High-Yield Isolation, Expansion, and Differentiation of Rat Bone Marrow–Derived Mesenchymal Stem Cells with Fibrin Microbeads

Abstract

Fibrin microbeads (FMB), made of extensively cross-linked dense and partially denatured fibrin, were used as a matrix for efficient isolation of mesenchymal stem cells (MSC) from rat bone marrow (BM). After 2 days of incubation of FMB with whole BM in suspension, a high number of cells of mesenchymal origin attached to the FMB. On the 14th day after their transfer to plastic, the yield of the cells isolated via FMB was approximately 3-4 times higher than that obtained by currently used protocols based solely on plastic adhesion. This implies that the number of MSC in BM may be higher than previously reported. FACS analyses and immunostaining showed the mesenchymal characteristics of these cells by positive staining for fibronectin, vimentin, CD49E, and CD29. Immediately after isolation, less than 20% of the cells still expressed the hematopoietic markers CD11b and CD45. Most of these cells were eventually eliminated after further expansion of the isolated cells on plastic. Cells isolated via FMB were expanded in culture for more than 4 months and could be defined as MSC along this time period based on their ability to differentiate into precursors of mesenchymal tissues, such as osteogenic, adipogenic, and chondrogenic cells. Similar differentiation plasticity was observed in clones derived from single cells from whole MSC populations isolated via FMB. Based on our results we propose that FMB can serve as a 3-dimensional biodegradable matrix for isolation, differentiation, and possibly implantation of MSC for tissue regeneration.

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

“…Therefore, we used FBS as the only source of growth factors for these cells. 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.…”

A review of fibrin and fibrin composites for bone tissue engineering

“…Made of extensively cross-linked and partially denatured fibrin, fibrin microbeads (FMBs) have been used to improve the biodegradability of matrices while retaining their high celladhesion properties. 97 In a typical procedure, FMBs are synthesized by oil-stirring of a fibrinogen and thrombin mixture at 65°C-75°C for 6-8 hours and stored as a dry powder. 98 At this high temperature, fibrinogen denatures while factor XIIIa can remain active and cross-link proteins.…”

“…Due to the conformational transformations of fibrinogen at these high temperatures, FMBs are highly haptotactic to various cell types, including osteogenic bone marrow-derived progenitors. 99,100 For bone tissue engineering studies, the obtained FMBs were incubated for 2 days in a suspension containing whole 97,98,101 Next, authors cultured FMB-MSCs constructs in osteogenic medium and obtained a bone-like structure which was implanted into a mouse skull defect. After 2 months, results showed that defects filled with bone-like tissue had a Ca/P ratio similar to that of the native bone while no repair was seen in the control animals without implants or when the defect was filled with FMBs alone.…”

Mesenchymal stem cells increase hippocampal neurogenesis and counteract depressive-like behavior

“…26 In brief, after the sacrifice of male FSL rats, the tibias and femurs were removed and cleaned of connective tissue. Marrow was flushed out of the cut bones after removal of the epiphysis and suspended in Dulbecco's modified Eagle's medium (DMEM; Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal bovine serum (Biological Industries), 100 units ml À1 of penicillin (Biological Industries), 100 mg ml À1 of streptomycin (Biological Industries) and 2 mM of L-glutamine (Biological Industries).…”