Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Matrigel”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed2005

Comparison of Matrigel and gelatin substrata for feeder-free culture of undifferentiated mouse embryonic stem cells for toxicity testing.

Murine embryonic stem (mES) cells have been used to evaluate cytotoxicity and developmental injury following exposure to embryotoxic agents. However, maintaining a homogeneous population of undifferentiated mES cells for this purpose has been complicated by the need for continuous co-culture with murine embryonic fibroblast (mEF) cells or limited passaging on plastic surfaces coated with gelatin. Here, we compare the synthetic basement membrane Matrigel with 0.1% gelatin substratum for feeder-free propagation of undifferentiated mES cells. Biomarkers of pluripotentiality, chromosome number, caspase-3 expression, and cardiomyocyte differentiation were monitored for mES cells cultured on Matrigel or 0.1% gelatin up to passage 7 (P7). Our results suggest that choice of substratum had no significant effect on population doubling time, cell viability, stage-specific embryonic antigen-1 (SSEA-1) expression, or early passage formation of beating cardiomyocytes (all P>or=0.09). In other comparisons, however, Matrigel supported significantly higher synthesis of alkaline phosphatase (7.7x10(-3)+/-0.8 vs 6.6x10(-3)+/-0.8 units/liter/cell, respectively, P=0.012), overall expression of activated caspase-3 following exposure to 5, 10, 50, 100 and 500 parts per billion (ppb) sodium arsenite (P<0.0001), and percent development to beating cardiomyocytes at P7 (P=0.01). Together, our findings suggest that Matrigel shows promise as a substrate for feeder-free propagation of undifferentiated mES cells for embryotoxicity endpoints.

Alkaline Phosphatase↗
PubMed1993

Enhancement of tumorigenicity of human breast adenocarcinoma cells in nude mice by matrigel and fibroblasts.

The failure of MCF7 cells to induce the formation of tumours after sub-cutaneous inoculation into athymic nude mice can be obviated by the simultaneous injection of an extract of basement membrane proteins (matrigel). Tumour growth is promoted and the latency period is low (2 to 4 weeks). In the absence of matrigel, the simultaneous inoculation of fibroblasts and MCF7 cells also resulted in the development of tumours, but with a longer latency period (about 2 months). The tumorigenic synergy between matrigel and fibroblasts was evidenced by co-inoculating MCF7 cells MDA-MB 231 cells with fibroblasts and matrigel. This co-inoculation decreased the delay of appearance of the tumours and/or accelerated the tumour growth, depending upon the number of fibroblasts injected. Repeated injections of fibroblasts conditioned medium, at the site of inoculum of tumour cells also enhanced tumour growth, suggesting the involvement of soluble factors secreted by fibroblasts. Histologically, tumours induced by co-inoculation of tumour cells and fibroblasts contained more stromal structures including vimentin-positive cells, fibronectin and interstitial collagens. These data suggest that human tumours may be reconstituted and grown in athymic nude mice using basement membrane components and fibroblasts as inductors.

Adenocarcinoma↗
PubMed1998

Frozen extracellular matrix preserves its thrombogenicity after thawing, while Matrigel induces a poor platelet response.

Two putative substitutes for fresh endothelial cell (EC) extracellular matrix (ECM), frozen ECM and Matrigel, were studied using a parallel-plate perfusion system and platelet deposition was evaluated morphometrically. Coverslips covered with ECM were stored frozen at-30 C for 0 (fresh ECM), 1, 2, 3 or 4 weeks. The ability of frozen ECM to support platelet adhesion after freezing was analyzed under three experimental approaches, perfusing blood: (i) at different shear rates; (ii) on a highly reactive ECM obtained from stimulated EC; and (iii) on ECM incubated with a monoclonal antibody against laminin (LM). Matrigel, alone or mixed with different fractions of wet cryoprecipitate, was layered on coverslips as a thin uniform coat, and perfused, as was done with the frozen ECM. Platelet deposition onto fresh ECM was 21.3 1.5%, 25.5 2.1% and 30.8 2.4% (at shear rates of 300, 800 and 1300/s, respectively) and 40.0 3.8% in PMA stimulated ECM perfused at 800/s. Values obtained on frozen ECM did not vary from those obtained using fresh ECM. Results from perfusion studies using ECM preincubated with an anti-laminin antibody and observations from immunofluorescence studies indicated that the presence and distribution of the adhesive proteins in frozen ECM were similar to those observed on fresh ECM. Platelet deposition on Matrigel was practically absent. Addition of a 20% cryoprecipitate fraction partially restored its thrombogenicity. Our results indicate that when ECM is kept frozen for up to 4 weeks, it behaves as fresh ECM in perfusion studies. On the contrary, Matrigel is not a suitable substrate to support platelet attachment under flow conditions.

