Early Stages Of Culture Research Articles

Simultaneous analysis of spatio-temporal gene expression for cephamycin biosynthesis in Streptomyces clavuligerus.

Linkage between structural and regulatory genes implies that a direct correlation should exist between the spatio-temporal distribution of their expression. Green fluorescent protein (GFP) and cyan fluorescent protein (CFP) were used as reporters to analyze simultaneously expression of lysine-epsilon-aminotransferase (LAT) and its corresponding genetic regulator, CcaR. The isogenic strain containing lat::gfp and ccaR::cfp in the chromosome produced cephamycin C at levels similar to wild type Streptomyces clavuligerus. Confocal laser scanning microscopy revealed that expression of both LAT and CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus mycelia. During the early culture stage only a part of the mycelia began to express LAT and CcaR at low levels. As the culture aged, expression levels and the population of mycelia expressing LAT and CcaR increased and were followed late in the growth cycle by a reduction of the mycelia population expressing LAT and CcaR. The approach provides a precise simultaneous temporal-spatial expression profile and corroborates the regulatory linkage between ccaR and lat in S. clavuligerus.

Endothelins are involved in regulating the proliferation and differentiation of mouse epidermal melanocytes in serum-free primary culture.

Mouse epidermal melanoblasts preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in serum-free melanoblast-defined medium (MDM). After 14 d, almost all keratinocytes that existed predominantly in the early stage of primary culture died, and pure cultures of melanoblasts were obtained. Epidermal melanoblasts dramatically increased in number in MDMDF consisting of MDM supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF). Epidermal melanocytes increased in number in MDMD consisting of MDM supplemented with DBcAMP. On the other hand, epidermal melanocytes were induced to differentiate in MDMM consisting of MDM supplemented with alpha-melanocyte-stimulating hormone (MSH). Pure cultured primary melanoblasts or melanocytes in MDMDF or MDMD were further cultured with MDMDF or MDMD supplemented with endothelin (ET)-1, -2, or -3 from 14 d. A dramatic increase in the number of melanoblasts or melanocytes was observed after 7 d; however, no increase in the number of melanoblasts or melanocytes was observed in the absence of ET-1, -2, or -3. The increase in the number of melanoblasts or melanocytes was comparable with that of melanoblasts or melanocytes cocultured with secondary keratinocytes in MDMDF or MDMD. Also, pure cultured primary melanoblasts in MDM were further cultured with MDMM supplemented with ET-1, -2, or -3 from 14 d. A dramatic increase in the percentage of melanocytes in the melanoblast-melanocyte population was observed after 7 d; however, no increase in the percentage of melanocytes was observed in the absence of ET-1, -2, or -3. The increase was comparable with that of melanocytes cocultured with secondary keratinocytes in MDMM. Moreover, anti-ET-1, -2, and -3 antibodies inhibited both the proliferation of melanoblasts or melanocytes in MDMDF or MDMD and the differentiation of melanocytes in MDMM in primary culture. These results suggest that ET-1, -2, and -3 are one member of the keratinocyte-derived factors that are involved in regulating the proliferation and differentiation of mouse epidermal melanocytes in primary culture.

Osteoprogenitor cells of mature human skeletal muscle tissue: an in vitro study

The presence of osteogenic progenitors in human skeletal muscle is suggested by the formation of ectopic bone in clinical and experimental conditions, but their direct identification has not yet been demonstrated. The aims of this study were to identify osteogenic progenitor cells in human skeletal muscle tissue and to expand and characterize them in culture. Specimens of gracilis and semitendinosus muscle were obtained from young adults and digested to separate the connective tissue and satellite cell fractions. The cells were cultured and characterized morphologically and immunohistochemically using antibodies known to be reactive with primitive osteoprogenitor cells, pericytes, intermediate filaments, and endothelial cells. Alkaline phosphatase activity and osteocalcin gene expression were also determined. In the early stages of culture, the connective tissue cells obtained were highly positive for primitive osteoprogenitor cell and for pericyte markers. Alkaline phosphatase activity was detectable at early stages of culture and rose as a function of time, whereas primitive osteoprogenitor cell markers declined and osteocalcin mRNA expression became detectable by reverse transcriptase-polymerase chain reaction (RT-PCR). It is shown that human skeletal muscle connective tissue contains osteogenic progenitor cells. Their identification as pericytes, perivascular cells with established osteogenic potential, suggests a cellular link between angiogenesis and bone formation in muscle tissue. These cells are easily cultured and expanded in vitro by standard techniques, providing an alternative source of osteogenic progenitor cells for possible cell-based therapeutic use in certain conditions.

