Pathology of mitosis after cellular recovery from a metaphase block]

The experiments in Chinese hamster fibroblast-like cells have shown that the c-mitosis inducing agents (colchicine, colcemid and low temperature) produce evident stathmokinetic and radiomimetic effects. By the reversibility moment of the former the latter becomes apparent, manifesting in bridge accumulation. This form of pathology is likely to be caused by the disturbance of the cellular nucleoprotein metabolism in c-mitosis.     

Cell cycle arrest and cellular differentiation mediated by a cell surface sialoglycopeptide

Cell cycling by a relatively wide variety of cell lines was shown to be reversibly inhibited by a cell surface sialoglycopeptide (SGP) isolated and purified from intact bovine cerebral cortex cells. Cell cycle arrest, mediated by the bovine SGP inhibitor, was shown to be completely reversible with mouse Swiss 3T3, mouse 1316 fibrosarcoma, mouse N2a neuroblastoma, bovine MDBK and monkey BSC-1 cells. These cell lines represented both fibroblast and epithelial-like cells, transformed and nontransformed cells, as well as their being derived from a broad array of species. In contrast to the others tested, human HL-60 leukemic cells were sensitive to the inhibitory effects of the SGP but did not reenter the mitotic cycle after the removal of the inhibitor. Instead, the mitotic arrest of HL-60 cells appeared to enhance entry into a terminal and irreversible state of cellular differentiation.     

Effect of cell surface trypsinization on ethanolamine base exchange enzymatic activity of astrocyte primary cultures and derived spontaneously transformed cell lines

Trypsinization of neonatal rat astrocyte primary cultures (normal cells) inhibited the activity of ethanolamine base exchange enzyme (EBEE) by 80%, whereas ethanolamine phosphotransferase (EPT) and choline base exchange (CBEE) enzymatic activities were not affected; subcellular fractionation demonstrated that trypsin treatment affected the intracellular EBEE activity. During trypsinization the enzyme was not taken up by cultured astrocytes but the cell surface was affected. In contrast, the same treatment did not alter EPT, CBEE and EBEE activities of spontaneously transformed cell lines derived from the primary cultures. However, treatment of the transformed cells with db-cAMP prior to trypsin, restored the pattern found in the primary culture, i.e. only EBEE activity was affected. These data suggest that a relationship exists between cell surface organization and intracellular EBEE activity in a culture system which possesses the property to control its own cell division or/and differentiation.     

Pharmacologic analysis of the stimulating effect of serotonin on proliferation in cell cultures]

Serotonin (10-7 M) stimulated cell proliferation in the primary cultures of mouse and human embryonic fibroblasts as well as in mouse L-cells and in monkey kidney cells (MA). The stimulatory effect is completely prevented by pretreatment of cultured cells with serotonin antagonists of tipindole, morphine or cyproheptadine type which do not affect cell proliferation. It is therefore assumed that the stimulatory action of serotonin on these cells is realized via specific serotonin receptors.     

Stratification, specialization, and proliferation of primary keratinocyte cultures. Evidence of a functioning in vitro epidermal cell system

A population of neonatal mouse keratinocytes (epidermal basal cells) was obtained by gentle, short-term trypsin separation of the epidermal and dermal skin compartments and discontinuous Ficoll gradient purification of the resulting epidermal cells. Over 4--6 wk of culture growth at 32--33 degrees C, the primary cultures formed a complete monolayer that exhibited entire culture stratification and upper cell layer shedding. Transmission and scanning electron microscopy demonstrated that the keratinocyte cultures progressed from one to two cell layers through a series of stratification and specialization phenomena to a six to eight cell layer culture containing structures characteristic of epidermal cells and resembling in vivo epidermal development. The temporal development of primary epidermal cell culture specialization was confirmed by use of two histological techniques which differentially stain the specializing upper cell layers of neonatal mouse skin. No detectable dermal fibroblast co-cultivation was demonstrated by use of the leucine aminopeptidase histochemical technique and routine electron microscope surveillance of the cultures. Incorporation of [3H]thymidine ([3H]Tdr) was greater than 85% into DNA and was inhibited by both 20 micron cytosine arabinoside (Ara-C) and low temperature. Autoradiography and 90% inhibition of [3H]Tdr incorporation by 2 mM hydroxyurea indicated that keratinocyte culture DNA synthesis was scheduled (not a repair phenomenon). The primary keratinocytes showed an oscillating pattern of [3H]Tdr incorporation into DNA over the initial 23--25 days of growth. Autoradiography demonstrated that the cultures contained 10--30% proliferative stem cells from days 2-25 of culture. The reproducibility of both the proliferation and specialization patterns of the described primary epidermal cell culture system indicates that these cultures are a useful tool for investigations of functioning epidermal cell homeostatic control mechanisms.     

