Multilayer rat hepatocyte aggregates formed on expanded polytetrafluoroethylene surface

Feasibility of using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE), for culturing hepatocytes on its surface was examined. Adult rat hepatocytes were attached to an ePTFE surface and cultured in a hormonally defined medium supplemented with or without fetal calf serum (FCS, 10%) or bovine serum albumin (BSA, 0.03–3%). When cultured in a FCS-suplemented medium, hepatocytes reorganized themselves into multilayer cell aggregates on an ePTFE surface. The morphological characteristics of hepatocytes were influenced by the modification of the ePTFE surface as well as the culture medium. Hepatocytes cultured on a polyvinylalcohol (PVA)-coated ePTFE surface formed many more multilayer cell aggregates than those cultured on an uncoated ePTFE surface. Such highly multilayered hepatocyte aggregates were also noted when the cells were cultivated in a BSA-supplemented medium. On the other hand, when cultured in a FCS- or BSA-free medium, hepatocytes formed cell monolayers on both PVA-coated and uncoated ePTFE surfaces as did the cells on a collagen-coated polystyrene surface. The hepatocytes in the aggregates exhibited high albumin expression capability and low DNA synthesis rate as compared with those in monolayer cultures. The multilayer hepatocyte aggregates, as immobilized on a PVA-coated ePTFE surface in a serum-supplemented medium, are shown to be not only morphologically, but functionally differentiated, and will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver. This revised version was published online in June 2006 with corrections to the Cover Date.     

Receptor-mediated formation of multilayer aggregates of primary cultured adult rat hepatocytes on lactose-substituted polystyrene.

We used a lactose-substituted polystyrene, poly-N-p-vinylbenzyl-D-lactonamide (PVLA), as a substratum for adult rat hepatocytes in primary culture. Spherical-shaped hepatocytes attached on PVLA substratum formed stable multilayer aggregates anchored on substratum through the stimulation of epidermal growth factor (EGF). The cells required calcium ion essentially to form the aggregates. The formation of multilayer aggregates was inhibited by colchicine, but not by cytochalasin B. The inhibition was also observed by added PVLA molecules in the culture medium and by treating surfaces of PVLA-coated dishes with allo A lectin. It was suggested that adult rat hepatocytes attached on PVLA substratum required the specific interaction between asialoglycoprotein receptors on the cell surface and PVLA substratum to form anchored multilayer aggregates.     

Isolation and characteristics of HeLa surface proteins

HeLa 71 and 65 cells grown in attached culture possess a coat of extracellular proteins that can be released from the cell by mild EDTA-detachment, with no significant effect on cellular integrity. This suggests that these surface proteins are weakly associated with the cell, possibly through divalent cations. The high affinity of surface proteins for critical divalent cations, shown by their high precipitability by Zn²⁺, Ca²⁺ and Mg²⁺, supports this assumption. Since surface proteins appear to be phosphoproteins, as suggested by significant incorporation of ³²Pi in vitro, it is possible that binding occurs through The amount of surface protein on HeLa 65 cells grown in suspension culture is greatly reduced compared with cells grown in monolayer culture. This may be related to impaired availability of Ca²⁺ in suspension culture medium. In monolayer grown HeLa cells surface proteins are predominantly distributed underneath the cells. The highest amount of these proteins is found on cells prior to growth initiation and steadily decreases as cells approach confluency. As shown by radioactive leucine protein labeling, surface proteins are primarily comprised of proteins synthesized within HeLa cells and released to the outer cell surface. The presence of serum proteins in surface protein matrix is physiologically significant.     

