Effects of cell adhesion to the substratum on the growth of chick embryo fibroblasts

Chick embryo fibroblasts were plated on Petri dishes that had not been treated for use in tissue culture (bacteriological dishes). On these dishes the cells grow at the same exponential rate as cells plated on tissue culture dishes, but their growth becomes inhibited sooner after plating, and therefore at a lower cell number per dish. The inhibition of cell growth on bacteriological dishes is correlated with the formation of cell clumps. Clump formation is reversible by mechanical transfer of the clumps to a tissue culture dish: the cells migrate out of the clumps, form a monolayer, and cell growth resumes.Clump formation was studied by time-lapse cinematography, and was found to be due to reduced adhesion of the cells to the bacteriological dish surface. This reduced adhesiveness of the substratum is due to a lower number of negatively-charged residues on the bacteriological dish surface, which can be measured by the binding of crystal violet. The number of negatively-charged residues, and therefore the adhesiveness of the substratum can be altered by treatment of the dishes with sulfuric acid. Serum components of the medium were found to affect cell adhesion to the bacteriological dishes, consequently altering the efficiency of cell attachment, the extent of cell growth and the pattern of clump formation.The cells in clumps were compared with those in confluent monolayers on tissue culture dishes. Growth-inhibited cells on both types of dish were found to be equally viable. Cells in clumps on bacteriological dishes were found to be inhibited in the G1 phase of the cell cycle, as are cells in density-inhibited monolayers. Infection by the oncogenic virus, Rous sarcoma virus, can release the cells from growth-inhibition on both types of dish. Cell-induced alterations of the medium are not involved in the growth inhibition of cells on bacteriological dishes.     

Adhesive and migratory behavior of normal and sulfate-deficient sea urchin cells in vitro

Dissociated cells from different stage embryos of the sea urchin Lytechinus pictus were compared in their adhesion to various substrates. Micromeres from 16-cell stage embryos bind to tissue culture and Petri dishes but not to Petri dishes coated with human plasma fibronectin. Other cell types did not adhere to any of the substrates tested. By hatched blastula stage, about 28% of the cells adhered to fibronectin as well as to tissue culture dishes. By the mesenchyme blastula stage, there was a further increase in the proportion of cells adhering to these substrates. At no stage did cells adhere to native rat tail collagen. Primary mesenchymal cells were isolated by their selective adhesion to tissue culture dishes in the presence of horse serum. These cells were then examined for their migratory capacity. Cell spreading and migration followed adhesion and occurred on fibronectin but not on the other substrates tested. Based on analysis of video tapes, greater than 60% of these cells moved faster than 1 micron/min. On the other hand, cells from sulfate-deprived embryos, in which primary mesenchyme migration is blocked in situ, failed to spread and migrated little on the same substratum. This defect was reversed by a 6 h pretreatment of the cells in normal sea water. Thus, the in vitro migratory behavior parallels that observed in vivo. These results support the hypothesis that the primary mesenchymal cells produce a sulfate-dependent component that is required for cell spreading and migration.     

Modulation by extracellular matrices of monooxygenase and CYP1A1 induction in Hep G2 cells in serum-free culture

The in vitro cellular functions of differentiated cells are influenced by culture conditions. Effects of several extracellular matrices (ECMs) on cytochrome P450-dependent monooxygenases (MFOs) induction and cytochrome P4501A1 (CYP1A1) gene expression were estimated in Hep G2 cells cultured in a serum-free medium. The cells were cultured on collagen type I- and II-, fibronectin-, and matrigel-coated dishes and MFO activities were induced by the addition of 3-methylcholanthrene (MC). The induction of ethoxycoumarin O-deethylase (ECOD) and alkoxyresorufin O-dealkylase activities as well as the expression of CYP1A1 mRNA were also determined. ECOD and methoxy- and ethoxyresorufin O-dealkylase activities in Hep G2 cells were enhanced by culturing the cells using a serum-free medium on fibronectin- or matrigel-coated dishes. ECOD activity on fibronectin-coated dishes was about 3-fold higher than that using a serum-supplemented medium on untreated dishes. Furthermore, both immobilized and soluble fibronectin enhanced the induction of MFOs. The expression of CYP1A1 mRNA using fibronectin-coated dishes was about 2-fold higher than that using a serum-supplemented medium on untreated dishes. These findings suggest that the gene expression in cultured cells is greatly influenced by ECMs. By using fibronectin-coated dishes to cell culture in a serum-free medium, reproducible and highly sensitive results can be obtained in experiments using cultured cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Delayed neural crest cell emigration from Sp and Spd mouse neural tube explants

