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

脂肪组织（fat）是成体干细胞（称为脂肪源性干细胞（ASCs））的丰富来源，在组织工程和再生医学中有着广泛的应用前景。ASCs可以被刺激增殖并分化为更特化的细胞类型，包括成熟的脂肪细胞、骨细胞、肌肉细胞和软骨细胞等。然而，细胞培养环境显著影响ASC反应。用传统的细胞培养方法在聚苯乙烯组织培养上扩增时，ASC增殖率下降，细胞迅速丧失多系分化能力。由于临床应用可能需要超过10亿个细胞的纯化干细胞群，因此迫切需要新的具有成本效益的细胞培养技术，能够从小组织活检中获得大量、特征明确的ASC群体，并保留干细胞潜力。本研究计划的重点是发展人类ASC扩增和多谱系（骨、软骨、脂肪）分化的生物反应器策略。为了密切复制ASCs的天然细胞外环境的组成，将使用电喷涂技术建立从人类脂肪中发现的天然细胞外基质（ECM）蛋白质中提取的组织特异性微载体的制造方法。将开发和优化ASC在这些微载体上增殖分化的动态培养系统。氧张力和与成熟细胞群共培养的影响将被系统地探索，以调整细胞的反应。细胞和分子生物学表征工具，包括基因和蛋白质表达分析，将用于深入了解细胞培养微环境对ASCs的影响。该项目将为HQP在干细胞、生物材料和组织工程等多学科领域提供宝贵的培训机会，受训者将发展广泛的技能，整合工程和生物学领域。总的来说，这项工作将促进基于asc的治疗在重建、骨科、心血管和神经外科等领域的临床应用。

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.