Use of blends of bioabsorbable poly(L-lactic acid)/poly(hydroxybutyrate- co-hydroxyvalerate) as surfaces for Vero cell culture.

Vero cells, a cell line established from the kidney of the African green monkey (Cercopithecus aethiops), were cultured in F-10 Ham medium supplemented with 10% fetal calf serum at 37 degrees C on membranes of poly(L-lactic acid) (PLLA), poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and their blends in different proportions (100/0, 60/40, 50/50, 40/60, and 0/100). The present study evaluated morphology of cells grown on different polymeric substrates after 24 h of culture by scanning electron microscopy. Cell adhesion was also analyzed after 2 h of inoculation. For cell growth evaluation, the cells were maintained in culture for 48, 120, 240, and 360 h. For cytochemical study, the cells were cultured for 120 or 240 h, fixed, processed for histological analysis, and stained with Toluidine blue, pH 4.0, and Xylidine ponceau, pH 2.5. Our results showed that cell adhesion was better when 60/40 and 50/50 blends were used although cells were able to grow and proliferate on all blends tested. When using PLLA/PHBV (50/50) slightly flattened cells were observed on porous and smooth areas. PLLA/PHBV (40/60) blends presented flattened cells on smooth areas. PLLA/PHBV (0/100), which presented no pores, also supported spreading cells interconnected by thin filaments. Histological sections showed that cells grew as a confluent monolayer on different substrates. Cytochemical analysis showed basophilic cells, indicating a large amount of RNA and proteins. Hence, we detected changes in cell morphology induced by alterations in blend proportions. This suggests that the cells changed their differentiation pattern when on various PLLA/PHBV blend surfaces.

Differential schwann cell migration in adult and old mice: an in vitro study.

The influence of aging on Schwann cell (SC) proliferation, migration and viability was studied in vitro. SCs were cultured in Ham F-10 medium enriched with 20% fetal calf serum (FCS), 40% FCS or collagen I gel plus 20% FCS. The migration of adult mice derived SCs was stimulated with FCS and collagen. With aging, SC migration, multiplication and viability decreased, indicating that ideal culturing conditions should be adjusted.

Morphological aspects of cultured rat seminiferous tubules in the presence of fetal calf serum and follicle-stimulating hormone.

Culturing seminiferous tubules allows the analysis of spermatogenesis under controlled conditions. Reproducing the specific microenvironment for germ cells is a challenge taken up by various studies. The difficulty in supplementing all nutrients and the physical disturbances during isolation procedures cause degeneration of many cells in the seminiferous epithelium. We tested some culture conditions in order to preserve an acceptable morphology of cells inside the tubules. Seminiferous tubules were cultured during fifteen days in HAM F10 medium supplemented with fetal calf serum (FCS) and/or follicle-stimulating hormone (FSH). The cellular morphology was analyzed using scanning and transmission electron microscopy. During the culture period cellular degeneration occurred progressively. Morphological modifications of the Sertoli cells and germ cells such as accumulation of lipid droplets, nuclear and cytoplasmatic vacuolization and the presence of cell debris were observed. The addition of FCS activated the myoid cells causing nuclear rounding and thickening of the tubular wall. The best results were obtained with a serum-free culture medium supplemented with FSH.

Dexamethasone and fetal calf serum effects in differentiation of Vero cells cultured on type I collagen gel.

Vero cells, a fibroblastic cell line, were cultured on a collagen I gel without fetal calf serum (FCS), with the addition of 10% FCS, 10% FCS plus dexamethasone (DEX) or 20% FCS. Our objective was to determine the effects of DEX on the differentiation pattern of fibroblastic cells cultured on a collagen substrate. We found that cells cultured with or without 10% FCS were capable of migrating into the collagen matrix. Near the cells in the gel, we found the deposition of extracellular granulations. Cytochemical data suggests that this material is glycosaminoglycans and/or proteoglycans. Surrounding the cells, a fibronectin deposition was found in the collagen. Thus, these cells make up a structure similar to a loose connective tissue. On the other hand, cells cultured with 10% FCS plus DEX or with 20% FCS did not invade the collagen matrix but formed multiple cell layers poor in fibronectin. On collagen I gel surface, we found an acellular layer rich in collagen IV, which appeared between the cells and the substrate. Thus, DEX or 20% FCS, furnished to the cells cultured on a collagen gel, block cell migration into the substrate and induce them to produce a basement membrane-like structure.

