GMP-Compliant Isolation and Expansion of Bone Marrow-Derived MSCs in the Closed, Automated Device Quantum Cell Expansion System

Abstract

The estimated frequency of MSCs in BM is about 0.001-0.01% of total nucleated cells. Most commonly, one applied therapeutic cell dose is about 1-5 million MSCs/kg body weight, necessitating a reliable, fast, and safe expansion system. The limited availability of MSCs demands for an extensive ex vivo amplification step to accumulate sufficient cell numbers. Human platelet lysate (PL) has proven to be a safe and feasible alternative to animal-derived serum as supplement for MSC cultivation. We have investigated the functionally closed automated cell culture hollow fiber bioreactor Quantum cell expansion system as an alternative novel tool to conventional tissue flasks for efficient clinical-scale MSC isolation and expansion from bone marrow using PL. Cells expanded in the Quantum system fulfilled MSC criteria as shown by flow cytometry and adipogenic, chondrogenic, and osteogenic differentiation capacity. Cell surface expression of a variety of chemokine receptors, adhesion molecules, and additional MSC markers was monitored for several passages by flow cytometry. The levels of critical media components like glucose and lactate were analyzed. PDGF-AA, PDGF-AB/ BB, bFGF, TGF-b1, sICAM-1, sVCAM-1, RANTES, GRO, VEGF, sCD40L, and IL-6 were assessed using a LUMINEX platform. Originally optimized for the use of fetal calf serum (FCS) as supplement and fibronectin as coating reagent, we succeeded to obtain an average of more than 100 ´ 10 6 of MSCs from as little as 18.8-28.6 ml of BM aspirate using PL. We obtained similar yields of MSCs/µl BM in the FCS-containing and the xenogen-free expansion system. The Quantum system reliably produces a cellular therapeutic dose in a functionally closed system that requires minimal manipulation. Both isolation and expansion are possible using FCS or PL as supplement. Coating of the hollow fibers of the bioreactor is mandatory when loading MSCs. Fibronectin, PL, and human plasma may serve as coating reagents.

Chondrogenic potential of hASCs expanded in flask or in a hollow-fiber bioreactor

“…Immunophenotyping showed that hASCs expanded in flask or HFBR retained a characteristic MSC immunophenotypic profile. In this sense hASCs from both cell expansion systems were remarkably similar, confirming data from the literature obtained with bone marrow-derived MSCs expanded in the same HFBR used in this study [43,44]. Concomitantly, the contamination of endothelial and hematopoietic cells was retained low enough to consider the cell therapy product for both cell expansion systems as pure [18].…”

Venous Blood Derivatives as FBS-Substitutes for Mesenchymal Stem Cells: A Systematic Scoping Review

“…The substitution of animal compounds is highly recommended for good manufacturing practices (GMP) (13,120) guidelines, eliminating the need for animal additives for regenerative therapies in humans (96)(97)(98)(99)(100)(101)(102). VBD-supplemented MSC were applied for sinus lift augmentation (97), regeneration of alveolar clefts (96), even as regeneration of large anterior mandibular defect (98) and radiation burn treatment (102).…”

Large-Scale Clinical Expansion of Mesenchymal Stem Cells in the GMP-Compliant, Closed Automated Quantum® Cell Expansion System: Comparison with Expansion in Traditional T-Flasks

“…Until now, few studies have been initiated to evaluate the system. Roberts et al demonstrated that human embryonic stem cells (hESC) culture can be scaled up through the use of the bioreactor [25] and two recently published studies evaluated the Quantum ® device to isolate and expand BM-MSC in FCS or platelet lysate (PL)-enriched media in two-step processes [26,27].…”