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PubMed1994

The primary and secondary metabolism of aflatoxin B1 by rat hepatocytes cultured on matrigel.

Rat hepatocytes on culturing rapidly lose the ability to metabolize many xenobiotics including the mycotoxin aflatoxin B1 (AFB1). Rat hepatocytes cultured for 5 days on plastic retain < 10% of the initial cytochrome P450 content compared with 34% in cells grown on matrigel alone and 74% in cells cultured on matrigel plus phenobarbital (PB). Although microsomal activation of AFB1 was preserved in matrigel cultured hepatocytes and increased by PB in all the culturing conditions examined, the AFB1 macromolecular binding capacity was reduced to < 10% of that present in the initial cultures. This lack of correlation between cytochrome P450 levels, microsomal activation, and AFB1 binding involved cytosolic glutathione-S-transferase activity (GST). This increased in matrigel cultured cells and was further enhanced by PB. Western blotting of the cytosols showed that the expression of the alpha class GST subunit Yc2 correlated with AFB1-GSH conjugate formation. By contrast, expression of the alpha class GST Ya-type subunit showed no similar close correlation. The expression of pi class GST Yf subunit, which is associated with dedifferentiation in hepatocytes, was suppressed by culturing on matrigel. The results obtained using hepatocytes cultured on matrigel indicate that despite limitations, this comprises a potentially useful model for the in vivo situation.

Aflatoxin B1↗
PubMed1992

Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components.

We have recently demonstrated the formation of interconnecting canalicular cell processes in bone cells upon contact with basement membrane components. Here we have determined whether growth factors in the reconstituted basement membrane (Matrigel) were active in influencing the cellular network formation. Various growth factors including transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), insulin-like growth factor 1, bovine fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) were identified in Matrigel. Exogenous TGF-beta blocked the cellular network formation. Conversely, addition of TGF-beta 1 neutralizing antibodies to Matrigel stimulated the cellular network formation. bFGF, EGF, and PDGF all promoted cellular migration and organization on Matrigel. Addition of bFGF to MC3T3-E1 cells grown on Matrigel overcame the inhibitory effect of TGF-beta. Some TGF-beta remained bound to type IV collagen purified from the Engelbreth-Holm-Swarm tumor matrix. These data demonstrate that reconstituted basement membrane contains growth factors which influence cellular behavior, suggesting caution in the interpretation of experiments on cellular activity related to Matrigel, collagen type IV, and possibly other extracellular matrix components.

Animals↗
PubMed2004

Dynamic regulation of gene expression by the Flt-1 kinase and Matrigel in endothelial tubulogenesis.

A nontubulogenic endothelial cell line, NP31, can be transformed by the active form of the Flt-1 kinase (BCR-FLTm1) into Tb3 cells, which show a tubulogenic property only when cultured in Matrigel. By utilizing this strict dependence of NP31 on BCR-FLTm1 and Matrigel for experimental angiogenesis, we performed microarray analyses under several conditions and found 97 genes whose dynamically regulated profiles of gene expression are divided into nine groups, in two major clusters. In one major cluster, gene expression is interdependently regulated by BCR-FLTm1 or Matrigel. The second major cluster contains genes whose expression patterns under BCR-FLTm1 influence are reversed by Matrigel. Based on these gene expression patterns in NP31 driven by BCR-FLTm1 and/or Matrigel, we propose a model in which sequential and alternate stimulation by BCR-FLTm1 and Matrigel induces cooperative regulation of subsets of genes. Microarray analyses of Tb3 under 11 different conditions revealed 5 candidate genes whose gene expression regulation is most closely associated with tubulogenesis.

Animals↗
PubMed2004

Quantification of angiogenesis by functional computed tomography in a Matrigel model in rats.

