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PubMed2005

Differentiation of a mouse submandibular gland-derived cell line (SCA) grown on matrigel.

SCA-9 cell line was developed from an induced tumor of mouse submandibular gland. We have studied some of the phenotypic characteristics of SCA cells cultured on different matrices. On plastic surface, the cells grow as a monolayer; on matrigel, they form branching structures and tubes, a phenomenon termed branching morphogenesis. EGF and HGF promoted cellular growth and branching morphogenesis which was inhibited by anti-EGF antibodies. We have performed RT-PCR and real-time quantitative RT-PCR of cells grown on plastic surface or on matrigel. Grown on plastic, the cells express EGF and renin 2, but no or only trace amounts of NGF. Growth on matrigel for 24 h resulted in a transient 21-fold increase in EGF mRNA and a 3371-fold increase in renin 2 mRNA. There was no change in NGF mRNA level. SCA-9 cells express mRNAs for receptors for the EGF family of ligands. On plastic, mainly ErbB1 and ErbB2 are expressed. Culture on matrigel resulted in 11-fold increase in mRNA levels for ErbB1 and ErbB2, and a 221-fold and 85-fold increase in the mRNA levels for ErbB3 and ErbB4, respectively. Small interfering RNAs siErbB3 and siErbB4 inhibited the growth of the cells grown on plastic or matrigel. Significant growth inhibition was seen also with siErbB1+siErbB3 and siErbB2+siErbB3. siErbB1 and siErbB2 also inhibited branching morphogenesis. Since SCA cells express EGF and receptors for EGF, EGF acts an autocrine regulator in promoting growth and branching morphogenesis. We conclude that SCA cells provide a useful model to analyze the mechanism of branching morphogenesis and the role of matrix in regulating expression of phenotypic characteristics of cultured cells.

Animals↗
PubMed1998

Matrigel augments xenograft transplantation of meningioma cells into athymic mice.

OBJECTIVE: We investigated the use of Matrigel to enhance the growth of human meningiomas in athymic (nude) mice. Tumor take and growth of xenograft meningioma cells in an in vivo model have previously been only partially successful. METHODS: The use of Matrigel has been reported to enhance tumorigenicity in a variety of solid tumors. This substance is derived from a mouse sarcoma and is a mixture of basement membrane proteins and growth factors. Meningioma cells obtained from human patients were placed in culture for 1 to 2 passages and then harvested and mixed with Matrigel and the mixture injected into the subcutaneous space in the flank of nude mice. Tumor volumes over time were measured at least three times a week and then harvested at 100 days postimplantation. Tumors were formalin-fixed, and histological examinations were performed. Immunohistochemistry was performed for human and mouse laminin, fibronectin, collagen Type IV, and epithelial membrane antigen. RESULTS: Tumors developed in all 40 mice studied. Growth of meningioma tumors was dependent on total number of cells injected and independent of the total volume of tumor cells and Matrigel matrix. Histologically and immunohistochemically, the xenograft tumors were very similar to the original human tumors. CONCLUSION: Matrigel is relatively easy to use and has a high rate of histologically confirmed meningioma tumor formation in an athymic mouse model. We plan to use this model for studying the growth of meningiomas in vivo.

Animals↗
PubMedDate not supplied

Differentiation of prostate epithelial cell cultures by matrigel/ stromal cell glandular reconstruction.

Three-dimensional epithelial culture models are widely used to emulate a more physiologically relevant microenvironment for the study of genes and signaling pathways. Prostate epithelial cells can grow into solid cell masses or acinus-like spheroids in Matrigel. To test if the ability to form acinus-like spheroids in Matrigel is dependent on how undifferentiated a cell is or whether it is tumor or nontumor, we established six novel epithelial cell lines. Primary prostate epithelial cells were immortalized using HPV16 E6 gene transduction and were named Shmac 2, 3, and 6 (nontumor); Shmac 4, Shmac 5, and P4E6 (tumor). All cell lines were phenotyped in monolayer culture, and their ability to form acinus-like spheroids in Matrigel investigated. The cell lines exhibited a wide range of population doubling times and all showed an intermediate phenotype in monolayer culture ((luminal)CK(+)/(basal)CK(+)/CD44(+)/PSA(+)/AR(-)). Only Shmac 5 cells formed acinus-like spheroids when cultured in Matrigel. Co-culture of the spheroids with fibroblasts advanced differentiation by inducing androgen receptor expression and epithelial polarization. Our findings indicate that tumor cells can form acinus-like spheroids in Matrigel.

