The Manufacture of GMP-Grade Bone Marrow Stromal Cells with Validated In Vivo Bone-Forming Potential in an Orthopedic Clinical Center in Brazil

Abstract

In vitro-expanded bone marrow stromal cells (BMSCs) have long been proposed for the treatment of complex bone-related injuries because of their inherent potential to differentiate into multiple skeletal cell types, modulate inflammatory responses, and support angiogenesis. Although a wide variety of methods have been used to expand BMSCs on a large scale by using good manufacturing practice (GMP), little attention has been paid to whether the expansion procedures indeed allow the maintenance of critical cell characteristics and potency, which are crucial for therapeutic effectiveness. Here, we described standard procedures adopted in our facility for the manufacture of clinical-grade BMSC products with a preserved capacity to generate bone in vivo in compliance with the Brazilian regulatory guidelines for cells intended for use in humans. Bone marrow samples were obtained from trabecular bone. After cell isolation in standard monolayer flasks, BMSC expansion was subsequently performed in two cycles, in 2- and 10-layer cell factories, respectively. The average cell yield per cell factory at passage 1 was of 21.93 ± 12.81 × 106 cells, while at passage 2, it was of 83.05 ± 114.72 × 106 cells. All final cellular products were free from contamination with aerobic/anaerobic pathogens, mycoplasma, and bacterial endotoxins. The expanded BMSCs expressed CD73, CD90, CD105, and CD146 and were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages in vitro. Most importantly, nine out of 10 of the cell products formed bone when transplanted in vivo. These validated procedures will serve as the basis for in-house BMSC manufacturing for use in clinical applications in our center.

Cell detachment rates and confluence of fibroblast and osteoblast cell culture using different washing solutions

“…The use of RL washing solution on osteoblast cells showed faster cell detachment than its use on fibroblast cells. This is in accordance with research which stated that RL solution can increase the expansion of osteoblast cells in bone marrow stromal cells (BMSCs) (Dias et al, 2019).…”

Reduced Osteogenic Differentiation Potential In Vivo in Acute Myeloid Leukaemia Patients Correlates with Decreased BMP4 Expression in Mesenchymal Stromal Cells

“…The subcutaneous xenotransplantation assay with AML-hMSCs and HD-hMSCs was performed as previously reported ( 10 ). All animal procedures were performed following the guidelines of the Institutional Animal Care and Use Committee (010/2020).…”

“…The implants obtained were processed for histology and immunohistochemistry analyses as previously reported ( 10 ). Rabbit anti-OSX antibody (sc-393325, Santa Cruz Biotechnology, USA) diluted 1：100 and goat anti-BMP-2/4 antibody (sc-6267, Santa Cruz Biotechnology, USA) diluted 1：100 were used.…”

“…MicroCT acquisition was performed as described in Dias ( 10 ). The deep learning segmentation tool available on DragonFly ( 11 ) was used to separate the newly formed bone from hydroxyapatite.…”

Mesenchymal stromal/stem cells: historical perspective and ongoing challenges

“…To achieve the needs for clinical application of MSCs, a variety of tools designed to improve isolation, differentiation, and in vitro expansion was developed (Mastrolia et al, 2019;Dias et al 2019). We are now able to expand MSCs from different sources in order to get sufficient number of cells to use in different clinical protocols, although the mechanisms of action for the distinct clinical conditions are still largely unknown (Ankrum & Karp, 2010, Mastrolia et al, 2019.…”