Hydrogel Environment Supports Cell Culture Expansion of a Grade IV Astrocytoma

Malignant astrocytomas are aggressive cancers of glial origin that can develop into invasive brain tumors. The disease has poor prognosis and high recurrence rate. Astrocytoma cell lines of human origin are an important tool in the experimental pathway from bench to bedside because they afford a convenient intermediate system for in vitro analysis of brain cancer pathogenesis and treatment options. We undertook the current study to determine whether hydrogel culture methods could be adapted to support the growth of astrocytoma cell lines, thereby facilitating a system that may be biologically more similar to in vivo tumor tissue. Our experimental protocols enabled maintenance of Grade IV astrocytoma cell lines in conventional monolayer culture and in the extracellular matrix hydrogel, Geltrex^?. Light and fluorescence microscopy showed that hydrogel environments promoted cellular reorganization from dispersed cells into multilayered aggregates. Transmission electron microscopy revealed the prevalence of autophagy and nuclear membrane distortions in both culture systems. Analysis of microarray Gene Expression Omnibus (GEO) DataSets highlighted expression of genes implicated in pathways for cancer progression and autophagy. A pilot quantitative polymerase chain reaction (qPCR) analysis of the autophagic biomarkers, Beclin 1 (BECN1) and microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), with two reference genes (beta actin, ACTB; glyceraldehyde 3-phosphate dehydrogenase, GAPDH), uncovered a relative increase of BECN1 and LC3B in hydrogel cultures of astrocytoma as compared to the monolayer. Taken together, results establish that ultrastructural and molecular characteristics of autophagy are features of this astrocytoma cell line, and that hydrogel culture systems can afford novel opportunities for in vitro studies of glioma.     

Lipid oxidation and signalling in programmed cell death

The pulmonary surfactant lines as a complex monolayer of lipids and proteins the alveolar epithelial surface. The monolayer dynamically adapts the surface tension of this interface to the varying surface areas during inhalation and exhalation. Its presence in the alveoli is thus a prerequisite for a proper lung function. The lipid moiety represents about 90% of the surfactant and contains mainly dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG). The surfactant proteins involved in the surface tension adaption are called SP-A, SP-B and SP-C. The aim of the present investigation is to analyse the properties of monolayer films made from pure SP-C and from mixtures of DPPC, DPPG and SP-C in order to mimic the surfactant monolayer with minimal compositional requirement. Pressure-area diagrams were taken. Ellipsometric measurements at the air-water interface of a Langmuir film balance allowed measurement of the changes in monolayer thickness upon compression. Isotherms of pure SP-C monolayers exhibit a plateau between 22 and 25 mN/m. A further plateau is reached at higher compression. Structures of the monolayer formed during compression are reversible during expansion. Together with ellipsometric data which show a stepwise increase in film thickness (coverage) during compression, we conclude that pure SP-C films rearrange reversibly into multilayers of homogenous thickness.

Lipid monolayers collapse locally and irreversibly if films are compressed to approximately 0–4 nm²/molecule. In contrast, mixed DPPG/SP-C monolayers with less than 5 mol% protein collapse in a controlled and reversible way. The pressure-area diagrams exhibit a plateau at 20 mN/m, indicating partial demixing of SP-C and DPPG. The thickness isotherm obtained by ellipsometry indicates a transformation into multilayer structures. In DPPC/DPPG/SP-C mixtures again a reversible collapse was observed but without a drastic increase in surface layer thickness which may be due to the formation of protrusion under the surface. Thus lipid monolayers containing small amounts of SP-C may mimic the lung surfactant.     

Establishment and characterization of a new human colon adenocarcinoma cell line: BCS-TC2

A new cell line designated as BCS-TC2 was established in culture from a primary human colon adenocarcinoma. This cell line has been in continuous culture over a 36-month period. The cells grow as a monolayer sheet, displaying areas with a multilayered pattern as well as single cells and free-floating aggregates. The morphological, immunological, and ultrastructural features of these cells are in agreement with their epithelial origin. The characterization of this cell line indicated a 38 hr doubling time, and a colony forming efficiency of 2% in semisolid media and 22% in liquid culture, at low cell densities. These cells produce low amounts of carcinoembryonic antigen in culture (0.1 ng of CEA/10⁶ cells). Sub-cutaneous injection into athymic mice shows that these cells have a non-tumorigenic capacity. Chromosomal analysis showed a karyotype 46 XX,-15, +der (15), inv (16) (p13::q13). BCS-TC2 cell line, which maintains in culture several characteristics of the original tumor, represents a useful model system for cell biology studies of primary and non-metastatic tumors.     

