Dental Pulp Derived Stem Cells: Optimizing Collection and Cryopreservation

描述（由申请人提供）：本申请的总体目标是了解从拔牙中恢复的干细胞/祖细胞的收集、处理和低温生物学特性，并最终获得这些数据，以便建立牙髓来源干细胞（DPSCs）冷冻库，用于研究和/或潜在移植或其他临床应用，作为根据21 CFR 1271中描述的现行良好组织规范（cGTP）处理的人类细胞产品。最近对这一令人兴奋的产后干细胞来源的研究发现，从酶消化的牙髓组织中获得了一群克隆性和高度增殖的细胞（Gronthos等，2000）。这些研究的结果表明，这些细胞可能有潜力用于干细胞介导的治疗和组织工程应用（Seo等，2005）。为了进一步研究这些细胞的潜在治疗用途，研究人员刚刚开始评估这些细胞及其各自的起源组织在低温保存处理中存活的能力。理想情况下，最佳的冷冻保存过程将是直接有效的，当应用于整个组织时，干细胞可以在解冻后提取。这样做的理由是为了保存临床样本，以便随后的干细胞恢复，因为有理由推测，在临床中对组织进行低温保存将比直接分离干细胞更实用，因为直接分离干细胞需要额外的设备和人员（Seo等人，2005）。这些细胞的冷冻银行将为它们的使用增加极大的灵活性，除其他外，允许将细胞运送给研究人员和/或临床医生，并有足够的时间进行供体鉴定和/或潜在测试。为了实现这一总体目标，我们建议从以下几个方面着手：(1)优化拔牙的收集、运输和加工；（二）牙源性干细胞冷冻保存的优化；（III）最终制定优化的cGTP协议，以开发用于分配和使用的牙齿干细胞库。与未冷冻的细胞相比，如果使用最灵敏的检测方法保留这些细胞90%的功能恢复，则认为开发DPSCs冷冻库的最终可行性是实现的。公共卫生意义：干细胞可以在体内发育成不同的组织类型。产后干细胞或成体干细胞提供了从胚胎中获取干细胞的另一种选择。这种干细胞存在于人类牙齿中，但在这些细胞能够直接用于临床之前，还需要进行更多的研究。一个冷冻的牙齿干细胞库将是促进这项研究的理想选择。该计划将优化处理和冷冻这些细胞的方法，以便于研究、测试和最终临床使用。

DESCRIPTION (provided by applicant): The overall goal of this application is to develop an understanding of the collection, processing and cryobiological characteristics of stem/progenitor cells recovered from extracted teeth with the ultimate culmination of these data to allow for a cryobank of Dental pulp derived stem cells (DPSCs) for research and/or potential transplant or other clinical utilization as a human cell product processed under current good tissue practice (cGTP) as described under 21 CFR 1271. Recent studies investigating this exciting source of post-natal stem cells have identified a population of clonogenic and highly proliferative cells derived from enzymatically digested Dental pulp tissue (Gronthos, et al., 2000). Results from these studies have indicated that these cells may have the potential to be utilized in stem cell mediated therapies and tissue engineering applications (Seo, et al., 2005). To allow further study and potential therapeutic use of these cells, investigators have just begun to evaluate the ability of these cells and their respective tissues of origin to survive cryopreservation processing. Ideally, the optimum cryopreservation process will be straight forward and effective when applied to the tissue as a whole, with the idea that stem cells could be extracted post-thaw. The rationale for this would be to preserve clinical samples for subsequent stem cell recovery as it is reasonable to speculate that cryopreservation of tissues in the clinic will be more practical than direct primary isolation of stem cells, which would require additional equipment and personnel (Seo et al., 2005). A cryobank of these cells would add great flexibility to their use by allowing, among other things, shipment of cells to investigators and/or clinicians and adequate time for donor characterization and/or potential testing. To achieve this overall goal, we propose to begin with the following Specific Aims: (I) Optimization of collection, transportation and processing of extracted teeth; (II) Optimization of cryopreservation of tooth derived stem cells; and (III) Final development of optimized, cGTP protocols for development of a tooth stem cell bank for distribution and use. Ultimate feasibility of developing a cryobank of DPSCs will be considered achieved if e90% functional recovery of these cells is retained using the most sensitive assay available when compared to their unfrozen counterparts. Public Health Significance: Stem cells can develop into different tissues types in the body. Post-natal, or adult stem cells offer an alternative to obtaining stem cells from embryos. Such stem cells exist in human teeth, but more research is required before these cells can have direct clinical use. A bank of frozen stem cells from teeth would be ideal to facilitate this research. This proposed project would optimize methods for processing and freezing these cells for ease of distribution for research, testing, and eventually clinical use.