Cell Culture Techniques↗
PubMed1998

Identification of anti-invasive but noncytotoxic chemotherapeutic agents using the tetrazolium dye MTT to quantitate viable cells in Matrigel.

Screening methods for chemotherapeutic agents usually rely on the cytotoxic properties of the drugs. However, agents that inhibit invasion may have more efficacy and cause fewer side effects. Various cellular invasion assays have been used to evaluate these types of compounds, including the modified Boyden chamber, monolayer wound models and Matrigel outgrowth assays. In this report, we have combined the use of the Matrigel outgrowth assay with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) visualization and cell viability dye to visualize invasive cells on Matrigel without magnification. Extraction of the dye's formazan byproduct allows cell viability to be assessed. Using several invasive and noninvasive cell lines, the utility of the method for various target cells was verified. Several established chemotherapeutic agents were also screened for their anti-invasive and/or cytotoxic effects when cultured on Matrigel. Our results suggest that this method may be an easy, inexpensive and nonradioactive alternative for both enumerating cells on Matrigel and screening various tumor cell lines treated with chemotherapeutic agent to look for compounds with noncytotoxic but anti-invasive properties.

Antineoplastic Agents↗
PubMed1994

Use of the Matrigel-based assay to measure the invasiveness of leukemic cells.

The reconstituted basement membrane (Matrigel)-based assay was used to quantify the invasive potential of hematopoietic cells including cultured human leukemic cells (KG-1, K-562, HEL, HL-60, and U-937), normal bone marrow (BM) cells, and normal polymorphonuclear leukocytes (PMNL). We found that (i) in contrast to 6- to 72-hour incubation periods typically used in assays with solid tumor cells, most of the invasive cell populations tested here required only 2 to 4 hours to cross the Matrigel layer; (ii) unlike that of PMNL, whose invasiveness was stimulated by the addition of FMLP, the invasive rate of cultured leukemic cells was not affected by this chemoattractant; (iii) the rate of invasion was inversely proportional to the Matrigel concentration per filter but varied with the Matrigel batch used; (iv) the most consistent results were obtained when 2.5 to 4 x 10(5) cells were added to the top portion of the blind well; and (v) of all leukemic cells tested, the least differentiated myeloblastic KG-1 cells exhibited the highest invasive potential, which was comparable to that of normal PMNL. We conclude that the Matrigel-based assay can be used as a model system in studies of mechanisms regulating movement of hematopoietic cells across basement membrane barriers.

Basement Membrane↗
PubMed1993

Heterotransplantation of early B-lineage acute lymphoblastic leukemia using a solubilized attachment matrix (Matrigel).

Maintenance of long term culture and conventional xenografting of early B-lineage acute lymphoblastic leukemia cells is most difficult. Matrigel, a solubilized attachment matrix shown to aid growth of anchorage dependent solid tumors, was studied in heterotransplantation. Material for xenografting was derived from 5 patient bone marrow aspirates and 5 cell lines previously established and maintained by intraocular inoculation in nude mice. Specimens were injected by 3 methods: intraocular (n = 397); s.c. in medium (n = 78); and s.c. in medium supplemented by Matrigel (n = 69). With intraocular injection, 6 of 10 cell sources grew with respective ingraftment rates of 29-76%. Using the conventional s.c. method, no tumors resulted. The addition of Matrigel produced s.c. ingraftment from 8 of 10 cell sources (ingraftment rate, 50-100%). Immunophenotype, histopathology, and karyotype of the cells derived after Matrigel dependent ingraftment correlated with the cells of origin. It is concluded that Matrigel enables establishment and maintenance of early B-lineage acute lymphoblastic leukemia cell growth in a s.c. xenograft model.

Animals↗
PubMed1993

Expression of multiple forms of cytochrome P450 mRNAs in primary cultures of rat hepatocytes maintained on matrigel.

