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，这是它们作为产品使用的绝对先决条件。许多再生医学产品依赖于向患者输送活细胞。目前，骨髓移植、输血和角膜移植等已确立的治疗干预措施就是例证；未来几代的产品可能包括包含供体干细胞的生物人工基质，例如骨替代和修复装置，以及人造“微型器官”，如胰腺或肝脏。目前干细胞产品的低温保存源于历史上的工作，使用DMSO作为低温保存剂，其最终的生物活性在很大程度上是未经证实的。DMSO可能对细胞有毒，导致解冻后存活率低，并且在长期培养过程中导致遗传和表观遗传不稳定（即多能性丧失）。先前已经尝试过对血细胞进行低温保存，但由于细胞完整性的丧失（主要是由于细胞膜的破坏和随后的整体细胞结构的丧失）而取得的成功有限。为了将海藻糖（一种不透膜的低温保护剂）输送到哺乳动物细胞中，已经研究了多种技术，包括显微注射、离子通道刺激、使用突变细菌毒素形成孔、流体期内吞作用和通过基因工程进行的内部海藻糖合成，但红细胞中达到的细胞内海藻糖浓度未超过50 mM，因此低于低温保护的阈值。与传统的将海藻糖加载到细胞中的方法相比，生物聚合物介导的细胞加载实现了细胞内海藻糖浓度的显著增加，最高可达251 mM，同时红细胞冷冻存活率提高了20.4%。该技术利用新型两亲性生物聚合物与外细胞膜相互作用，使冷冻保护剂能够渗透并保留在细胞中。这些细胞渗透聚合物（CPPs）的膜渗透是快速的，并且通过缓冲洗涤完全可逆。海藻糖的细胞摄取取决于聚合物分子结构、浓度、pH值、外部海藻糖浓度、孵育温度和时间。这些参数的优化赋予细胞渗透保护。总的来说，在-80℃的单次冻融循环中，细胞的总回收率为82.6%，而在PBS中冷冻的细胞的回收率仅为0.8%。本研究旨在探索CPP介导的保存剂装载到干细胞中，通过冷冻和干燥来检验保存剂，并得出制备最稳定干细胞的综合加工路线。

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.