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，除了评估临床反应的明显需要外，研究人员还必须分析免疫干预导致的免疫反应性变化。因此，有必要在治疗前后定期收集患者的PBMC。这些PBMC被冷冻保存以保存PBMC的表型和功能，直到稍后的时间点PBMC可以解冻并评估免疫治疗是否有效。此外，后续剂量的制备得益于易于获得的PBMC来源，该来源将对类似于实验室接收产品当天的体外操作产生反应。由于涉及到免疫治疗，目前很少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协议，这些协议本身经常提供细胞活力和解冻后功能的不一致。评估免疫治疗试验的结果并在实验室之间进行比较也很困难。目前的建议将重点放在冷冻保存介质上，并比较静态和控制速率冷冻技术。在4个具体目标中，我们将研究：1)冷冻保存的最佳培养基。人血清+ DMSO将与Plasmalyte-A进行比较，Plasmalyte-A是FDA批准的商业血清无电解质或补液。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，批次之间是一致的。第二个具体目标是评估不同的解冻方法。具体目标3将比较静态冻结技术和控制速率冻结。第四个特异性目标将通过比较不同时间间隔的PBMC亚群功能，使用细胞免疫测定来监测特异性目标1和3。本研究旨在为外周血单核细胞（PBMC）的低温保存提供一种更广泛应用的方法。更标准化的方法将允许更准确地评估免疫反应，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的时间内完成。

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.