Bioprocessing Research For Cellular Products

该项目旨在开发成功储存人类细胞所需的新型保存平台技术，这是将其用作产品的绝对先决条件。许多再生医学产品依赖于将活细胞输送给患者。目前，骨髓移植、输血和角膜移植等已建立的治疗干预措施就是例证；未来几代产品可能包括包含捐赠者干细胞的生物人工基质，例如骨替代和修复设备，以及诸如胰腺或肝脏的人造‘微型器官’。目前干细胞产品的冷冻保存是历史性工作的结果，使用DMSO作为低温保存剂，对于最终的生物活性在很大程度上是未经证实的。DMSO对细胞有毒性，解冻后存活率低，在长期培养过程中遗传和表观遗传不稳定(即丧失多能性)。以前曾尝试过对血细胞的冷冻保存，但由于细胞完整性的丧失，主要是由于细胞膜的破裂和随之而来的整体细胞结构的丧失，所以成功有限。已经研究了多种技术将海藻糖(一种膜不渗透的冷冻保护剂)输送到哺乳动物细胞中，包括显微注射、离子通道刺激、利用突变细菌毒素形成孔、液相内吞和通过基因工程合成内部海藻糖，但红细胞内的海藻糖浓度不超过50 mM，因此低于低温保护的阈值。与传统的细胞负载海藻糖方法相比，生物聚合物介导的细胞负载显著增加了高达251 mM的细胞内海藻糖浓度，并伴随着高达20.4%的红细胞冷冻存活率提高。该技术利用新型两亲性生物聚合物与外部细胞膜相互作用，使冷冻保护剂能够穿透和保留到细胞内。通过用缓冲液清洗，这些细胞渗透聚合物(CPP)的膜渗透作用快速且完全可逆。细胞对海藻糖的摄取依赖于聚合物的分子结构、浓度、pH、外界海藻糖浓度、孵育温度和时间。这些参数的优化提供了细胞渗透保护。总体而言，在-80oC的单次冷冻-解冻循环中，细胞回收率达到82.6%，相比之下，在PBS中冷冻的细胞回收率仅为0.8%。该方案旨在探索CPP介导的干细胞保存剂的负载，考察冷冻和干燥保存方法，并为制备最稳定的干细胞提供完整的加工路线。

This project aims to develop novel preservation platform technologies required for the successful banking of human cells, an absolute prerequisite for their use as products. Many regenerative medicine products rely on the delivery of live cells to patients. At present this is exemplified by established therapeutic interventions such as bone marrow transplantation, blood transfusion and corneal grafting; future generations of products may include bio-artificial matrices that incorporate donor stem cells, for example bone replacement and repair devices, and artificial 'mini-organs' such as pancreas or liver. Current cryopreservation of stem cell based products results from historic work, using DMSO as a cryopreserving agent which is largely unsubstantiated with respect to final biological activity. DMSO can be toxic to cells, lead to low viabilities post thaw and both genetic and epigenetic instability (i.e loss of pluripotency) over long term culture. Cryopreservation of blood cells has been attempted previously, with limited success due to loss of cell integrity, primarily due to the breakdown of the cell membrane and consequent loss of overall cell structure. A variety of techniques have been investigated for delivering trehalose, a membrane impermeable cryoprotectant, into mammalian cells, including microinjection, ion channel stimulation, pore formation using mutant bacterial toxins, fluid phase endocytosis, and internal trehalose synthesis via genetic engineering but intracellular trehalose concentrations achieved in erythrocytes has not exceeded 50 mM and is therefore below thresholds for cryoprotection. Biopolymer mediated cell loading achieves substantially increased intracellular trehalose concentrations of up to 251 mM and a concomitant improvement of erythrocyte cryosurvival of up to 20.4 % as compared with conventional methods of loading trehalose into cells. The technology utilizes novel amphiphilic biopolymers that interact with the external cell membrane to enable penetration and retention of cryoprotectant agents into the cells. Membrane permeabilisation by these Cell Permeating Polymers (CPPs) is rapid and completely reversible via washing with buffer. Cellular uptake of trehalose is dependent on polymer molecular structure, concentration, pH, external trehalose concentration, incubation temperature and time. Optimization of these parameters imparts cellular osmoprotection. Overall, a total cell recovery through a single freeze-thaw cycle at -80oC of 82.6 % has been achieved, which compares with a recovery of only 0.8 % for cells frozen in PBS. This proposal aims to explore the CPP mediated loading of preservation agents into stem cells, to examine preservation by freezing and dessication and to arrive at integrated processing routes for the preparation of optimally stable stem cells.