Cell proliferation and cell differentiation in tissue cultures of adult muco-cilliary epithelia.

Explants and monolayers from a variety of muco-ciliary epithelia were cultivated in vitro and the kinetics of their proliferation and differentiation described. New epithelial lining and epithelial-like monolayer sheets of cells formed in which the migration cells were all originally undifferentiated cycling stem cells. The divided and differentiated in ML growth into cell types characteristic of the tissue source: however, the control mechanisms which regulate cell division and cell differentiation in the tissues were lost outside the tissue framework. Cell division and cyto-differentiation in ML growths both in ciliated and in mucus-producing cells, were not always mutually exclusive.     

Cell viability and proliferation capability of long-term human dental pulp stem cell cultures

Background aimsEvaluation of cell viability is one of the most important steps of the quality control process for therapeutic use of cells. The aim of this study was to evaluate the long-term cell viability profile of human dental pulp stem cell (hDPSC) subcultures (beyond 10 passages) to determine which of these passages are suitable for clinical use and to identify the cell death processes that may occur in the last passages.MethodsFour different cell viability assays were combined to determine the average cell viability levels at each cell passage: trypan blue exclusion test, water-soluble tetrazolium 1 (WST-1), LIVE/DEAD Viability/Cytotoxicity Kit and electron probe x-ray microanalysis (EPXMA). Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and caspase 4 and BCL7C Western blotting, and cell proliferation was analyzed by WST-1 and proliferating cell nuclear antigen protein detection.ResultshDPSCs showed high average cell viability levels from passages 11–14, with adequate cytoplasmic and mitochondrial functionality at these subcultures. A non-significant trend to decreased cell proliferation was found from passages 16–20. EPXMA and TUNEL analyses suggested that a pre-apoptotic process could be activated from passages 15–20 (P < 0.001), with a correlation with caspase 4 and BCL7C expression.ConclusionshDPSCs corresponding to passages 11–14 show adequate cell function, proliferation and viability. These cells could be considered as potentially useful for clinical applications.     

First results of the investigation of the stability and tissue integration of a degradable, elastomeric copolymer in an animal model]

The stability and tight integration into adjacent tissue of a novel, degradable, elastic copolymer were examined in an animal model. The biomaterial was used for the reconstruction of a gastric wall defect in Sprague-Dawley rats (n=42) to test the polymeric material under the extreme chemical, enzymatical and mechanical conditions of the stomach. In the control group (n=21) the same defect of the gastric wall was primarily closed without biomaterial implantation. In the baseline group (n=21) the animals were kept under standard conditions without any surgical procedure. The implantation periods were 1 week, 4 weeks and 6 months. The animals' weight was determined preoperatively and before explantation. After explantation, air was pumped into the stomach and the pressure was measured by using a pressure-gauge in order to test whether the surgically produced union of the stomach wall and the polymer patch was gas-tight. After 1 week of implantation time a statistically significant increase of the body weight of the animals was found only in the baseline group. Four weeks and 6 months after the abdominal surgical procedure, a statistically significant increase of the animals' weight was found in the implantation group, the control and the baseline group. Gastrointestinal complications like fistula, perforation or peritonitis did not occur in any of the animals. The measurement of the stomach pressure after maximal gas insufflation did not show significant differences between the implantation group, the control and the baseline group in any of the time periods investigated. Despite very high strains of the gastric wall, no gas leakage was detected. There was a tight connection between the polymer and the adjacent stomach wall in all animals investigated. An adequate mechanical stability of the biomaterial was detectable under the extreme pathophysiological conditions of the stomach milieu. A fast and unfavourable degradation of the degradable polymer was not found in any of the animals. Further investigations are needed to analyse the mechanisms of the tissue integration of the biomaterial as well as the degradation kinetic of the polymer and the process of the tissue remodeling. The knowledge of these processes is necessary to adapt the novel biomaterial and thus prepare it for the use and implantation in different body locations and to develop novel therapeutical options in medicine.     

