Good Manufacturing Practice-Compliant Expansion of Marrow-Derived Stem and Progenitor Cells for Cell Therapy

Abstract

Ex vivo expansion is being used to increase the number of stem and progenitor cells for autologous cell therapy. Initiation of pivotal clinical trials testing the efficacy of these cells for tissue repair has been hampered by the challenge of assuring safe and high-quality cell production. A strategy is described here for clinical-scale expansion of bone marrow (BM)-derived stem cells within a mixed cell population in a completely closed process from cell collection through postculture processing using sterile connectable devices. Human BM mononuclear cells (BMMNC) were isolated, cultured for 12 days, and washed postharvest using either standard open procedures in laminar flow hoods or using automated closed systems. Conditions for these studies were similar to long-term BM cultures in which hematopoietic and stromal components are cultured together. Expansion of marrow-derived stem and progenitor cells was then assessed. Cell yield, number of colony forming units (CFU), phenotype, stability, and multilineage differentiation capacity were compared from the single pass perfusion bioreactor and standard flask cultures. Purification of BMMNC using a closed Ficoll gradient process led to depletion of 98% erythrocytes and 87% granulocytes, compared to 100% and 70%, respectively, for manual processing. After closed system culture, mesenchymal progenitors, measured as CD105 + CD166 + CD14 − CD45 − and fibroblastic CFU, expanded 317-and 364-fold, respectively, while CD34 + hematopoietic progenitors were depleted 10-fold compared to starting BMMNC. Cultured cells exhibited multilineage differentiation by displaying adipogenic, osteogenic, and endothelial characteristics in vitro. No significant difference was observed between manual and bioreactor cultures. Automated culture and washing of the cell product resulted in 181 × 10 6 total cells that were viable and contained fibroblastic CFU for at least 24 h of storage. A combination of closed, automated technologies enabled production of good manufacturing practice (GMP)-compliant cell therapeutics, ready for use within a clinical setting, with minimal risk of microbial contamination.

Angiogenic and Osteogenic Potential of Bone Repair Cells for Craniofacial Regeneration

“…Through cell surface marker characterization of BRCs, it was determined that the SPP ex vivo cell expansion processing of bone marrow aspirate was capable The cell-processing system employed to produce these cells utilizes an SPP protocol. 20,22,25,35 Gastens et al 35 examined this system and its ability to expand bone-marrowderived cells to produce clinical-scale cell numbers. These initial studies compared cell phenotypes of cells produced with this closed SPP system to phenotypes of cells cultured with an open system utilizing conventional tissue culture parameters.…”

“…This served as an indication that the BRC product was highly enriched with cells possessing mesenchymal and angiogenic potential and is in accordance with previous reports demonstrating enrichment of theses cell types with this cell-processing protocol. 26,35 Although the cell product is highly enriched for vascular and mesenchymal cells as indicated by cell surface marker expression and in vitro differentiation capacity, it is clear that in vitro osteogenic differentiation of cell populations does not guarantee bone-forming capacity in vivo. Meijer et al performed a clinical study evaluating the repair of jawbone defects in six subjects treated with autologous cells expanded using an open system, 39 similar to the protocol used in traditional tissue culture of mesencyhmal stem cells.…”

Therapeutic Potentials of Mesenchymal Stem Cells Derived from Human Umbilical Cord

“…However, in addition to ethical and political concerns, their clinical application is also severely limited by their lack of accessibility, technique difficulties in purification and manipulation as well as concerns for formation of teratoma. In contrast, adult stem cells (ASCs) which can be easily harvested from various tissues, such as skin [19], bone marrow [20] and adipose [21], might be used clinically to treat disorders of vulnerable vital organs with fewer concerns than ESCs. However, their application becomes less preferable when considering limited numbers, decreased growth and differential capacities with increasing age as well as invasive harvesting procedures [22].…”

A novel composition for the culture of human adipose stem cells which includes complement C3

“…Growth medium was replaced with DMEM supplemented with 100 nM dexamethasone, 50 lM L-ascorbic acid-2-phosphate, 10 mM b-glycerol phosphate, 10% FBS, and Pen-Strep. After three rounds of induction for 3 days each, the cells were lysed with 500 lL/10 cm 2 of 0.5 N HCl, followed by mixing at 500 rpm for 4 h at 4°C to solubilize the calcium, and centrifugation at 1,000g for 5 min (Gastens et al 2007). 20 lL of the supernatant was used for calcium estimation per manufacturer's instructions (Calcium Quantitative Kit, Pointe Scientific Inc).…”