Freshly isolated rat hepatocytes rapidly lose their cytochrome P450 (P450) proteins and mRNAs, with no evidence of subsequent restoration, after placement into traditional systems of primary culture on type I collagen. We examined the patterns of expression of 10 constitutively expressed P450 mRNAs in rat hepatocytes cultured for up to 5 days on a matrix of Matrigel, a reconstituted basement membrane that allows inducible expression of some P450s, and compared these patterns with those seen in hepatocytes cultured on type I collagen (Vitrogen). mRNA for each P450 was detected on Northern blots in samples prepared from freshly isolated male rat hepatocytes, and the amount of each mRNA decreased markedly during the first 2 days of culture in cells maintained on either matrix (decreases of 53-97% on Matrigel and 62 to > 99% on Vitrogen), in contrast to the level of NADPH-P450 oxidoreductase mRNA, which increased during this interval. On subsequent days, hepatocytes cultured on Matrigel expressed the individual P450 mRNAs in one of the following four patterns. 1) P450 1A2, 2A2, 2E1, and 3A1/2 mRNAs remained low throughout the 5-day culture period. 2) 2A1 mRNA increased between days 2 and 4 but then decreased on day 5. 3) 2B1/2, 2C6, 2C7, and 4A1 mRNAs increased continually between days 2 and 5, 4) The total mRNAs detected with a 2D probe remained at constant levels between culture days 2 and 5. Our results show that rat hepatocytes cultured on Matrigel in serum-free medium spontaneously reexpress the mRNAs for several constitutive P450s in form-specific patterns, and they suggest that the Matrigel culture system will be useful for identifying the underlying regulatory mechanisms.

Animals↗
PubMed1992

Malignant transformation of NIH-3T3 cells after subcutaneous co-injection with a reconstituted basement membrane (matrigel).

NIH-3T3 cells are non-tumorigenic when injected into athymic mice. If these cells are mixed with an extract of basement-membrane proteins (matrigel) and injected s.c., they form locally invasive and highly vascularized tumors. Cells cultured from the NIH-3T3-matrigel-induced tumors showed a transformed phenotype and lacked contact inhibition. When cultured in a gel of matrigel, they proliferated and formed branched and invasive colonies. In contrast, the parental NIH-3T3 cells cultured on matrigel remained as cell aggregates and were not invasive. I.V. injections of the tumor-derived NIH-3T3 cells produced many colonies on the surface of the lungs, whereas the parental NIH-3T3 cells were not metastatic. Zymographic analysis of the conditioned media obtained from both the tumor-derived and parental NIH-3T3 cells demonstrated higher amounts of the 72-kDa gelatinase (type-IV collagenase) enzyme in the tumor-derived cells. Also, tumor-derived NIH-3T3 cells, but not parental NIH-3T3 cells, secreted the 92-kDa type-IV collagenase. These studies suggest that the interaction of pre-malignant NIH-3T3 cells with extracellular matrix components may contribute to the process of tumor progression.

Animals↗
PubMed1992

Matrigel promotes retinoblastoma cell growth in vitro and in vivo.

Cells derived from retinoblastomas grow slowly in vitro and only very rarely form tumors in nude mice. Matrigel, a mixture of components normally found in basement membranes, promotes the growth of Y-79 and WERI-Rb1 retinoblastoma (Rb) cells when added to suspension cultures of the 2 Rb cell lines. It also substantially increases cell adhesion in vitro. Y-79 cells, seeded into a Matrigel matrix, form round colonies over a 3-week period similar to those of control, weakly metastatic murine melanoma cells. In vivo, s.c. co-injection of Matrigel with either Y-79 or WERI-Rb 1 cells into nude mice promotes retinoblastoma tumor formation. Transplantation of as few as 1,000 cells allows for xenografting under these conditions, while no tumors were observed in the absence of Matrigel, even at 10 x 10(6) cells/inoculum. The tumors produced have the expected morphology and express an mRNA for a highly specific retina/retinoblastoma marker protein, the interphotoreceptor retinoid-binding protein. Thus, the xenografts obtained maintain the original morphological and molecular characteristics of the injected cells and represent a useful model for in vivo studies of retinoblastoma growth and treatment.

