Culture and Use of Mesenchymal Stromal Cells in Phase I and II Clinical Trials

Present in numerous tissues, mesenchymal stem cells/multipotent stromal cells (MSCs) can differentiate into different cell types from a mesoderm origin. Their potential has been extended to pluripotency, by their possibility of differentiating into tissues and cells of nonmesodermic origin. Through the release of cytokines, growth factors and biologically active molecules, MSCs exert important paracrine effects during tissue repair and inflammation. Moreover, MSCs have immunosuppressive properties related to non-HLA restricted immunosuppressive capacities. All these features lead to an increasing range of possible applications of MSCs, from treating immunological diseases to tissue and organ repair, that should be tested in phase I and II clinical trials. The most widely used MSCs are cultured from bone marrow or adipose tissue. For clinical trial implementation, BM MSCs and ADSCs should be produced according to Good Manufacturing Practices. Safety remains the major concern and must be ensured during culture and validated with relevant controls. We describe some applications of MSCs in clinical trials

Translation of a standardized manufacturing protocol for mesenchymal stromal cells: A systematic comparison of validation and manufacturing data

Background Many data are available on expansion protocols for mesenchymal stromal cells (MSCs) for both experimental settings and manufacturing for clinical trials. However, there is a lack of information on translation of established protocols for Good Manufacturing Practice (GMP) from validation to manufacturing for clinical application. We present the validation and translation of a standardized pre-clinical protocol for isolation and expansion of MSCs for a clinical trial for reconstitution of alveolar bone. Methods Key parameters of 22 large-scale expansions of MSCs from bone marrow (BM) for validation were compared with 11 expansions manufactured for the clinical trial “Jaw bone reconstruction using a combination of autologous mesenchymal stromal cells and biomaterial prior to dental implant placement (MAXILLO1)” aimed at reconstruction of alveolar bone. Results Despite variations of the starting material, the robust protocol led to stable performance characteristics of expanded MSCs. Manufacturing of the autologous advanced therapy medicinal product MAXILLO-1-MSC was possible, requiring 21 days for each product. Transport of BM aspirates and MSCs within 24 h was guaranteed. MSCs fulfilled quality criteria requested by the national competent authority. In one case, the delivered MSCs developed a mosaic in chromosomal finding, showing no abnormality in differentiation capacity, growth behavior or surface marker expression during long-term culture. The proportion of cells with the mosaic decreased in long-term culture and cells stopped growth after 38.4 population doublings. Conclusions Clinical use of freshly prepared MSCs, manufactured according to a standardized and validated protocol, is feasible for bone regeneration, even if there was a long local distance between manufacturing center and clinical site. Several parameters, such as colony forming units fibroblasts (CFU-F), percentage of CD34+ cells, cell count of mononuclear cells (MNCs) and white blood cells (WBCs), of the BM may serve as a predictive tool for the yield of MSCs and may help to avoid unnecessary costs for MSC manufacturing due to insufficient cell expansion rates.publishedVersio

Comparison of Enzymatic and Non-Enzymatic Means of Dissociating Adherent Monolayers of Mesenchymal Stem Cells

The dissociation of adherent mesenchymal stem cell (MSC) monolayers with trypsin and enzyme-free dissociation buffer was compared. A significantly lower proportion of viable cells were obtained with enzyme-free dissociation buffers compared to trypsin. Subsequently, the dissociated cells were re-seeded on new cell culture dishes and were subjected to the MTT assay 24 h later. The proportion of viable cells that reattached was significantly lower for cells obtained by dissociation with enzyme-free dissociation buffer compared to trypsin. Frozen–thawed MSC displayed a similar trend, yielding consistently higher cell viability and reattachment rates when dissociated with trypsin compared to enzyme-free dissociation buffer. It was also demonstrated that exposure of trypsin-dissociated MSC to enzyme-free dissociation buffer for 1 h had no significant detrimental effect on cell viability

