玻璃化法结合三维灌注技术在神经组织长期低温保存的作用研究

Regenerative medicine using tissue engineering and stem cell technologies aims to treat currently non-curable diseases. Neural tissue regeneration in particular has been the focus of much research in the last decade, for its unprecedented potential to treat neurodegenerative diseases such as Parkinson's disease, or spinal cord injuries. The translation and commercialization of tissue engineering products needs long-term and stable cryopreservation of the engineered tissues with living cells and functional structures to ensure the off-the-shelf availability. .In contrast with the conventional slow freezing, vitrification, suppressing intracellular and extracellular ice formation, has the outstanding advantage of preserving the extracellular structures which is critical to neuronal tissues. Vitrification would be ideal to preserve natural and engineered neural tissues where there is no ice formation and hence no ice induced damage to the extracellular matrix and cell-cell connections. Dimethylsulfoxide (DMSO) has been used extensively as a permeating cryoprotecting agent (CPA) for cryopreservation of cells and tissues. However, DMSO used at a higher concentration such as the common 10% (v/v) has been associated with neurological toxicity, particularly in the developing brain. Cryopreserved cells treated with DMSO may cause other adverse effect including gene mutation. The use of serum must be eliminated for clinical applications. Therefore, it is necessary to find new cryprotecting/vitrification solutions with low cytotoxicity and efficient protocols with well-defined clinical approved formula, which is the objective of this proposed project. In this project, brain slice models are used for the screening and testing of the vitrification solutions and optimization of vitrification protocols. This is because neurons in the brain slices reside in the physiological environment of the brain consisting of natural extracellular matrix, and neuronal connectivity. Hence neural functions can be detected and any loss due to vitrification can be quantified. Different formula of vitrification solutions were evaluated in terms of cell viability, K+/Na+ ratio, structure integrity, immunocytochemistry, electrophysiological properties, and synaptic transmission. A parallel perfused system will be used for the continuous addition/removal and freezing protocols of developed vitrification solutions optimization. To make the work more clinically relevant, human adipose-derived stem cells are used to construct 3D neural tissue models. Electrospun nanofibers of PLLA-Collagen will be used as the scaffolds. The multiple parallel perfused microbioreactor platform, TissueFlex, will be used to conduct the 3D hADSC culture and differentiation to form neuronal model tissues. The vitrification of these neural-like tissues using the optimized vitrification solutions and freezing/thawing protocols will be performed to validate the developed methodology and technology.

干细胞组织工程可望成为治疗神经损伤和退行性疾病的有效手段，神经干细胞及其三维构件、分化或部分分化的神经组织长期保存，是制约该技术临床应用的重要技术瓶颈之一。传统冷冻保护剂中的二甲基亚砜对神经系统有毒性，冷冻时形成的冰晶会破坏组织结构，使组织丧失功能。本课题旨在保证高细胞活率的同时，重点研制能够保持突触和细胞间连接结构完整、低DMSO浓度、无血清且成分明确的新型神经组织冷冻保护剂及玻璃化长期低温保存神经网络的方法，利用平行式灌注反应器优化冷冻保护剂的配方和导入（导出）及降温过程。以大鼠脑片为模型优化玻璃化保护剂配方和降温解冻方案，以人脂肪干细胞三维灌注式培养和控制分化技术建立具有神经功能的工程化类神经组织模型，并研究新型保护剂和冻存方法对工程化类神经组织模型的玻璃化保存。本项目的研究将实现神经组织从冷冻到使用的一步化，为干细胞组织工程技术治疗神经系统疾病从基础到临床应用提供重要的实验依据。

利用静电纺丝技术制备新型复合纳米生物支架材料，创新性地利用中药活性成分梓醇结合脂肪干细胞、纳米材料以及三维灌注培养技术构建体外神经组织模型。以低DMSO浓度、无血清且成分明确的新型神经组织冷冻保护剂对神经干细胞进行了低温冻存，并研究了黏附在不同基质包被的表面上人骨髓间充质干细胞的低温保存，检测了基质材料、冷却速率及不同冷冻保护剂对黏附细胞的活率、细胞骨架肌动蛋白丝形态等的影响。为神经组织低温保存以及类神经组织模型体外构建的临床应用奠定实验基础。