Splotch (Sp) and splotch-delayed (Spd) are allelic mutations on chromosome 1 of the mouse. Embryos homozygous for either allele have neural tube defects (NTDs) and deficiencies in neural crest cell (NCC) derived structures. The fact that Spd mouse mutants sometimes have deficiencies in NCC derivatives in the absence of an NTD led to the hypothesis that neurulation and the release of NCCs may depend on a regulatory event that is common to both processes. Therefore, it may be possible to understand the cause of NTDs in these mutants by examining the basis of aberrant NCC derivatives. Caudal neural tubes were excised from day 9 Sp and Spd embryos and placed into gelatin-coated tissue culture dishes, or 3-dimensional basement membrane matrigel, and cultured for 72 hours. A cytogenetic marker was used to genotype the embryos. In planar cultures, no morphological differences were observed between NCCs from neural tube explants of Spd mutants compared to those from heterozygous or wild-type embryos. However, there appeared to be a delay in the release of NCCs from the neural tube in both Sp and Spd mutants, which was particularly evident in Sp. After 24 hours in culture, the extent of NCC outgrowth, as well as the number of NCCs emigrating from explanted neural tubes, was significantly lower in Sp and Spd mutant cultures than in controls. No differences were observed in the mitotic indices among cells which had emigrated. By 72 hours, mutant cultures and their non-mutant counterparts were similar in terms of outgrowth, cell number, and migratory capability. After 24 hours in 3-dimensional basement membrane matrigel, cell outgrowth from Sp explants was also significantly less than controls. The pattern of NCC outgrowth in both types of culture conditions indicates a 24 hour delay in mutant cultures compared to controls. This stems from a delay in the release of NCCs from the neural tube, suggesting that the defect lies within the neuroepithelium with respect to the release of NCCs.     

Enhanced binding of fibronectin-coated latex beads to quiescent 3T3-L1 cells is correlated with escape from growth arrest

The possible involvement of fibronectin receptors in growth stimulation was investigated by an analysis of fibronectin-coated latex bead binding to 3T3-L1 cells under various conditions. 3T3-L1 cells, growth-arrested in a medium with a low concentration of calf serum, bound few fibronectin-coated beads. After addition of serum at concentrations of 1.0% or higher, there was a rapid and transient increase in the number of cells with bound beads and a subsequent increase in the incorporation of bromodeoxyuridine (BrdU) into cell nuclei. Incorporation of BrdU was observed in about 60% of the cells with bound beads. Fibroblast growth factor and platelet-derived growth factor at concentrations of 5 ng/ml or higher also enhanced binding of fibronectin-coated beads to cells. Stimulation of bead binding by epidermal growth factor and insulin was weak. Fibroblast growth factor, but not epidermal growth factor, increased the incorporation of BrdU into nuclei. These results indicate a relationship between stimulation of cell proliferation in quiescent cells and increased binding by cells of fibronectin-coated latex beads.     

