Novel Technique for Isolating Human Bone Marrow Stem Cells Using Hyaluronic Acid Hydrogel.
Centrifugation based on density gradients is a general methodology for isolating human bone marrow (hBM)-derived mesenchymal stem cells (hBMSCs). The mononuclear cell (MNC) layer can be obtained using a density gradient solution in the conventional protocol, but it is not suitable for direct transplantation due to the possible toxicity of this solution. The results obtained are also influenced by the skill level when applying the technique, which involves time-consuming processes. We have developed a novel protocol for isolating hBMSCs using hyaluronic acid (HA), which is the most widely used injectable biomaterial in clinical settings and a major component of the extracellular matrix. Laying hBM over the HA and then applying centrifugation yielded three separate layers, with the HA layer, including MNCs being the most superficial one. Increasing the volume of HA and/or its crosslinking rate enhanced the yield of MNCs from hBM, and the cell yield was also significantly higher for a lower centrifugal acceleration (530 g) than for a higher one (1500 g). Isolated hBMSCs by HA exhibited similar biological characteristics such as in terms of their proliferation rate, fibroblast-like morphology, cell-cycle status, immunophenotype, and multipotency. The use of either type of hBMSC confirmed the regenerative potential of bone and bone marrow-like tissue in ectopic transplantation models. This is the first report of a novel protocol for isolating hBMSCs that utilize HA. We suggest that this novel isolation technique can be used for the direct application of autogenous MSCs with advantages of being less time-consuming and involving steps that are easier to perform.
1 Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University , Seoul, Republic of Korea.
Jung-Seok
JS
Lee
1 Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University , Seoul, Republic of Korea.
Seul-Ki
SK
Kim
1 Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University , Seoul, Republic of Korea.
Jae-Kook
JK
Cha
2 Department of Neurosurgery, Naeun Hospital , Anyang, Republic of Korea.
Byung-Joo
BJ
Jung
2 Department of Neurosurgery, Naeun Hospital , Anyang, Republic of Korea.
Seong-Bok
SB
Choi
1 Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University , Seoul, Republic of Korea.
Eun-Young
EY
Choi
1 Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University , Seoul, Republic of Korea.|3 Department of Applied Life Science, BK21 PLUS Project, College of Dentistry, Yonsei University , Seoul, Republic of Korea.
Chang-Sung
CS
Kim
D000328:Adult; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000737:chemistry; D005260:Female; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry*; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008875:Middle Aged; D012038:Regeneration; D023822:Tissue Engineering / Q000379:methods*
D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D006820:Hyaluronic Acid