Isolation and Maintenance of Mesenchymal Stem Cell

Mesenchymal stem cells (MSCs) hold great promise in regenerative medicine due to their remarkable ability to differentiate into various cell types and their potential for tissue repair. Among the different sources of MSCs, adipose tissue has emerged as a rich and easily accessible source of isolation. Isolation and maintenance of adipose-derived mesenchymal stem cells (AD-MSCs) have become essential techniques in stem cell research and therapeutic applications.

In this protocol, Creative Biolabs will delve into the materials required, the isolation procedure, and important notes to consider when working with AD-MSCs.

Materials Required

• Adipose tissue sample
• Dulbecco's modified eagle's medium (DMEM)
• Fetal bovine serum (FBS)
• Trypsin-EDTA solution
• Phosphate-buffered saline (PBS)
• Antibiotics

Procedure

Isolation of AD-MSCs

Wash the adipose tissue sample with PBS and mince the tissue into small fragments. Digest the tissue fragments with collagenase or other appropriate enzymes for a specific period. Inactivate the enzymes by adding a culture medium supplemented with FBS. Centrifuge the cell suspension to separate the stromal vascular fraction (SVF) from the adipocyte layer. Discard the adipocyte layer and resuspend the SVF in a fresh culture medium.

Primary Culture

Plate the SVF cells onto a sterile culture dish or flask and incubate. After 24-48 hours, remove non-adherent cells and debris by gently washing the culture dish with PBS. Replace the medium with fresh medium containing FBS and continue the culture. Monitor cell growth daily and change the medium every 2-3 days.

Passaging of AD-MSCs

Once the cells reach 70-80% confluence, they are ready for passaging. Aspirate the culture medium and wash the cells with PBS. Add an appropriate volume of trypsin-EDTA solution to cover the cells and incubate for a few minutes until the cells detach. Neutralize trypsin activity by adding culture medium containing FBS. Centrifuge the cell suspension and resuspend the pellet in fresh culture medium. Plate the cells into new culture dishes or flasks at a desired density and continue the culture.

Notes

• The success of AD-MSC isolation depends on the quality of the adipose tissue sample. Proper handling and transportation to the laboratory are crucial to maintaining cell viability.
• The choice of collagenase or other enzymes for tissue digestion should be based on their effectiveness in isolating MSCs while minimizing cell damage.
• During the primary culture, it is important to remove non-adherent cells and debris to promote the growth of AD-MSCs.
• AD-MSCs should be passaged at appropriate intervals to prevent over-confluence and maintain their multipotency. Passage number should be monitored and recorded.
• Maintain strict aseptic techniques throughout the isolation and maintenance process to prevent contamination, as it can compromise the quality and functionality of AD-MSCs.
• It is essential to perform phenotypic characterization and functional validation of isolated AD-MSCs to ensure their identity and potency.

By following a carefully designed protocol, researchers can obtain a population of multipotent cells with the potential to contribute to tissue repair and regeneration. Creative Biolabs can provide valuable insights for the isolation and maintenance of AD-MSCs and contribute to the advancement of regenerative therapies.

If you have any questions about the protocol and need help, please do not hesitate to contact us.

Reference

1. Bourin P, et al. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy, 2013, 15(6): 641-648.

For Research Use Only. Not For Clinical Use.