Polydimethylsiloxane Films Modified with Chitosan/Pectin Multilayers as Scaffolds for Mesenchymal Stem Cells

Solid (smooth) and porous films of polydimethylsiloxane (PDMS) have been obtained; the effect of their structure on the adhesion of mesenchymal stem cells (MSCs) on their surface was found. It is shown that modification of these films with a (chitosan/pectin)₄ multilayer increased the efficiency of viable cell adhesion. A (3-aminopropyl)triethoxysilane–glutaraldehyde system was used to enhance the binding of the polysaccharide layer to the hydrophobic surface of PDMS. It was found that MSCs formed a monolayer culture of the fibroblast-like cells with high viability on porous PDMS modified with (chitosan/pectin)₄.     

Proliferation of pulmonary endothelial cells: Time-lapse cinematography of growth to confluence and restitution of monolayer after wounding

A fundamental characteristic of vascular endothelium is that it exists as a monolayer, a condition that must be met in both vascular growth and repair. Maintenance of the monolayer is important both for the exchange of nutrients and for interactions between blood solutes and endothelial enzymes and transport systems. We have used time-lapse cinematography to compare proliferative behavior of bovine pulmonary endothelial cells in (1) establisment of a monolayer from a low-density seed (7.5 × 10⁴ cells in a 60 mm dish) and (2) restitution of a confluent monolayer (approx. 2.9 × 10⁶ cells in a 60 mm dish) following a mechanical wound (removal of cells from an area 5 × 15 mm by scraping). Culture 2 was not refed after wounding. In culture 2, approx. 30% of the cells accounted for repopulation (confluence in 40 hr). In culture I, all cells entered into division. Participating cells of culture 2 began division immediately (69 divisions/filmed area in 10 hr, vs. four divisions in culture I). Interdivision times (IDT) were longer and relatively constant in culture I until near confluence; none were < 10 h, whereas in 2, 24% of the IDT's were ≤ 10 hr. Remarkably, IDTs of culture 2 decreased steadily until confluence was re-established. Cell migration in culture 1 was multidirectional while direction of migration in culture 2 was always into the wound area. Mean migration rate (MIG) in culture 2 was related to the site of origin of the cells, those dividing farthest from the unwounded area had fastest MIGs. Neither culture formed more than a single layer of cells. Although the cell kinetics of cultures 1 and 2 differed, the same goal, confluence, was achieved in either case.     

Arabinogalactan proteins are involved in cell aggregation of cell suspension cultures of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L.

Arabinogalactan proteins (AGPs) are glycoproteins present at cell surfaces. Although exact functions of AGPs remain elusive, they are implicated in plant growth and development. The aim of this study was to evaluate the role of AGPs in the process of cell aggregation of Beta vulgaris L. suspension cultures. It was observed that B. vulgaris suspension cultures accumulated AGPs in parallel form to its cell growth. The AGPs maximum content in the stationary phase was 0.330 mg g⁻¹ dry weight (DW) in the cell wall (CW) and 1.534 mg g⁻¹ DW in the culture medium (CM), generating cell aggregates >500 μm (93.21% DW). The addition of tunicamycin (TM) caused a reduction of AGPs content in CW and CM of 46 and 64%, respectively. These changes were associated with inhibition of growth and the reduction of the cell aggregates >500 μm (50.0% DW). When TM was removed from the CM, cell growth, aggregation, and AGPs content on CW and CM were recovered. Precipitation of AGPs with Yariv reagent generated a reduction of 61.14% of AGPs content in CW and a total inhibition of AGPs secretion in CM. This Yariv treatment generated a reduction in the cell aggregates >500 μm of 51.31% of DW. When the Yariv reagent was removed from the culture, cells did not recover their AGPs accumulation. In addition, cell cultures did not recover their ability to grow and aggregate. These results indicate that AGPs are molecules required in the cellular aggregation process of B. vulgaris L. suspension cultures.     