RATIONALE AND OBJECTIVES: The aim was to evaluate functional computed tomography (fCT) in the quantification of angiogenesis by comparing the tissue perfusion parameters measured by CT perfusion (CTP) software with histologic vascular parameters in a Matrigel model in rats. It was hypothesized that tissue perfusion parameters and histologic vascular parameters are related. MATERIALS AND METHODS: In vivo angiogenesis assays were performed using Matrigel supplemented with escalating doses (0 ng [control group], 250 ng, and 1,000 ng) of recombinant rat vascular endothelial growth factor (VEGF164) subcutaneously injected into the backs of Sprague Dawley rats. On day 7, rats with Matrigel plug underwent fCT following a bolus injection of iodinated contrast medium. Using CTP software, fCT parameters were generated (blood flow [BF], blood volume [BV], mean transit time, and permeability-surface area product) and functional maps on the basis of a distributed parameter tracer kinetic model, the adiabatic approximation to the tissue homogeneity model. The animals were then sacrificed. Matrigel plug was sectioned into slices corresponding to the CT scan plane and stained with CD31 immunohistochemical stain. Histologic vascular parameters, including microvascular density (MVD), vessel number (VN), vascular area, and vascular perimeter, were measured. CTP and histologic parameters were correlated. RESULTS: The Matrigel plugs with the 1,000-ng VEGF group exhibited a higher MVD than the 250-ng VEGF and control groups (P < .05). VN differed significantly between the control versus the 250-ng VEGF groups and 250-ng versus 1,000-ng VEGF groups (P < .05), with the highest VN in the 250-ng VEGF group. BF, mean transit time, and permeability-surface area product each differed significantly to VEGF levels. Changes in BF and BV did not correspond with increases in MVD or VN; however, in the 250-ng VEGF group, there was a strong positive correlation (r = 0.9) between BV and VN, vascular area, and vascular perimeter, which was not seen in the control or 1,000-ng VEGF group. All fCT parameters significantly correlated with each other (P < .05), with strong correlations between BF and mean transit time (r = -0.7) and between BF and permeability-surface area product (r = 0.7) and a weak correlation between BF and BV (r = 0.3). CONCLUSION: These results validate the VEGF-induced endothelial cell in a rat Matrigel model. In addition, histologic vascular parameter MVD does not correlate with fCT parameters measured by CTP software.

Angiogenesis Inducing Agents↗
PubMed1997

Culture with Matrigel inhibits development of mouse zygotes.

PURPOSE: It was reported that Matrigel improved hatching of mouse blastocysts produced in vitro from F1 hybrid-derived zygotes. We investigated whether Matrigel would be similarly beneficial with outbred strain-derived embryos, which exhibit a "two-cell" block similar to the developmental blocks of other species. METHODS: Mouse embryo development was assessed with or without Matrigel in KSOM medium, which supports the development of blocking strain zygotes in vitro, and in human tubal fluid (HTF) medium, which normally does not but which is used for human IVF. RESULTS: Matrigel severely inhibited the development of zygotes to blastocysts in KSOM and did not improve culture in HTF. There was no effect on development from the two-cell stage. We were not able to replicate the previous finding of Matrigel's beneficial effect on hatching of F1-derived zygotes. CONCLUSIONS: Matrigel may be a deleterious addition to embryo culture or coculture systems.

Animals↗
PubMed1994

Effects of inoculation site and Matrigel on growth and metastasis of human breast cancer cells.

The co-injection of extracellular matrix components, such as Matrigel, with human tumour cells into nude mice has been reported to facilitate tumour formation and growth, but it is unknown whether such components exert similar effects on tumour progression and metastasis. Metastatic behaviour is known to be enhanced when tumour cells are implanted orthotopically, and it is inferred that full and efficient expression of this phenotype may involve some interactions with local connective tissue matrix. It was therefore decided to investigate whether manipulation of the mesenchymal environment by co-injection of extracellular matrix components, in the form of Matrigel, with human breast cancer cells into orthotopic or ectopic sites could augment their metastatic performance, as well as their growth at the site of inoculation. Standard aliquots of 10(6) cells of the polyclonal human breast carcinoma cell line MDA-MB-435, and of four clonal cell lines, two metastatic and two non-metastatic derived from it, were injected with and without Matrigel, orthotopically or subcutaneously into nude mice. The latent period of tumour formation at the inoculation site as well as final tumour size and metastatic performance at autopsy, 140 days after inoculation, were then assessed. The prevalence of metastasis of the parent, polyclonal, cell line and of its metastatic clones was increased if the cell inoculum was mixed with Matrigel. Non-metastatic clones were not induced to become metastatic by this treatment, but local tumour growth at the site of inoculation was enhanced in all experimental groups receiving Matrigel. Orthotopic inoculation acted synergistically with Matrigel to maximise both tumour growth and metastatic behaviour. The composition of the local extracellular matrix at the site of tumour growth influenced expression of the metastatic phenotype by cells which are constitutionally capable of this behaviour, but did not induce it in ones which are not. Previous reports that local tumour growth is facilitated by enrichment of the mesenchymal matrix are confirmed. The mechanisms by which such effects are exerted are worthy of study, to ascertain whether they might be subject to clinical manipulation designed to retard tumour growth and dissemination.