Scanning and transmission electron microscopy of transformed Vero cells, with altered in vitro growth characteristics.

The Vero lineage, established from kidney cells of the green. African monkey, presented fibroblasts-like cells and growth in monolayers. Maintained in culture, the Vero cells presented behavioural and morphologic alterations, associated with cellular transformation. The morphological alterations were investigated using scanning and transmission electron microscopy. The study of proliferation and determination of the cellular doubling time was obtained from the growth curve. The initial population presented growth in a monolayer, while the altered cells grew in multilayers forming cellular aggregates, with flattened cells on the surface and globular cells in the inner region of the aggregate, together with extracellular matrix material. The cell surface of the altered population presented innumerable structures similar to little vesicles, microvilli and cytoplasmic prolongations. The cellular proliferation of both populations was very similar. Our results indicate that morphological and growth changes probably resulted from cellular transformation of the initial Vero cells. These transformed cells presented several characteristics associated with neoplastic growth, and can be used as a model for tumor cells studies in vitro.

Behavioural differences and cytogenetic analysis of a transformed cellular population derived from a Vero cell line.

Arising from a Vero cell line, an altered population was spontaneously obtained, which grew in multiple layers. Cytogenetic analyses revealed a higher polyploidy index (19%) than occurred in the initial population (5%). A modal chromosome number of 56 was found for the altered population, while the initial cell culture had only 54 chromosomes. Karyotypic studies showed that the extranumerary chromosomes were homologues of the 8th and the 27th pairs. The altered population probably resulted from a transformation of the initial line, and their transformed phenotypes may have been associated with the cytological differences.

PolyHEMA and polyHEMA-poly(MMA-co-AA) as substrates for culturing Vero cells.

Poly (2-hydroxyethyl methacrylate), polyHEMA, is known to prevent cellular attachment and spreading. This hydrogel is used to culture cells not dependent on anchorage. Blending polyHEMA with a copolymer of methyl methacrylate and acrylic acid introduces negative charges to the hydrogel and improves its mechanical characteristics. PolyHEMA and the blend were tested for attachment and proliferation of Vero cells. Dense and porous samples of the hydrogels were used. Attachment assays included cellular quantification with MTT photometry and cellular morphology with the scanning electron microscopy after 2 h culture. Proliferation assays were carried out with 5 and 10 days culture. Cellular morphology included cytochemistry of resin sections and scanning electron microscope observations. Hydrogels allowed a few cells to attach and proliferate. The cells growing on the surface of hydrogels were organized in various layers and showed a differential morphology. Cells located inside the pores remained rounded. The hydrogels showed the possibility of inducing differentiated phenotypic expression.

Growth and differentiation of Vero cells cultivated in three-dimensional type 1 collagen.

Vero cells grown in a three-dimensional arrangement of type 1 collagen were phenotypically altered. Such alterations consisted of variations in cellular form and reactivity to toluidine blue. After 22 days of incubation the Vero cells formed three-dimensional arrangements in both tubular form and cellular mass. A reduction in the collagenous substrate was observed in the culture and the alteration noted in the birefringence of the extracellular matrix was attributed to enzymatic action. This model demonstrates a good in vitro system for the study of collagenase activation and for in vitro tests with collagen.

Alterations in the growth and adhesion pattern of Vero cells induced by nutritional stress conditions.