Spreading of human fibroblasts in serum-free medium: Inhibition by dithiothreitol and the effect of cold insoluble globulin (plasma fibronectin)

We have tested the effect of dithiothreitol (DTT) treatment on the initial spreading of human fibroblasts in serum-free medium in tissue culture dishes. Cell spreading was inhibited following treatment of these cells with 10 mM DTT. Inhibition occurred when the cells were treated at 37°C but not at 4° and was reversible metabolically but not by the addition of sulfhydryl oxidizing reagents. The inhibition was overcome when DTT-treated human fibroblasts were plated on cold insoluble globulin (plasma fibronectin)—coated dishes. Under these conditions spreading appeared to be completely normal, including the formation of focal adhesions. Analysis of the fibronectin concentrations in the human fibroblasts following DTT treatment indicated that there was little decrease in the absolute level of activity as determined in a biological assay for BHK cell spreading on culture dishes. Analysis of the fibronectin distribution on the DTT-treated human fibroblasts by indirect immunofluorescence using a specific anti-CIG antiserum revealed that fibronectin was no longer deposited onto the culture dish surfaces. Even when the DTT-treated human fibroblasts spread in the presence of fetal calf serum, the cell fibronectin remained for the most part in a perinuclear location. These results indicate that DTT treatment of human fibroblasts prevents the normal translocation of fibronectin from a perinuclear location to the surface of the culture dish. This study further supports our hypothesis that the initial spreading in serum-free medium of fibroblasts from cell strains depends upon secretion of fibronectin onto the culture dish surface.     

Enhanced chondrogenic marker expression of human mesenchymal stem cells by interaction with both TGF-β3 and hyaluronic acid

This study was designed to evaluate the additive effects of transforming growth factor-beta3 (TGF-β3) and hyaluronic acid (HA) on chondrogenic differentiation of human mesenchymal stem cells (hMSCs). The hMSCs were cultured on collagen type I-, HA-, or fibronectin-coated cell culture dishes with or without TGF-β3 added to the culture medium. Four weeks after cell culture, chondrogenic differentiation of hMSCs was determined by evaluating the expression of cartilage-specific markers using real-time polymerase chain reaction, immunocytochemistry, and Western blot analysis. hMSCs cultured on HA-coated dishes with TGF-β3 supplementation revealed a prominent increase in collagen type II, aggrecan, and Sox9. When hMSCs were cultured without TGF-β3 supplementation, only hMSCs cultured on HA-coated dishes showed prominent expression of the cartilage-specific markers. This study shows that chondrogenic differentiation of hMSCs can be enhanced additively by interactions with both a specific cell-adhesion matrix and a soluble growth factor.     

Optimization of the differentiation of human preadipocytes in vitro

This study aimed at developing an optimal protocol for proliferation and differentiation of preadipocytes that is a prerequisite for constructing an ideal biohybrid composed of viable adipose precursor cells in a three-dimensional matrix. Such an implant could represent an adequate solution for correcting soft tissue defects, e.g., extensive deep burns or tumor resections. Preadipocytes were isolated from human subcutaneous adipose tissue samples and cultured in Dulbecco's modified eagle medium (DMEM)/Ham's F12 medium (F12) or OPTIMEM medium with or without the addition of human serum (hS) or fetal calf serum (FCS). The advantages of fibronectin-coated culture dishes for preadipocyte yield after isolation and differentiation were evaluated. After culture expansion, differentiation was induced by insulin, isobutylmethylxanthine, pioglitazone, dexamethasone, and transferrin in the absence of serum. The extent of differentiation was assayed by measuring the activity of glycerophosphate dehydrogenase as well as counting of differentiated versus undifferentiated cells. Our results show that fibronectin coating does not only strongly increase the yield of preadipocytes after isolation from adipose tissue but also significantly enhances differentiation of precursor cells to mature adipocytes. For optimal cell expansion, DMEM/F12 is more promoting than OPTIMEM and culturing with FCS shows a slightly better proliferation compared with hS supplementation. Differentiation, in contrast, is significantly improved when hS is used instead of FCS during proliferation. Our results smooth the way for autologous preadipocyte culturing and show that hS for preadipocyte culturing opens new and promising perspectives for adipose tissue engineering by optimizing in vitro expansion in cell culture and inducing substantial differentiation.     

Creation of human cardiac cell sheets using pluripotent stem cells

Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets, the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study, we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14days produced around 8×10(7) cells/100ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation, cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating, even after cell sheets were detached from culture dishes. Furthermore, extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue.     