Mycelial pellet intrastructure and visualization of mycelia and intracellular lipid in a culture of Mortierella alpina.

The intrastructure of mycelial pellets of Mortierella alpina, which accumulate fatty acids in mycelia, was visualized following labeling with fluorescein isothiocyanate (FITC) and Nile red using fluorescence microscopy. The pellet was an ellipse shape, but its intrastructure was shaped as a doughnut with a cave inside. Using three-dimensional image analysis, it was shown that the lipid was produced on the edge of the pellet, which corresponded to the area where the mycelial density was high. The cavity ratio of the pellet section was determined on the basis of the FITC fluorescence intensity, and in the early culture stage remained at 0.2 in a 10-kl fermentor culture, but finally increased to 0.35. Mycelial pellet volume paralleled the cavity ratio. Application of the technique used here allows analysis of the intrastructure of fungal pellets and new types of fungal biological study.

A Possible Role of Glutathione and Glutathione Disulfide in Tracheary Element Differentiation in the Cultured Mesophyll Cells of Zinnia elegans

We investigated the relationship between the cellular redox state of GSH or GSSG and tracheary element (TE) differentiation using a Zinnia experimental system, in which isolated mesophyll cells transdifferentiate to TEs. TE differentiation was suppressed by the application of L-buthionine sulfoximine (BSO), a potent inhibitor of GSH biosynthesis, at the early stage of cell culture. Application of GSSG at the early culture stage promoted the differentiation, but that of GSH or GSSG at an advanced period of culture suppressed the differentiation. Application of GSH and GSSG nullified the TE differentiation-suppressing effect of BSO. The results suggest that changes in the redox states of GSH and GSSG have a role in TE differentiation.

Nuclear fusion leads to chromosome doubling during mannitol pretreatment of barley (Hordeum vulgare L.) microspores

A cytological study of barley microspores during pretreatment of the uninucleate stage to the early culture stage was conducted utilizing six genotypes. Among the three main pretreatments investigated, microspores completed the first mitotic division during 28 d cold pretreatment of spikes, with or without leaf sheath attached, and during 0.3 M mannitol pretreatment of anthers at 25 degrees C. However, during a 4 d pretreatment in 0.3 M mannitol at 4 degrees C this first mitotic division was blocked or delayed and subsequently most often occurred during the first day on culture medium. The first mitotic division of most microspores pretreated in 0.3 M mannitol was mostly symmetrical (55-60%), whereas it was asymmetric (94%) during the 28 d cold pretreatment of spikes. Following the first mitotic division during the mannitol pretreatment at 25 degrees C, closely associated daughter nuclei often appeared to fuse via membrane coalescence, leading to a high frequency of large uninucleate microspores. Based upon nuclear size, the frequencies of fused uninucleate microspores in genotypes GBC 778, GBC 777 and Igri were estimated to be 87%, 54% and 75%, respectively, after a 4 d mannitol pretreatment at 25 degrees C. Chromosome numbers in dividing nuclei and relative densitometry measurements of nuclear DNA in microspores from cv. Igri confirmed the apparent fused nature of large nuclei in uninucleate microspores. The high frequency of fused nuclei indicates that nuclear fusion occurred between both symmetric and asymmetric nuclei. Microspores of cv. Igri cultured on filter paper following three different pretreatments provided an average of about 12 000 embryo-like structures (ELS) per plate. In samples, 85-97% of these ELS regenerated green shoots. The frequency of doubled haploids (74-83%) following all pretreatments was similar to the frequencies of fused nuclei. The pretreatment of spikes in 0.3 M mannitol at 4 degrees C for 4 d is preferred as it appears to provide genotype independent induction and suspension of nuclear division, as well as regenerating green plants in a shorter time than cold alone.

Homologous expression of recombinant manganese peroxidase genes in ligninolytic fungus Pleurotus ostreatus.

Pleurotus ostreatus is a white-rot fungus known as an efficient degrader of lignin and also various organo-pollutants. Using a DNA-mediated transformation system, a molecular breeding approach to isolate over-producers of a manganese peroxidase isozyme, MnP3, was carried out. Recombinant mnp3 constructs under the control of P. ostreatus sdi1 expression signals were introduced into the wild-type P. ostreatus strain by co-transformation with a carboxin-resistant vector plasmid, pTM1. One of the recombinants obtained by a mating between two monokaryotic transformants, TMG9-C1, showed a several times higher level of MnP activity than the wild-type control in the early stage of liquid culture. Predominant transcription of the recombinant mnp3 in the strain was demonstrated by RT-PCR experiments. This is the first report of a genetically modified P. ostreatus strain with an expression system for recombinant genes.