Animals↗
PubMed1993

TPA-enhanced invasion of Matrigel associated with augmentation of cell motility but not metalloproteinase activity in a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1.

We previously found that the enhanced activity to invade Matrigel upon stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) was one of the major properties of a highly metastatic variant (L-10) of a human rectal adenocarcinoma cell line RCM-1. To clarify the mechanism of this enhancement, we examined the effect of TPA on 2 major biological factors involved in tumor cell invasion: cell motility and matrix-degrading metalloproteinase activity. The enhanced invasiveness was inhibited by protein-kinase-C inhibitors. TPA markedly enhanced both haptotactic response to type-IV collagen and motility on tissue-culture glass substrate of L-10 cells in a dose-response manner quite similar to that of TPA-enhanced invasion of Matrigel. On the other hand, TPA showed little enhancement of metalloproteinase production, which was assessed by gelatin- and casein-zymography, and of type-IV collagenolytic activity. Addition of TIMP (tissue inhibitors of metalloproteinase)-I inhibited TPA-enhanced invasion of Matrigel by only up to 13%. Thus, TPA treatment of L-10 cells enhanced invasion of Matrigel in association with augmentation of cell motility but did not enhance metalloproteinase activity.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Stimulation of angiogenesis as an explanation of Matrigel-enhanced tumorigenicity.

Matrigel, a reconstituted extract of basement membrane, enhances the growth of different human cancer cell lines when transplanted into nude mice. Here that stimulation was confirmed in the BALB/c murine mammary-tumor cell line M3MC, as well as in human colon (SW948) and mammary (MDA-MB-468) carcinoma cell lines transplanted in nude and SCID mice, respectively. Subcutaneous and intra-mammary fat-pad inoculations of Matrigel alone generated an angiogenic response which was macroscopically evident by day 9. Histological analysis of the local host reaction occurring at the site of injection revealed an early peripheral fibroblast response, followed by mononuclear cell infiltration, solid and hollow fibroblast cords projections from the edge to the center of the Matrigel plug, and finally capillary ingrowths. Conditioned media obtained from the gels generated in vivo, acted as very strong chemoattractants for mouse lung capillary endothelial cells, stimulating their motility between 38 and 82 times with respect to the control. Our results suggest an important role of host cells recruited by Matrigel, which could favor angiogenesis of the area and thus facilitate the growth of tumor cells co-inoculated with the basement membrane extract.

Adenocarcinoma↗
PubMed1992

Morphological assessment of hepatoma cells (HepG2) microencapsulated in a HEMA-MMA copolymer with and without Matrigel.

Hepatoma cells (HepG2), an anchorage-dependent cell line, were microencapsulated in a HEMA-MMA polyacrylate membrane to which the cells do not adhere. This environment was altered by the coencapsulation of Matrigel, a reconstituted extracellular matrix derived from the Engelbreth-Holm-Swarm (EHS) mouse tumor basement membrane, to provide sites for cell attachment. The effect on the cells of these two capsule microenvironments during a 2-week in vitro culture period was assessed by examining the spatial arrangement, morphology, and viability of the cells using light microscopy and scanning electron microscopy (SEM). In preparation for microscopy, dissolution of the polymer was prevented by the use of frozen sections embedded in a water-soluble compound. Similarly, freeze cleavage of conductively stained capsules permitted SEM observation of the capsule interior along with ultrastructural detail of the cells. In the absence of Matrigel, cells in HEMA-MMA capsules were found to form aggregates in intracapsular pockets with central necrosis occurring at day 7 in large aggregates. The coencapsulation of HepG2 cells with Matrigel, resulted in an initially uniform distribution of essentially individual cells with aggregates appearing later within the Matrigel. Many cells within these capsules had remained viable when examined up to day 14 with only limited cellular necrosis, implying a favorable environment for microencapsulated HepG2 cells.

Biocompatible Materials↗
PubMed1996

TGF-beta and basement membrane matrigel stimulate the chondrogenic phenotype in osteoblastic cells derived from fetal rat calvaria.

