Silicone is a biomaterial that is widely used in many areas because of its high optical clarity, its durability, and the ease with which it can be cast. However, these advantages are counterbalanced by strong hydrophobicity. Gelatin cross-linking has been used as a hydrophilic coating on many biomaterials but not on silicone rubber. In this study, two gelatin glutaraldehyde (GA) cross-linking methods were used to coat a hydrophilic membrane on silicone rubber. In method I, gelatin and GA were mixed in three different proportions (64:1, 128:1, and 256:1) before coating. In method II, a newly formed 5% gelatin membrane was cross-linked with a 2.5% GA solution. All coatings were hydrophilic, as determined from the measurement of contact angle for a drop of water on the surface. Bovine coronary arterial endothelial cells were shown to grow well on the surface modified by method II at 72 h. In method I, the cells grew well for gelatin–GA proportions of 64:1 and 128:1 at 72 h. No cell attachment on untreated silicone rubber was observed by the third d of seeding. The results indicated that both methods of gelatin–GA cross-linking provided a hydrophilic surface on silicone for endothelial cell adhesion and growth in vitro.

We characterized bovine aortic endothelial cells (BAEC) continuously cultured in the rotating wall vessel (RWV) bioreactor for up to 30 d. Cultures grew as large tissue-like aggregates (containing 20 or more beads) after 30 d. These cultures appeared to be growing in multilayers around the aggregates, where single beads were covered with confluent BAEC, which displayed the typical endothelial cell (EC) morphology. The 30-d multibead aggregate cultures have a different and smoother surface when viewed under a higher-magnification scanning electron microscope. Transmission electron microscopy of these large BAEC aggregates showed that the cells were viable and formed multilayered sheets that were separated by an extracellular space containing matrix-like material. These three-dimensional cultures also were found to have a basal production of nitric oxide (NO) that was 10-fold higher for the RWV than for the Spinner flask bioreactor (SFB). The BAEC in the RWV showed increased basal NO production, which was dependent on the RWV rotation rate: 73% increase at 8 rpm, 262% increase at 15 rpm, and 500% increase at 20 rpm as compared with control SFB cultures. The addition of l-arginine to the RWV cultures resulted in a fourfold increase in NO production over untreated RWV cultures, which was completely blocked by L-NAME [N(G)-nitro-L-arginine-methylester]. Cells in the SFB responded similarly. The RWV cultures showed an increase in barrier properties with an up-regulation of tight junction protein expression. We believe that this study is the first report of a unique growth pattern for ECs, resulting in enhanced NO production and barrier properties, and it suggests that RWV provides a unique model for investigating EC biology and differentiated function.

During implantation, complex embryo–endometrium interactions result in blastocyst adhesion. To study the mechanisms of implantation, an effective assay for monitoring adhesiveness between embryos and endometrial epithelium is essential. In this study, we describe a simple and reliable method to quantify embryo–endometrium adhesion in vitro. Murine blastocysts or BeWo trophoblast spheroids were cocultured with monolayers of RL95-2 endometrial epithelial cells (EEC) grown in 96-well plates. At the end of coculture, the wells were filled with medium, and the plate was sealed with an adhesive film, inverted, and centrifuged at 25 × g for 5 min. After centrifugation, the plate was kept inverted and directly examined microscopically to determine whether the blastocysts or spheroids were attached to EEC monolayers. Our assay demonstrated that blastocysts recovered at 1200–1400 h on d 4 were more adherent to EEC than those recovered earlier, consistent with the timing of intrauterine embryo activation. Serum also enhanced blastocyst–EEC adhesion. Spheroid–EEC adhesion was inhibited by blocking Ca² influx with extracellular Ca² chelators (ethylenediaminetetraacetic acid or ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) or a Ca² channel blocker (verapamil) but not by interfering with Ca² release from intracellular stores using chelating (1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) or depleting (thapsigargin) agents. Using 96-well plates for coculture, centrifugation, and examination to minimize transfer procedures, our assay system is readily applicable to investigate implantation mechanisms.

A stem cell population isolated from murine skeletal muscle has recently been shown to differentiate into hematopoietic cells after transplantation in vivo. In the present study, we tested the hypothesis that this cell population would also, under appropriate culture conditions, differentiate into skeletal muscle cells in vitro. Lower-extremity skeletal muscle tissue isolated from 3- to 4-wk-old mice was dissected free from bone and vessels, enzymatically digested, and flow cytometrically sorted to yield CD45⁻Sca-1 c-Kit⁻ (S ) cells. These cells were further sorted into CD34 and CD34⁻ fractions and examined for skeletal, cardiac, and hematopoietic lineage–specific messenger RNA (mRNA) transcripts immediately after isolation and after a 10- to 14-d culture period. Freshly isolated S CD34 cells lacked expression of skeletal-, cardiac-, or hematopoietic-specific mRNA transcripts, whereas S CD34⁻ cells expressed c-met, a marker for skeletal muscle satellite cells. During 10–14 d in culture, both S CD34 and S CD34⁻ cell populations underwent a period of attachment followed by elongation and, ultimately, fusion to create large multinucleated contractile myotubes expressing skeletal muscle lineage mRNA transcripts but not hematopoietic or cardiac lineage transcripts. We conclude that murine skeletal muscle possesses two populations of progenitor cells that can be directly isolated. One population expressing the phenotype S CD34⁻ may contain satellite cells, whereas the S CD34 population is devoid of satellite cell markers. Both populations possess the ability to differentiate into skeletal muscle cells in vitro.