Aggregation of biopharmaceuticals in human plasma and human serum

Analytical methods based on light microscopy, 90° light-scattering and surface plasmon resonance (SPR) allowed the characterization of aggregation that can occur when antibodies are mixed with human plasma. Light microscopy showed that aggregates formed when human plasma was mixed with 5% dextrose solutions of Herceptin^® (trastuzumab) or Avastin^® (bevacizumab) but not Remicade^® (infliximab). The aggregates in the plasma-Herceptin^®-5% dextrose solution were globular, size range 0.5–9 μm, with a mean diameter of 4 μm. The aggregates in the plasma-Avastin^®-5% dextrose samples had a mean size of 2 μm. No aggregation was observed when 0.9% NaCl solutions of Herceptin^®, Avastin^® and Remicade^® were mixed with human plasma. 90° light-scattering measurements showed that aggregates were still present 2.5 h after mixing Herceptin^® or Avastin^® with 5% dextrose-plasma solution. A SPR method was utilized to qualitatively describe the extent of interactions of surface-bound antibodies with undiluted human serum. Increased binding was observed in the case of Erbitux^® (cetuximab), whereas no binding was measured for Humira^® (adalimumab). The binding of sera components to 13 monoclonal antibodies was measured and correlated with known serum binding properties of the antibodies. The data presented in this paper provide analytical methods to study the intrinsic and buffer-dependent aggregation tendencies of therapeutic proteins when mixed with human plasma and serum.     

Media and culturing protocol using a full 25 factorial design for the production of submerged aggregates by the potential bio-herbicide Plectosporium alismatis against weed species of Alismataceae

The potential bio-herbicide Plectosporium alismatis produces drying and UV tolerant micro-sclerotia-like structures named aggregates, effective against the weed Alisma plantago-aquatica. In this study, we evaluated (i) optimal liquid culture conditions that supported the high dry weight, conidia and aggregate yields and (ii) stress-tolerance and effectiveness of aggregates. Using a full factorial 2⁵ design, we studied the impact of agitation (A), glucose concentration (B), Tween 80 concentration (C), malt extract concentration (D), and inoculum density (E), on P. alismatis growth yields. The inoculum density (E), the agitation rate (A), and 3-factor interactions involving E and/or A had significant effects on aggregate yields (average 213 aggregates mL^?1; main effect of E: +112.87; main effect of A: ?74.81), but no significant effect on conidial yields and dry weight. The agitation was maintained at 100?rpm and increasing numbers of conidia were inoculated into the culture media. Maximal aggregate yields (3.6?×?10³ aggregates mL^?1) were obtained with an initial conidial density of 4?×?10⁶ conidia mL^?1. While freshly-harvested, dried and/or UV exposed aggregates germinated at high rates (respectively: 100%; 99%; 76%; 85%), sporogenesis significantly decreased after stress exposure. In bioassays using leaf discs of A. plantago-aquatica, chlorosis and/or necrosis was observed after 8 days incubation, regardless of whether aggregates had been exposed to desiccation and/or UV-radiations prior to application on leaf discs. These data provide a cultural protocol for the production of high numbers of UV and drying resistant aggregates effective against weed species of Alismatacae.     

The location of cytochrome c on the surface of ultrathin lipid multilayer films using x-ray diffraction.