Primary cultures of fetal rat calvarial cells contain a spectrum of osteogenic phenotypes including undifferentiated mesenchymal cells, osteoprogenitor cells, and osteoblasts. We recently demonstrated that rat calvarial osteoblast-like cells grown on basement membrane undergo profound morphological changes resembling a canalicular network in bone. In the present study, we examined the effect of reconstituted basement membrane Matrigel on chondroblastic versus osteoblastic differentiation of different cell subpopulations obtained by five consecutive enzymatic digestions of rat calvarial cell populations. We found that the appearance of canalicular cell processes decreased with the later digests. When cells from the fourth and fifth digest were grown on top of Matrigel for 7 days, the majority of the cell aggregates displayed chondrocytic characteristics but none of the cells became hypertrophic. When individual chondroblastic cell aggregates were subsequently transferred from Matrigel to plastic, they started expressing types I and X collagens, alkaline phosphatase, and osteocalcin. Within the next 7 days (days 8-14 of the experiment), the majority of cells increased in size, and at day 17 on plastic (day 24 of the experiment) mineralized bone nodules formed. The chondroblastic differentiation of calvarial cells grown on Matrigel could be inhibited by a specific transforming growth factor-beta 1 (TGF-beta 1) but not by a TGF-beta 2 antibody. Addition of recombinant TGF-beta 1 to similar cultures promoted the appearance of chondroblastic cell aggregates. The cartilage phenotype could not, on the contrary, be promoted by growing the cells on other extracellular matrices such as a collagen I gel. We suggest that TGF-beta 1 in concert with the basement membrane extracellular matrix induces chondroblastic differentiation of rat calvarial osteoprogenitor cells.

Alkaline Phosphatase↗
PubMed2001

Effects of src kinase and TGFbeta1 on the differentiation and morphogenesis of MDCK cells grown in three-dimensional collagen and Matrigel environments.

This study attempted to analyse in detail the effect of src kinase on the growth and differentiation of MDCK cells in different extracellular matrix (ECM) environments. A method was developed to label the membrane proteins in situ and the distribution of cytoskeletal and junctional proteins was visualized in three-dimensional cell complexes, using optical sections generated by confocal microscopy. Independently of the ECM, non-transformed MDCK cells formed differentiated cell cysts with one or a few lumina, with the apical side facing the lumen; ZO-1 was expressed at the tight junctions close to the apical side and beta-catenin, E-cadherin and fodrin along the entire lateral walls. The phenotype of src kinase activated MDCK cells was strongly dependent on the ECM and varied from an irregular cluster in collagen I, to tubular structures in laminin or proteoglycans, and finally to a polarized cell cyst in Matrigel. In collagen I, E-cadherin and beta-catenin were seen partially along the lateral walls and partially in the cytoplasm of src-transformed MDCK cells; fodrin was released into the cytoplasm and ZO-1 was not visualized. When the src-transformed cells were cultivated in Matrigel, their junctional proteins were recruited to the cell membranes and ZO-1 reappeared at the apical face. Thus, the components of Matrigel could overcome the deleterious effect of src on the polarity of MDCK cells. TGFbeta1, together with its receptors and other soluble factors in Matrigel, were responsible for the induction of differentiation. The results show that tyrosine phosphorylation sensitizes the epithelial MDCK cells to ECM and TGFbeta1.

Cadherins↗
PubMed1999

In vitro endothelial differentiation of long-term cultured murine embryonic yolk sac cells induced by matrigel.

The yolk sac of an early mammalian embryo contains progenitors of hematopoietic cells and vascular endothelial cells. We established a cell line, YS4, from murine embryonic yolk sac 10 years ago. The line has been successfully cultured since then. To determine whether these long-term cultured yolk sac cells still have the potential to differentiate into endothelial cells, an in vitro model of yolk sac cell differentiation into tubeforming endothelial cells was established in the present study by culturing the yolk sac cells on basement membrane proteins (Matrigel). The results indicate that upon plating onto Matrigel, YS4 cells attach quickly, align in tandem, and form a complete network of capillary structures within 12 h. By using antibodies against the known components of Matrigel in a tube formation inhibition assay, we found that extracellular matrix proteins such as laminin, collagen IV, vitronectin, and fibronectin are the most important components in the Matrigel which induce the yolk sac cells to undergo endothelial differentiation. New basement membrane proteins are also required for the endothelial differentiation process, as indicated by the fact that base membrane protein synthesis inhibitor, D609, can block the differentiation process. Furthermore, our experiments revealed the involvement of several signal transduction pathways, such as protein kinase A, C and protein tyrosine kinase in this differentiation process.

