Seeding density modulates migration and morphology of rabbit chondrocytes cultured in collagen gels

The cultures of rabbit chondrocytes embedded in collagen gels were conducted to investigate the cell behaviors and consequent architectures of cell aggregation in an early culture phase. The chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) underwent a transition to spindle-shaped morphology, and formed the loose aggregates with a starburst shape by means of possible migration and gathering. These aggregates accompanied the poor production of collagen type II, while the cells seeded at 1.6 x 10(6) cells/cm(3) exhibited active proliferation to form the dense aggregates rich in collagen type II. Stereoscopic observation was performed at 5 days to define the migrating cells in terms of a morphology-relating parameter of sphericity determined for individual cells in the gels. The frequency of migrating cells decreased with increasing seeding density, while the frequency of dividing cells showed the counter trend. The culture seeded at 1.0 x 10(5) cells/cm(3) gave the migrating cell frequency of 0.25, the value of which was 25 times higher than that at 1.6 x 10(6) cells/cm(3). In addition, the analysis of mRNA expression revealed that the chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) showed appreciable down-regulation in collagen type II relating to differentiation and up-regulation in matrix metalloproteinases relating to migration, as compared to the cells seeded at 1.6 x 10(6) cells/cm(3). These data supports the morphological analyses concerning the cell migration and aggregate formation in the cultures with varied seeding densities. It is concluded that the seeding density is an important factor to affect the cell behaviors and architecture of aggregates and thereby to modulate the quality of cultured cartilage.     

Phenotypic comparison between mesothelial and microvascular endothelial cell lineages using conventional endothelial cell markers, cytoskeletal protein markers and in vitro assays of angiogenic potential

Endothelial and mesothelial cells are mesodermally derived simple squamous epithelial cells. A controversy concerning the ontogenetic origin of neoplasms derived from these cell types, commonly cited in the literature, is whether Kaposi's sarcoma is a mesothelioma or an angioma. To assess the similarities and differences between these cell types, pulmonary microvascular endothelial cells (PMVEC) and pericardial mesothelial cells (PMC) were cultured in vitro. PMVEC and PMC were found to be difficult to distinguish from one another by histological criteria alone. Both cell types formed contact-inhibited, and 'cobblestone', monolayers typical of simple epithelial cells. PMVEC and PMC demonstrated positive immunoreactivity to Factor VIII-related antigen and angiotensin-converting enzyme (ACE) antigen. They also showed uptake of 1,1'-dioctacecyl-1,3,3,3',3-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein (DiI-Ac-LDL) in 4 h. Both PMVEC and PMC expressed low ACE activities when compared to macrovessel endothelial cells. PMVEC and PMC shared similar isoform profiles for vimentin and actin. Both cell types expressed the simple epithelial keratins, cytokeratins 8 and 19, though PMC contained 50% more cytokeratins than PMVEC. Additionally, PMC contained cytokeratin 18, an intermediate filament protein not detectable in PMVEC. PMC formed 15 times as many epithelial ringlets or "stomata" as PMVEC. PMVEC but not PMC could be induced in vitro to differentiate into branching tube-like structures in response to their culture environment. Reorganization of PMVEC into vessel-like structures was more rapid and complete than PMC when embedded in three-dimensional collagen I lattices, cultured on Matrigel or exposed to a shaped-pulsed electromagnetic field. The angiogenic response of PMVEC to specialized culture conditions in vitro may reflect their phenotypic differentiation state characterized by anastomosing vascular structures in vivo, whereas PMC remain differentiated into monolayer sheet-like structures.     