X-ray diffraction and spectroscopic techniques were used to characterize ultrathin fatty acid multilayers having a bound surface layer of cytochrome c. Three to six monolayers of arachidic acid were deposited onto an alkylated glass surface, using the Langmuir-Blodgett method. These fatty acid multilayer films were stored either in a 1 mM NaHCO3 pH 7.5 solution or a buffered 10 microM cytochrome c solution, pH 7.5. After washing extensively with buffer, these multilayer films were assayed for bound cytochrome c by optical spectroscopy. It was found that the cytochrome c bound only to the odd-numbered monolayer films (which have hydrophilic surfaces). The theoretical number of cytochrome c molecules bound to the ultrathin multilayer films having three or five monolayers was calculated as N = 1.2 x 10(13)/cm2 (assuming a hexagonally close-packed monolayer of protein), which would produce an optical density of 0.002 at a wavelength of 550 nm; for a three or five monolayer ultrathin film that was incubated with cytochrome c, OD550 approximately equal to 0.002. The protein was released from the film when treated with greater than 100 mM KCl solution, as would be expected for an electrostatic interaction. Meridional x-ray diffraction data were collected from the arachidic acid films with and without a bound cytochrome c layer. A box refinement technique, previously shown to be effective in deriving the profile structures of nonperiodic ultrathin films, was used to determine the multilayer electron density profiles. The electron density profiles and their autocorrelation functions showed that bound cytochrome c resulted in an additional electron dense feature on the multilayer surface, consistent with a bound cytochrome c monolayer. The position of the bound protein relative to the multilayer surface was independent of the number of fatty acid monolayers in the multilayer. Future studies will use these methods to investigate the structures of membrane protein complexes bound directly to the surface of multilayer films.     

Measurement of fresh and dry densities of suspended plant cells and estimation of their water content

A new method that considers the osmotic pressure of a culture broth for measurement of fresh cell density was developed. The water content of suspended plant cells was calculated using the value of the density obtained by the method. In the method, the optical density (O.D.) of cells and cell aggregates in a sedimentation equilibrium state in various solutions of known density and equal osmotic pressure to that of the culture broth was measured. The fresh cell density and density distribution of heterogeneous cell aggregates could be measured quantitatively. It was found that the fresh cell density of Catharanthus roseus (a dicotyledonous plant) had a relatively narrow distribution (1.010-1.028 g/cm³) with light cell aggregates; however, Oryza sativa (a monocotyledonous plant) had a wide distribution (1.030-1.064 g/cm³) with relatively heavy cell aggregates. In both cell lines, the distribution of fresh cell density at the logarithmic growth phase was more localized in the heavier density area compared with that at the stationary phase, and the small cell aggregates group had a heavier density distribution than the large cell aggregates group. The dry cell density was measured by a pycnometer method designed for plant cells using tridecane as immersion liquid. The water contents [% (v/v)] of C. roseus (92.2% at 7 d and 95.0% at 12 d) and O. sativa (82.6% at 7 d and 89.8% at 12 d) were calculated by using the values of both the fresh cell density and the dry cell density. The maximum cell concentration in suspension culture was calculated from the value of the solid content of cells on the assumption that the volume of water existing in the spaces among cells is half the total volume of the culture vessel. The result showed that the maximum cell concentration in a suspension culture of C. roseus was about half that of the O. sativa culture.     

Orientation and lateral mobility of cytochrome c on the surface of ultrathin lipid multilayer films.