Journal Article↗
PubMed1997

Epithelial-stromal interactions modulating penetration of matrigel membranes by HPV 16-immortalized keratinocytes.

The role of epithelial-stromal interactions in the progression of human papillomavirus-associated squamous intraepithelial lesions to invasive cervical cancer is poorly understood. Using the Matrigel artificial basement membrane assay as a model of keratinocyte invasion, the effects of selected growth factors on penetration of human papillomavirus 16-immortalized keratinocytes through Matrigel were studied. Also studied in this model were the effects of conditioned media from fibroblast lines derived from normal cervical tissues (normal fibroblasts) and adjacent cervical cancer biopsies (tumor-associated fibroblasts) and from primary keratinocytes. Addition of basic fibroblast growth factor, transforming growth factor-alpha, and hepatocyte growth factor/scatter factor or conditioned media from tumor-associated fibroblasts to the Matrigel resulted in near-doubling of penetration of human papillomavirus 16-immortalized keratinocytes, whereas transforming growth factor-beta, platelet derived growth factor-B, or conditioned media from primary keratinocytes decreased penetration 10-fold. Antibodies to basic fibroblast growth factor abrogated the stimulatory effects of conditioned media from tumor-associated fibroblasts on keratinocyte penetration, whereas antibodies to transforming growth factor-beta abrogated the inhibitory effects of conditioned media from normal fibroblasts on keratinocyte penetration. S1 nuclease protection and enzyme-linked immunosorbent assay showed increased expression of transforming growth factor-beta and decreased expression of basic fibroblast growth factor in normal compared with tumor-associated fibroblasts. Messenger RNA in situ hybridization of five cervical cancer biopsies demonstrated basic fibroblast growth factor expression in stromal cells surrounding nests of invading keratinocytes. Epithelial-stromal interactions mediated by growth factors such as transforming growth factor-beta and basic fibroblast growth factor modulate penetration of human papillomavirus 16-immortalized keratinocytes through Matrigel in vitro and these interactions may also be operative in vivo.

Basement Membrane↗
PubMed2006

Growth of ameloblast-lineage cells in a three-dimensional Matrigel environment.

Enamel organ epithelial cells grow in culture as two distinct cell populations--either stellate-shaped or polygonal-shaped cells. The polygonal cells have an ameloblast cell phenotype and are difficult to grow in culture beyond two passages. This study was designed to determine the effects of a Matrigel three-dimensional (3D) environment on polygonal cells, as compared with stellate cells, derived from porcine tooth enamel organ. Enamel organs were dissected free from the unerupted molars of 30-kg pigs and then grown in LCH-8e media, either with or without serum. Cells grown in serum-free media were primarily polygonal shaped, whereas cells grown in media containing serum were stellate shaped. Both types of cells were grown in a 3D Matrigel matrix. In addition, polygonal-shaped cells were mixed with hydroxyapatite powder and transplanted subcutaneously into nude mice. Polygonal-shaped epithelial cells formed cell groups, similar to epithelial pearls, both in vitro and in vivo. The stellate-shaped cells, in contrast, did not form similar structures, but remained suspended in the Matrigel and gradually disappeared from the culture. These results suggest that a Matrigel environment, rich in basement membrane and matrix proteins, selects for polygonal-shaped ameloblast-lineage cells and induces the formation of epithelial pearls.

