More Human BM-MSC With Similar Subpopulation Composition and Functional Characteristics Can Be Produced With a GMP-Compatible Fabric Filter System Compared to Density Gradient Technique

Abstract

BackgroundMesenchymal stromal cells (MSCs), multipotent progenitors that can be isolated from a variety of different tissues, are becoming increasingly important as cell therapeutics targeting immunopathologies and tissue regeneration. Current protocols for MSC isolation from bone marrow (BM) rely on density gradient centrifugation (DGC), and the production of sufficient MSC doses is a critical factor for conducting clinical MSC trials. Previously, a Good Manufacturing Practice (GMP)–compatible non-woven fabric filter device system to isolate MSCs was developed to increase the MSC yield from the BM. The aim of our study was to compare high-resolution phenotypic and functional characteristics of BM-MSCs isolated with this device and with standard DGC technology.MethodsHuman BM samples from 5 donors were analyzed. Each sample was divided equally, processing by DGC, and with the filter device. Stem cell content was assessed by quantification of colony-forming units fibroblasts (CFU-F). Immunophenotype was analyzed by multicolor flow cytometry. In vitro trilineage differentiation potential, trophic factors, and IDO-1 production were assessed. Functionally, immunomodulatory potential, wound healing, and angiogenesis were assayed in vitro.ResultsThe CFU-F yield was 15-fold higher in the MSC preparations isolated with the device compared to those isolated by DGC. Consequently, the MSC yield that could be manufactured at passage 3 per mL collected BM was more than 10 times higher in the device group compared to DGC (1.65 × 109 vs. 1.45 × 108). The immunomodulatory potential and IDO-1 production showed donor-to-donor variabilities without differences between fabric filter-isolated and DGC-isolated MSCs. The results from the wound closure assays, the tube formation assays, and the trilineage differentiation assays were similar between the groups with respect to the isolation method. Sixty-four MSC subpopulations could be quantified with CD140a+CD119+CD146+ as most common phenotype group, and CD140a+CD119+CD146+MSCA-1–CD106–CD271– and CD140a+CD119+CD146–MSCA-1–CD106–CD271– as most frequent MSC subpopulations. As trophic factors hepatocyte growth factor, epidermal growth factor, brain-derived neurotrophic factor, angiopoietin-1, and vascular endothelial growth factor A could be detected in both groups with considerable variability between donors, but independent of the respective MSC isolation technique.ConclusionThe isolation of MSCs using a GMP-compatible fabric filter system device resulted in higher yield of CFU-F, producing substantially more MSCs with similar subpopulation composition and functional characteristics as MSCs isolated by DGC.

Small volume bone marrow aspirates with high progenitor cell concentrations maximize cell therapy dose manufacture and substantially reduce donor hemoglobin loss

“…For the following passages, the projected MSC yield was calculated according to the formula N = N 0 × 2 n , where N is the projected MSC yield at end of the respective passage, N 0 is the projected MSC yield at P0, and n are the cumulative population doublings (cPDs) of the respective passage, i.e., number of cumulative doublings occurring in this passage. For the calculation, we used representative cPDs for MSCs as reported previously [ 19 ].…”

Turning Trash Into Treasure: A Simple Protocol for Human Mesenchymal Stromal Cell Isolation From Used Bone Marrow Collection Kits

“…However, this methodology requires skilled manual dexterity and entails a prolonged duration for the isolation of cells (Grisendi et al, 2010;Yamamoto et al, 2015;Zhu & Murthy, 2013). Alternatively, the evaluation of the bone marrow collection kits for Use premixed commercialized differentiation kits hMSC isolation has been a subject of high interest (Anasiz, 2018;Capelli et al, 2009;Lopes et al, 2020;Otsuru et al, 2013;Spohn et al, 2021). For clinical indications, harvested bone marrow from a suitable donor is pooled and filtered through the bone marrow collection kit to remove residual fat, bone chips, and clots (McDaniel et al, 2017).…”

Translating mesenchymal stem cell and their exosome research into GMP compliant advanced therapy products: Promises, problems and prospects

“…Recently, a fully automated and closed ficoll system has been applied to harvest cells from bone marrow (BM), and a recent report analyzing a GMP-compatible nonwoven fabric filter system revealed that it could potentially increase isolated cell numbers without altering the properties of MSCs. 22 Nevertheless for different cell types and production processes, it is usually necessary to customize different automation equipment, greatly increasing capital investment and time consumption. 23 Furthermore, calibration of custom equipment and verification of GMP compliance are timeconsuming and labor-intensive processes.…”

“…For well‐defined procedures, especially those for late‐stage clinical trials, it is preferable to maintain the entire manufacturing process in a closed system. Recently, a fully automated and closed ficoll system has been applied to harvest cells from bone marrow (BM), and a recent report analyzing a GMP‐compatible nonwoven fabric filter system revealed that it could potentially increase isolated cell numbers without altering the properties of MSCs 22 . Nevertheless for different cell types and production processes, it is usually necessary to customize different automation equipment, greatly increasing capital investment and time consumption 23 .…”