Serum deprivation and NGF induce and modulate voltage-gated Na(+) currents in human astrocytoma cell lines.

Glial tumor cells derived from primary tissue express large voltage-gated Na(+) currents, whereas glioma cell lines usually lack this feature. We studied the effect of serum deprivation on the expression of Na(+) currents in two astrocytoma cell lines (1321N1 and A172). Serum deprivation for more than 2 days sufficed to induce large Na(+) currents in both cell lines; 300 nM of the specific blocker of voltage-gated Na(+) channels, tetrodotoxin, blocked these currents by about 85%. During serum deprivation, the cells also underwent morphological changes that were characterized by cell rounding and outgrowth of processes. Treatment with 100 ng/ml nerve growth factor (NGF) promoted these morphological changes and also accelerated the development of Na(+) currents. In 1321N1 cells, NGF increased the Na(+) current density after short serum deprivation (3--6 d) and changed several gating properties after longer serum deprivation (9--13 d). In comparison with cells from the early culture stage (3--6 d), the steady-state inactivation of the Na(+) current was shifted by -24 mV in NGF-treated cells from the late (9--13 d) culture stage. In untreated cells, this shift was only -13 mV. NGF accelerated the kinetics of inactivation and shifted the current-voltage relationship in cells from the late culture stage by -14 mV. In A172 cells, most of these effects were present already after short serum deprivation either in presence or absence of NGF. It is concluded that in astrocytoma cells, Na(+) currents are induced by serum deprivation and are modulated by NGF. This result supports the idea that NFG controls Na(+) currents in these cells by autocrine stimulation.

Caspase activation in high-pressure--induced apoptosis of murine erythroleukemia cells.

Murine erythroleukemia cells were subjected to physicochemical stresses such as high pressure (60--110 MPa), heating (42--45 degrees C), and ultraviolet (UV) irradiation (5--25 kJ/cm(2)). After exposure to these stresses, the cells were cultured at 37 degrees C and atmospheric pressure. The number of the cells treated at 100 MPa, 45 degrees C, or 20 kJ/cm(2) remained constant or decreased at an early stage of culture. The nuclear morphology, agarose gel electrophoresis, and flow cytometry of these cells showed that they undergo apoptosis. The activity of caspase-3 was observed in cells subjected to each stress. In particular, caspase-3 was most readily activated by high pressure under our conditions. The caspase-3 activity and apoptosis exhibited a similar pressure dependence. It is important that both caspase-3 activation and apoptosis induced by high pressure were significantly suppressed by a caspase-3 inhibitor. These results suggest that high-pressure-induced apoptosis is also characterized by the activation of caspase-3, as seen with heat- and UV-induced apoptosis.

Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration

Various aspects of transformation and regeneration processes were examined in efforts to improve the efficiency of production of transgenic cotton (Gossypium hirsutum L.). Green fluorescent protein (GFP) proved to be a valuable tool in elucidating the timing and localization of transient gene expression and in visualizing conversion of transient events to stable transformation events. By day 4 after infection, there was maximal transient activity in the cells at the cut edge of Agrobacterium-infected cotyledon disks. We were able to visualize conversion of some of these events to stable transformation by day 8. The effects of Agrobacterium strains, acetosyringone, and temperature on stable transformation were also evaluated. Strain LBA4404 proved to be significantly better than EHA105. Acetosyringone increased significantly the stable transformation efficiency in cotton. Cocultivation at 21 °C, compared to 25 °C, consistently resulted in higher transformation frequencies. GFP expression in stably transformed callus was useful in studying the efficiency of selection during early stages of culture. We found that the survival of individual callus lines on selection medium was influenced by their original size and initial transgene expression status. Larger-size calluses and calluses expressing the transgene (GFP) had a higher rate of survival. Survival could be improved by an additional two-week culture on medium high in cytokinin and low in auxin before transfer to a medium to induce embryogenesis. However, this treatment delayed embryogenesis. Various other important aspects of the regeneration process are described and an overall scheme for producing transgenic cotton is presented.