The pattern of growth, adhesion and protein synthesis in Vero cells submitted to nutritional stress conditions was investigated. The control cells presented a characteristic pattern, with monolayer growth, while the stressed cells presented multilayered growth, with aggregate or spheroid formation which detached on the flask surface and continued their growth in another region. In the soft agar assay, with reduced amount of nutrients, only the stressed cells presented growth, indicating physical and nutritional independence. A 44-kDa protein was observed in stressed cells and was absent in non-stressed cells. The adhesion index and fibronectin synthesis and distribution were altered in stressed cells. After confluence, control cells presented fibronectin accumulation in lateral cell-cell contact regions, while this fibronectin accumulation pattern was not observed in stressed cells. These alterations may be responsible for the multilayered growth and decreased adhesion index observed in stressed cells which were transformed by nutritional stress conditions.

Behaviour of fibroblastic cells cultured in collagen I using the sandwich technique.

Vero cells, a fibroblastic lineage derived from fibroblastic kidney cells of the African green monkey, were cultivated by means of the sandwich technique, involving glass coverslip/collagen and collagen/collagen with varied foetal calf serum concentrations in the culture medium. The cells, cultured on coverslips, then received a type I collagen gel layer on top, and migrated from the coverslip to the collagen layer. When the cells were cultivated on collagen followed by a covering of type I collagen gel, the cells migrated into both collagen layers. Cellular morphology was similar, independent of the type of sandwich and serum concentration used. Cells in contact with collagen either migrated into the layer or formed a basal lamina separating them from the collagen matrix. The formation of a basal lamina with laminin and collagen IV deposition was most noticeable when 20% foetal calf serum was used in the culture medium. Cellular infiltration into the collagen gel was most evident with the use of 10% foetal calf serum. The gel contraction was similar for the two serum concentrations employed.

Foetal calf serum and dexamethasone effects on Vero cell growth and differentiation.

Vero cells were cultured without foetal calf serum (FCS), with 10% FCS, 10% FCS plus dexamethasone (DEX) or 20% FCS for 48, 120 or 240 h. The cells were analysed by a growth curve, cytochemical and immunocytochemical (anti-cellular fibronectin or anti-collagen IV) methods. In 48 h Vero cells produced fibronectin and collagen IV. All samples showed basophilic cytoplasm indicating high protein synthesis. The growth of metachromatic multicellular masses was induced by DEX. The Vero cells produced collagen IV with 10 and 20% FCS, and also cells which did not have this activity (without FCS or with 10% FCS + DEX). The multicellular masses induced by DEX were rich in fibronectin. DEX induced differentiation and the expression of collagen IV and fibronectin in Vero cells. This work was carried out to evaluate the possible therapeutic effects of glucocorticoids as inducers of cell differentiation.

Morphology of fibroblastic cells cultured on poly(HEMA-co-AA) substrates.

Fibroblastic cells in culture are characteristically elongated and grow in monolayers. This growth pattern can be modified by different factors, such as substrate interaction. It is characteristic of hydrogels made of poly(2-hydroxyethylmethacrylate) (polyHEMA) that they inhibit cellular attachment and spreading. Vero cells were cultured on porous samples of polyHEMA and the copolymer poly(HEMA-co-AA) with 7.5% (w/w) and 15% (w/w) acrylic acid. Cultures were maintained for 2 and 10 days in HAM F10 medium with 10% foetal calf serum. Hydrogel samples were processed for light microscopy and scanning electron microscopy. The round Vero cells proliferated on the hydrogels and were principally located inside the pores. Some cells were aggregated, but no extracellular matrix was found. The copolymer with 15% (w/w) acrylic acid was the most suitable substrate and should be used in future tests of morphological differentiation and induction of cellular function.

Importance of uniform heparin coating on biopolymers.

Cardiopulmonary surgeries need connectors for extracorporeal circulation. The patient's blood in contact with the tube surfaces modifies its plasmatic proteins, promotes platelet aggregation, and activates the complement system, unleashing thrombus formation. Thus, it becomes necessary for an anticoagulant to keep the circuit free from these events. Heparin is the anticoagulant used even after reports about its disadvantages. Platelet adherence seems to be very dependent on the quality from the surfaces that can promote cellular proliferation, aggregation, and thrombosis. In this study, we compare the quality of the heparin-coated and uncoated surfaces. We used a blood cell culture and scanning electron microscopy (SEM) to visualize the platelet aggregation. It was concluded that there are groove areas that permit platelet adherence, and if they are not coated totally by the heparin, aggregation still occurs although in lower scale than on the uncoated tubes.