Cell surface galactosyltransferase acts as a general modulator of rat acinar cell proliferation

Summary Several physiological parameters were examined for inducing acinar cell proliferation and corresponding increased expression of 1–4 galactosyltransferase. In this study, dietary changes causing acinar cell proliferation included the following: the introduction of animals to a liquid diet (causing gland atrophy) followed by reintroduction of solid chow, gustatory stimulation provided by the introduction of 0.5% citric acid to animal drinking water, and removal of the submandibular gland with subsequent reliance on the parotid gland for salivary protein. Alterations in growth factor levels were produced by injecting animals with a chronic (3 day) regimen of either nerve growth factor or epidermal growth factor. Parotid gland proliferation could be blocked in all cases except EGF by the injection of propranolol, a -adrenoceptor antagonist, or the galactosyltransferase specific modifier protein, -lactalbumin. EGF-induced proliferation could, however, be prevented by treating the animals with monoclonal antibody to EGF receptor or galactosyltransferase modifier protein a-lactalbumin. These results for normal acinar cell proliferation suggest a direct role for cell surface 1–4 galactosyltransferase in signalling and maintaining active cell growth.     

Acid mucopolysaccharide metabolism, the cell surface, and primary mesenchyme cell activity in the sea urchin embryo

The relationship between ³⁵SO₄ incorporation into acid mucopolysaccharides and the appearance and activity of the primary mesenchyme cells has been studied in the sea urchin, Lytechinus pictus. The ratio of the uptake of ³⁵SO₄ to its incorporation into cetylpyridinium chloride precipitable material varies over a wide range during early development, with the smallest ratio, therefore the greatest sulfation activity, being found at the early mesenchyme blastula stage. The types of mucopolysaccharides produced have not been identified, but are heterogeneous. At the mesenchyme blastula stage nearly 90% of the polysaccharides produced become sulfated. When embryos develop in sulfate-free sea water to the mesenchyme blastula stage there is a 70% decrease in the incorporation of ³H-acetate into polysaccharides and a 13-fold decrease in the ratio of sulfated to nonsulfated polysaccharides produced. Embryos raised in sulfate-free sea water develop normally to the mesenchyme blastula stage at which time there is an accumulation in the blastocoel of primary mesenchyme cells that do not migrate. The surface of the primary mesenchyme cells of sulfate-deficient embryos has a smooth appearance in the scanning electron microscope, while the surface of these cells in control embryos is rough, possibly reflecting the presence of an extracellular coat. It is suggested that there is a correlation between sulfated polysaccharide synthesis, cell surface morphology and cell movement.     

Proteolytically degradable hydrogels with a fluorogenic substrate for studies of cellular proteolytic activity and migration

We have developed proteolytically degradable hydrogels with covalently immobilized fluorogenic protease substrates to visualize extracellular proteolytic activity and cell migration in three dimensions. Dye quenched-bovine serum albumin (DQ-BSA), a quenched, proteolytically activated fluorogenic substrate, was conjugated to poly(ethylene glycol) (PEG)-monoacrylate, and the product (DQ-BSA-PEG) was then covalently incorporated into proteolytically degradable and cell adhesive PEG hydrogels via photopolymerization. The DQ-BSA-PEG substrate in solution and incorporated into hydrogels exhibited significantly enhanced fluorescence after exposure to enzymes. Fibroblasts seeded within this hydrogel spread in three dimensions and extended lamellipodia. Cell migration and proteolytic activity were visualized using confocal microscopy. Proteolytic activity was concentrated near cell surfaces and remained present in the tracks where cell migration had occurred.     