Ameloblasts↗
PubMed1996

Whole cell potassium currents in fetal rat alveolar type II cells cultured on Matrigel matrix.

Patch-clamp studies were performed on fetal rat alveolar type II cells isolated at 19 days of gestation and cultured on either plastic for 7 days or Matrigel matrix (40-50 microliters/cm2) for 10 days. Before study, cells cultured on Matrigel matrix were dissociated from alveolar-like structures with enzymes, replated, and washed with cold buffer at a constant flow rate to remove residual gel. This wash significantly improved obtaining of successful seals. Potassium current-voltage relationships and maximum whole cell K+ conductance (normalized to membrane capacitance) were significantly changed with time in cells cultured on plastic, but no significant change occurred in cells cultured on Matrigel matrix. Application of 20 mM tetraet hyl ammonium, 2mM 4-aminopyridine, and 5mM BaCl2 significantly inhibited K+ currents, showing differences in channel sensitivity to these agents and a voltage-dependent blockage between culture groups or with time in culture. To conclude, we have developed a new method by which epithelial cells cultured on Matrigel matrix can be successfully studied with the use of patch-clamp techniques. Furthermore, these studies show that fetal type II cells have voltage-activated K+ channels and that channel density and their sensitivity to channel blockers are modulated by the substratum on which the cells are cultured.

Animals↗
PubMed1997

Cell cycle in alveolar epithelial type II cells: integration of Matrigel and KGF.

The regulation of cell cycle control in alveolar epithelial type II cells (AEC2) in response to peptide growth factors and extracellular matrix signals is not well understood. Herein, we have determined that, in adult rat AEC2 in primary culture on Engelbreth-Holm-Swarm biomatrix (Matrigel) in the presence of keratinocyte growth factor, the expression of key cell cycle control elements, including cyclins A and D and cyclin-dependent kinases (cdk) 1 and 4, is increased and that retinoblastoma protein (pRb) phosphorylation is also increased, with a corresponding decrease in the expression of p53 and the cdk inhibitors (cdkis) p21WAF1/CIP1 and p27KIP-1 compared with cells cultured on plastic. The Matrigel biomatrix-KGF culture conditions were also associated with an enhanced proliferative response, as measured by fluorescent-activated cell sorter analysis, thymidine incorporation into DNA, and proliferating cell nuclear antigen expression. This enhanced proliferation occurred with neither a soluble extract of Matrigel biomatrix nor with other simple biological matrices. We conclude that coordinated induction of key cyclins and cdks, with the concomitant suppression of key negative cell cycle regulators, occurs in AEC2 on Matrigel biomatrix in the presence of KGF. We speculate that the balance between cyclin and cdk activation and cdki suppression in AEC2 serves to integrate the combined influences of biomatrix and KGF signaling on pRb phosphorylation, thereby controlling transit through S phase of the cell cycle. Conversely, AEC2 express high levels of cdkis and p53 at rest in G1 phase. The latter finding may explain the quiescent state of normal adult AEC2 in vivo.

Animals↗
PubMed2005

Epidermal growth factor mediates detachment from and invasion through collagen I and Matrigel in Capan-1 pancreatic cancer cells.

BACKGROUND: Pancreatic adenocarcinoma is a highly invasive neoplasm. Epidermal growth factor (EGF) and its receptor are over expressed in pancreatic cancer, and expression correlates with invasion and metastasis. We hypothesized that EGF receptor and integrin signalling pathways interact in mediating cellular adhesion and invasion in pancreatic cancer, and that invasiveness correlates temporally with detachment from extracellular matrix. METHODS: We tested this hypothesis by investigating the role of EGF in mediating adhesion to and invasion through collagen I and Matrigel in the metastatic pancreatic adenocarcinoma cell line Capan-1. Adhesion and invasion were measured using in vitro assays of fluorescently-labeled cells. Adhesion and invasion assays were also performed in the primary pancreatic adenocarcinoma cell line MIA PaCa-2. RESULTS: EGF inhibited adhesion to collagen I and Matrigel in Capan-1 cells. The loss of adhesion was reversed by AG825, an inhibitor of erbB2 receptor signalling and by wortmannin, a PI3K inhibitor, but not by the protein synthesis inhibitor cycloheximide. EGF stimulated invasion through collagen I and Matrigel at concentrations and time courses similar to those mediating detachment from these extracellular matrix components. Adhesion to collagen I was different in MIA PaCa-2 cells, with no significant change elicited following EGF treatment, whereas treatment with the EGF family member heregulin-alpha elicited a marked increase in adhesion. Invasion through Matrigel in response to EGF, however, was similar to that observed in Capan-1 cells. CONCLUSION: An inverse relationship exists between adhesion and invasion capabilities in Capan-1 cells but not in MIA PaCa-2 cells. EGF receptor signalling involving the erbB2 and PI3K pathways plays a role in mediating these events in Capan-1 cells.

