Cryopreserved bone marrow aspirate concentrate as a cell source for the colony-forming unit fibroblast assay.

Autor: Berger DR; Regenexx, LLC, Broomfield, Colorado, USA., Aune ET; Regenexx, LLC, Broomfield, Colorado, USA., Centeno CJ; Regenexx, LLC, Broomfield, Colorado, USA; Centeno-Schultz Clinic, Broomfield, Colorado, USA., Steinmetz NJ; Regenexx, LLC, Broomfield, Colorado, USA. Electronic address: nsteinmetz@regenexx.com.
Jazyk: angličtina
Zdroj: Cytotherapy [Cytotherapy] 2020 Sep; Vol. 22 (9), pp. 486-493. Date of Electronic Publication: 2020 Jun 19.
DOI: 10.1016/j.jcyt.2020.04.091
Abstrakt: Purpose: The prevalence of connective tissue progenitor cells within a cell-based therapy is often quantified using the colony-forming unit fibroblast (CFU-F) assay. The present study investigates the feasibility of using cryopreserved bone marrow aspirate concentrate (BMAC) as an alternative cell source to fresh BMAC for CFU-F quantification.
Methods: Freshly prepared and corresponding cryopreserved BMAC samples from patients receiving autologous cell therapy (n = 98) were analyzed using the CFU-F assay for comparison. Cultures were established by directly plating BMAC at low cell densities and maintained for a 2-week growth period. Colonies were enumerated to determine CFU-F frequency, and a subset of cultures was imaged and analyzed to quantify colony area and density.
Results: A nonlinear relationship was observed between plating density and CFU-F frequency over a wide range in plating densities (~30-fold). Cryopreserved BMAC yielded recoverable (77 ± 23%) and viable (73 ± 9%) nucleated cells upon thawing. After cryopreservation, CFU-F frequencies were found to be significantly lower (56.6 ± 34.8 vs. 50.3 ± 31.7 colonies per million nucleated cells). Yet the number of CFU-F in fresh and cryopreserved BMAC were strongly correlated (r = 0.87) and had similar area and densities. Further, moderate correlations were observed between the number of CFU-F and nucleated cells, and both the mean colony area and density were negatively correlated with patient age. Notably, no relationship was found between CFU-F frequency and age, regardless of whether fresh or cryopreserved BMAC was used.
Conclusions: Freshly prepared and cryopreserved BMAC yielded correlated results when analyzed using the CFU-F assay. Our findings support the cryogenic storage of patient BMAC samples for retrospective CFU-F analyses, offering a potential alternative for characterizing BMAC and furthering our understanding of progenitor cells in relation to clinical outcome.
(Copyright © 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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