Isolation,Culture and Callus Formation of lpomoea batatas Protoplasts

Numerous viable protoplasts from stem callus cells of Ipomoea batatas tissue culture have been isolated by enzyme treatment involving cellulase EA3 867 (2.0%), CaC12·2H2O (20 mM) and mineral constituent of medium A at pH5.4 in 0.8 M mannitol in 5 hours at 25±1℃. The protoplasts were cultured at a density of 1-2 × 105/ml in solid agar medium E supplemented with 2, 4-D (0.1mg/l) and kinetin (0.1 mg/l), or NAA (0.3 mg/l) and kinetin (0.1 mg/l) in petri dishes, and placed in a controlled growth cabinet maintained at 27 ℃, and illuminated with floureseent light. They regenerated new cell wails after 7 days of culture. The first cell division was observed after 10 days. Ceil division continued thereafter, and after 40 days of culture small white calli (size about 0.2–0.3 mm) were visible in the petri dishes small calli were inoculated in the same nutrients as the protoplasts culture media, but without mannitol. They developed into large calli.     

Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness

To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.     

Integrin binding and cell spreading on extracellular matrix act at different points in the cell cycle to promote hepatocyte growth.

This study was undertaken to determine the importance of integrin binding and cell shape changes in the control of cell-cycle progression by extracellular matrix (ECM). Primary rat hepatocytes were cultured on ECM-coated dishes in serum-free medium with saturating amounts of growth factors (epidermal growth factor and insulin). Integrin binding and cell spreading were promoted in parallel by plating cells on dishes coated with fibronectin (FN). Integrin binding was separated from cell shape changes by culturing cells on dishes coated with a synthetic arg-gly-asp (RGD)-peptide that acts as an integrin ligand but does not support hepatocyte extension. Expression of early (junB) and late (ras) growth response genes and DNA synthesis were measured to determine whether these substrata induce G0-synchronized hepatocytes to reenter the growth cycle. Cells plated on FN exhibited transient increases in junB and ras gene expression (within 2 and 8 h after plating, respectively) and synchronous entry into S phase. Induction of junB and ras was observed over a similar time course in cells on RGD-coated dishes, however, these round cells did not enter S phase. The possibility that round cells on RGD were blocked in mid to late G1 was confirmed by the finding that when trypsinized and replated onto FN-coated dishes after 30 h of culture, they required a similar time (12-15 h) to reenter S phase as cells that had been spread and allowed to progress through G1 on FN. We have previously shown that hepatocytes remain viable and maintain high levels of liver-specific functions when cultured on these RGD-coated dishes. Thus, these results suggest that ECM acts at two different points in the cell cycle to regulate hepatocyte growth: first, by activating the G0/G1 transition via integrin binding and second, by promoting the G1/S phase transition and switching off the default differentiation program through mechanisms related to cell spreading.     

不同粘附剂对大鼠肝实质细胞体外培养的影响

摘要 目的：比较鼠尾胶与多聚赖氨酸对大鼠肝实质细胞体外培养的影响。方法：分别采用鼠尾胶与多聚赖氨酸包被同一块培养板,然后将从大鼠肝脏中分离出来的肝实质细胞,接种到包被好的培养板中。于接种前（0 h）,接种后体外培养24 h、72 h显微镜下观察细胞贴壁与形态变化情况。结果：接种前（0 h）可见新鲜分离的肝实质细胞呈圆形,明亮,有立体感,轮廓完整,层次清楚;体外培养24 h后两种粘附剂包被的同一块培养板中,均可观察到肝实质细胞正常生长,且细胞形态由圆形转变为多角形,并且融合聚集,胞体变平整,贴壁情况区别不大;培养72 h后细胞间开始出现连接,大部分肝细胞呈现出双核或多核,并且多聚赖氨酸包被的培养板中可见大量肝细胞呈岛屿状,已完全贴壁于培养板上。结论：多聚赖氨酸作为包被材料更有利于肝实质细胞贴壁生长以及保持细胞固有形态。     

Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix

The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tension-dependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (greater than 500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (less than 100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern-regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical loads. Thus, we propose that FGF-stimulated endothelial cells may be "switched" between growth, differentiation, and involution modes during angiogenesis by altering the adhesivity or mechanical integrity of their ECM.     

