Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada.
Cryobiology. 2010 Aug;61(1):133-41. doi: 10.1016/j.cryobiol.2010.06.007. Epub 2010 Jun 22.
Dental pulp stem cells (DPSCs) are of interest to researchers and clinicians due to their ability to differentiate into various tissue types and potential uses in cell-mediated therapies and tissue engineering. Currently DPSCs are cryopreserved in suspension using Me(2)SO. However, preservation as two and three dimensional constructs, along with the elimination of toxic Me(2)SO, may be required. It was shown that intracellular ice formation (IIF), lethal to cells in suspensions, may be innocuous in cell monolayers due to ice propagation between cells through gap junctions that results in improved post-thaw recovery. We hypothesized that innocuous IIF protects confluent DPSC monolayers against injury during cryopreservation. The objective was to examine the effects of IIF on post-thaw viability of both confluent monolayers and suspensions of DPSCs. Confluent DPSC monolayers were assessed for the expression of gap junction protein Connexin-43. IIF was induced on the cryostage and in the methanol bath at different subzero temperatures. Membrane integrity and colony-forming ability were assessed post-thaw. Confluent DPSC monolayers expressed Connexin-43. In cell suspensions, 85.9+/-1.7% of cells were damaged after 100% IIF. In cell monolayers, after 100% IIF, only 25.5+/-5.5% and 14.8+/-3.3% of cells were damaged on the cryostage and in the methanol bath respectively. However, DPSC monolayers exposed to 100% IIF showed no colony-forming ability. We conclude that confluent monolayers of DPSCs express the gap junction-forming protein Connexin-43 and upon IIF retain membrane integrity, however lose the ability to proliferate.
牙髓干细胞（DPSCs）因其能够分化为多种组织类型以及在细胞介导的治疗和组织工程中的潜在用途而引起研究人员和临床医生的关注。目前，DPSCs 以悬浮液的形式使用 Me(2)SO 进行冷冻保存。然而，可能需要将其保存为二维和三维结构，并消除有毒的 Me(2)SO。已经表明，在悬浮液中对细胞致命的细胞内冰形成（IIF）在细胞单层中可能是无害的，因为冰通过间隙连接在细胞之间传播，从而导致解冻后恢复更好。我们假设无害的 IIF 可保护贴壁 DPSCs 单层免受冷冻保存过程中的损伤。目的是检查 IIF 对贴壁单层和 DPSCs 悬浮液解冻后活力的影响。评估贴壁 DPSCs 单层中间隙连接蛋白 Connexin-43 的表达。在不同的亚零温度下，在 cryostage 和甲醇浴中诱导 IIF。解冻后评估细胞膜完整性和集落形成能力。贴壁 DPSCs 单层表达 Connexin-43。在细胞悬浮液中，100% IIF 后 85.9+/-1.7%的细胞受损。在细胞单层中，100% IIF 后，在 cryostage 和甲醇浴中分别只有 25.5+/-5.5%和 14.8+/-3.3%的细胞受损。然而，暴露于 100% IIF 的 DPSCs 单层没有集落形成能力。我们得出结论，贴壁的 DPSCs 单层表达间隙连接形成蛋白 Connexin-43，并且在 IIF 后保留细胞膜完整性，但丧失增殖能力。
