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，这是通过建立的治疗干预措施，如骨髓移植，输血和角膜移植;未来几代的产品可能包括生物人工基质，纳入供体干细胞，例如骨置换和修复设备，以及人造“微型器官”，如胰腺或肝脏。目前基于干细胞的产品的冷冻保存来自历史工作，使用DMSO作为冷冻保存剂，其在最终生物活性方面基本上未经证实。DMSO可能对细胞有毒，导致解冻后的低活力以及长期培养中的遗传和表观遗传不稳定性（即多能性丧失）。以前曾尝试过血细胞的冷冻保存，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，成功有限。已经研究了将海藻糖（一种膜不可渗透的冷冻保护剂）递送到哺乳动物细胞中的各种技术，包括显微注射、离子通道刺激、使用突变细菌毒素的孔形成、液相内吞作用和通过基因工程的内部海藻糖合成，但是在红细胞中实现的细胞内海藻糖浓度不超过50 mM，因此低于冷冻保护阈值。与将海藻糖装载到细胞中的常规方法相比，生物聚合物介导的细胞装载实现了高达251 mM的细胞内海藻糖浓度的显著增加以及高达20.4%的红细胞冷冻存活的伴随改善。该技术利用新型两亲性生物聚合物，其与外部细胞膜相互作用，以使冷冻保护剂能够渗透和保留到细胞中。通过这些细胞渗透聚合物（CPP）的膜渗透是快速的，并且通过用缓冲液洗涤是完全可逆的。海藻糖的细胞摄取取决于聚合物分子结构、浓度、pH、外部海藻糖浓度、孵育温度和时间。这些参数的优化赋予了细胞凋亡保护。总体而言，在-80 ° C下通过单个冻融循环实现了82.6%的总细胞回收率，相比之下，在PBS中冷冻的细胞的回收率仅为0.8%。该提案旨在探索CPP介导的将保存剂加载到干细胞中，通过冷冻和干燥检查保存，并获得用于制备最佳稳定干细胞的综合处理路线。

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.