Biocompatible Materials↗
PubMed2001

Lysophosphatidic acid receptor-selective effects on Jurkat T cell migration through a Matrigel model basement membrane.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) from platelets and mononuclear phagocytes mediate T cell functions through endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) specific for LPA (Edg-2, -4, and -7) or S1P (Edg-1, -3, -5, -6, and -8). Jurkat leukemic T cells with the SV40 virus large T Ag (Jurkat-T cells) express Edg-3>-2>-4 Rs, as assessed by RT-semiquantitative PCR and Western blots with anti-Edg R mAbs. Jurkat-T cells expressing predominantly Edg-2 R (Jurkat-T-2 cells) and Edg-4 R (Jurkat-T-4 cells) were developed by cotransfection with the respective sense plasmids and a mixture of antisense plasmids for the other Edg Rs, and hygromycin selection. Migration of Jurkat-T-4 cells, but not Jurkat-T-2 cells, through a layer of Matrigel on a 5-um pore polycarbonate filter was stimulated up to 5-fold by 10(-9) to 10(-6) M LPA and by 30-300 ng/ml of anti-Edg-4 R Ab, but not anti-Edg-2 R Ab. LPA and anti-Edg-4 R Ab also enhanced by up to 4-fold the expression of matrix metalloproteinase by Jurkat-T-4 cells, but not Jurkat-T-2 cells, as assessed by cleavage of [(3)H]-type IV human collagen in the Matrigel. Enhancement of matrix metalloproteinase-dependent trans-Matrigel migration of Jurkat-T cells by the chemokine RANTES was suppressed by anti-Edg-2 R Abs, but was stimulated by anti-Edg-4 R Abs. The opposite effects of Edg-2 and Edg-4 LPA receptors on trans-Matrigel migration and some other T cell functions provide receptor-selective mechanisms for regulation of T cell recruitment and immune contributions.

Antibodies, Monoclonal↗
PubMed1993

Identification of the 72-kDa (MMP-2) and 92-kDa (MMP-9) gelatinase/type IV collagenase in preparations of laminin and Matrigel.

EDTA inhibitable type IV collagenolytic activity copurified with laminin preparations from the Engelbreth-Holm-Swarm (EHS) tumor. Several gelatinolytic and type IV collagenolytic matrix metalloproteinase (MMP) species were visualized in EHS laminin from three different sources by gelatin and type IV collagen substrate gel electrophoresis. Incubation with 4-aminophenylmercuric acetate and trypsin suggested that laminin contained both active and latent MMPs. EHS-derived reconstituted basement membrane, Matrigel, was found to possess an MMP profile identical to that of laminin. The presence of 72-kDa (MMP-2) and 92-kDa (MMP-9) gelatinases/type IV collagenases was demonstrated in laminin and Matrigel preparations by Western blot analysis. A rough quantitation of MMP-2 and MMP-9 in 30 micrograms of laminin and 100 micrograms of Matrigel was between 0.3 and 0.6 ng. The presence of these contaminants must be considered in experiments addressing the effects of EHS laminin or Matrigel on cell behavior and, in particular, stimulation of cellular proteolytic activity.