Vero cell growth and differentiation on poly(L-lactic acid) membranes of different pore diameters.

In the last few years, the demand has increased for research on polymeric materials, which can be used as substitutes for injured tissues and organs or to improve their regeneration. In this work, we studied poly(L-lactic acid) (PLLA) membranes, a resorbable biomaterial, which were either dense or had different pore diameters (less than 45 microm, between 180 and 250 microm, and between 250 and 350 microm), in relation to stimulation of cell adhesion, growth, and differentiation in vitro. We used Vero cells, a fibroblastic cell line, as the biological model of investigation. We found that cells attached slowly to all PLLA membranes studied. On the other hand, once the adhesion occurs, the cells are able to grow and differentiate on the different polymers. The cells grew to form a confluent monolayer and were capable of producing collagen Type IV and fibronectin on different PLLA membranes. This behavior indicates that cells try to create a better environment to stimulate their growth. This also indicates that Vero cells alter their differentiation pattern once they are producing extracellular matrix molecules related to epithelial differentiation.

Adhesion and morphology of fibroblastic cells cultured on different polymeric biomaterials.

Cell adhesion is influenced by the physical and chemical characteristics of the materials used as substrate for cell culturing. In this work, we evaluated the influence of the morphological and chemical characteristics of different polymeric substrates on the adhesion and morphology of fibroblastic cells. Cell growth on poly (L-lactic acid) [PLLA] membranes and poly(2-hydroxy ethyl methacrylate) [polyHEMA], poly(2-hydroxy ethyl methacrylate)-cellulose acetate [polyHEMA-CA] and poly(2-hydroxy ethyl methacrylate)-poly(methyl methacrylate-co-acrylic acid) [polyHEMA-poly(MMA-co-AA)] hydrogels of different densities and pore diameters was examined. Cells adhered preferentially to more negatively charged substrates, with polyHEMA hydrogels being more adhesive than the other substractes. The pores present in PLLA membranes did not interfere with adhesion, but the cells showed a distinctive morphology on each membrane.

Use of blends of bioabsorbable poly(L-lactic acid)/poly(hydroxybutyrate- co-hydroxyvalerate) as surfaces for Vero cell culture

Vero cells, a cell line established from the kidney of the African green monkey (Cercopithecus aethiops), were cultured in F-10 Ham medium supplemented with 10 percent fetal calf serum at 37°C on membranes of poly(L-lactic acid) (PLLA), poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and their blends in different proportions (100/0, 60/40, 50/50, 40/60, and 0/100). The present study evaluated morphology of cells grown on different polymeric substrates after 24 h of culture by scanning electron microscopy. Cell adhesion was also analyzed after 2 h of inoculation. For cell growth evaluation, the cells were maintained in culture for 48, 120, 240, and 360 h. For cytochemical study, the cells were cultured for 120 or 240 h, fixed, processed for histological analysis, and stained with Toluidine blue, pH 4.0, and Xylidine ponceau, pH 2.5. Our results showed that cell adhesion was better when 60/40 and 50/50 blends were used although cells were able to grow and proliferate on all blends tested. When using PLLA/PHBV (50/50) slightly flattened cells were observed on porous and smooth areas. PLLA/PHBV (40/60) blends presented flattened cells on smooth areas. PLLA/PHBV (0/100), which presented no pores, also supported spreading cells interconnected by thin filaments. Histological sections showed that cells grew as a confluent monolayer on different substrates. Cytochemical analysis showed basophilic cells, indicating a large amount of RNA and proteins. Hence, we detected changes in cell morphology induced by alterations in blend proportions. This suggests that the cells changed their differentiation pattern when on various PLLA/PHBV blend surfaces.