Growing Cells in Geltrex™ Reduced Growth Factor Basement Membrane Matrix

实验概要

Basement  membranes are continuous sheets of specialized extracellular matrix  that form an interface between endothelial, epithelial, muscle, or  neuronal cells and their adjacent stroma. Basement membranes are  degraded and regenerated during development and wound repair. They not  only support cells and cell layers, but they also play an essential role  in tissue organization that affects cell adhesion, migration,  proliferation, and differentiation.

Basement  membranes provide major barriers to invasion by metastatic tumor cells.  Basement Membrane Matrix is a soluble form of basement membrane  purified from Engelbreth-Holm-Swarm (EHS) tumor. The extract gels at  37°C to form a reconstituted basement membrane. The major components of  the Basement Membrane Matrix include laminin, collagen IV, entactin, and  heparin sulfate proteoglycan. Basement Membrane Matrix can be used for  promotion and maintenance of a differentiated phenotype in a variety of  cell cultures including primary epithelial cells, endothelial cells, and  smooth muscle cells. It has been employed in angiogenesis assays,  neurite outgrowth assays, and tumor cell invasion assays.

实验步骤

There  are many applications for Basement Membrane Matrix which require  different thicknesses and concentrations. In general, a protein  concentration > 9 mg/ml, is used for differentiation studies of  primary cells. Extract diluted below 9 mg/ml does not form a gel, and  will only support the propagation of primary cells, but not their  differentiation. For applications such as endothelial cell  differentiation into capillary-like structures (Tube Assay) a thin gel  is needed. For applications such as the differentiation of rat aorta  tissue into capillary-like structures (Ring Assay), or cell invasion  assays, a thick gel is needed. Some applications, such as propagation of  primary cells, only need a protein layer and not a protein matrix;  therefore, the layer method should be used.

1.        Thin Gel Method:

1)        Thaw Geltrex. See Precautions.

2)        Mix Geltrex by slowly pipetting solution up and down; be careful not to introduce air bubbles.

3)        Place 50 µl per cm² onto the growth surface.

4)        Place coated object at 37°C for 30 minutes.

5)        Coated objects are ready for use.

2.        Thick Gel Method:

1)        Thaw Geltrex. See Precautions.

2)        Mix Geltrex by slowly pipetting solution up and down; be careful not to introduce air bubbles.

3)        Place 150-200 µl per cm² onto the growth surface.

4)        Place coated object at 37°C for 30 minutes.

5)        Coated objects are ready for use

3.        Thin Layer Method (non-gelling):

1)        Thaw Geltrex. See Precautions.

2)        Mix Geltrex by slowly pipetting solution up and down; be careful not to introduce air bubbles.

3)         Dilute the extract to desired concentration in cold serum-free medium.  Empirical determination of the optimal coating concentration for your  application may be required. A protein concentration of 0.1 mg/ml is a  recommended starting concentration for the propagation of primary cells.

4)        Add a sufficient amount of solution to cover the entire area onto growth surface.

5)        Place coated object at 37°C for 60 minutes or until dry.

6)        Coated objects are ready for use.

4.        Thin Gel Method (non-gelling) for hESC Applications:

1)        Thaw Geltrex. See Precautions.

2)        Mix Geltrex by slowly pipetting solution up and down; be careful not to introduce air bubbles.

3)         Dilute 1 ml of Geltrex into 29 ml DMEM/F12 medium (SKU: 10565).  Empirical determination of the optimal coating concentration for your  application may be required. Volumes can be adjusted accordingly.

4)         Add a sufficient amount of diluted Geltrex solution to cover the entire  area onto growth surface (1.5 ml for 35 mm dish, 3 ml for 60 mm dish).

5)        Coat the dish and place at room temperature for a minimum of 60 minutes.

6)         For long term storage, seal each dish with parafilm to minimize  dehydration of the Geltrex solution. The coated dish is stable for two  weeks when stored at 2 to 8°C. Do not allow coated surface to dry out  and maintain a storage temperature of 2 to 8°C to avoid premature  gelling.

7)        At time of use, aspirate Geltrex™ coating and immediately plate cells in pre-equilibrated cell culture medium.