Density-related expression of caldesmon and vinculin in cultured rabbit aortic smooth muscle cells

Quantitative immunoblotting techniques were used to study the effects of seeding density on the expression of caldesmon and vinculin variants, which are sensitive markers of vascular smooth muscle cell (SMC) phenotypic modulation in culture. Rabbit aortic SMC were seeded at different densities: 13 x 10(4) cells/cm2 (high density), 3 x 10(4) cells/cm2 (medium density), and 0.2 x 10(4) cells/cm2 (low density) and cultured in the presence of 5% fetal calf serum. Irrespective of cell density and growth phase, caldesmon150 was gradually and irreversibly substituted by caldesmon77, but at high seeding density this substitution proceeded at a slower rate. The fraction of meta-vinculin (smooth muscle variant of vinculin) was reduced after seeding SMC in culture, but was reestablished when the cells reached confluency. Thus, high SMC seeding density is essential but not sufficient to keep vascular SMC cultured in the presence of serum in the contractile phenotype.     

Composition of cell-polymer cartilage implants

Cartilage implants for potential in vivo use for joint repair or reconstructive surgery can be created in vitro by growing chondrocytes on biodegradable polymer scaffolds. Implants 1 cm in diameter by 0.176 cm thick were made using isolated calf chondrocytes and polyglucolic acid (PGA). By 6 weeks, the total amount of glycosaminoglycan (GAG) and collagen (types I and II) increased to 46% of the implant dry weight; there was a corresponding decrease in the mass of PGA. Implant biochemical and histological compositions depended on initial cell density, scaffold thickness, and the methods of cell seeding and implant culture. Implants seeded at higher initial cell densities reached higher GAG contents (total and per cell), presumably due to cooperative cell-to-cell interactions. Thicker implants had lower GAG and collagen contents due to diffusional limitations.Implants that were seeded and cultured under mixed conditions grew to be thicker and more spatially uniform with respect to the distribution of cells, matrix, and remaining polymer than those seeded and/or cultured statically. Implants from mixed cultures had a 20-40-mum thick superficial zone of flat cells and collagen oriented parallel to the surface and a deep zone with perpendicular columns of cells surrounded by GAG Mixing during cell seeding and culture resulted in a more even cell distribution ad enhanced nutrient diffusion which could be related to a more favorable biomechanical environment for chondrogenesis. Cartilage with appropriate for and function for in vivo implantation ca thus be created by selectively stimulating the growth and differentiated function of chondrocytes (i.e., GAG and collagen synthesis) through optimization of the in vitro culture environment. (c) 1994 John Wiley & Sons, Inc.     

Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells

Tissue engineering may offer patients new options when replacement or repair of an organ is needed. However, most tissues will require a microvascular network to supply oxygen and nutrients. One strategy for creating a microvascular network would be promotion of vasculogenesis in situ by seeding vascular progenitor cells within the biopolymeric construct. To pursue this strategy, we isolated CD34(+)/CD133(+) endothelial progenitor cells (EPC) from human umbilical cord blood and expanded the cells ex vivo as EPC-derived endothelial cells (EC). The EPC lost expression of the stem cell marker CD133 but continued to express the endothelial markers KDR/VEGF-R2, VE-cadherin, CD31, von Willebrand factor, and E-selectin. The cells were also shown to mediate calcium-dependent adhesion of HL-60 cells, a human promyelocytic leukemia cell line, providing evidence for a proinflammatory endothelial phenotype. The EPC-derived EC maintained this endothelial phenotype when expanded in roller bottles and subsequently seeded on polyglycolic acid-poly-l-lactic acid (PGA-PLLA) scaffolds, but microvessel formation was not observed. In contrast, EPC-derived EC seeded with human smooth muscle cells formed capillary-like structures throughout the scaffold (76.5 +/- 35 microvessels/mm(2)). These results indicate that 1) EPC-derived EC can be expanded in vitro and seeded on biodegradable scaffolds with preservation of endothelial phenotype and 2) EPC-derived EC seeded with human smooth muscle cells form microvessels on porous PGA-PLLA scaffolds. These properties indicate that EPC may be well suited for creating microvascular networks within tissue-engineered constructs.     

