STEMmatrix™ BME (basement membrane extract) is a soluble, hPSC-qualified basement membrane matrix extracted from mouse Engelbreth-Holm-Swarm (EHS) tumors that is rich in extracellular matrix proteins (e.g. collagen IV, entactin, heparan sulphate proteoglycans, laminin) and growth factors (e.g. EGF, bFGF, IGF-1, TGF-β, VEGF). When used with feeder-free human pluripotent stem cell (hPSC) maintenance media, such as mTeSR™1, mTeSR™ Plus, or eTeSR™, STEMmatrix™ BME can be used to successfully maintain human embryonic stem cell and induced pluripotent stem cell lines in the undifferentiated state. These cells retain characteristic morphology and expression of undifferentiated cell markers such as OCT4 and TRA-1-60, and have the capacity to differentiate into all three germ layers.

Follow this step-by-step protocol to learn how to coat cultureware with STEMmatrix™ BME for your hPSC cultures. The process is very similar to coating plates with other popular basement membrane matrices (e.g. Corning® Matrigel® or Gibco™ Geltrex™).

Materials and Reagents

• STEMmatrix™ BME (Coming Soon!)
• DMEM/F-12 with 15 mM HEPES (Catalog #36254)
• 50 mL polypropylene conical tube (e.g. Falcon® Conical Tubes, 50 mL, Catalog #38010)
• Tissue culture-treated cultureware (e.g. Falcon® 6-Well Flat-Bottom Plate, Tissue Culture-Treated, Catalog #38016)
• Cell culture medium
  • mTeSR™1 (Catalog #85850)
  • mTeSR™ Plus (Catalog #100-0276)
  • eTeSR™ (Catalog #100-1215)

Protocol

1. Thaw STEMmatrix™ BME by submerging the vial in a container of ice maintained at 2 - 8°C overnight.
   Note: It is recommended to prepare aliquots of STEMmatrix™ BME and store at -20°C until use. Do not exceed the shelf life of the product. After thawing aliquots, use immediately. Do not refreeze.
2. Swirl the vial to ensure that STEMmatrix™ BME appears evenly dispersed. Spray the top of the vial with 70% ethanol and air dry. Keep STEMmatrix™ BME on ice and handle using sterile technique.
   Note: Ensure that STEMmatrix™ BME, media, and all consumables are ice-cold. STEMmatrix™ BME will start to gel at 4°C. Keep on ice when thawing and handling to prevent it from gelling. To liquefy, submerge the vial in ice for 1 - 2 hours.
3. Calculate the volume of STEMmatrix™ BME required to achieve a final concentration of 100 µg/mL in 25 mL of STEMmatrix™ BME working solution. For the lot-specific protein concentration, refer to the certificate of analysis (CoA), available at www.stemcell.com/coa.
   Example Dilution Calculation:

   Dilution equation: C₁V₁ = C₂V₂

   • C₁: Final concentration of STEMmatrix™ BME working solution = 100 µg/mL
   • V₁: Final volume of STEMmatrix™ BME working solution = 25 mL
   • C₂: Stock concentration of STEMmatrix™ BME = 9.8 mg/mL

   • V₂: Required volume of stock STEMmatrix™ BME = [100 µg/mL x 25 mL] / 9.8 mg/mL = 255.1 µL
   • Required volume of dilution medium = 25 mL - 255.1 µL = 24.75 mL
4. Aliquot STEMmatrix™ BME in pre-chilled tubes, according to the calculated volume in step 3. Switch pipette tips if clogged by STEMmatrix™ BME. Keep tubes on ice.
   Note: If not used immediately, store at -20°C. Do not exceed the shelf life of the product. Thaw aliquots on ice.
5. Prepare a working solution of STEMmatrix™ BME as follows:
   1. Add the calculated volume of cold DMEM/F-12 with 15 mM HEPES to a 50 mL conical tube and place on ice.
   2. Add one aliquot of thawed STEMmatrix™ BME to the cold DMEM/F-12. Mix thoroughly.
6. Immediately add the diluted STEMmatrix™ BME solution to cultureware; see Table 1 for recommended coating volumes. Gently rock the cultureware back and forth to spread the solution evenly across the surface.
   Note: If the cultureware surface is not completely and evenly coated by the STEMmatrix™ BME solution, it should not be used for hPSC culture.
7. Incubate at room temperature (15 - 25°C) for at least 1 hour before use. Do not let the STEMmatrix™ BME solution evaporate.
   Note: If not used immediately, seal the cultureware to prevent evaporation of the STEMmatrix™ BME solution (e.g. with Parafilm®) and store it at 2 - 4°C for up to 1 week after coating. When ready to use, allow stored coated cultureware to reach room temperature for 30 minutes before proceeding to the next step.
8. Gently tilt the cultureware onto one side and allow the excess STEMmatrix™ BME solution to collect at the edge. Remove the excess solution using a serological pipette or by aspiration. Ensure that the coated surface is not scratched.
9. Coated cultureware is now ready for use. Add your desired cell culture medium (e.g. 2 mL/well if using a 6-well plate) and proceed with your cell culture protocol.
   
   

   Table 1. Recommended Volumes for Coating Cultureware

   Cultureware	Volume of Diluted Matrix
   24-well plate	0.3 mL/well
   12-well plate	0.5 mL/well
   6-well plate	1 mL/well
   100 mm dish	8mL/dish
   T-25 cm2 flask	3 mL/flask
   T-75 cm2 flask	8 mL/flask