Animals↗
PubMed2001

Prostate epithelial cell lines form spheroids with evidence of glandular differentiation in three-dimensional Matrigel cultures.

Normal (PNT2-C2) and metastatic (PC-3) prostate cell lines were grown in Matrigel to observe the effects on morphology and phenotype in comparison to monolayer culture. In monolayer cultures, PNT2-C2 showed typical round/cuboidal epithelial morphology, with tight cell associations, whereas in Matrigel they formed smooth spheroids, tightly packed with cells. In both monolayer and Matrigel, PNT2-C2 had a differentiated luminal epithelial phenotype with high expression of cytokeratin 8, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), E-cadherin and desmoglein. In contrast, PC-3 cells possessed an epithelial/mesenchyme morphology in monolayer with loose cell to cell contact and pseudopodial extensions. Immunohistochemical phenotyping indicated the cells were undifferentiated, expressing high levels of vimentin, beta1 integrin, CD44 and low expression of cytokeratin 8. In Matrigel they formed smooth and irregular spheroids, which had a lumen surrounded by a single cell layer. Matrigel also influenced the expression of PSA, PSMA and CD44. These results indicate that Matrigel culture can induce morphological differentiation of prostate cancer cells which initially had a basal phenotype.

Cell Differentiation↗
PubMed2005

Influence of Matrigel-overlay on constitutive and inducible expression of nine genes encoding drug-metabolizing enzymes in primary human hepatocytes.

1. Previous studies reported that rat hepatocytes overlaid with extracellular matrix components (Matrigel) maintain the expression and responsiveness of drug-metabolizing enzymes. However, whether Matrigel provides similar advantages in human hepatocytes remains largely uncertain.2. The influence of Matrigel-overlay on the constitutive and phenobarbital- and oltipraz-inducible expression of nine biotransformation enzymes, cytochrome P450s 1A1, 1A2, 2B6, 3A4, and glutathione S-transferases A1, A2, M1, T1, P1, in primary human hepatocytes was evaluated.3. Hepatocytes from five livers were maintained on a rigid collagen substratum with or without Matrigel overlay and treated for 48?h with two doses of each inducer. Quantitative RT-PCR, and for selected genes, immunoblot and enzyme activity analyses, demonstrated that human hepatocytes overlaid with Matrigel showed consistently higher constitutive and inducible expression of biotransformation genes. 4. Phenobarbital-mediated responsiveness of cytochrome P450 2B6, a potential indicator of hepatocyte differentiation status, was markedly higher in overlaid relative to non-overlaid hepatocytes. 5. It is concluded that an Matrigel overlay facilitates the maintenance and induction of xenobiotic metabolizing enzymes in primary cultures of human hepatocytes.

Adult↗
PubMed2004

Monocytes and macrophages form branched cell columns in matrigel: implications for a role in neovascularization.