Effect of cell-seeding density on the proliferation and gene expression profile of human umbilical vein endothelial cells within ex vivo culture

Background aimsCharacterization of endothelial cell–biomaterial interaction is crucial for the development of blood-contacting biomedical devices and implants. However, a crucial parameter that has largely been overlooked is the cell-seeding density.MethodsThis study investigated how varying cell-seeding density influences human umbilical vein endothelial cell (HUVEC) proliferation on three different substrata: gelatin, tissue culture polystyrene (TCPS) and poly-l-lactic acid (PLLA).ResultsThe fastest proliferation was seen on gelatin, followed by TCPS and PLLA, regardless of seeding density. On both TCPS and gelatin, maximal proliferation was attained at an initial seeding density of 1000 cells/cm². At seeding densities above and below 1000 cells/cm², the proliferation rate decreased sharply. On PLLA, there was a decrease in cell numbers over 7 days of culture, below a certain threshold seeding density (c. 2500–3000 cells/cm²), which meant that some of the cells were dying off rather than proliferating. Above this threshold seeding density, HUVEC displayed slow proliferation. Subsequently, quantitative real-time polymerase chain reaction (RT-qPCR) analysis of eight gene markers associated with adhesion and endothelial functionality (VEGF-A, integrin-α5, VWF, ICAM1, ICAM2, VE-cadherin, endoglin and PECAM1) was carried out on HUVEC seeded at varying densities on the three substrata. A significant downregulation of gene expression was observed at an ultralow cell-seeding density of 100 cells/cm². This was accompanied by an extremely slow proliferation rate, probably because of an acute lack of intercellular contacts and paracrine signaling.ConclusionHence, this study demonstrates that seeding density has a profound effect on the proliferation and gene expression profile of endothelial cells seeded on different biomaterial surfaces.     

High-density seeding of myocyte cells for cardiac tissue engineering

Tissue engineering of 1- to 5-mm-thick, functional constructs based on cells that cannot tolerate hypoxia for prolonged time periods (e.g., cardiac myocytes) critically depends on our ability to seed the cells at a high and spatially uniform initial density and to maintain their viability and function. We hypothesized that rapid gel-cell inoculation in conjunction with direct medium perfusion through the seeded scaffold would increase the rate, yield, viability, and uniformity of cell seeding. Two cell types were studied: neonatal rat cardiomyocytes for feasibility studies of seeding and cultivation with direct medium perfusion, and C2C12 cells (a murine myoblast cell line) for detailed seeding studies. Cells were seeded at densities corresponding to those normally present in the adult rat heart ([0.5-1] x 10(8) cells/cm(3)), into collagen sponges (13 mm x 3 mm discs), using Matrigel as a vehicle for rapid cell delivery. Scaffolds inoculated with cell-gel suspension were seeded either in perfused cartridges with alternating medium flow or in orbitally mixed Petri dishes. The effects of seeding time (1.5 or 4.5 h), initial cell number (6 or 12 million cells per scaffold), and seeding set-up (medium perfusion at 0.5 and 1.5 mL/min; orbitally mixed dishes) were investigated using a randomized three-factor factorial experimental design with two or three levels and three replicates. The seeding cell yield was consistently high (over 80%), and it appeared to be determined by the rapid gel inoculation. The decrease in cell viability was markedly lower for perfused cartridges than for orbitally mixed dishes (e.g., 8.8 +/- 0.8% and 56.3 +/- 4%, respectively, for 12 million cells at 4.5 h post-seeding). Spatially uniform cell distributions were observed in perfused constructs, whereas cells were mainly located within a thin (100-200 microm) surface layer in dish seeded constructs. Over 7 days of cultivation, medium perfusion maintained the viability and differentiated function of cardiac myocytes, and the constructs contracted synchronously in response to electrical stimulation. Direct perfusion can thus enable seeding of hypoxia-sensitive cells at physiologically high and spatially uniform initial densities and maintain cell viability and function.     

