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

Abstract

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.

Colony Forming Potential and Protein Composition of Commercial Umbilical Cord Allograft Products in Comparison With Autologous Orthobiologics

“…It should be noted that while we used culture conditions that have reliably generated CFU-Fs from bone marrow, the lack of CFU-Fs does not preclude the existence of stem cells within UC allograft products, as laboratory variables–including choice of basal media, growth factors or other culture supplements, and plating density, and environmental conditions, such as oxygen levels–can affect colony formation. 1 Despite these variables, the present study reached similar conclusions to previously analyzed amniotic fluid allograft products; all UC allograft products failed to form CFU-Fs using conventional methods, contradicting claims that these are stem cell therapies. 26 On the other hand, BMC from middle-aged patients undergoing routine orthobiologic procedures consistently produces CFU-Fs, with frequencies ranging from tens to hundreds of colonies per million cells plated.…”

“…26 On the other hand, BMC from middle-aged patients undergoing routine orthobiologic procedures consistently produces CFU-Fs, with frequencies ranging from tens to hundreds of colonies per million cells plated. 1…”

“…The aspirated marrow was manually processed into BMC for a same-day procedure, as previously described. 1 A small volume of BMC (~0.1 mL), typically reserved for routine quality control, was set aside for cellular analysis (n = 5). Likewise, small volumes of leukocyte-poor PRP derived from whole blood (~6-fold increase in platelet concentration; n = 3) and plasma derived from bone marrow aspirate (~2-fold increase in platelet concentration; n = 3), collectively referred to as plasma preparations (Appendix Table A1, available in the online version of this article), were manually processed 2 and maintained in ultra–low temperature storage (–80°C) before protein analysis.…”

Mesenchymal stromal cell-based therapy for cartilage regeneration in knee osteoarthritis

“…MSCs are spindle-shaped and adherent cells, capable of proliferation, self-renewal, and differentiating into cells of multi-lineage. One of the characteristic features of MSCs is adhering to tissue culture plastic and generating colonies when plated at low densities [ 78 ]. MSCs growing from individual foci, or colonies from the microscopic view, and these colonies generated from progenitor cells have been called the colony-forming unit fibroblast [ 79 ].…”

Image Guided Injection of Anterior Cruciate Ligament Tears with Autologous Bone Marrow Concentrate and Platelets: Midterm Analysis from A Randomized Controlled Trial

“…Please see our prior publication for a detailed description of the bone marrow aspiration and concentration protocol. 20,45 This isolation produced 2-5 mL of BMC, which was sterilely transported from the clinic-based laboratory to the clinic procedural suite. A total nucleated cell (TNC) count of the injectate was performed and recorded by lab staff with a cell counter (TC10; Bio-Rad Laboratories, Hercules, CA, USA).…”