcinoma, is able to differentiate if galactose replaces glucose in the culture medium. We have isolated a clone (HT29-18) from this cell line which displays differentiated properties of the parent cell line. HT29-18 cells grown in glucose-containing medium form multiple layers of round cells without specific cell-cell adhesion. In contrast, when grown in galactose-containing medium, they form a monolayer with tight junctions and exhibit a well differentiated brush border at their apical membrane, which faces the culture medium. The polarized properties of HT29-18 cells grown in galactose-containing medium were demonstrated by immunofluorescent techniques with antibodies against 2 plasma membrane proteins. Class I histocompatibility antigens (HLA) and transferrin receptors, 2 well characterized integral membrane proteins, are uniformly distributed on the cell surface of undifferentiated HT29-18 cells, but acquire a polarized distribution during differentiation, localized on the basolateral membranes and absent from the apical surface. Binding of 125I-labeled transferrin was used to determine transferrin receptor distribution on apical and basolateral membranes. Functional tight junctions in the differentiated cultures were demonstrated, as the monolayer was impermeable to a permeation dye (ruthenium red) as well as to antibodies. The sealing of these tight junctions is, as in vivo, Ca++-dependent as they could be opened by a short incubation in Ca++-free medium.     

A Yolk-granule Component Acts as an Adhesive Material for Dissociated Gastrula Cells of the Newt, Cynops pyrrhogaster

Yolk granules were collected from full-grown ovarian oocytes of the newt, Cynops pyrrhogaster , and dissolved in 3% acetic acid or 8 M urea solution. Culture dishes were then coated with either of the yolk-granule solutions. The yolk-coated surfaces acted as adhesive substrata for dissociated gastrula cells, which showed active locomotion when spread on the surfaces. Divalent cation was required for cell adhesion and spreading on the yolk-coated surface. Trypsin and glycosidase digestions of dissociated cells or the yolk-coated surfaces inhibited cell adhesion and spreading. Addition of concanavalin A to the culture medium inhibited cell spreading on the yolk-coated surfaces, while high concentration (50 mM) of the saccharides, D-galactose, D-lactose and D-mannose, had a slightly inhibitory effect on cell spreading.
Two yolk components (30-kD and 108-kD proteins) were isolated from yolk granules and applied to the culture dish surfaces. Surfaces coated with the 30-kD protein showed strong adhesiveness for dissociated gastrula cells.     

Effects of growth media on cell cycle progression in CHO cells exposed to the radioprotector WR-1065

Abstract. WR-1065 (2-[(aminopropyl)amino]ethanethiol) reduces cytotoxic and mutagenic effects caused by exposure of cells to radiation and chemotherapeutic drugs, but the mechanisms involved are not fully known. We have observed an accumulation of cells in G, in WR-1065 treated Chinese hamster ovary cells grown in a-minimal essential medium, while others have found no cell cycle effects in WR-1065 treated Chinese hamster ovary cells grown in McCoy's 5A medium. To determine if the two types of media had an effect on cells treated with WR-1065, we examined survival and cell cycle progression. Population doubling times of 12 h were observed for cells grown in both media. Incubation of AA8 cells grown in McCoy's 5A medium with 4 mM WR-1065 30 min prior to and during irradiation with ^13'Cs gamma-rays resulted in a protection factor of 2.2, in close agreement with the value of 2.0 we previously obtained for AA8 cells grown in α-minimal essential medium. Treatment with WR-1065 caused an alteration in the cell cycles of cells grown in both media. An increase in the G₂ population and a decrease in the G₁ population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065, with a redistribution of the cells throughout the cell cycle occurring following removal of the drug. These data suggest that exposure of cells to WR-1065 is the cause of perturbations in cell cycle progression, and is not affected by the type of medium the cells are grown in.     

Isolation of an adhesion-mediating protein from chick neural retina adherons

Adherons are high molecular weight glycoprotein complexes which are released into the growth medium of cultured cells. They mediate the adhesive interactions of many cell types, including those of embryonic chick neural retina. The cell surface receptor for chick neural retina adherons has been purified, and shown to be a heparan sulfate proteoglycan (Schubert, D., and M. LaCorbiere, 1985, J. Cell Biol., 100:56-63). This paper describes the isolation and characterization of a protein in neural retina adherons which interacts specifically with the cell surface receptor. The 20,000-mol-wt protein, called retinal purpurin (RP), stimulates neural retina cell-substratum adhesion and prolongs the survival of neural retina cells in culture. The RP protein interacts with heparin and heparan sulfate, but not with other glycosaminoglycans. Monovalent antibodies against RP inhibit RP-cell adhesion as well as adheron-cell interactions. The RP protein is found in neural retina, but not in other tissues such as brain and muscle. These data suggest that RP plays a role in both the survival and adhesive interactions of neural retina cells.     

Analysis of Vero cell growth behavior on microcarrier by means of environmental scanning electron microscopy

By using environmental scanning electron microscopy, the morphological changes of Vero cells attached to and grown on the microcarrier Cytodex-3 were observed, and their behavior of adhesion, spreading and proliferation was analyzed. The effect of exogenous fibronectin/ laminin on adhesion and spreading of MCC/Vero cell was studied. The images of ESEM showed that expansion of cell growth was directed toward vacancy space. The growth curve and cell concentration change during the whole culture process were obtained from the statistical counting method based on ESEM images and the crystal violet method. The growth rate of Vero cells increases with increasing the concentration of cell inoculation, that is, the specific growth rate increases quickly with increasing the concentration of cell inoculation. When serum concentration in medium #199 ranged from 5% to 10%, experimental results indicated that serum concentration is one of the important factors influencing cell growth, particularly in the cell adhesio     

Analysis of Vero cell growth behavior on microcarrier by means of environmental scanning electron microscopy

By using environmental scanning electron microscopy, the morphological changes of Vero cells attached to and grown on the microcarrier Cytodex-3 were observed, and their behavior of adhesion, spreading and proliferation was analyzed. The effect of exogenous fibronectin/ laminin on adhesion and spreading of MCC/Vero cell was studied. The images of ESEM showed that expansion of cell growth was directed toward vacancy space. The growth curve and cell concentration change during the whole culture process were obtained from the statistical counting method based on ESEM images and the crystal violet method. The growth rate of Vero cells increases with increasing the concentration of cell inoculation, that is, the specific growth rate increases quickly with increasing the concentration of cell inoculation. When serum concentration in medium #199 ranged from 5% to 10%, experimental results indicated that serum concentration is one of the important factors influencing cell growth, particularly in the cell adhesion and spreading stage.