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Colony Forming Unit Assays.

Patrice Penfornis1, Radhika Pochampally2,3

  • 1Cancer Institute, University of Mississippi Medical Center, Jackson, MS, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 30, 2016
PubMed
Summary

This study presents a simple protocol to isolate 80-100 million high-viability mesenchymal stem/stromal cells (MSCs) from bone marrow in 14-20 days. The protocol also includes assays to ensure the quality of these regenerative medicine building blocks.

Keywords:
Colony forming unit assayCultureExpansionIsolationMSCs

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Area of Science:

  • Regenerative Medicine
  • Cell Biology
  • Tissue Engineering

Background:

  • Mesenchymal stem/stromal cells (MSCs) show promise in tissue regeneration, immune modulation, and wound healing, with over 350 clinical trials globally.
  • MSCs are being investigated for various conditions including ischemic, inflammatory, autoimmune, and degenerative disorders.
  • Numerous protocols exist for isolating MSCs from different sources like adipose and bone marrow.

Purpose of the Study:

  • To provide a straightforward protocol for isolating a large number of human MSCs from a small bone marrow sample.
  • To detail a method for achieving high cell viability (>90%) and yield (80-100 million cells).
  • To include quality control assays for assessing MSC preparations.

Main Methods:

  • Utilized double stack culture chambers for cell expansion.
  • Processed a small volume (1-3 mL) of bone marrow aspirate.
  • Incubation period of 14-20 days for cell production.
  • Incorporated two distinct colony-forming unit (CFU) assays for quality assessment.

Main Results:

  • Successfully produced 80-100 million human MSCs from 1-3 mL bone marrow aspirate.
  • Achieved an average cell viability exceeding 90%.
  • Demonstrated the utility of CFU assays in evaluating cell preparation quality.

Conclusions:

  • The presented protocol offers an efficient and reliable method for generating clinical-grade MSCs.
  • High cell viability and yield are achievable with the described technique.
  • CFU assays are crucial for ensuring the quality and consistency of MSC preparations for therapeutic applications.