Takebe Yusuke, Tatehara Seiko, Fukushima Tatsuhiro, Tokuyama-Toda Reiko, Yasuhara Rika, Mishima Kenji, Satomura Kazuhito
1 Department of Oral Medicine and Stomatology, Tsurumi University School of Dental Medicine , Yokohama, Japan .
2 Division of Pathology, Department of Oral Diagnostic Sciences, School of Dentistry, Showa University , Tokyo, Japan .
Tissue Eng Part C Methods. 2017 May;23(5):251-261. doi: 10.1089/ten.TEC.2016.0519.
Dental pulp stem cells (DPSCs) are an attractive cell source for use in cell-based therapy, regenerative medicine, and tissue engineering because DPSCs have a high cell proliferation ability and multidifferentiation capacity. However, several problems are associated with the collection and preservation of DPSCs for use in future cell-based therapy. In particular, the isolation of DPSCs for cryopreservation is time consuming and expensive. In this study, we developed a novel cryopreservation method (NCM) for dental pulp tissues to isolate suitable DPSCs after thawing cryopreserved tissue. Using the NCM, dental pulp tissues were cultured on adhesion culture dishes for 5 days and then cryopreserved. After thawing, the cryopreserved dental pulp tissue fragments exhibited cell migration. We evaluated each property of DPSCs isolated using the NCM (DPSCs-NCM) and the explant method alone without cryopreservation (DPSCs-C). DPSCs-NCM had the same proliferation capacity as DPSCs-C. Flow cytometry (FACS) analysis indicated that both DPSCs-NCM and DPSCs-C were positive for mesenchymal stem cell markers at the same level but negative for hematopoietic cell markers. Moreover, both DPSCs-NCM and DPSCs-C could differentiate into osteogenic, chondrogenic, and adipogenic cells during culture in each induction medium. These results suggest that DPSCs-NCM may be mesenchymal stem cells. Therefore, our novel method might facilitate the less expensive cryopreservation of DPSCs, thereby providing suitable DPSCs for use in patients in future cell-based therapies.
牙髓干细胞（DPSCs）是一种有吸引力的细胞来源，可用于基于细胞的治疗、再生医学和组织工程，因为DPSCs具有高细胞增殖能力和多分化能力。然而，在为未来基于细胞的治疗收集和保存DPSCs方面存在几个问题。特别是，用于冷冻保存的DPSCs的分离既耗时又昂贵。在本研究中，我们开发了一种用于牙髓组织的新型冷冻保存方法（NCM），以便在解冻冷冻保存的组织后分离出合适的DPSCs。使用NCM，将牙髓组织在贴壁培养皿上培养5天，然后进行冷冻保存。解冻后，冷冻保存的牙髓组织碎片表现出细胞迁移。我们评估了使用NCM分离的DPSCs（DPSCs-NCM）和仅采用外植体方法且未进行冷冻保存的DPSCs（DPSCs-C）的各项特性。DPSCs-NCM与DPSCs-C具有相同的增殖能力。流式细胞术（FACS）分析表明，DPSCs-NCM和DPSCs-C对于间充质干细胞标志物均呈同等水平的阳性，但对于造血细胞标志物呈阴性。此外，在每种诱导培养基中培养期间，DPSCs-NCM和DPSCs-C均可分化为成骨细胞、软骨细胞和脂肪细胞。这些结果表明DPSCs-NCM可能是间充质干细胞。因此，我们的新方法可能有助于以较低成本冷冻保存DPSCs，从而为未来基于细胞的治疗中患者提供合适的DPSCs。
