Growing Cells in Geltrex™ Reduced Growth Factor Basement Membrane Matrix

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Introduction

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.
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Precautions

Thaw Geltrex™ Basement Membrane Matrix at 2-8°C overnight. Refrigerator temperatures may vary; therefore, thaw extract on ice in a refrigerator. Basement Membrane Matrix gels in 5-10 minutes above 15°C; therefore when working from a full 5 ml vial, it is unnecessary to keep it on ice if used within 5 minutes and the environmental temperature does not exceed 25°C. It is also unnecessary to prechill pipette tips, tubes, plates, or other objects that may come in contact with the extract. Since smaller volumes warm more quickly, partial tubes and aliquots should be kept on ice to prevent premature gelling.
 
When working with volumes of Geltrex <5ml, aliquot to appropriate required working volumes and store at -20 to -80°C.
 
Avoid multiple freeze/thaw cycles.

Protocol

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.


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.

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

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.

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.

Features and Source

Features and Source

  • Provides a 3D matrix for growing a large number of cell types including stem cells and primary cells
  • Reduction in specific growth factors
  • Formulated in Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamycin sulfate and no phenol red

Source

Murine Engelbreth-Holm-Swarm (EHS) tumor
5012     4-Apr-2010
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