The Production of Low-Alcohol Wines by Aerobic Yeasts

Aerobic yeasts of the genera Pichia and Williopis are commonly regarded as spoilage yeasts of beer and wine by causing turbidity, a surface film of yeast growth and often an excessive estery flavour. However, their ability to utilise sugars oxidatively for cell growth with the production of estery and other flavours of wine with only minimal production of ethanol suggests a method for the production of low-alcohol wines of pleasant “fermented” flavour without the need for additional equipment to remove alcohol by dialysis, reverse osmosis or distillation, or without the excessive sweetness remaining from arrested fermentation. Three strains of Pichia and one of Williopsis were examined for their ability to produce approximately 3%(v/v) ethanol and a good estery and fruity flavour. With normal anaerobic fermentation conditions, or with gentle stirring to prevent formation of a surface film, excessive amounts of alcohol were produced from grape juice of 15% or 20% (w/v) initial sugar concentration. However, an acceptably flavoured wine of alcohol content < 3% was produced by agitation and aeration during fermentation. The ethanol formed in the early stages of culture was oxidised to a final level < 3%, with the production of cell mass and an acceptable flavour.

Plant regeneration from embryogenic suspension cultures of dune reed

Embryogenic callus, derived from mature seeds of dune reed (Phragmites communisTrinius) was used to establish suspension culture. Green shoot-forming type and albino shoot-forming type embryogenic callus of dune reed were selected carefully by the difference of shape and color of callus growing under light and mechanically dispersed before suspending in liquid MS medium supplemented with 1.0 mg l−12,4-D. They were subcultured every 5 days to remove mucilaginous material in the early culture stage. Both fine albino and green shoot-forming cell suspension lines of dune reed were composed of rapidly growing small cell aggregates that were densely cytoplasmic and potentially embryogenic. Globular somatic embryos were continuously produced in each liquid medium containing 1.0 mg l−1 2,4-D. The cell aggregates in fine albino cell suspension line (size below 300 μm) were smaller than that of green shoot-forming cell suspension line (size between 300 and 800 μm). Following transfer to a differentiation medium, both suspension cultures formed regenerating plants with normal roots and albinotic or green shoots, respectively.

Biphasic effects of leukemia inhibitory factor on osteoblastic differentiation.

Leukemia inhibitory factor (LIF) is a cytokine produced by multiple cell types including osteoblasts and which is active on bone metabolism. We have previously shown that in a bone nodule forming in vitro model of osteogenesis, the fetal rat calvaria (RC) cell model, LIF inhibits osteoblast differentiation, acting on late osteoprogenitors and/or early osteoblasts. These results are in contrast to in vivo experiments, in which LIF has been found to increase bone formation. To resolve this discrepancy, we have tested the effect of LIF on rat bone marrow (RBM) stromal cell cultures, an in vitro model encompassing earlier osteoprogenitor stages. LIF inhibited cell growth in early, proliferating RBM cultures, but increased the culture saturation density. The effect of LIF on bone nodule formation in this model was cell density dependent and biphasic. Continuous treatment with LIF reduced the number of bone nodules present in confluent, more mature cultures, and the inhibitory effect was strongest when cells were plated at higher cell density than lower. In contrast, during the early stages of RBM culture, nodule numbers were higher in LIF-treated dishes than in controls, and this effect was greater in lower density cultures. Acute LIF treatment restricted to early time points increased the final number of bone nodules formed in mature RBM cell cultures, but not in RC cell cultures. Our results indicate that LIF exerts complex, stage-specific effects on osteoprogenitor recruitment, differentiation, and bone formation, and that the effects are cell nonautonomous, in the rat bone marrow stromal cell model. J. Cell. Biochem. Suppl. 36: 63-70, 2001.

Improvement of metabolic performance of cultured hepatocytes by high oxygen tension in the atmosphere.

Maintaining metabolic functions of cultured hepatocytes at higher levels is an essential requirement for the development of a bioartificial liver. We investigated the effect of oxygen tension (10--40%) of the medium on immobilization efficiency and metabolic functions of cultured hepatocytes obtained from a rat for up to 4 days. Immobilization efficiencies of cultures in 10% oxygen showed a significantly lower value from those for the other conditions. The ammonium metabolic rate and the albumin secretion rate were significantly improved with an increase of dissolved oxygen tension for up to 2 days. These values remained similar in the later stage of the culture. The urea secretion rate showed similar values in all conditions. In conclusion, higher oxygen tension improved immobilization efficiency and metabolic functions of cultured rat hepatocytes in the earlier stage of culture for up to 2 days.