Animals↗
PubMed1993

Growth and metastasis of human breast carcinomas with Matrigel in athymic mice.

Immunodeficient athymic mice with human tumor xenografts provide an important in vivo experimental model for cancer research. However, only a limited number of tumor types grow in these animals. For human breast carcinomas, the incidence of tumor-take is 6-15%. Recently, increased incidence of xenograft development in mice has been reported for various human tumors when the tumors were coinjected with Matrigel. We studied the development of human breast carcinoma xenografts in athymic mice with and without coinjection of Matrigel. Tumors developed in only 7.3% of enzyme-dispersed tumors injected subcutaneously in saline solution alone. None of these tumors metastasized to distant sites. On the other hand, 50% of enzyme-dispersed tumors coinjected with Matrigel developed xenografts; four out of five of these tumors metastasized to distant sites. Our data from the recent study suggest that, in athymic mice, Matrigel not only enhanced breast tumor growth but also facilitated tumor metastasis.

Animals↗
PubMed1991

Sulfation and glucuronidation of acetaminophen by cultured hepatocytes reproducing in vivo sex-differences in conjugation on Matrigel and type 1 collagen.

The sulfate and glucuronide conjugation of acetaminophen (APAP) by hepatocytes cultured on Matrigel or type 1 collagen was compared to APAP metabolism in vivo. The metabolic fate of low (15 mg/kg), medium (125 mg/kg), and high (300 mg/kg) doses of APAP injected intraperitoneally were determined in male and female rats. Males excreted more APAP as the sulfate conjugate than females, which correlated with the twofold greater APAP sulfotransferase activity in the male vs. females (301 +/- 24 vs. 156 +/- 18 pmol.mg-1 protein.min-1). Also, as sulfate conjugation became saturated, there was a dose-related shift in APAP metabolism from sulfate to glucuronide conjugation in both sexes. After death, the livers of the same animals were perfused with collagenase and the hepatocytes cultured in modified Waymouth's medium on either Matrigel or rat-tail collagen, with various doses of APAP (0, 0.125, 0.25, 0.5, and 1.0 mM). Sex differences in APAP sulfation and glucuronidation persisted in culture for up to 4 days, with sulfation predominating in the male similar to in vivo. With increasing APAP concentration (dose), there was a saturation of sulfate conjugation and a shift to glucuronidation as observed in vivo. Sex differences in APAP sulfation and glucuronidation were no longer significant by Day 4 in culture. Sulfation, and to a lesser extent, glucuronidation, were more stable on Matrigel than collagen. We concluded that APAP metabolism of freshly isolated hepatocytes could replicate in vivo sex differences in conjugation, and that Matrigel was superior to collagen as substrate.

Acetaminophen↗
PubMed1992

Platelet adhesion to collagen-coated wells: analysis of this complex process and a comparison with the adhesion to matrigel-coated wells.

The mechanisms of platelet adhesion to collagen type III-coated wells and Matrigel-coated wells were analyzed. The adhesion of 51Cr-labeled platelets to collagen-coated wells showed a biphasic pattern. The early stage of adhesion was inhibited by antibodies against platelet glycoprotein(GP)s Ia/IIa and VI. The later stage of platelet adhesion was inhibited by an antibody against the GPIIb/IIIa complex and a concomitant release of 14C-labeled serotonin was observed. The percentage of adhered platelets was increased when a higher platelet concentration was added in the reaction medium. These results indicated that the adhesion assay of platelets to collagen-coated wells was composed of two reactions: the first one is the platelet-collagen interaction that depends on GPIa/IIa and GPVI on the platelet surface; and the second reaction is the platelet-platelet interaction, platelet aggregation, which depends on GPIIb/IIIa. The adhesion of platelets to Matrigel-coated wells was indicated to involve platelet-Matrigel interactions that were partly dependent on the laminin in the Matrigel solution.

Antibodies, Monoclonal↗