Linear arrays of cells, or cell columns, have been observed in the extracellular matrix prior to neovascularization, but their nature and significance remains elusive. Based on the emerging evidence implicating a role for monocytes and macrophages (MC/MPH) in vasculogenesis, we hypothesized that MC/MPH also can form linear or branched columns, facilitating the co-migration and the spatial arrangement of other cell types. To test this hypothesis, we studied the distribution of MC/MPH effected by chemotactic migration in novel in vitro and in vivo models of development. We induced transversal and lateral migration of THP-1 monocytoid cells in Matrigel in vitro. The effect of this process on co-localization of other micro-objects was assessed using erythrocytes and micron-sized plastic beads. In vivo, we analyzed MC/MPH infiltration in subcutaneously implanted Matrigel plugs containing angiogenic factors and across a microporous filter comprising the wall of a chamber filled with Matrigel, also placed subcutaneously in mice. In vitro, we found that migrating THP-1 cells induced the lasting degradation of Matrigel and produced cell columns, a process amplified by monocyte chemoattractant protein-1 (MCP-1). We also report the co-localization of erythrocytes with THP-1 cells in cell columns. Endothelium-free tunnels containing MC/MPH, neutrophils, or erythrocytes were also observed in the Matrigel-filled chambers. In free subcutaneous Matrigel plugs, we found MC/MPH-based columns harboring isolated Tie-2+ cells (a marker of endothelial progenitor phenotype), as well as fibroblasts, dendritic cells, and adypocytes. Many of these cell columns displayed conspicuous branching. Our data demonstrate formation of branched MC/MPH cell columns in vitro and in vivo, a previously unrecognized pattern of penetration of extracellular matrices by inflammatory cells. Thus, monocytes and macrophages influence the distribution of neovessels as well as their branching points. These cells are the "architects of development," assisting organogenesis, tumorigenesis, and wound healing by patterning the tissular space.

Adipocytes↗
PubMed1991

Enhanced tumor growth of both primary and established human and murine tumor cells in athymic mice after coinjection with Matrigel.

Previously we found that the reconstituted basement membrane matrix Matrigel, when premixed with human small-cell lung carcinoma cells and injected subcutaneously into athymic mice, permitted tumor growth, whereas cells injected in the absence of Matrigel did not form tumors. In the present study, we examined additional cell types and determined some of the underlying mechanisms involved in the promotion of tumor formation by Matrigel. The tumor cell lines that we studied included transformed mouse Englebreth-Holm-Swarm tumor cells (T-EHS), human submandibular carcinoma A253 cells, mouse melanoma B16F10 cells, human epidermoid carcinoma KB cells, and human primary renal cell carcinoma cells. When coinjected subcutaneously with Matrigel, these cell lines formed rapidly proliferating tumors. Primary biopsy specimens of human colon carcinoma, when dispersed and coinjected with Matrigel, also formed tumors. Only A253, KB, and B16F10 cells formed small tumors in the absence of Martrigel, but a fivefold to tenfold increase in tumor size was observed in the presence of Matrigel. These data demonstrate a useful method for improving the growth of human tumors in athymic mice.

Animals↗
PubMed1992

Role of the SIKVAV site of laminin in promotion of angiogenesis and tumor growth: an in vivo Matrigel model.

BACKGROUND: Angiogenesis (vascularization) has a critical role in tumor growth and metastasis, and peptides containing the SIKVAV amino acid sequence (Ser-Ile-Lys-Val-Ala-Val) have been shown to stimulate many angiogenic activities in vitro. The use of model systems to identify agents that stimulate or inhibit angiogenesis may lead to the development of new antitumor strategies. PURPOSE: Our purpose was to use an in vivo murine model system to study the angiogenic activity of a synthetic peptide derived from the laminin A protein chain and containing the SIKVAV amino acid sequence. We also examined the ability of the peptide to enhance tumor growth in vivo. METHODS: The SIKVAV-containing peptide was mixed with Matrigel, a reconstituted basement membrane extract used to assay stimulation of angiogenesis. The mixture was subcutaneously injected into C57BL/6 mice. At various times after injection, the Matrigel plug was excised, and angiogenic activity was assessed by histologic examination and immunohistochemical staining with an antibody to the von Willebrand factor (vWF), an endothelium-specific antigen. In other experiments, the mixture of peptide and Matrigel was co-injected with B16F10 murine melanoma cells into C57BL/6 mice, and the resultant tumors were assessed for size and vascularization. RESULTS: When co-injected with Matrigel at doses as low as 10 micrograms, the SIKVAV-containing peptide stimulated angiogenesis fourfold greater than that seen in controls, and maximum angiogenic activity was observed 2 weeks after injection. This peptide was angiogenic in a dose-dependent manner up to a 100-micrograms dose. When co-injected with Matrigel and B16F10 melanoma cells, the peptide enhanced tumor growth by approximately 2.5-fold, and tumor vascularization was significantly increased (P = .027) over that observed after injection with melanoma cells and Matrigel alone. CONCLUSIONS: These data demonstrate that the laminin-derived SIKVAV-containing peptide is angiogenic in a new in vivo model system and can enhance tumor vascularization and growth.