Immunofluorescence analysis of cytokine and granule protein expression during eosinophil maturation from cord blood–derived CD34 + progenitors

Background: In allergic inflammation and asthma, eosinophils are major effector cells. They have been shown to synthesize at least 23 cytokines, some of which are stored intracellularly in their unique crystalloid granules together with cationic granule protein. Little is known about the synthesis and storage of cytokines relative to cationic granule proteins in maturing eosinophils during eosinophilopoiesis. Objective: Our purpose was to analyze the expression of eosinophil-derived mediators, major basic protein (MBP), eosinophil cationic protein (ECP), IL-6, and RANTES, during early stages of eosinophil maturation in CD34 + cell-derived colonies. Methods: Purified human cord blood CD34 + cells were grown in methylcellulose cultures in the presence of recombinant human IL-3 and IL-5. By confocal laser scanning microscopy, the coexpression of eosinophil granular proteins MBP and ECP was determined concurrently with IL-6 and RANTES during eosinophil maturation on days 16, 19, 23, and 28 of culture. Results: Immunoreactivity against MBP, ECP, IL-6, and RANTES was not detectable in freshly purified CD34 + cells. Maturing eosinophils (>95%) exhibited positive immunostaining for all these proteins between days 16 and 28 of culture. At early stages of culture, discrete immunostaining was observed around the periphery but not in the center of granular structures. By day 28 cultured eosinophil-like cells showed evidence of the acquisition of crystalloid granule-like structures, analogous to those observed in mature peripheral blood eosinophils. Conclusions: Eosinophils express and store cytokines simultaneously with cationic granule proteins during the process of maturation. We propose that the storage of cytokines during the development of eosinophils is an early event and it may be integral to inflammatory responses involving these cells. The results of this study suggest a potential immunoregulatory function for maturing eosinophils. (J Allergy Clin Immunol 2000;105:1178-84.)

In vitro cytokine mRNA expression in normal human peripheral blood mononuclear cells.

Normal human peripheral blood mononuclear cells (PBMC) were analyzed for basic profiles of mRNA expression of distinct genes during incubation in a standard cell culture system. Human PBMC from healthy adult blood donors. Steady-state mRNA expression was measured using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Shortly after isolation and cell seeding, mRNA levels of monocyte-derived cytokines (IL-1alpha, IL-6, and TNF-alpha) were significantly increased, whereas lymphocyte-derived cytokines (IL-4, IFN-gamma) were not affected. Expression levels of monokines returned to basal within 24 h. At later stages of culture, the mRNA levels of all genes studied gradually increased and were significantly elevated after 96 h of incubation. Monokine and lymphokine mRNAs respond differently to cell culture even under control conditions. With regard to the enhanced mRNA expression of distinct cytokines in the early stages of culture, human PBMC should not be used for gene expression studies in vitro within 24 h of isolation.

Physical characterization of the MUC5AC mucin: a highly oligomeric glycoprotein whether isolated from cell culture or in vivo from respiratory mucous secretions

We have isolated the high-M(r) mucins from growth medium of the early stage of an HT-29 cell culture by gel chromatography and isopycnic density gradient centrifugation. The mucins (buoyant density 1.34-1.44 g/ml) were reactive with an anti-peptide antiserum (MAN-5ACI) raised against a sequence from within the MUC5AC mucin. Similar antisera raised against the MUC2 and MUC5B mucins were not reactive. The MUC5AC reduced-mucin subunits exhibited a homogeneous charge distribution on anion-exchange chromatography, but appeared as two bands, one major and one more minor, after agarose gel electrophoresis. The unreduced mucins had an average M(r) in excess of 40 MDa and were visualized in the electron microscope as large, fine filamentous threads (many microns in length) that after reduction were greatly reduced in size (number average length 570 nm). Agarose gel electrophoresis of unreduced MUC5AC mucins identified a major band just entering the gel with evidence of a 'ladder' of faster-migrating minor bands. Partial reduction of the mucins increased the proportion of the faster bands and at least 16 could be discriminated. M(r) measurements showed that these bands differed by single monomer units. The mucins behaved as very stiff extended structures in solution and this characteristic might explain the poor separation of different-sized oligomers in sedimentation-rate experiments. The cell-culture mucin preparation had similar characteristics of charge and buoyant density to MUC5AC mucins from respiratory secretions in vivo. In addition the MUC5AC mucin from respiratory tract secretions exhibited similar behaviour, reduced and unreduced on agarose gel electrophoresis, indicating that the mucin has a similar molecular phenotype in vivo and in vitro.

