Optimisation of a potency assay for the assessment of immunomodulative potential of clinical grade multipotent mesenchymal stromal cells

Abstract

Clinical use of multipotent Mesenchymal Stromal Cell (MSC)-based medicinal products requires their production in compliance with Good Manufacturing Practices, thus ensuring that the final drug product meets specifications consistently from batch to batch in terms of cell viability, identity, purity and potency. Potency relates to the efficacy of the medicine in its target clinical indication, so adequate release tests need to be defined and validated as quality controls. Herein we report the design and optimisation of parameters affecting the performance of an in vitro cell-based assay for assessing immunomodulatory potential of clinical grade MSC for human use, based on their capacity to inhibit proliferation of T lymphocytes under strong polyclonal stimuli. The resulting method was demonstrated to be reproducible and relatively simple to execute. Two case studies using clinical grade MSC are presented as examples to illustrate the applicability of the methodology described in this work.

Characterization of a Cytomegalovirus-Specific T Lymphocyte Product Obtained Through a Rapid and Scalable Production Process for Use in Adoptive Immunotherapy

“…Peripheral blood mononuclear cells (PBMC) were collected from the peripheral blood of 9 CMV + seropositive healthy donors with donor informed consent and the corresponding approval of the ethics committee (complies with good clinical practice CPMP/ICH/135/95 and Real Decreto 1090/2015). PBMC isolation, cryopreservation, and thawing were performed as described in previous studies (23). Positive serology for CMV IgG was confirmed using chemiluminescence (Abbot, Abbot Park, Illinois, USA).…”

“…A cytotoxicity assay was performed to detect the manufactured lymphocytes' ability to attack either autologous or allogeneic lymphoblasts with or without presentation of CMV pp65 peptides (Miltenyi). Lymphoblasts were labeled with CFSE (CellTrace TM CFSE Cell Proliferation Kit; Invitrogen, Waltham, MA, USA), as already reported (23). Allogeneic and autologous non-pulsed lymphoblasts were stained with a high concentration of CFSE (2.5 µM), while pp65 peptide pool-pulsed lymphoblasts were labeled with a low concentration of CFSE (0.25 µM) (25).…”

Osteogenic commitment of Wharton’s jelly mesenchymal stromal cells: mechanisms and implications for bioprocess development and clinical application

“…BM-MSC (n = 3) and WJ-MSC (n = 3) (passage 3-5) were isolated according to 'Good Manufacturing Practice for Advanced Therapy Medicinal Products' (GMP for ATMPs, European Commission Guidelines of 2017. 11.22) and further expanded in Dulbecco's modified Eagle's medium (DMEM) (31885-023; Gibco) containing 2 mM glutamine and supplemented with 10% human serum B (hSerB)-'expansion medium' [37,38]. All cell cultures were maintained at 37°C and 5% CO 2 in humidified incubators, and media were changed every 3-4 days.…”

“…Immunophenotypic characterisation of MSC was performed using the following antibodies: mouse anti-human CD45-fluorescein isothiocyanate (CD45-FITC) (Clone HI30; 555482; BD Pharmingen), anti-human CD105phycoerythrin (CD105-PE) (Clone 43A4E1; 130-117-696; Miltenyi Biotec), anti-human HLA-DR-FITC (Clone L243; 347363; BD Biosciences), anti-human CD90-PE (Clone F15-42-1-5; IM1840U; Beckman Coulter), anti-human CD31-FITC (Clone WM59; 555445; BD Pharmingen) and anti-human CD73-PE (Clone AD2; 550257; BD Pharmingen). Cells were stained for 15 min at room temperature (RT), washed and re-suspended in phosphate-buffered saline (PBS) (14190-094; Gibco) as described elsewhere [38]. Acquisition and data analysis were performed using a FACSCalibur cytometer and the CellQuest Pro software (Becton Dickinson), respectively.…”

Production and quality testing of multipotent mesenchymal stromal cell therapeutics for clinical use

“…Assays to assess the immunomodulation capacities of MSC include: Inhibition of mitogen/alloantigen‐induced T‐cell proliferation Suppression of CD154 activation marker expression on CD4 T cells CD200 expression as an indicator or surrogate marker for suppression (however, blocking did not impair suppressive strength) Inducible indoleamine 2,3‐dioxygenase 1 and programmed death ligand 1 expression TNF‐α expression in lipopolysaccharide‐stimulated macrophages MSC‐induced interleukin (IL)‐10 release from blood cells …”