We have previously shown that cytochrome c can be electrostatically bound to an ultrathin multilayer film having a negatively charged hydrophilic surface; furthermore, x-ray diffraction and absorption spectroscopy techniques indicated that the cytochrome c was bound to the surface of these ultrathin multilayer films as a molecular monolayer. The ultrathin fatty acid multilayers were formed on alkylated glass, using the Langmuir-Blodgett method. In this study, optical linear dichroism was used to determine the average orientation of the heme group within cytochrome c relative to the multilayer surface plane. The cytochrome c was either electrostatically or covalently bound to the surface of an ultrathin multilayer film. Horse heart cytochrome c was electrostatically bound to the hydrophilic surface of fatty acid multilayer films having an odd number of monolayers. Ultrathin multilayer films having an even number of monolayers would not bind cytochrome c, as expected for such hydrophobic surfaces. Yeast cytochrome c was covalently bound to the surface of a multilayer film having an even number of fatty acid monolayers plus a surface monolayer of thioethyl stearate. After washing extensively with buffer, the multilayer films with either electrostatically or covalently bound cytochrome c were analyzed for bound protein by optical absorption spectroscopy; the orientation of the cytochrome c heme was then investigated via optical linear dichroism. Polarized optical absorption spectra were measured from 450 to 600 nm at angles of 0 degrees, 30 degrees, and 45 degrees between the incident light beam and the normal to the surface plane of the multilayer. The dichroic ratio for the heme alpha-band at 550 nm as a function of incidence angle indicated that the heme of the electrostatically-bound monolayer of cytochrome c lies, on average, nearly parallel to the surface plane of the ultrathin multilayer. Similar results were obtained for the covalently-bound yeast cytochrome c. Furthermore, fluorescence recovery after photobleaching (FRAP) was used to characterize the lateral mobility of the electrostatically bound cytochrome c over the monolayer plane. The optical linear dichroism and these initial FRAP studies have indicated that cytochrome c electrostatically bound to a lipid surface maintains a well-defined orientation relative to the membrane surface while exhibiting measurable, but highly restricted, lateral motion in the plane of the surface.     

Aggregated chondrocytes as a model system to study cartilage metabolism

Differentiated chondrocytes, isolated from chick embryo cartilage, were cultured in monolayer, as aggregate or pellet. Aggregation of chondrocytes was accomplished by incubating 2 × 10⁵ cells in a 5-μl drop of culture medium. Under all three conditions, the cells remained healthy and proliferated during culture. However, matrix production, as indicated by incorporation of [³⁵S]sulphate into glycosaminoglycans, was greater in aggregated chondrocytes than in monolayers or pellets. In addition, aggregates consisting of a well defined number of cells, could easily be manipulated for experiments. Therefore aggregates provide a favourable model system to study factors modulating the metabolism of chondrocytes.     

Evidence for an interaction between canine synovial cell proteoglycans and link proteins

Link proteins are glycoproteins which stabilize aggregates of proteoglycans and hyaluronic acid in cartilage. We recently identified link proteins in canine synovial cell cultures. We now find that link proteins and proteoglycans extracted from these cells under dissociative conditions sediment in the high-buoyant-density fractions of an associative cesium chloride density gradient, suggesting that link proteins interact with high-bouyant-density proteoglycans. In gradients containing [³⁵S]sulfate-labeled synovial cell extracts, 76% of the labeled sulfate and 54% of the uronic acid is found in the high-buoyant-density fractions. Under associative conditions, Sepharose 2B elution profiles of the crude synovial cell extract, synovial cell high-buoyant-density fractions, and culture medium indicate that synovial cell proteoglycans are present in monomeric form, rather than in aggregates. Synovial cell link proteins co-elute with the [³⁵S]sulfate-labeled material under the same conditions. These proteoglycans do not interact in vitro with exogenous hyaluronic acid. Dermatan sulfate, chondroitin sulfate and heparan sulfate are the major cell-associated sulfated glycosaminoglycans synthesized by cultured canine synovial cells, while hyaluronic acid is found in the culture medium. Although the proteoglycans synthesized by cultured synovial cells interact with link proteins, these data indicate that they do not interact with hyaluronic acid to form aggregates.     