Donor Variability in Growth Kinetics of Healthy hMSCs Using Manual Processing:Considerations for Manufacture of Cell Therapies

Human mesenchymal stromal cells (hMSCs) are excellent candidates for cell therapy but their expansion to desired clinical quantities can be compromised by ex vivo processing, due to differences between donor material and process variation. The aim of this article is to characterize growth kinetics of healthy baseline "reference" hMSCs using typical manual processing. Bone-marrow derived hMSCs from ten donors are isolated based on plastic adherence, expanded, and analyzed for their growth kinetics until passage 4. Results indicate that hMSC density decreases with overall time in culture (p < 0.001) but no significant differences are observed between successive passages after passage 1. In addition, fold increase in cell number dropped between passage 1 and 2 for three batches, which correlated to lower performance in total fold increase and expansion potential of these batches, suggesting that proliferative ability of hMSCs can be predicted at an early stage. An indicative bounded operating window is determined between passage 1 and 3 (PDL < 10), despite the high inter-donor variability present under standardized hMSC expansion conditions used. hMSC growth profile analysis will be of benefit to cell therapy manufacturing as a tool to predict culture performance and attainment of clinically-relevant yields, therefore stratifying the patient population based on early observation

Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial

Background: ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis. Methods: Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5–10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority. Findings: With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging). Interpretation: Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic. Funding: EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876).The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/FP7-HEALTH-2009); REBORNE Project (GA: 241876

Mesenchymal stromal cells’ therapy for polyglutamine disorders: where do we stand and where should we go?

Polyglutamine (polyQ) diseases are a group of inherited neurodegenerative disorders caused by the expansion of the cytosine-adenine-guanine (CAG) repeat. This mutation encodes extended glutamine (Q) tract in the disease protein, resulting in the alteration of its conformation/physiological role and in the formation of toxic fragments/aggregates of the protein. This group of heterogeneous disorders shares common molecular mechanisms, which opens the possibility to develop a pan therapeutic approach. Vast efforts have been made to develop strategies to alleviate disease symptoms. Nonetheless, there is still no therapy that can cure or effectively delay disease progression of any of these disorders. Mesenchymal stromal cells (MSC) are promising tools for the treatment of polyQ disorders, promoting protection, tissue regeneration, and/or modulation of the immune system in animal models. Accordingly, data collected from clinical trials have so far demonstrated that transplantation of MSC is safe and delays the progression of some polyQ disorders for some time. However, to achieve sustained phenotypic amelioration in clinics, several treatments may be necessary. Therefore, efforts to develop new strategies to improve MSC's therapeutic outcomes have been emerging. In this review article, we discuss the current treatments and strategies used to reduce polyQ symptoms and major pre-clinical and clinical achievements obtained with MSC transplantation as well as remaining flaws that need to be overcome. The requirement to cross the blood-brain-barrier (BBB), together with a short rate of cell engraftment in the lesioned area and low survival of MSC in a pathophysiological context upon transplantation may contribute to the transient therapeutic effects. We also review methods like pre-conditioning or genetic engineering of MSC that can be used to increase MSC survival in vivo, cellular-free approaches-i.e., MSC-conditioned medium (CM) or MSC-derived extracellular vesicles (EVs) as a way of possibly replacing the use of MSC and methods required to standardize the potential of MSC/MSC-derived products. These are fundamental questions that need to be addressed to obtain maximum MSC performance in polyQ diseases and therefore increase clinical benefits.Portuguese Foundation for Science and Technology: SFRH/BD/148877/2019; CENTRO01-0145-FEDER-000008 CENTRO-01-0145FEDER-022095 POCI-01-0145-FEDER-016719 POCI-01-0145-FEDER-029716 POCI01-0145-FEDER-016807 POCI-01-0145-FEDER016390 UID4950/2020 CENTRO-01-0145-FEDER-022118info:eu-repo/semantics/publishedVersio