Guide to Using Cultrex™ BME as a Cell Culture Matrix

This video provides best practices and protocols for working with Cultrex BME, including thawing, aliquoting, thin layer method, thick layer method, and dome formation. R&D Systems’ Cultrex BME portfolio provides consistent and soluble extracellular matrices used globally for cell culture applications that require attachment or a three dimensional matrix for complex tissue development. Cultrex BME matrices are available for a variety of in vitro applications including pluripotent stem cell, spheroid, and organoid culture. Techniques in this video apply generally when using Cultrex BME. Cultrex BME should be maintained at -80 °C for long-term storage. To thaw Cultrex BME, remove a vial from the -80 °C freezer, submerge it in ice, and place it in a refrigerator overnight. We recommend placing pipettes, cryovials, and other materials needed to aliquot or dilute Cultrex BME in a -20 °C freezer so that they are chilled prior to use. The next morning, remove the matrix from the refrigerator while keeping it on ice. Gently swirl the matrix on ice to ensure a homogeneous mixture before aliquoting or downstream use.

Aliquoting Cultrex BME will extend the lifetime and preserve the quality of the matrix by limiting freeze-thaw cycles that compromise matrix composition and polymerization. First, place the thawed Cultrex BME (submerged in ice) in a cell culture hood.  Fluctuations or temporary exposure of Cultrex BME to temperatures above 2-8 °C can cause changes in protein polymerization that could impact the matrix consistency or cell behavior. To ensure the matrix stays cold, we recommend chilling all pipettes and pre-labeled vials in the -20 °C freezer prior to aliquoting. Mix Cultrex BME by pipetting up and down to achieve a homogeneous solution. Aliquot the matrix at a dilution recommended by the manufacturer or that suits your desired application. Use reverse pipetting techniques to minimize bubble formation when pipetting Cultrex BME. You can store aliquots of Cultrex BME at -80 °C for up to six months.

The thin coating method is commonly used for the monolayer culture of adherent cells as well as to expand embryonic and induced pluripotent stem cells.  We recommend Cultrex Ultimatrix BME or Stem Cell Qualified Reduced Growth Factor BME for thin coating. Using pre-chilled pipette tips, mix thawed Cultrex BME by pipetting the solution up and down. Avoid introducing bubbles during this process. Dilute Cultrex BME to the desired concentration in cold serum-free medium. Optimal coating concentration may vary by cell type or application and should be empirically determined. Add dilute matrix to the vessel for coating. Sufficient volume should be added to cover the growth surface of the vessel. Swirl the vessel to encourage full surface coating. Incubate the coated object for one hour at room temperature or at 37 °C for 30 to 45 minutes. Incubation at 4 °C overnight results in a thicker coating, which can be beneficial for some cell lines. Once solidified, aspirate the remaining solution. It is important to make sure the Cultrex BME coating does not dry out, so move on to the next step immediately or seal the plate with parafilm until you are ready to culture.

The thick layer method uses undiluted Cultrex BME to promote the formation of 3-D structures including spheroids. Cells can be plated on top of the concentrated matrix or embedded within the matrix. Using cooled pipette tips, mix thawed Cultrex BME by pipetting the solution up and down. Avoid introducing bubbles during this process. To create a thick layer for growing cells on, either dispense Cultrex BME directly into the culture vessel or if you are embedding cells in the matrix, mix the cell suspension with Cultrex BME prior to adding into the culture vessel. Add enough volume to cover the growth surface. Incubate the plate at 37 °C for 30 minutes to solidify the matrix. Once solidified, aspirate any unpolymerized material and rinse with serum-free medium. Add the appropriate cell culture media to each well, while being careful not to disrupt the matrix surface.

Cultrex Ultimatrix BME and Organoid Qualified Cultrex Reduced Growth Factor BME Type 2 are used to generate domes for organoid culture. Prepare your cell culture vessel by pre-incubating it in the 37 °C incubator. The pre-warmed plate promotes proper dome formation when plating. Using cooled pipette tips, mix thawed Organoid Qualified Cultrex BME or Cultrex Ultimatrix BME by pipetting the solution up and down. Avoid introducing bubbles during this process. Dilute the organoid solution directly in ice-cold Cultrex BME and mix thoroughly. A final matrix concentration of greater than eight milligrams per milliliter is recommended to ensure dome formation. Slowly dispense organoid and matrix mixture onto the center of the pre-warmed culture dish. Use reverse pipetting techniques to minimize bubble formation when creating domes. Depending on your organoids, domes can be arranged as a single dome per well or six to eight domes per well. Incubate the plate at 37 °C for 30 minutes to solidify the matrix. Inverting the plate while incubating may help retain dome formation and prevent settling of organoids on the plates. Once the domes are polymerized, add growth media into the culture wells, being careful not to disturb the Cultrex BME domes. Your Cultrex BME domes are ready for continued culture.