Growth and differentiation of the cultured secretory cells of the cow oviduct on reconstituted basement membrane

Isolated bovine oviduct epithelial cells were cultured on plastic precoated with matrigel. The epithelial cells seeded on 10 mg/ml matrigel often organized themselves into hollow tubes or spheres with microvilli directed towards the lumen. This is the first report of describing the spontaneous tube formation of oviduct epithelial cells in vitro. The epithelial cells growing on this substratum became fully differentiated with the formation of junctional complexes and the production of secretory vesicles which migrated to apical plasmalemma. Epithelial cells seeded on 2 mg/ml matrigel (dry film) formed a subconfluent monolayer in 15-20 days after seeding. The histoarchitecture of the secretory cells growing on the matrigel dry film closely resembled that of intact epithelial cells. Occasional ciliated cells containing large numbers of mitochondria were observed in cell colonies growing on 2 mg/ml matrigel but they possessed very few intact cilia. The monolayer linearly incorporated 35S-methionine into proteins up to 8 hr in presence of estradiol or progesterone. The fluorography of the newly synthesized proteins indicated that the cell extracts of estradiol-stimulated cells contained an additional protein of approximate molecular weight of 60 kd as compared to the extracts of cells incubated without steroids or incubated with progesterone.     

A novel matrine derivative WM622 inhibits hepatocellular carcinoma by inhibiting PI3K/AKT signaling pathways

Nasopharyngeal carcinoma (NPC) is a unique subtype of head and neck cancer, with tendency to spread to regional lymph nodes and distant organs at early stage. Vimentin, a major cytoskeletal protein constituent of the intermediate filament, plays a critical role in the epithelial–mesenchymal transition. Overexpression of vimentin is considered to be a critical prerequisite for metastasis in numerous human cancers. Therefore, targeting vimentin for cancer therapy has gained a lot of interest. In the present study, we detected vimentin expression in NPC tissues and found that overexpression of vimentin is associated with poor prognosis of NPC patients. Silencing of vimentin in NPC CNE2 cells by RNAi suppresses cells migration and invasion in vitro. However, blocking vimentin did not affect cell proliferation of CNE2 cells. In addition, the in vivo metastatic potential of CNE2 cells transfected with Vimentin shRNA was suppressed in a nude mouse model of pulmonary metastasis. Silencing of Vimentin in CNE2 cells leads to a decrease of microvessel density and VEGF, CD31, MMP2, and MMP9 expressions in pulmonary metastatic tumors. Importantly, we found that it is easier for the tumor cells from the high vimentin-expressing pulmonary metastatic tumors to reinvade the microvessel and to form stable tumor plaques attached to the endothelial cells, which resemble the resource of circulating tumor cells and are very hard to eliminate. However, depletion of vimentin inhibits the formation of vascular tumor plaques. Our findings suggest that RNAi-based vimentin silencing may be a potential and promising anti-metastatic therapeutic strategy for NPC.     

Differential control of cytokeratins and vimentin synthesis by cell- cell contact and cell spreading in cultured epithelial cells

The expression of cytokeratins and vimentin was investigated in Madin- Darby bovine epithelial cells (MDBK) in culture under conditions of varied cell spreading and cell-cell contact. When extensive cell-cell contact was achieved by seeding cells at high density in monolayer, or in suspension culture in which multicellular aggregates formed, the cells synthesized high levels of cytokeratins and low levels of vimentin. In contrast, in sparse monolayer and suspension cultures where cell-cell contact was minimal, the cells synthesized very low levels of cytokeratins. The level of vimentin synthesis was high in sparse monolayer culture and was low in both sparse and dense suspension cultures. The ratio of cytokeratin to vimentin synthesis was not affected during the cell cycle, or when cell growth was inhibited by ara C and in serum-starvation-stimulation experiments. The variations in the synthesis of cytokeratins and vimentin under the various culture conditions were also reflected at the level of mRNA activity in a cell-free in vitro translation system and as determined by RNA blot hybridization with cDNA to vimentin and cytokeratins. The results suggest that control of cytokeratin synthesis involves cell- cell contact, characteristic of epithelia in vivo, while vimentin synthesis responds to alterations in cell spreading.     

Organ and species specific differences in cytoskeletal protein profiles of cultured microvascular endothelial cells.