Amino Acid Sequence↗
PubMed1995

Can Matrigel substitute for Vero cells in promoting the in-vitro development of mouse embryos?

The influences of Vero cells and the basement membrane substratum for these cells (Matrigel) on the rate of hatched blastocyst formation from mouse zygotes in vitro were compared. Zygotes obtained from C57BL/6 x BALB/c F1 females pretreated with pregnant mare's serum gonadotrophin/human chorionic gonadotrophin mated with BDF1 males were cultured (120 h) in human tubal fluid medium supplemented 0.5% with bovine serum albumin. The rates of early hatching and hatched blastocyst formation at 96 and 120 h of culture were expressed as the percentage of 2-cell embryos visualized after the initial 24 h. The rate of total blastocyst formation did not differ between treatment groups. However, < 10% of embryos cultured for 96 h in medium alone advanced to the hatching stage compared with 35-40% of blastocysts cultured with Vero cells or with Matrigel alone. Similarly, by 120 h of culture, only 20% of embryos cultured in medium alone developed to hatching or hatched blastocysts compared with > 70% for those embryos co-cultured with Vero cells or with Matrigel. In conclusion, Vero cells improved the rate of development of mouse embryos to hatched blastocysts during serum-free culture. Similar improvements were seen in the presence of Matrigel alone; Matrigel is the basement membrane substratum used for the Vero cells. Further studies on the means whereby Matrigel promotes early embryonic development (e.g. appropriate combination of basement membrane-associated growth factors) may lead to a safe, defined medium preparation for the stimulation of in-vitro development of human embryos.

Animals↗
PubMed2002

Matrigel-coated stents reduce intimal thickening in a large animal vascular stent model.

BACKGROUND: Restenosis within vascular stents is primarily due to intimal thickening secondary to intimal hyperplasia (IH) which occurs maximally around stent struts. Dedifferentiation of vascular smooth muscle cells (VSMC) with subsequent migration and proliferation is believed to be a key event in IH formation. Matrigel (basement membrane protein) has been shown to inhibit dedifferentiation of VSMC in vitro. Our aim was to test the in vivo effect of Matrigel on IH formation using a novel sheep vascular stent model. METHODS: Twenty vascular stents were implanted in the renal arteries of ten sheep. The left renal artery of each sheep was used to deploy uncoated stent and the right renal artery was used to deploy Matrigel-coated stent. Five sheep were analysed at four weeks and five at eight weeks after stent implantation. The sheep were sacrificed at the end of the study periods and the stented renal artery segments were examined by histology. Luminal, intimal and medial areas were determined using computer-assisted morphometric analysis. RESULTS: All stent sites were widely patent without thrombosis. No luminal stenosis was seen angiographically. IH was quantified from histology cross-sections and expressed as an intima to media (I/M) ratio. The ratio was significantly reduced in the matrigel-coated sites at eight weeks (uncoated 0.49+/-0.23; Matrigel-coated 0.32+/-0.12; p value <0.05). CONCLUSIONS: The sheep renal artery vascular stent model is feasible for the study of stent biology. IH was reduced by Matrigel-coated stents.

Animals↗
PubMed2006

In vitro culture on Matrigel favors the long-term maintenance of rat zona glomerulosa-cell differentiated phenotype.

Zona glomerulosa (ZG) cells cultured on plastic within few days dedifferentiate losing their capacity to secrete aldosterone (ALDO) in appreciable amounts. Evidence indicates that extracellular matrix modulates the secretory behavior of adrenocortical cells cultured in vitro. Hence, we compared the morphology and function of rat ZG cells grown on plastic and Matrigel basement membrane matrix (hereinafter Matrigel) for up to 12 days. At day 3, no significant differences were observed between cells cultured on plastic and Matrigel. Starting from day 6, ZG cells cultured on plastic lost their ultrastructural differentiated features (mitochondria with tubular cristae, smooth endoplasmic reticulum cisternae and lipid droplets), exhibiting a fibroblast-like appearance. The mRNA expression of the main steroidogenic enzymes, as evaluated by real-time polymerase chain reaction, the baseline secretion of ALDO and other post-pregnenolone hormones, as evaluated by high pressure liquid chromatography, and the secretory response to ACTH, angiotensin-II and K(+), as evaluated by radioimmunoassay, displayed a time-dependent decrease. Matrigel was found to maintain unchanged both the ultrastructure and the expresion of steroidogenic enzymes of ZG cells until day 12 of culture. Baseline and agonist-stimulated steroid-hormone secretion decreased with the duration of culture on Matrigel, but was always higher than that of ZG cells grown on plastic. Hence, our study clearly indicates that the culture on Matrigel favors the maintenance of rat ZG-cell differentiated phenotype, allowing the conclusion that this technique is suitable for long-term in vitro investigations.