Quantitation of cell area on glass and fibronectin-coated surfaces by digital image analysis.

By using digital image processing and analysis, two procedures were developed to rapidly measure the projected area of a field of adherent 3T3 fibroblasts without staining of cell borders. The cell area of newly attached and rounded cells with well-resolved borders was obtained by a gray value thresholding procedure. For cells that had undergone an appreciable degree of spreading, cell boundaries were less distinct and a nonlinear spatial Sobel filter was used, followed by thresholding. For both procedures, linear relations were observed between cell areas obtained from image analysis and cell areas obtained by tracing. The areas of a population of traced cells were not statistically different from the area distribution obtained by using the standard curves for the processed images. Uncertainty in the estimated mean area depended only upon the number of cells examined. Approximate numbers of cells required to obtain estimates of the mean are calculated. As an application of these procedures, cell areas were measured for 3T3 cells attached to glass and fibronectin-coated surfaces and were found to be significantly larger for cells spreading on fibronectin-coated glass than on glass alone. Increased cell area during spreading on fibronectin-coated surfaces was proportional to increased cell adhesivity after exposure to a shear stress of 58 dyn/cm2.     

A fibronectin matrix is required for differentiation of murine erythroleukemia cells into reticulocytes

Erythroid differentiation of murine erythroleukemia (MEL) cells is far more extensive when the cells are attached to fibronectin-coated dishes than in suspension culture. Cells induced in suspension culture for 4 d become arrested at a late erythroblast stage and do not undergo enucleation. Incubation of cells in suspension beyond 4 d results in lysis. In contrast, cells induced by DMSO on fibronectin-coated dishes for 7 d differentiate into enucleating cells, reticulocytes, and erythrocytes. As determined by quantitative immunoblotting, cells induced in suspension culture accumulate approximately 33% of the amount of the major erythroid membrane protein Band 3 present in erythrocyte, whereas cells induced on fibronectin-coated dishes accumulate 80-100% of the amount present in erythrocytes. Both suspension-induced cells and cells induced on fibronectin-coated dishes accumulate approximately 90% of the amount of spectrin and ankyrin present in erythrocytes. As revealed by immunofluorescence microscopy during enucleation of MEL cells, both Band 3 and ankyrin are sequestered in the cytoplasmic fragment of the emerging reticulocyte. Enucleated and later-stage cells detach from the fibronectin matrix, due to the loss of the surface fibronectin receptor; this mimics the normal release of reticulocytes from the matrix of the bone marrow into the blood. Thus a fibronectin matrix provides a permissive microenvironment within which erythroid precursor cells reside, proliferate, migrate, and express their normal differentiation program.     

Effects of rapamycin on number activity and eNOS of endothelial progenitor cells from peripheral blood

The aim of this investigation is to determine whether rapamycin treatment has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days in culture, attached cells were stimulated with rapamycin (in a series of final concentrations: 0.1, 1.0, 2.0 and 5.0 g/ml) for 6, 12, 24 and 48 h. EPCs were characterized as adherent cells, double positive for DiLDL uptake and lectin binding by direct fluorescence staining. EPC proliferation and migration were determined using the MTT assay and a modified version of the Boyden chamber assay, respectively. An EPC adhesion assay was performed by replating the cells on fibronectin-coated dishes; adherent cells were then counted. Tube formation activity was assayed by using a tube formation assay kit and endothelial nitric oxide synthase (eNOS) was assayed by Western blot analysis. Incubation of isolated human MNCs with rapamycin decreased the number of EPCs present; rapamycin also decreased EPCs proliferative, migratory, adhesive, tube formation capacity and eNOS production in a concentration- and time-dependent manner. Rapamycin was found to decrease the number, proliferative, migratory, adhesive and tube formation capacities of the EPCs, and also was found to decreases eNOS in the EPCs.     