1. Using two-dimensional gel electrophoresis and immunoblotting techniques we systematically document the structural diversity of cytoskeletal proteins in tight and leaky cultured microvascular endothelial cells (MEC). Bovine pulmonary and eel rete mirabile MEC primarily express cytokeratins 8 and 19. Cytokeratins 8 and 18 were found to be prominent in rat pulmonary MEC. Bovine retinal MEC contained cytokeratins 8, 18 and 19. Bovine adrenal MEC contain vimentin as their sole intermediate filament protein. 2. Four principal actin isoforms were resolved in micro/macrovascular endothelial cells as well as in vascular smooth muscle cells. Retinal pericytes expressed three principal actin isoforms. 3. These results indicate that MEC are diverse, highly differentiated cells displaying a large repertoire of cytoskeletal protein profiles suited for specific tissue functions.     

小鼠角膜上皮祖细胞系TKE2的表型研究

目的：观察小鼠角膜上皮祖细胞系TKE2在扩增以及分化状态下的角蛋白及干细胞标志物的表达情况。方法小鼠角膜上皮祖细胞系TKE2在无血清培养基Keratinocyte-SFM （KSFM）以及含10﹪胎牛血清（FBS）的DMEM培养基中培养,约70﹪融合时进行角蛋白10、12、14、15、16（K10、K12、K14、K15、K16）以及Connexin43、ABCG2的免疫荧光染色,以及Ki67、P63、PCNA的免疫细胞化学染色。结果无血清培养状态下的TKE2细胞呈克隆样生长,克隆内所有细胞呈ABCG2、K14、Ki67、PCNA以及P63阳性,K15阳性细胞散在分布,K16阳性细胞呈片状分布于克隆中央区,K10、K12以及Connexin43染色为阴性。在含有10﹪胎牛血清的DMEM中培养2 d后,细胞明显增大, ABCG2、K15、P63、Ki67以及PCNA转为阴性,克隆内只有少量细胞呈K16、K14阳性染色, K10、K12、Connexin43仍为阴性。结论 TKE2细胞具有角膜上皮干细胞特性,可以作为角膜缘上皮干细胞表型维持和分化诱导研究的良好工具。     

Cytokeratin filament modulation in pulmonary microvessel endothelial cells by vasoactive agents and culture confluency.

Recently, bovine pulmonary microvascular endothelial cells (PMV) were shown to contain cytokeratin 8 and 19 intermediate filaments (Patton et al., 1990). In this study, we examine the effect of culture contiguity and vasoactive agents on the content and assembly of cytokeratins in PMV. Immunofluorescent staining of PMV cultures show a progressive increase in cytokeratin filament assembly. In freshly plated PMV, keratin appears as hazy staining (less than 4 hr) and later organizes into keratin 'plaques' (4 days) associated with cell-cell contacts; post confluent (greater than 7 days) PMV cultures contain fully assembled cytokeratin filaments which extend to the cell periphery and approach filaments in apposed cells. Vimentin filaments are also present in freshly plated PMV cultures but unlike cytokeratins, become less filamentous at confluency. This cell density-dependent modulation of cytokeratins is also demonstrated by densitometric analysis of autoradiographs of 35S-methionine labeled keratins in which PMV keratin content is elevated at high cell densities, while vimentin content remains constant. Desmoplakins I and II, components of desmosomes, could not be demonstrated in PMV by immunoblotting. PMV treated with permeability modulating agents (4 x 10(-3) M EGTA, 1 microM cytochalasin B, 1 microM bradykinin, 1 microM A23187, and 1 microM PMA) exhibit border retraction and altered keratin filament staining. From these studies we conclude: 1) cytokeratin 8 and 19 containing intermediate filaments are present in confluent PMV cultures with vimentin but without desmosomes, 2) the state of assembly of PMV cytokeratin and vimentin filaments appears to be oppositely affected by culture contiguity, and 3) treatment of monolayers with vasoactive agents alters the state of assembly of cytokeratin filaments. We speculate that modulation of cytokeratin assembly in PMV may be involved in regulation of pulmonary microvascular structure and function.     

The leukocyte-endothelial cell interactions are modulated by extracellular matrix proteins.