Adhesion,proliferation, and adipogenesis in primary rat cell cultures: effects of collagenous substrata,fibronectin, and serum

Summary The effects of collagenous substrata, fibronectin, and fetal bovine serum on the adhesion, proliferation, and adipogenesis of rat stromal-vascular cells are reported. There was no effect on initial stromal-vascular cell-attachment by fetal bovine serum or fibronectin. The number of cells attached to a hydrated collagen-gel was almost twice (P<0.04) the number attached to dried collagen-gel or dried denatured collagen-gel. Total number of cells after 5 days in culture was similar among the collagenous substrata and among the treatments with or without fibronectin in the growth media. Total number of cells increased significantly (P<0.02) with 10% FBS. Adipocytic formation was inhibited by hydrated collagen-gel (P<0.02) compared to dried collagen-gel or dried, denatured collagenous substrata. An interaction occurred between dried, denatured gel and fetal bovine serum so that total formation of adipocytes increased by increasing the level of fetal bovine serum (P<0.07). Adipocytic formation was inhibited by hydrated collagen-gel at all levels of fetal bovine serum. The percentage of cells that converted to adipocytes was significantly lower (P<0.01) on hydrated collagen-gel compared to dried, denatured or dried collagen-gel. Percentage of conversion was not significantly different among levels of fetal bovine serum, although this percentage increased as fetal bovine serum level increased. Adipocytic conversion was not different between fibronectin-treated or untreated cells. Morphology of stromal vascular cells was similar on dried collagen and dried, denatured collagen-gel, but tended to remain bipolar on hydrated collagen-gel. These studies indicate that fetal bovine serum in combination with the extracellular matrix (dried, denatured collagen) increased the differentiation of rat stromal-vascular cells into adipocytes, and that hydrated collagen inhibited differentiation.     

Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices

The cell seeding density and spatial distribution in a 3-D scaffold are critical to the morphogenetic development of an engineered tissue. A dynamic depth-filtration seeding method was developed to improve the initial cell seeding density and spatial distribution in 3-D nonwoven fibrous matrices commonly used as tissue scaffolds. In this work, trophoblast-like ED27 cells were seeded in poly(ethylene terephthalate) (PET) matrices with various porosities (0.85-0.93). The effects of the initial concentration of cells in the suspension used to seed the PET matrix and the pore size of the matrix on the resulting seeding density and subsequent cell proliferation and tissue development were studied. Compared to the conventional static seeding method, the dynamic depth-filtration seeding method gave a significantly higher initial seeding density (2-4 x 10(7) vs 4 x 10(6) cells/cm3), more uniform cell distribution, and a higher final cell density in the tissue scaffold. The more uniform initial cell spatial distribution from the filtration seeding method also led to more cells in S phase and a prolonged proliferation period. However, both uniform spatial cell distribution and the pore size of the matrices are important to cell proliferation and morphological development in the seeded tissue scaffold. Large-pore matrices led to the formation of cell aggregates and thus might reduce cell proliferation. The dynamic depth-filtration seeding method is better in providing a higher initial seeding density and more uniform cell distribution and is easier to apply to large tissue scaffolds. A depth-filtration model was also developed and can be used to simulate the seeding process and to predict the maximum initial seeding densities in matrices with different porosities.     

Demonstration of the oncogenic activity of cell cultures using antithymocytic serum]

The authors examined 10 cell strains of different origin as to their effect on mice by means of antithymocytic (ATC) serum. In dependence on the strain used the tumors developed in different number and with different growth tendency. In control animals not treated by the ATC serum, small ganglions developed in some cases which, however, disappeared in 2--3 days. Both strains of diploide cells WI 38 and LEP and primary cultures of chicken fibroblast from embryos of SPF chickens did not develop any tumors. The antithymocytic serums from calfs were found to be less toxic for mice than the rabbit sera.     

A role of activated Sonic hedgehog signaling for the cellular proliferation of oral squamous cell carcinoma cell line

Sonic hedgehog (Shh) is a secreted morphogen crucial for appropriate cellular proliferation during mammalian development. The activated Shh signaling is known to predispose to human tumors such as medulloblastoma and basal cell carcinoma, while a role of Shh signaling in the other common tumors is still controversial. Here we showed the overexpression of Shh in five cell lines among 14 human oral squamous cell carcinoma (OSCC) cell lines. One of the Shh-expressing OSCC cell lines HSQ-89 showed the inhibition of G1/S transition and apoptotic cell death by treatment with Cyclopamine, a steroidal alkaloid that blocks the intracellular Shh signaling. Furthermore, we found that treatment with Y-27632, a specific inhibitor of Rho-associated kinase, mimicked the effect of Cyclopamine on the cell cycle progression of HSQ-89. Our study revealed the involvement of activated Shh signaling in the cellular proliferation of OSCC cells, indicating Shh signaling might be a good therapeutic target for OSCC.