Cryopreparation of PBMC for Immunotherapy and Immune Assessment Studies.

描述（由申请人提供）：世界各地正在进行许多免疫治疗试验，从疫苗到T细胞转移和抗体研究。在这些试验中，除了明显需要评估临床应答外，研究者还必须分析免疫干预导致的免疫应答变化。因此，需要在治疗之前和之后定期从患者收集PBMC。将这些PBMC冷冻保存以保存PBMC表型和功能，直到稍后的时间点，此时PBMC可以解冻并评估以确定免疫疗法是否有效。此外，后续剂量的制备受益于易于获得的PBMC来源，这些PBMC将对与实验室收到产品当天相似的体外操作产生反应。由于细胞冷冻保存与免疫治疗有关，因此几乎没有标准化的技术可用于细胞冷冻保存。大多数实验室都有自己的方案，这些方案本身往往在解冻时提供细胞活力和功能的不一致性。评估免疫治疗试验的结果并从实验室到实验室进行比较也很困难。目前的建议将重点放在冷冻保存介质和比较静态与控制速率冷冻技术。在4个具体目标中，我们将研究：1）最佳的冷冻保存培养基。将人血清+ DMSO与Plasmalyte-A（一种FDA批准的市售无血清电解质或再水化液）进行比较。Plasmalyte A与血清的比较很重要，因为如果它起作用，将显著降低成本，因为每升成本不到血清成本的1%。此外，这是FDA批准的产品，批次之间一致。第二个具体目标是评估不同的解冻方法。具体目标3将比较静态冷冻技术和受控速率冷冻。第四个具体目标将通过使用细胞免疫测定在不同时间间隔比较PBMC亚群功能来监测具体目标1和3。该提案的目标将是制定一个策略，外周血单核细胞（PBMC）冷冻保存，可以更普遍地应用。更标准化的方法将允许更准确地评估免疫应答，可以在不同中心的研究之间进行比较。此外，利用PBMC的其他临床研究也可以从这样的研究中受益（例如HIV和移植的研究）。这四个具体目标将在两年时间内完成。

DESCRIPTION (provided by applicant): There are many immunotherapy trials being conducted throughout the world ranging from vaccines to T cell transfer and antibody studies. In these trials, aside from the obvious need to assess clinical responses, Investigators must analyze changes in immune responsiveness which results from the immune intervention. Thus, there is a need to collect PBMC from patients at regular intervals both before and following the therapy. These PBMC are cryopreserved to save the PBMC phenotype and function until a later time point when the PBMC can be thawed and assessed to determine if the immunotherapy was effective. In addition, preparation of subsequent doses benefits from a source of readily obtainable PBMC which will respond to in vitro manipulation similar to the day the product was received by the lab. There are few standardized techniques available for cell cryopreservation as it relates to immunotherapy. Most labs have their own protocols which themselves often provide inconsistencies in cell viability and function upon thawing. It is also difficult to evaluate results of immunotherapy trials and make comparisons from lab to lab. The current proposal will focus on cryopreservation medium and compare static vs. controlled rate freezing techniques. In 4 Specific aims, we will examine: 1) optimum medium for cryopreservation. Human serum + DMSO will be compared to Plasmalyte-A, an FDA approved commercial serum free electroyte or rehydration fluid. The comparison of Plasmalyte A to serum is important because if it works, significant cost reduction will occur since the cost per liter is less than 1% of the cost of serum. In addition, this is an FDA approved product which is consistent from batch to batch. The second specific aim will evaluate different methods of thaw. Specific Aim 3 will compare static freeze techniques to controlled rate freezing. The fourth specific aim will monitor Specific aims 1 and 3 by comparing PBMC subset function at various time intervals using assays of cellular immunity. The goal of the proposal will be to develop a strategy for peripheral blood mononuclear cell (PBMC) cryopreservation which can be more universally applied. A more standardized approach will allow a more accurate assessment of immne responsivenss which can be compared between studies in different centers. In addition, other clinical studies utilizing PBMC can also benefit from such a study (studies of HIV and transplantation for instance). The 4 specific aims will be done over a two year period of time.