BACKGROUND: Endothelium is supported, in normal conditions, by a basement membrane composed, among others, by collagen IV and laminin. Changes in the basement membrane composition could induce changes in endothelial cell modifying their interactions with leukocytes. METHODS AND RESULTS: Isolated polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC) were added to cultured human umbilical endothelial cells (HuVEC) previously seeded on collagen IV, collagen I or gelatin. Adhesion of leukocytes to HUVEC and specific cytotoxicity were analysed. PMN adhesion and cytotoxicity were lower whereas those from PBMC were higher when HuVEC were seeded on collagen I, as compared with cells seeded on collagen IV. To analyse the mechanisms involved in these phenomena, P-selectin, ICAM-1, VCAM-1 and MCP- 1 expression were evaluated in HuVEC seeded on the different ECM components. P-selectin and mRNA expression of VCAM-1 were lower in cells seeded on collagen I. By contrast, MCP-1 expression was higher in collagen I. Collagen I-dependent effects were partially prevented when collagen I was treated with pepsin. ILK activity was lower in cells seeded on collagen I, whereas ERK 1/2 activity was enhanced. ILK overexpression reduced ERK 1/2 phosphorylation and this could promote the reduction in P-selectin and the increase in MCP-1. CONCLUSION: Collagen I decreased ILK activity and this would induce an increase in ERK 1/2 activity in HuVEC. As a consequence, the P-selectin content is diminished and, by contrast, the MCP-1 content is increased. The final effect is a lower recruitment of PMN and a higher adhesion of PBMC.     

Identification of Cell Types in the Developing Goat Mammary Gland

The goat was chosen as the model system for investigating mammary gland development in the ruminant. Histological and immunocytochemical staining of goat mammary tissue at key stages of development was performed to characterize the histogenesis of the ruminant mammary gland. The mammary gland of the virgin adult goat consisted of a ductal system terminating in lobules of ductules. Lobuloalveolar development of ductules occurred during pregnancy and lactation which was followed by the regression of secretory alveoli at involution. The ductal system was separated from the surrounding stroma by a basement membrane which was defined by antisera raised against laminin and Type IV collagen. Vimentin, smooth-muscle actin and myosin monoclonal antisera as well as antisera to cytokeratin 18 and multiple cytokeratins stained a layer of myoepithelial cells which surround the ductal epithelium. Staining of luminal epithelial cells by monoclonal antibodies to cytokeratins was dependent on their location along the ductal system, from intense staining in ducts to variable staining in ductules. The staining of epithelial cells by monoclonals to cytokeratins also varied according to the developmental status of the goat, being maximal in virgin and involuting glands, lowest at lactation and intermediate during gestation. In addition, cuboidal cells, situated perpendicular to myoepithelial cells and adjacent to alveolar cells in secretory alveoli, were also stained by cytokeratin monoclonal antibodies and antisera to the receptor protein, erbB-2, in similar fashion to luminal epithelial cells. These results demonstrate that caprine mammary epithelial cell differentiation along the alveolar pathway is associated with the loss of certain types of cytokeratins and that undifferentiated and secretory alveolar epithelial cells are present within lactating goat mammary alveoli.     

Characterization of spatial growth and distribution of chondrocyte cells embedded in collagen gels through a stereoscopic cell imaging system

The stereoscopic image analysis of fluorescence-labeled chondrocyte cells for cytoplasm and nucleus was performed for the quantitative determination of spatial cell distribution as well as cell aggregate size in the collagen-embedded culture. The three-dimensional histomorphometric data indicated that the cells in the gels formed aggregates by cell division, and the size of aggregates increased with elapsed culture time. In the culture seeded at 2.0 x 10(6) cells/cm(3), the cells showed a semilunar shape that is a typical chondrocytic morphology, and formed the dense cell aggregates producing collagen type II. From the quantitative analysis of aggregate size, in addition, it was found that the cell division caused the aggregate growth with an increase of cell number in respective aggregates at 7 days, and some of aggregates made coalescence at 14 days. In the gel surface region, further coalescence of aggregates accompanied with cell division produced larger cell clusters, creating cell layers on the gel surface at the end of culture (21 days). In the culture seeded at 2.0 x 10(5) cells/cm(3), the different manner of aggregation was observed. At 14 days, the loose clusters of spindle-shaped cells emerged in the deeper region of gels, suggesting that the cell migration and gathering occurred in the gels. This loose-clustered aggregates did not produce collagen type II. Our results suggest that the seeding density is a factor to cause different mechanisms of cell distribution accompanied with the formation of aggregates as well as collagen type II.     

