Department of Hard Tissue Research, Graduate School of Oral Medicine, Matsumoto Dental University, Shiojiri, Japan.
Department of Oral and Maxillofacial Surgery, School of Dentistry, Matsumoto Dental University, Shiojiri, Japan.
Tissue Eng Part C Methods. 2021 Apr;27(4):253-263. doi: 10.1089/ten.TEC.2021.0001.
Spontaneously formed spheroids from mouse compact bone-derived mesenchymal stromal cells (CB-MSCs) possess enhanced stemness and superior plasticity. In this study, the effect of cryopreservation on viability, stemness, and osteogenic differentiation capability of spontaneous CB-MSC spheroids were investigated. CB-MSCs were isolated from mouse femur and tibia. Spheroids were cryopreserved with various concentrations of dimethyl sulfoxide (DMSO). After thawing, the number of living and dead cells was measured. The expression levels of stem cell markers and osteogenic marker genes were analyzed. The cryopreserved and noncryopreserved spheroids were transplanted in mice with a beta-tricalcium phosphate as a scaffold to evaluate the bone-forming capability. The percentage of living cells was highest when 5% DMSO was used as a cryoprotectant, confirmed by the number of dead cells. The expression of stem cell marker genes and osteogenic differentiation capability were maintained after cryopreservation with 5% DMSO. The cryopreserved spontaneous CB-MSC spheroids showed remarkable new bone formation , identical to that of the noncryopreserved spheroids even without osteogenic induction. The cryopreserved spontaneous CB-MSC spheroids retained stemness and osteogenic differentiation capability and highlight the utility of spontaneous CB-MSC spheroids as ready-to-use tissue-engineered products for bone tissue engineering.
自发形成的小鼠致密骨源性间充质基质细胞（CB-MSCs）球体具有增强的干性和更好的可塑性。在这项研究中，研究了冷冻保存对自发形成的 CB-MSC 球体活力、干性和成骨分化能力的影响。从小鼠股骨和胫骨中分离 CB-MSCs。用不同浓度的二甲基亚砜（DMSO）冷冻保存球体。解冻后，测量活细胞和死细胞的数量。分析干细胞标志物和成骨标志物基因的表达水平。将冷冻保存和未冷冻保存的球体移植到β-磷酸三钙作为支架的小鼠中，以评估其成骨能力。当使用 5% DMSO 作为冷冻保护剂时，活细胞的百分比最高，这可以通过死细胞的数量来证实。用 5% DMSO 冷冻保存后，干细胞标志物基因的表达和成骨分化能力得以维持。冷冻保存的自发形成的 CB-MSC 球体表现出显著的新骨形成，与未冷冻保存的球体相同，甚至无需成骨诱导。冷冻保存的自发形成的 CB-MSC 球体保留了干性和成骨分化能力，突出了自发形成的 CB-MSC 球体作为可立即用于骨组织工程的组织工程产品的实用性。