Adrenocorticotropic Hormone↗
PubMed1995

Tumor formation by a human pre-B leukemia cell line in scid mice is enhanced by matrigel and is associated with induction of CD10 expression.

We have previously demonstrated the engraftment of human pre-B acute lymphoblastic leukemia (ALL) cells injected intravenously into irradiated scid mice. We now report on the ability of the reconstituted extracellular matrix, Matrigel, to promote the formation of subcutaneous tumors in non-irradiated scid mice by a CD10- pre-B ALL cell line termed G2. Lymphatic tumors infiltrating the dermis were seen in all eight mice sacrificed 10-13 weeks after the co-injection of G2 cells and Matrigel but in only 2/8 mice injected with leukemic cells alone. Infiltration of bone marrow, spleen, thymus, lung and liver was observed earlier and was more extensive in the Matrigel-treated group. The tumor cells derived from Matrigel-treated mice could be passaged in vitro and their colony-forming ability was higher than that of the original G2 line. When re-injected intravenously into non-irradiated scid mice, the tumor cells invaded the thymus earlier than did the G2 cells. The expression of CD10/neutral endopeptidase was induced at high levels in all tumors, in Matrigel or non Matrigel-treated animals. This up-regulation was transient as the tumor variants grown in vitro or in vivo lost expression of CD10. However, 6-8 weeks later, induction of CD10 was observed on both tumor variants and parental G2 cells growing in the thymus and at a lower level on cells in bone marrow and spleen. Culturing G2 cells in vitro at high density or in the presence of documented growth-promoting cytokines such as IL-3, IL-6, IL-7, and GM-CSF did not stimulate the expression of CD10 mRNA. The induction of this surface endopeptidase was thus associated with growth of leukemic cells in the specific microenvironments provided by the lymphoid tumors and the thymus in scid mice. The function of CD10 might be related to the hydrolysis of peptides which are critical in regulating interactions between adjacent pre-B cells, the stromal microenvironment and the transduction of growth and/or differentiation signals.

Animals↗
PubMed1994

Reorganization of endothelial cord-like structures on basement membrane complex (Matrigel): involvement of transforming growth factor beta 1.

The formation of capillary-like network structures by cultured vascular endothelial cells on reconstituted basement membrane matrix, Matrigel, models endothelial cell differentiation, the final step of angiogenesis (Kubota et al., 1988; Grant et al., 1989). When endothelial cells derived from bovine aorta and brain capillaries were plated on Matrigel, DNA synthesis was suppressed and a network of capillary-like structures rapidly formed in 8-12 h. With time, the network broke down, resulting in dense cellular cords radiating from multiple cellular clusters in 16-24 h. Finally, multicellular aggregates of cells were formed as the network underwent further retraction. Network regression was prevented when either dithiothreitol (DTT) or anti-TGF-beta 1 antibodies were added during the assay. The addition of exogenous TGF-beta 1 promoted the regression of endothelial cells into the clusters. This response to TGF-beta 1 was blocked by potent serine threonine protein kinase inhibitors, H-7 and HA100. TGF-beta 1 was released from polymerized Matrigel by incubation with Dulbecco's modified eagle's medium (DMEM) in the absence of cells. The Matrigel-conditioned DMEM inhibited endothelial DNA synthesis even in the presence of anti-TGF-beta 1 antibodies. These results suggest that TGF-beta 1 and possibly other soluble factors from Matrigel may be important for differentiation and remodeling of endothelial cells in a capillary network with possible implications for wound healing and development.