Differential mitogenic responses of human macrovascular and microvascular endothelial cells to cytokines underline their phenotypic heterogeneity

A variety of growth factors promote the complex multistep process of angiogenesis. The mitogenic activity of vascular endothelial growth factors (VEGFs) and placental growth factors (PlGFs), known as cytokines acting predominantly on endothelial cells, was tested on human umbilical vein endothelial cells (HUVEC) and microvascular endothelial cells (MIEC) and compared with the potency of the universally acting basic fibroblast growth factor (FGF-2). The cells were seeded at different cell numbers and incubated with various doses of growth factors for a period of 24-72 h in culture medium +/- serum. Proliferation was determined by measuring the optical density after staining the cells with the tetrazolium salt WST-1. VEGF121 and VEGF165 increased the number of HUVEC and MIEC at low and high seeding densities various doses and incubation times. The efficiency of FGF-2 was less pronounced at high seeding densities of the cells under serum-free conditions. PlGF-1 and PlGF-2 stimulated mitogenesis on HUVEC only at low cell numbers and after a short incubation time by 125 +/- 3% and 102 +/- 5% (P < 0.001), respectively. Longer incubation times with the lower seeding density in the absence of FCS did not induce a significant stimulatory effect of the PlGFs. MIEC responded stronger to all growth factors. In particular under serum free conditions, PlGF-1 and PlGF-2 effectively stimulated cell proliferation by 247 +/- 54% (P < 0.01) and 288 +/- 40% (P < 0.05) at low cell numbers, and by 81 +/- 13% (P < 0.05) and 49 +/- 13% (P < 0.01), respectively, at high cell numbers. The addition of fetal calf serum caused a reduced proliferative response of all growth factors on both cell types related to the controls. In conclusion, MIEC and HUVEC differ in their proliferative response to VEGFs, PlGFs and FGF-2.     

Hepatocyte behavior within three-dimensional porous alginate scaffolds

A potential approach to facilitate the performance of implanted hepatocytes is to enable their aggregation and re-expression of their differentiated function prior to implantation. Here we examined the behavior of freshly isolated rat adult hepatocytes seeded within a novel three-dimensional (3-D) scaffold based on alginate. The attractive features of this scaffold include a highly porous structure (sponge-like) with interconnecting pores, and pore sizes with diameters of 100-150 microm. Due to their hydrophilic nature, seeding hepatocytes onto the alginate sponges was efficient. DNA measurements showed that the total cell number within the sponges did not change over 2 weeks, indicating that hepatocytes do not proliferate under these culture conditions. Nearly all seeded cells maintained viability, according to the MTT assay. Within 24 h post-seeding, small clusters of viable cells, were seen scattered within the sponge. More than 90% of the seeded cells participated in the aggregation; the high efficiency is attributed to the non-adherent nature of alginate. The spheroids had smooth boundaries and by day 4 in culture reached an average diameter of 100 microm, which is at the same magnitude of the sponge pore size. The cells appeared to synthesize fibronectin which was deposited on the spheroids. No laminin or collagen type IV were detected in the deposit. The 3-D arrangement of hepatocytes within the alginate sponges promoted their functional expression; within a week the cells secreted the maximal albumin secretion rate of 60 microg albumin/10(6) cells/day. Urea secretion rate did not depend on cell aggregation and was similar to that obtained when hepatocytes were cultured on collagen type I coated dishes (100 microg/10(6) cells/day). Our studies show that alginate sponges can provide a conducive environment to facilitate the performance of cultured hepatocytes by enhancing their aggregation.