Physical characterization of the MUC5AC mucin: a highly oligomeric glycoprotein whether isolated from cell culture or in vivo from respiratory mucous secretions

We have isolated the high-Mr mucins from growth medium of the early stage of an HT-29 cell culture by gel chromatography and isopycnic density gradient centrifugation. The mucins (buoyant density 1.34-1.44 g/ml) were reactive with an anti-peptide antiserum (MAN-5ACI) raised against a sequence from within the MUC5AC mucin. Similar antisera raised against the MUC2 and MUC5B mucins were not reactive. The MUC5AC reduced-mucin subunits exhibited a homogeneous charge distribution on anion-exchange chromatography, but appeared as two bands, one major and one more minor, after agarose gel electrophoresis. The unreduced mucins had an average Mr in excess of 40 MDa and were visualized in the electron microscope as large, fine filamentous threads (many microns in length) that after reduction were greatly reduced in size (number average length 570 nm). Agarose gel electrophoresis of unreduced MUC5AC mucins identified a major band just entering the gel with evidence of a ‘ladder’ of faster-migrating minor bands. Partial reduction of the mucins increased the proportion of the faster bands and at least 16 could be discriminated. Mr measurements showed that these bands differed by single monomer units. The mucins behaved as very stiff extended structures in solution and this characteristic might explain the poor separation of different-sized oligomers in sedimentation-rate experiments. The cell-culture mucin preparation had similar characteristics of charge and buoyant density to MUC5AC mucins from respiratory secretions in vivo. In addition the MUC5AC mucin from respiratory tract secretions exhibited similar behaviour, reduced and unreduced on agarose gel electrophoresis, indicating that the mucin has a similar molecular phenotype in vivo and in vitro.

Two-dimensional manipulation of differentiated Madin-Darby canine kidney (MDCK) cell sheets: the noninvasive harvest from temperature-responsive culture dishes and transfer to other surfaces.

A renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, adheres, spreads, and proliferates to confluency on our developed temperature-responsive culture dishes grafted with a poly(N-isopropylacrylamide) (PIPAAm) at 37 degrees C. In addition to other cell types, including hepatocytes and endothelial cells, MDCK cell sheets noninvasively were harvested from PIPAAm-grafted dishes merely by reducing the temperature. However, during the early stage of culture (up to 3 weeks), confluent MDCK cell detachment is greatly repressed. In the present study, we succeeded in the rapid harvest of confluent MDCK cell sheets and intact transfer to other culture dishes by utilizing hydrophilically modified poly(vinylidene difluoride) (PVDF) membranes as supporting materials. Immunocytochemistry with anti-beta-catenin antibody revealed that the functional cell-cell junctions were well organized in the transferred MDCK cell sheets. The viability assay showed that the transferred cells were not damaged during the two-dimensional cell-sheet manipulation. By transmission electron microscopy it was confirmed that the harvested MDCK cells retained differentiated phenotypes and had many microvilli and tight junctions at the apical and lateral plasma membranes, respectively. This two-dimensional cell-sheet manipulation technique promises to be useful in tissue engineering as well as in the investigation of epithelial cell sheets.

Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts.

Adult human osteoblastic cells were grown in a native type I collagen gel. Proliferation and viability analyses showed that cells rapidly stopped dividing and became blocked in the G0G1 phase (91% on day 13). Carboxyfluorescein diacetate cell staining and flow cytometry showed that osteoblasts were viable for the first 16 days and then viability decreased (58% viable cells on day 22). Osteoblasts were able to retract the matrix. Betaglycerophosphate (betaGP) stimulated the deposition of mineral particles in the collagen network, and electron probe microanalysis showed that they were principally calcium and phosphorus, with a Ca/P ratio of about 1.7. Various times of betaGP supply were tested. We compared 10 mM betaGP added only once at day 0, or continuously from day 0, day 8, or day 21. Mineralization was observed in conditions where betaGP was added at day 0. Furthermore, 10 mM betaGP added once during gel preparation was sufficient to induce mineralization with mineral accumulation up to day 15 whereas the speed of the gel contraction decreased. In every condition, cultures expressed high alkaline phosphatase (ALP) levels as early as day 3, which decreased afterwards. These kinetics might explain why the other conditions did not prove favorable to the mineralization process. The model was used to study the influence of blocking gel retraction. Blocking retraction delayed the ALP activity decrease, but had no effect on mineralization. In conclusion, human adult osteoblasts cultured in native collagen gel stopped proliferation and underwent mineralization very early. This model should be used to investigate the influence of effectors on the early stages of culture.