Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing

Abstract

Mesenchymal stem cells (MSC) have great therapeutic potential for the repair of nonhealing bone defects due to their proliferative capacity, multilineage potential, trophic factor secretion, and lack of immunogenicity. However, a major barrier to the translation of cell-based therapies into clinical practice is ensuring their survival and function upon implantation into the defect site. We hypothesized that forming MSC into more physiologic 3-dimensional spheroids, rather than employing dissociated cells from 2-dimensional monolayer culture, would enhance their survival when exposed to a harsh microenvironment while maintaining their osteogenic potential. MSC spheroids were formed using the hanging drop method with increasing cell numbers. Compared to larger spheroids, the smallest spheroids which contained 15,000 cells exhibited increased metabolic activity, reduced apoptosis, and the most uniform distribution of proliferating cells. Spheroids were then entrapped in fibrin gels and cultured in serum-free media and 1% oxygen. Compared to identical numbers of dissociated MSC in fibrin gels, spheroids exhibited significantly reduced apoptosis and secreted up to 100-fold more VEGF. We also observed that fibrin gels containing spheroids and those containing an equivalent number of dissociated cells exhibited similar expression levels of early and late markers of osteogenic differentiation. These data demonstrate that MSC spheroids exhibit greater resistance to apoptosis and enhanced proangiogenic potential, while maintaining similar osteogenic potential to dissociated MSC entrapped in a clinically relevant biomaterial, supporting the use of MSC spheroids in cell-based approaches to bone repair.

Measurement of oxygen tension within mesenchymal stem cell spheroids

“…We and others have demonstrated that MSCs exhibit increased overall function and improved survival when formed into three-dimensional spheroids [6][7][8][9]. Compared with an equal number of dissociated MSCs, 15 000 cell MSC spheroids exhibited similar caspase 3/7 activity yet secreted up to 100 times the amount of vascular endothelial growth factor.…”

“…MSC spheroids were formed using the hanging drop technique over 48 h with 15 000, 30 000 or 60 000 cells per 25 ml droplet [6]. This range was selected due to previous reports of resulting sizes below and above the diffusion limit of oxygen, known increases in trophic factor secretion, and to examine spheroids of similar sizes reported in the literature [9,20,21].…”

“…Larger spheroids formed with more MSCs had increased caspase activity coupled with reduced metabolic activity and proliferation [6]. Spheroid size must be carefully considered due to limitations in the diffusive length of nutrient transport, a feature that may render cells in the core of spheroids with radii greater than 200 mm vulnerable to hypoxia and cell death [6]. Some have speculated that the presence of a hypoxic core within the spheroid may pre-programme the cells to promote survival and enhance their trophic factor secretion [10].…”

“…Compared with an equal number of dissociated MSCs, 15 000 cell MSC spheroids exhibited similar caspase 3/7 activity yet secreted up to 100 times the amount of vascular endothelial growth factor. Larger spheroids formed with more MSCs had increased caspase activity coupled with reduced metabolic activity and proliferation [6]. Spheroid size must be carefully considered due to limitations in the diffusive length of nutrient transport, a feature that may render cells in the core of spheroids with radii greater than 200 mm vulnerable to hypoxia and cell death [6].…”

Advances in multicellular spheroids formation

“…Human colon Matrigel -rich in growth factors, signal factors for cell proliferation -expensive and animal derived PC-3M, PrCa, NCI-H600 [74], HepG2 [75], A235, B16F10 [76] agar -damage on cells by forces from gel formation inside the agar -excellent mechanical properties human foreskin dermal fibroblasts [77], cortical cell [78] alginate -rapid formation of hydrogel scaffolds -strong chemicals required to break hydrogel networks to harvest MCSs human fat-derived stromal vascular fraction cells [79], human colon cancer cells (HCT116) [80] fibrin -promoting cell attachment -high cell viability human mesenchymal stem cell [81], B16-F1…”