Improved expansion of human bone marrow-derived mesenchymal stem cells in microcarrier-based suspension culture

Abstract

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have potential clinical utility in the treatment of a multitude of ailments and diseases, due to their relative ease of isolation from patients and their capacity to form many cell types. However, hBM-MSCs are sparse, and can only be isolated in very small quantities, thereby hindering the development of clinical therapies. The use of microcarrier-based stirred suspension bioreactors to expand stem cell populations offers an approach to overcome this problem. Starting with standard culture protocols commonly reported in the literature, we have successfully developed new protocols that allow for improved expansion of hBM-MSCs in stirred suspension bioreactors using CultiSpher-S microcarriers. Cell attachment was facilitated by using intermittent bioreactor agitation, removing fetal bovine serum, modifying the stirring speed and manipulating the medium pH. By manipulating these parameters, we enhanced the cell attachment efficiency in the first 8 h post-inoculation from 18% (standard protocol) to 72% (improved protocol). Following microcarrier attachment, agitation rate was found to impact cell growth kinetics, whereas feeding had no significant effect. By serially subculturing hBM-MSCs using the new suspension bioreactor protocols, we managed to obtain cell fold increases of 10³ within 30 days, which was superior to the 200-fold increase obtained using the standard protocol. The cells were found to retain their defining characteristics after several passages in suspension. This new bioprocess represents a more efficient approach for generating large numbers of hBM-MSCs in culture, which in turn should facilitate the development of new stem cell-based therapies.

Suspension-Expansion of Bone Marrow Results in Small Mesenchymal Stem Cells Exhibiting Increased Transpulmonary Passage Following Intravenous Administration

“…High yield MSC expansion on similar microbead carriers has been previously demonstrated for both rat 23,24 and human cells. 25,26 Unlike these prior reports, we expanded these cultures by adding media and microbead carriers, while retaining the nonadherent cellular fractions. The rationale behind this approach is to allow for the retention of Green fluorescent cells were isolated by plating from bioreactor MSCs injected rat arterial blood, but not from the monolayer MSCs experiments (inset).…”

Effects of hypoxia on proliferation of human cord blood-derived mesenchymal stem cells

“…Early studies paid exquisite attention to the balance of nutrients, growth factors (Chen et al 2009), and pH buffers (Yuan et al 2014) used to grow cells in vitro. Very little attention was given to the oxygen concentration present in culture media.…”

PCL microspheres tailored with carboxylated poly(glycidyl methacrylate)–REDV conjugates as conducive microcarriers for endothelial cell expansion

“…First used 70 to cultivate mammalian cell lines for large-scale vaccine production, 17 microcarriers have been explored extensively for the culture of embryonic stem cells, 18 mesenchymal stem cells, [19][20][21] osteoblasts, 22 chondrocytes, 23 and hepatocytes. First used 70 to cultivate mammalian cell lines for large-scale vaccine production, 17 microcarriers have been explored extensively for the culture of embryonic stem cells, 18 mesenchymal stem cells, [19][20][21] osteoblasts, 22 chondrocytes, 23 and hepatocytes.…”