STEM CELL PRESERVATION WITH NONTOXIC SUGARS

说明（由申请人提供）：可采用冷冻造血干细胞移植治疗的骨髓疾病包括白血病、淋巴瘤和再生障碍性贫血。在冷冻保存领域，目前的观点是在移植前应去除冷冻保护剂。二甲基亚砜（DM80）是最流行的冷冻保护剂，在细胞水平上有许多记录在案的作用，同时经常有患者有害副作用的临床报告。在目前使用的冷冻保存程序中，使用无细胞毒性糖的能力应该能够避免DMSO。因此，在本提案中，我们将使用人类干细胞/祖细胞开发保存方法，利用新的细胞膜渗透技术与通常不渗透的糖相结合。预计通过该技术冷冻保存的细胞将直接用于患者。该技术还可用于提供各种细胞的稳定长期储存，包括成熟血细胞、间充质干细胞、基于细胞的生物传感器和一些涉及基因治疗的医学疗法。

DESCRIPTION (provided by applicant): Diseases of the bone marrow that may be treated with cryopreserved hemopoletic stem cell transplantation indude the leukemias, lymphomas and aplastic anemia. In the field of cryopreservation, current opinion is that cryoprotective agents should be removed prior to transplantation. In the case of dimethylsulfoxide (DM80), the most popular cryoprotectant, there are many documented effects at the cellular level along with frequent clinical reports of detrimental side effects in patients. The ability to use non-cytotoxic sugars should enable the avoidance of DMSO in currently employed cryopreservation procedures. Therefore, in this proposal we will use human stem/progenitor cells to develop preservation methods utilizing a new cell membrane permeabilization technology in combination with sugars that are normally impermeable. It is anticipated that cells cryopreserved by this technology will be directly infusible into patients. This technology may also be leveraged to provide stable long-term storage of a variety of cells, including mature blood cells, mesenchymal stem cells, cell-based biosensors and some medical therapies involving gene therapy. The following specific aims will be addressed in this Phase I research study: #1 Optimization of H5 pore and divalent metal ion (zinc for pore opening and dosing) concentrations; #2 Optimization of postpermeabilization pore removal; #3 Comparison of controlled rate cryopreservation at four cooling rates following permeabilization with trehalose or sucrose; and #4 Evaluation of residual H5 cytotoxicity and the long-term hematopoietic potential of treated cells. These aims will be accomplished using human CD34+ cord blood cells and a panel of in vitro assays, and by transplantation in vivo into NOD/SCID mice. The technical innovations in this proposal are based upon the concept of reversible permeabilization of cell membranes using engineered pores to load the cells with disaccharides prior to freezing. The permeabilization technology employs a genetically engineered pore forming protein. The opening and dosing of the pore is regulated by divalent metal ions. Preliminary feasibility data is submitted demonstrating cryopreservation of a porated human hemopoietic progenitor cell line with trehalose. A Phase-Il SBIR study wilt be proposed to optimize hemopoletic stem and progenitor cell preservation if mice treated with porated human cells demonstrate good survival and hematopoietic reconstitution.