Growth and survival signalling in B16F10 melanoma cells in 3D culture

The two‐way communication between the ECM (extracellular matrix) and the cytoplasm via the integrins has many functions in cancer cells, including the suppression of apoptosis. As cells in a 3D (three‐dimensional) architecture resemble the in vivo situation more closely than do cells in more conventional 2D cultures, we have employed a substratum that prevents cell adhesion and induces cell aggregation to determine why highly metastatic B16F10 melanoma cells resist anoikis. We compared the behaviour of B16F10 cells in 2D [on tPS (tissue culture polystyrene)] and 3D culture {on polyHEMA [poly(2‐hydroxyethylmethacrylate)]} configurations. For this, we analysed cell morphology, proliferation, apoptosis and the activation status of several proteins involved in cell proliferation and survival [RhoA, FAK (focal adhesion kinase), Akt, ERK1/2 (extracellular‐signal‐regulated kinase 1/2)]. B16F10 cells in 3D architecture were able to proliferate as cell aggregates for 3 days, after which the number of cells decreased. The normal Swiss 3T3 cells used as an anoikis‐sensitive control did not proliferate on the anti‐adhesive substratum. Rho A was activated in B16F10 aggregates throughout their time in culture, whereas it was not in Swiss 3T3 aggregates. An absence of apoptotic activity was correlated with the proliferation of B16F10 cells in aggregates: caspase 3 was significantly activated only after 3 days in culture on polyHEMA. FAK and Akt were transiently activated, and their inactivation was correlated with the induction of apoptosis. ERK1/2 were activated throughout the 3D culture. No survival protein was activated in Swiss 3T3 aggregates. Data obtained from cells in 3D culture suggest that B16F10 cells are resistant to anoikis through the activation of the FAK and Akt signalling pathways.     

Directed differentiation of human mesenchymal stem cells toward a cardiomyogenic fate commitment through formation of cell aggregates

Cell morphology is known to modulate the multipotential lineage commitment of stem cells. We provide a new strategy to induce the early lineage commitment of human mesenchymal stem cells (hMSCs) toward a cardiomyogenic fate through the formation of cell aggregates. A surface-immobilized polyamidoamine dendrimer with fifth generation of dendron structure was used during the culturing of hMSCs. These hMSCs cultured on the G5 surface formed aggregates through active migration and division. More than 22% of cardiac troponin-T (cTnT)-positive (cTnT⁺) cells in aggregates formed on the dendrimer surface; the population formed on the dendrimer surface was higher than that in conventional culture vessel. When cell aggregate was reseeded onto a fresh G5 surface, single cells migrated out of the aggregates, proliferated, and formed new aggregates. This passage method, accompanied with repetitive aggregate dispersion and formation, was applied to cultures over 40 days. The proportion of cTnT⁺ cells increased to 62% by the end of third passage. Our results suggest that culturing hMSCs on G5 surface results in directed commitment of the hMSCs toward a cardiomyocyte-like fate.     

Response of articular chondrocytes to pituitary fibroblst growth factor (FGF)

Rabbit chondrocytes from pooled articular joints have been delineated by their time of attachment of culture flasks after initiation of primary monolayer culture, either attached (48-AT) or floating (48-F) after 48 hours. A general population of chrondrocytes (attached after 72 hours, 72-AT) was also studied. The growth-promoting activity of pituitary fibroblast growth factor (FGF) and its effect on sulfated-proteoglycan synthesis was studied on each chondrocyte population in secondary monolayer culture. ³H-thymidine incorporation during a 1-hour pulse was stimulated by FGF (100 ng/ml) in each chondrocyte population. The response of AT-72 chondrocytes to FGF required an additional fetal bovine serum supplement, while 48-F cells resonded independent of serum. The response of 48-AT chondrocytes to FGF (100 ng/ml) during a 1-hour pulse with ³H-thymidine was increased in low serum (0.5–2.0%) rather than when high serum (8–10%) was present in the culture medium. FGF reduced ³⁵SO₄ incorporation into sulfated-proteoglycans in the 48-AT and 48-F chondrocyte populations, but not in the 72-AT population. The reduction in ³⁵SO₄ incorporation in the 48-AT and 48-F chondrocytes was not characterized by alterations in the hydrodynamic size of the sulfated-proteoglycans as measured by Sepharose CL-2B chromatography nor by changes in the types of sulfated-glycosaminoglycans produced. These results indicated that FGF produced quantitative rather than qualitative alterations in chondrocyte sulfated-proteoglycan synthesis. The latter appears uncoupled from the growth-promoting activity of FGF on chondrocytes.