Dynamic Culture Systems with Tissue-specific Microcarriers for Human Adipose-derived Stem Cell Expansion and Delivery

脂肪组织是成体干细胞的丰富来源，被称为脂肪来源干细胞(ASCs)，在组织工程和再生医学中有着广泛的应用前景。ASCs可以被刺激增殖和分化为更专业的细胞类型，包括成熟的脂肪、骨、肌肉和软骨细胞以及其他血统。然而，细胞培养环境对ASC的反应有很大影响。用传统的细胞培养方法扩增后，ASC的增殖率下降，细胞迅速失去多向分化能力。由于临床应用可能需要超过10亿个细胞的纯化干细胞群体，因此迫切需要新的高性价比细胞培养技术，以便能够从小组织活检中获得具有保存干细胞潜力的大量、特征良好的ASC群体。这项研究计划专注于开发生物反应器策略，以促进人ASC的扩增和多谱系(骨、软骨、脂肪)的分化。为了紧密复制ASCs天然细胞外环境的组成，将利用电喷雾技术建立从人体脂肪中发现的天然细胞外基质(ECM)蛋白衍生的组织特异性微载体的制造方法。将开发和优化在这些微载体上进行ASC增殖和分化的动态培养系统。我们将系统地探索氧气紧张和与成熟细胞群体共培养的影响，以调整细胞的反应。将利用细胞和分子生物学表征工具，包括基因和蛋白质表达的分析，深入了解细胞培养微环境对ASCs的影响。该计划将在干细胞、生物材料和组织工程等多学科领域为HQP提供宝贵的培训机会，受训者将发展广泛的技能，将工程学和生物学领域结合在一起。总体而言，这项工作将推进基于ASC的疗法在重建、矫形、心血管和神经外科等应用中的临床翻译。

Adipose tissue (fat) is a rich source of adult stem cells, termed adipose-derived stem cells (ASCs), which hold great promise for a range of applications in tissue engineering and regenerative medicine. ASCs can be stimulated to proliferate and differentiate into more specialized cell types, including mature fat, bone, muscle, and cartilage cells, amongst other lineages. However, the cell culture environment dramatically impacts the ASC response. ASC proliferation rates decline and the cells rapidly lose their multilineage differentiation capacity when expanded using traditional cell culture methods on tissue culture polystyrene. As clinical applications may require purified stem cell populations in excess of 1 billion cells, there is a significant need for new cost-effective cell culture technologies that enable the procurement of large, well-characterized ASC populations, with preserved stem cell potential, from small tissue biopsies. This research program focuses on the development of bioreactor strategies for human ASC expansion and multilineage (bone, cartilage, fat) differentiation. To closely replicate the composition of the native extracellular environment of the ASCs, methods for the fabrication of tissue-specific microcarriers derived from the natural extracellular matrix (ECM) proteins that are found in human fat will be established using electro-spraying. Dynamic culture systems for ASC proliferation and differentiation on these microcarriers will be developed and optimized. The effects of oxygen tension and co-culturing with mature cell populations will be systematically explored to tune the cell response. Cell and molecular biology characterization tools, including analyses of gene and protein expression, will be employed to develop an in-depth understanding of the impact of the cell culture microenvironment on the ASCs. This program will provide invaluable training opportunities for HQP in the multidisciplinary areas of stem cells, biomaterials, and tissue engineering, and the trainees will develop extensive skills that integrate the fields of engineering and biology. Overall, this work will advance the clinical translation of ASC-based therapies in applications including reconstructive, orthopedic, cardiovascular, and neurological surgery.