Animals↗
PubMedDate not supplied

Tumor cell invasion through matrigel is regulated by activated matrix metalloproteinase-2.

We tested the hypothesis that there is a correlation between tumor cell efficiency in activation of matrix metalloproteinase-2 (MMP-2) and invasion through basement membrane-like Matrigel barriers. To generate cells capable of MMP-2 activation, we stably transfected three human tumor cell lines, HT-1080 fibrosarcoma, MCF7 breast carcinoma, and U251.3 glioma with cDNA encoding the full length human membrane-type matrix metalloproteinase-1. Our results show a bimodal correlation between the extent of MMP-2 activation and Matrigel invasion by tumor cells. Cell transfectants characterized by a partial activation of MMP-2 were the most invasive while those with an extensive conversion of MMP-2 proenzyme into enzymatically active forms were the least efficient in invading Matrigel. Modulation of MMP-2 activation by exogenous TIMP-2 reverted the rate of Matrigel invasion by cell transfectants to control levels. We conclude that the regulation of activated MMP-2 in the tumor cells, microenvironment may be critical in facilitating tumor cell invasiveness.

Collagen↗
PubMed1998

Reduced viability of human vascular endothelial cells cultured on Matrigel.

Optimal vascular homeostasis requires efficient control of both proliferation and elimination of vascular endothelial cells. Programmed cell death, or apoptosis, is the main mechanism controlling cell elimination, and it is an essential component of vascular formation. Human vascular endothelial cells die in vitro, if prevented from obligatory survival factors like growth factors or attachment and cell spreading, but very little is known about the mechanisms controlling endothelial cell elimination. Signaling from the extracellular matrix affects the behavior and functions of human umbilical vein endothelial cells (HUVECs), and we have recently demonstrated the beneficial effects of plating on the reconstituted extracellular matrix Matrigel, on the inducible nitric oxide production of freshly isolated HUVECs. In this work we observed that cultured HUVECs formed typical capillary-like structures on Matrigel, but unexpectedly, after 24-48 hours their viability was gradually lost. Viability was measured with an assay based on mitochondrial reduction of reagent XTT. No decrease in viability was seen in freshly isolated HUVECs or in cultured fibroblasts during this time. It is known that cells often turn into apoptosis if they receive conflicting information from their surroundings, and apparently signaling from Matrigel to HUVECs, while at their in vitro proliferating phenotype, resulted in launching of the apoptotic machinery. Thus, proliferating and differentiated phenotypes of endothelial cells seemed to have different sensitivity to signals that induce apoptosis.

Apoptosis↗
PubMed1995

Culture of marmoset blastocysts on matrigel: a model of differentiation during the implantation period.

BACKGROUND: The objective was to develop an experimental model for studying the differentiation of trophoblast and inner cell mass (ICM) during the early stages of implantation in primates. METHODS: Marmoset monkey blastocytes were used in these studies. Ovulation was timed by plasma progesterone assays in ovarian cycles initiated by administering a luteolytic agent to mating marmosets. Embryos were recovered from the uterus usually at the eight-cell stage and cultured in minimum essential medium containing fetal calf serum, insulin, and transferrin. The embryos that formed hatched blastocysts by about day 11 after ovulation were transferred for further development in Matrigel-coated culture chambers. After 2, 4, and 6 days of development, two blastocysts were processed at each interval and serially sectioned for light and electron microscopy. RESULTS: All blastocysts adhered to the Matrigel at their embryonic pole within 24 hours. Adherent polar cytotrophoblast was differentiating to syncytiotrophoblast at all time intervals, but syncytium was not detected in mural trophoblast until day 4 after attachment. By day 2 syncytial microvilli and processes had penetrated the Matrigel surface, whereas by days 4 and 6 cytotrophoblast that was differentiating to syncytiotrophoblast had invaded the matrix. Since all blastocysts maintained their structural integrity progressive differentiation of the ICM, endoderm and presumptive mesoderm was observed. A small amniotic cavity was observed at 2 days and by 6 days a distinct cavity separated polarized epiblast and amnion cells. Visceral and parietal endoderm were present at 2 days, and a completed primary yolk sac was observed by 4 days after attachment. In all blastocysts a basal lamina lined the inner surface of mural and polar trophoblast and the basal surface of the differentiating ICM. CONCLUSIONS: The developmental time sequence of the cultured blastocysts closely resembled the time frame reported for marmoset embryos implanting in utero. An effective model for studying trophoblast invasion and differentiation of embryonic germ cell layers has been established.