Animals↗
PubMed2002

Growth of purified lacrimal acinar cells in Matrigel raft cultures.

The objective of this study was to develop a tissue culture system which closely mimics the in situ lacrimal gland for improved study of lacrimal acinar cell physiology. Highly purified preparations of lacrimal acinar cells from adult female New Zealand White rabbits were isolated and grown in suspension culture in the form of Matrigel 'rafts', i.e., aggregates of acinar cells enclosed within a Matrigel coating. The rafts were seeded onto Matrigel-coated culture plates and their growth was followed for up to 28 days. Immunohistochemistry was used to demonstrate the cellular sites of prolactin (PRL), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), secretory component (SC) and major histocompatibility complex class-II molecules (MHC-II) within the acinar cells. By 3 days the cultures contained numerous, well-formed acini enclosed within the Matrigel. The acinar epithelial cells demonstrated histotypic polarity, with large, pale-staining, secretory granules aggregated adjacent to the lumen, and exocytotic release of secretory material into the lumen. From 5-10 days the pale-staining secretory granules decreased in number, while the lumenal contents of the acini increased in staining density. Throughout the culturing period as the pale-staining, secretory granules decreased in number, smaller more densely stained, secretory granules increased in number. The number of cells and size of acinar clusters increased steadily throughout the culturing period, and acini frequently achieved dimensions in excess of 0.5 mm. Increases in the size of acinar clusters were often accompanied by an increase in the size of the lumen. Frequently the lumen and its contents bulged asymmetrically towards one edge of the acinus. Immunhistochemistry demonstrated PRL and EGF within the lumens and within the apical cytoplasm of the acinar cells. Acini were strongly immunopositive for SC throughout the 28 day culture period, whereas immunopositivity for MHC-II molecules was strong initially, but diminished dramatically by 21 days. Immunostaining for FGF-2 was most intense on days 1 and 3, with staining throughout the cytoplasm, but became progressively more localized to the periphery of the acini as the culture period lengthened. In cultures of 1-28 days duration, Western blots of cell lysates demonstrated a major band (approximately 40 kDa) for PRL in 3-28 day preparations; a major band (approximately 80 kDa) for SC in 3 day and 7 day preparations that decreased in intensity in 14-28 day preparations; and a major band (approximately 23 kDa) for MHC-II protein in 1-21 day preparations that decreased in intensity in 28 day preparations. Lysosomes increased in number with time in culture, becoming a dominant cytoplasmic feature in 21 and 28 day cultures. Carbachol stimulation of 4 day rafts resulted in increased release of beta-hexosaminidase and SC from the rafts. The authors conclude that Matrigel rafts containing purified lacrimal gland acinar cells offer a highly advantageous system for study of lacrimal acinar cell function and one that correlates well with the in situ gland.

Animals↗
PubMed2001

The effect of a reconstituted basement membrane (matrigel) on a human salivary gland myoepithelioma cell line.

We have already demonstrated that a reconstituted basement membrane (Matrigel) is a key modulator of morphogenetic changes and cytodifferentiation of pleomorphic adenoma cells in culture. Myoepithelioma is considered to be a neoplasm closely related to pleomorphic adenoma and should experience similar induction processes. Thus, the aim of this study was to investigate whether Matrigel would influence myoepithelioma cells. We used a cell line derived from a human salivary gland plasmacytoid myoepithelioma (M1 cells) grown in a three-dimensional preparation of Matrigel. Phenotype differences were assessed using conventional light microscopy technique (haematoxylin and eosin) and phase and differential interference contrast (Nomarski). Immunofluorescence was carried out to detect smooth-muscle actin, laminin and type-IV collagen. M1 cells exhibited all proteins studied, showing a myoepithelial differentiation. M1 cells grown inside Matrigel presented morphological changes and changes in smooth-muscle actin status. By growing M1 cells inside Matrigel, it was possible to reproduce the tumour architecture with no duct-like structures. Based on our findings, we suggest that myoepithelioma would be derived from a cell with a commitment to myoepithelial differentiation. We also suggest that the mechanical properties of the matrix environment will likely regulate smooth-muscle actin expression in myoepithelioma.

Actins↗