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.

Figure 1

Standardization of cell isolation. (a) Schematic representation of the protocol used for determining the optimal initial seeding densities. (b) Fold increase in cell number (BMSCs in passage 1). n = 4; p = 0.015.

Figure 2

Schematic representation of the in vitro bone marrow stromal cells manufacturing in a GMP-compliant semiclosed system. After bone marrow dissociation from bone spicules, nucleated cells were seeded in T-75 flasks for BMSC isolation (P0), followed by expansion in 2-layer cell factories (P1) and subsequent expansion in 10-layer cell factories (P2).

Figure 3

In vitro osteogenic, adipogenic, and chondrogenic potential of BMSC products. Representative images of (a) a noninduced BMSC layer, (b) mineralized nodules visualized by Von Kossa staining, and (c) intracellular lipid accumulation stained with Oil Red O. (d–i) Chondrogenic differentiation: (d) H&E staining, (e, f) Masson Trichrome staining, and (g-i) immunofluorescence staining of collagen II in representative BMSC micromass pellet cultures with differentiation towards the chondrogenic lineage. Cartilage matrix deposition (blue) in the extracellular matrix was assessed by Masson Trichrome staining and confirmed by immunofluorescence staining for collagen II. Representative images of n = 10 experiments. For the immunofluorescence analysis, n = 3.

Figure 4

Osteogenic potential of BMSCs in vivo. In vivo transplantation assays were performed by combining BMSCs with HA/TCP followed by subcutaneous transplantation into immunocompromised mice. (a, b) H&E staining. (a) Negative control (HA/TCP transplantation without BMSC). (b) BMSCs formed ectopic ossicles that were sometimes populated by host hematopoietic marrow (asterisk). The arrowheads indicate osteocytes. HA = hydroxyapatite/tricalcium phosphate particles. (c–f) The human origin of the woven bone by immunohistochemical analysis. (c, e) Positive control of lamin A/C and collagen I stains, respectively, in human skin. (d, f) Expression of human lamin A/C and collagen I within the woven bone (for the immunohistochemistry analysis, n = 3).