The basement membrane is a thin layer of extracellular protein and glycosaminoglycans separating the epithelium from underlying tissue that functions to anchor the epithelium to the supporting connective tissue. Basement membrane extracts (BME) are crude ECM mixtures that may include laminin, collagen IV, entactin, and heparan sulfate proteoglycan, and which are isolated from ECM-rich Engelbreth-Holm-Swarm (EHS) sarcoma tumors in mice. Basement membrane extracts provide a coating substrate for numerous cell types and recapitulates the natural ECM environment found in tissues. ECM Gel is a biological 3D hydrogel isolated from the basement membrane of EHS sarcoma tumors. The product will undergo activated polymerization at 20-40 °C, forming a reconstituted basement membrane. ECM Gel supports numerous in vitro assays such as cell adhesion, neurite outgrowth, cell invasion, cell migration, and angiogenesis tube formation with multiple cell types including epithelial cells, endothelial cells, muscle cells, nerve cells, tumor cells, and stem cells. ECM Gel is cited in over 1000 publications, and is offered in numerous pack sizes in either standard (E1270) or growth factor reduced (E6909) formats. Growth factor-reduced ECM Gel has lower levels of bFGF, EGF, IGF-1, TGF-β, PDGF and NGF cytokines compared to normal ECM Gel.
Figure 1.Structure of the basement membrane. The basement membrane consists of a complex mixture of extracellular matrix proteins (collagen, laminin, fibronectin) and glycosaminoglycans separating the epithelium from underlying connective tissue. ECM Gel Matrix is a basement membrane extract (BME) isolated from EHS tumors rich in ECM proteins.
Figure 2.Neurite outgrowth of PC12 rat neuronal cells using ECM Gel Matrix. A) Normal morphology of PC12 cells (88022401) cultured on uncoated plasticware. B, C) PC12 cells grown on ECM Gel Matrix form neurites with complex 3D neural networks after 24-48 hours of culture.
Figure 3.Cell invasion assay using ECM Gel Matrix. Analysis of HT-1080 (85111505) cell invasion through an ECM Gel coated Millicell® insert using a 96-well Cell Invasion Assay (ECM550). Invading cells were visualized by crystal violet staining (purple). NIH3T3 cells (93061524) were used as a non-invasive control.
Figure 4.Angiogenesis tube formation using ECM Gel Matrix. HUVEC cells stained green using Calcein-AM (17783) after induction of angiogenesis.
Expansion of human pluripotent stem cells (ES and iPS Cells) requires cultureware that is coated with prequalified ECM proteins. Below are general guidelines for the coating of 6-well plates using the Stem Cell Qualified ECM Gel Matrix (CC131). All procedures should be performed under aseptic conditions in a biological safety cabinet.
IMPORTANT: Do not allow the plates to dry out.
Figure 5.Growth of PBMC-derived human iPS cells grown in serum-free media on Stem Cell Qualified ECM Gel Matrix (A) or Matrigel® (B) after 5 passages. Pluripotent morphology of iPSCs grown on ECM Gel is identical to Matrigel® substrates. Cell growth of PBMC-derived human iPSCs grown on Stem Cell Qualified ECM Gel Matrix is comparable to Matrigel® as analyzed by cell counting over 7 pasages (C).
The ECM gel is produced from whole EHS tumors (not separated cells). It is the product of a total extraction of the tumors.
ECM gel is the biological equivalent to these products, manufactured by MilliporeSigma.
Product E1270 is extracted from Engelbreth-Holm-Swarm mouse sarcomas with no exogenous additives.
The principal components of basement membranes are laminin, collagen IV, entactin, and heparan sulfate proteoglycan (HSPG). The first three are usually present in approximately equimolar amounts, while HSPG is less abundant, but as ECM Gel is a biological extract, precise concentrations of components cannot we cannot state fixed values of relative component concentrations.
The approximate concentration of CaCl2 in the ECM solution is 0.265 mg/mL.
Our ECM gel is supplemented with gentamicin.
The DMEM used in the preparation of E1270 does not contain sodium pyruvate.
We do not test product E1270 for virus presence. However, several product lots were tested in the past and found free of LDEV, and our raw material supplier Harlan added the LDEV test to their SPF tests list thus the source (mice) are cleaned for this virus. Moreover, by risk assessment, RNA viruses should not survive the manufacturing procedure. Thus, we assume the product is LDEV free.
We would not recommend thawing and then refreezing the product ECM gel. Alternatively, gels can be cast in plates and stored them at 2-8° C. Seal stored gels securely to prevent dessication and cracking.
ECM gel may be diluted twofold with cold Dulbecco’s Modified Eagle Medium (DMEM). This means ECM gel may be diluted with an equal volume of cold DMEM.
We know that product does not degrade after 7 days of co-incubation with cells.
Dispase (D4818) is a Bacillus-derived neutral protease that is recommended for recovering cells cultured in ECM gel (Cat. No. E1270). Dispase will yield a single cell suspension far more effectively than trypsin, collagenase, or other proteolytic enzymes. Dispase is gentle and will not damage cells harvested for sub-cultivation or other study.
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