Amnion↗
PubMed2006

Alginate hydrogel and matrigel as potential cell carriers for neurotransplantation.

Development of biosynthetic conduits carrying extracellular matrix molecules and cell lines expressing neurotrophic growth factors represents a novel and promising strategy for spinal cord and peripheral nerve repair. In the present in vitro study, the compatibility and growth-promoting effects of (i) alginate hydrogel, (ii) alginate hydrogel complemented with fibronectin, and (iii) matrigel were compared between olfactory ensheathing cells (OECs), Schwann cells (SCs), and bone marrow stromal cells (BMSCs). Neurite outgrowth from embryonic dorsal root ganglia (DRG) neurons was used to assess the efficacy of the hydrogels alone or in combination with cultured cells to promote axonal regeneration. The result showed that alginate hydrogel transformed OECs, SCs, and BMSCs into atypical cells with spherical shape and inhibited their metabolic activity. Combination of alginate hydrogel with fibronectin promoted only OECs proliferation. Alginate hydrogel also inhibited outgrowth of DRG neurites, although this effect was attenuated by addition of fibronectin, SCs, or BMSCs. In contrast, matrigel stimulated cell proliferation, preserved the typical morphological features of the cultured cells and induced massive sprouting of DRG neurites. Addition of cultured cells to matrigel did not further improve DRG neurite outgrowth. The present findings suggest that addition of extracellular matrix should be considered when engineering biosynthetic scaffolds on the basis of alginate hydrogels.

Alginates↗
PubMed1993

Primary culture of flow cytometry-sorted rat mammary epithelial cell (RMEC) subpopulations in a reconstituted basement membrane, Matrigel.

We studied the growth of rat mammary epithelial cell (RMEC) subpopulations cultured in Matrigel. Mammary glands were removed, minced, and enzymatically dispersed. Mammary organoids (ductal and endbud fragments) were collected, enzymatically monodispersed, filtered, and labeled with fluorescein isothiocyanate-peanut agglutinin (PNA) and phycoerythrin-anti-Thy-1.1 monoclonal antibody. By flow cytometry, we separated four RMEC subpopulations: cells negative to both reagents (B-), PNA-positive cells (PNA+), Thy-1.1-positive cells (Thy-1.1+), and cells positive to both reagents (B+). The ultrastructural characteristics of sorted cells were examined by transmission electron microscopy. Thy-1.1+ cells appeared to be myoepithelial cells with actin filaments. PNA+, B-, and B+ cells were morphologically similar. Sorted RMEC subpopulations, unsorted mixed cells, and undispersed organoids were seeded in Matrigel and cultured in either a complete hormone medium (CHM) with 10% fetal bovine serum, progesterone, estradiol, hydrocortisone, insulin, and prolactin or a serum-free medium containing EGF, insulin, hydrocortisone, and human transferrin. Several types of colonies were observed: stellate, ductal, webbed, squamous, and lobuloductal colonies. At the immunocytochemical and electron micrograph levels, casein proteins were predominantly localized near the apical surfaces of the cells or in the lumina of ductal or lobuloductal colonies. Laminin and collagen IV were detected only in the basal lamina of ductal structures cultured in CHM. RMEC and subpopulations from immature virgin rats developed the capacity to synthesize and secrete lipid and casein when grown in Matrigel in serum medium or under defined serum-free conditions.

Animals↗
PubMed1996

A rapid and sensitive fluorometric screening assay using YO-PRO-1 to quantify tumour cell invasion through Matrigel.