Estrogen-independent growth of mouse vaginal epithelium in organ culture.

A serum-free vaginal explant culture system was established to investigate the in vitro effect of estrogen on the growth of mouse vaginal epithelium. Vaginal explants were isolated from 40-day-old, ovariectomized BALB/cCrgl mice and cultured in a basal unsupplemented medium or in basal medium plus various doses of 17 beta-estradiol. Explants were processed for histology at the end of culture periods or were given 4-hour pulses of tritiated thymidine at various times and processed for autoradiography. Vaginal epithelium increased 3- to 5-fold in thickness and 2-fold in the number of epithelial cell layers during 72 hours of culture without estrogen; addition of estrogen did not significantly influence epithelial growth. Keratinization of vaginal epithelium occurred within 48 hours of culture in the absence of estrogen, and again addition of estrogen did not accelerate its appearance. Covering the explants with collagen decreased the estrogen-independent growth of vaginal epithelium. Autoradiography showed that ca. 70-90% of basal epithelial cells entered S phase during the initial 4 hours of culture and that this number declined rapidly after 48 hours to ca. 20%. Addition of 1.8 nM 17 beta-estradiol significantly decreased the labelling index of basal cells at 48 hours, but did not affect the labelling index at 24 and 72 hours. Stromal cells were not labelled at any time. Thus, DNA synthesis, cellular proliferation, and differentiation (keratinization) of vaginal epithelium in organ culture occurred without estrogen and were not stimulated by the addition of estrogen.     

Cell-to-substratum adhesion and guidance of axonal elongation

The behavior of axonal growth cones on surfaces with patterned variations in substratum was observed. Cells from sensory ganglia of 8-day-old chicken embryos were cultured on plastic petri dishes, plastic tissue culture dishes, and polyornithine-coated tissue culture dishes, all of which contained gridlike patterns of palladium (Pd) deposition.The results indicated that growth cones elongated on the Pd-shadowed areas vs areas lacking Pd deposits depending on the relative adhesivity of the growth cones to the substrata. In petri dishes, growth cones stay on the Pd; in tissue culture dishes, they cross from one surface to the other; and in polyornithine-coated dishes, they elongate for great distances on the Pd-free areas. Analyses of time-lapse movies showed that, on Pd-shadowed polyornithine dishes, growth cones often approach the Pd-coated areas and microspikes touch the Pd surface. Yet, the axon tip continues to elongate on the Pd-free polyornithine surface.The conclusion is offered that interactions between microspikes and the substratum adjacent to the growth cone are important determinants of the directions and pathways of axonal elongation.     

Modulation of the morphology and glycosaminoglycan biosynthesis of human monocytes, induced by culture substrates

Monocytes were isolated from human blood and cultured in vitro on plastic culture dishes or on fibronectin-coated dishes. After 5 days in vitro, the cells on plastic dishes displayed marked morphological changes compared with day 1, with an epithelioid appearance resembling that of foreign-body cells. This transition was inhibited in cells cultured on fibronectin-coated dishes. 35S-labelled polysaccharides were isolated from the culture media after 24h incubation periods with inorganic [35S]sulphate. The cells cultured for 5 days on a plastic substrate synthesized, and secreted into the medium, an oversulphated galactosaminoglycan previously shown to contain 4,6-di-O-sulphated N-acetylgalactosamine units [Kolset, Kjellén, Seljelid & Lindahl (1983) Biochem. J. 210, 661-667]. In contrast, 35S-labelled polysaccharide produced by cells cultured on plastic for 1 day only, or on fibronectin for either 1 or 5 days, contained only minor amounts of such disulphated sugar units. These findings indicate that the formation of oversulphated chondroitin sulphate is coupled to the conversion of monocytes into epithelioid cells. Furthermore, they suggest that the overall process is induced by contact with artificial substrates, and that it may be regarded as the equivalent of a foreign-body reaction in vivo.