GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSC

Abstract

BackgroundMesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components.Methods and FindingsWe established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform.ConclusionsThe five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion.

Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions

“…(5,6). The increasing use of MSCs as Advanced Therapy Medicinal Products (ATMP) has led to production processes that need to meet Good Manufacturing Practices (GMP) (7,8).…”

Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation

“…23 To avoid the selection of a specific subset of BMSC we used the conventional method of plastic adherence purification, which is the gold standard method for GMP isolation and large-scale expansion of bone marrow-derived mesenchymal stem cells. 24 We cultivated BMSC with bFGF to stimulate cell proliferation, but we used low bFGF dosage to avoid the trigger of osteogenic program. [25][26][27] After initial plating, BMSC cultures were expanded to 70-80% confluency.…”

Cell therapy for bone fracture repair: A comparative preclinical review of mesenchymal stromal cells from bone marrow and from adipose tissue

“…Alternative harvesting sites are femoral heads (from total hip arthroplasty of an elderly patient population), sternum and less frequently vertebrae. The commonly applied preparation method for the generation of BM-MSC is density gradient centrifugation, but other methods rely on their properties to adhere to plastic [1,[61][62][63][64][65][66] . Ad-MSC are isolated from lipoaspirates obtained during liposuction, lipoplasty or lipectomy procedures.…”