A new quantitative assay for the study of tumour cell invasion in vitro is described. Employing the novel fluorescent dye YO-PRO-1, cells that penetrate Matrigel-coated transwells are counted on the basis of dye-bound cellular nucleic acid content. Following transmigration, the cells in the lower compartments are lysed by freezing in water. After a brief incubation with YO-PRO-1, nucleic acid or DNA content is measured as fluorescence intensity in 96-well microplates and quantitated by a cell- or DNA-calibration curve. Using standard curves, a linear relationship between fluorescence intensity and cell number was found in the range tested (from 100 to 80 000 cells). The mean relative intra- and inter-assay variability of the cell quantitation in this range was 3.5 and 4.2%, respectively. When applied to Matrigel invasion studies, as few as 400 cells could be counted. The quantitation could be performed within 3 h. HCT 116, MDA MB 231 and HT 29 cells were investigated as examples of tumour cells with different invasive abilities in the 48-h Matrigel invasion assay. Using YO-PRO-1, 6.5 +/- 0.6% invasive HCT 116 cells and 52.6 +/- 4.5% MDA MB 231 cells (percentage of the inoculated cell population) were measured. HT 29 cells were practically non-invasive. These results were confirmed by visual scoring of DAPI-stained nuclei. In conclusion, the main advantages of the assay are its sensitive, reproducible and rapid quantitation of tumour cell invasion in vitro and the applicability to extended sample numbers by measuring in 96-well microplates.

Basement Membrane↗
PubMed1994

Role of plasminogen activator and of 92-KDa type IV collagenase in glioblastoma invasion using an in vitro matrigel model.

The invasive nature of human gliomas represents a major factor in preventing their total resection. The exact nature of the underlying mechanisms of tumor cell invasion are still unclear. In this study, we have quantitatively assayed a glioblastoma cell line for its ability to migrate through a polycarbonate filter coated with matrigel which contains a complex of multiple basement membrane components. At 48 h the glioblastoma cell line (U251) showed a rate of invasiveness of 42% and also dependent on the concentration of matrigel. The U251 cell line produced a urokinase type plasminogen activator and a 92-KDa type IV collagenase. Both enzymes were inhibited by the addition of uPA and 92-KDa type IV collagenase antibodies. Those same antibodies reduced the invasion rate of U251 cells from 42% to 12 and 21%, respectively. Similarly, the addition of epsilon-aminocaproic acid (a plasmin inhibitor) or tissue inhibitor of metalloprotease (TIMP2, a collagenase inhibitor) reduced the invasiveness of U251 cells from 42% to 14% and 10%, respectively. Additionally, the other two glioblastoma cell lines (LG11, UWR1) and astrocytes showed a rate of invasiveness at 41%, 61% and 12%, respectively. Finally, the addition of hyaluronic acid to the matrigel, a constituent of brain extracellular matrix, enhanced the rate of invasion. These findings provide evidence for the role of serine proteases and metalloproteases in facilitating the invasion of extracellular matrix components by glioblastoma cell line and suggest a therapeutic role for protease inhibitors in attempting to minimize the invasive propensity of gliomas.

Aminocaproic Acid↗
PubMed1992

Protein kinase C regulates endothelial cell tube formation on basement membrane matrix, Matrigel.

Human umbilical vein endothelial cells differentiate within 12 h to form capillary-like networks of tube structures when the cells are plated on Matrigel, a mixture of basement membrane proteins. Nothing is known about the intracellular signaling events involved in this differentiation. As a first step to define the process, we investigated the possible role of protein kinase C activation by beta-phorbol 12-myristate 13-acetate (PMA) in regulating the formation of the tube structures. In this model, PMA increased tube formation several-fold in a dose-dependent manner with half-maximum stimulation of tube formation at approximately 5 nM PMA. In the absence of serum, essentially little or no tubes were formed on Matrigel unless PMA was added to the medium. Only active phorbol analogs increased tube formation, while the protein kinase C inhibitor, H-7, blocked tube formation. The protein kinase C activators and inhibitors were effective only when added at or just after plating of the cells and did not affect already formed tubes. This study suggests that protein kinase C is involved in the early events of in vitro endothelial cell tube formation on Matrigel.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