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除的牙齿中回收的干细胞/祖细胞的收集、处理和低温生物学特性，并最终达到这些数据的顶峰，以便建立牙髓干细胞(DPSCS)的冷藏库，用于研究和/或潜在的移植或其他临床用途，作为根据21 CFR 1271所述的当前良好组织实践(CGTP)处理的人类细胞产品。最近对这种令人兴奋的出生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和高度增殖的细胞(Gronthos等人，2000年)。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等人，2005年)。为了对这些细胞进行进一步的研究和潜在的治疗用途，研究人员刚刚开始评估这些细胞及其各自来源的组织在低温保存过程中存活的能力。理想情况下，最佳的冷冻保存过程将是直接和有效的，当应用于整个组织时，其想法是在解冻后可以提取干细胞。这样做的理由是为随后的干细胞回收保存临床样本，因为可以合理地推测，在临床上冷冻保存组织将比直接初步分离干细胞更实用，后者需要额外的设备和人员(Seo等人，2005年)。这些细胞的冷藏库将增加其使用的极大灵活性，尤其是允许将细胞运送给研究人员和/或临床医生，并允许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，我们建议从以下具体目标开始：(I)优化拔除牙齿的收集、运输和加工；(Ii)优化牙源性干细胞的冷冻保存；以及(Iii)最终制定优化的CGTP方案，以开发供分配和使用的牙齿干细胞库。如果与未冷冻的细胞相比，使用最灵敏的方法保留了这些细胞90%的功能恢复，那么开发DPSCs冷冻库的最终可行性将被认为是实现的。公共卫生意义：干细胞可以在体内发育成不同的组织类型。出生后或成年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，但在这些细胞可以直接用于临床之前，还需要更多的研究。一个冰冻的牙齿干细胞库将是促进这项研究的理想选择。这个拟议的项目将优化处理和冷冻这些细胞的方法，以便于分配用于研究、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.