Recent studies have indicated that maternal skeletal metabolism undergoes significant changes during gestation. The agents that are responsible for eliciting these changes in bone turnover during pregnancy have yet to be defined. We therefore sought to investigate whether chaperonin 10 (Cpn10), a homolog of early-pregnancy factor, or human placental lactogen (PL) were capable of influencing the synthesis of type I collagen by human osteoblasts in vitro. Both Cpn10 and PL are major components of the maternal circulation during pregnancy, but how they might contribute to bone metabolism has not been determined. Type I collagen represents the most abundant component of bone tissue, accounting for approximately 90% of the organic compartment. Both Cpn10 and PL were capable of stimulating the synthesis of type I collagen by human osteoblasts in culture. The inclusion of 17β-estradiol or prolactin, however, failed to influence the ability of cells to mobilize type I collagen. These novel findings support a role for PL and Cpn10 in the metabolism of bone tissue during pregnancy. Maternal bone collagen metabolism is clearly an important event during pregnancy, and the identification of the factors responsible will aid our understanding of the regulation of skeletal metabolism during gestation.

Components of fetal calf serum (FCS) are known to contribute to growth and maintenance of cultured cells. Fetal calf serum supplementation of media also may contribute to the cytotoxicity of other substances to cells grown in vitro. Semicarbazide-sensitive amine oxidase (SSAO) enzyme, present in FCS, metabolizes primary amines and contributes to amine cytotoxicity in vascular smooth muscle cells (VSMC). In cell culture experiments, the media used may greatly affect enzymic activities such as SSAO. In these studies, the SSAO activity in FCS, cultured rat aortic VSMC, and rat plasma was determined in the presence and absence of various culture media. Semicarbazide-sensitive amine oxidase activity in FCS (5–20 μl) was significantly enhanced (∼1.5- to 2-fold) in the presence of various culture media, with Dulbecco modified Eagle medium (DMEM), causing the greatest enhancement. Dulbecco modified Eagle medium enhanced the SSAO activity of cultured VSMC in two of the four passages but reduced activity in two passages. Activity in rat plasma was reduced by ∼25% in the presence of DMEM. The concentrations of various media components, such as glucose, sodium pyruvate, pyridoxine·HCl, and l-glutamine, were not correlated with enhancement. This study identifies an important enhancement effect of culture media on the FCS enzyme, SSAO, although the media components responsible for the enhancement are yet to be identified.

An elevation of the intracellular levels of adenosine 3′,5′-cyclic monophosphate (cAMP) induces terminal differentiation in neuroblastoma (NB) cells in culture; however, genetic alterations during differentiation have not been fully identified. To investigate this, we used Mouse Genome U74A microarray containing ∼6000 functionally characterized genes to measure changes in gene expression in murine NB cells 30 min and 4, 24, and 72 h after treatment with cAMP-stimulating agents. Based on the time of increase in differentiated functions and their status (reversible versus irreversible) after treatment with cAMP-stimulating agents, the induction of differentiation in NB cells was divided into three distinct phases: initiation (about 4 h after treatment when no increase in differentiated functions is detectable), promotion (about 24 h after treatment when an increase in differentiated functions occurs, but they are reversible upon the removal of cAMP), and maintenance (about 72 h after treatment when differentiated functions are maximally expressed, but they are irreversible upon the removal of cAMP). Results showed that alterations in expression of genes regulating cell growth, proliferation, apoptosis, and necrosis occurred during cAMP-induced differentiation of NB cells. Genes that were upregulated during the initiation, promotion, or maintenance phase were called initiators, promoters, or maintainers of differentiation. Genes that were downregulated during the initiation, promotion, or maintenance phase were called suppressors of initiation, promotion, or maintenance phase. Genes regulating growth may act as initiators, promoters, maintainers, or suppressors of these phases. Genes regulating cell proliferation may primarily act as suppressors of promotion. Genes regulating cell cycle may behave as suppressors of initiation or promotion, whereas those regulating apoptosis and necrosis may act as initiators or suppressors of initiation or promotion. The fact that genetic signals for differentiation occurred 30 min after treatment with cAMP, whereas cell-cycle genes were downregulated at a later time, suggests that decision for NB cells to differentiate is made earlier and not at the cell-cycle stage, as commonly believed.

Ontogeny-specific differences in hematopoietic behavior may be influenced by unique adhesive interactions between hematopoietic cells and the microenvironment, such as that mediated by vascular cell adhesion molecule-1 (VCAM-1, CD 106). Although VCAM-1 is variably expressed during vertebrate development, we hypothesized that VCAM-1 expression might be linked to the enhanced capacity of the fetal liver microenvironment to support hematopoiesis. To test this we used immortalized murine stromal cell lines derived from midgestation fetal liver and adult bone marrow to compare the functional expression of VCAM-1. Molecular analysis of VCAM-1 expression was performed on stromal cell lines using Northern blot analysis, immunoprecipitation studies, and solid-phase enzyme-linked immunosorbent assay. Hematopoietic studies were performed by coculturing fetal liver cells with stromal cell lines, and the functional readout was determined by high-proliferative potential colony–forming cell (HPP-CFC) adherence assays. In contrast to our initial hypothesis, we observed greater expression of VCAM-1 messenger ribonucleic acid and protein on an adult marrow stromal cell line. In functional studies, anti–VCAM-1 antibody inhibited the binding of nearly half of the HPP-CFCs to adult marrow stroma but had a minimal effect on their binding to fetal liver stroma, despite the greater adherence of HPP-CFCs to fetal stroma. We conclude that VCAM-1 influences the hematopoietic supportive capacity of immortalized murine stroma derived from adult bone marrow. Our studies suggest that cellular interactions other than those mediated by VCAM-1 are involved in the increased adhesive capacity of immortalized murine stroma derived from fetal liver.