Fluorinated Chitosan Microgels to Overcome Internal Oxygen Transport Deficiencies in Microtissue Culture Systems.
Fluorinated Chitosan Microgels to Overcome Internal Oxygen Transport Deficiencies in Microtissue Culture Systems.
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DOI：
10.1002/adbi.201900250
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发表时间：
2020-08
影响因子：
4.1
通讯作者：
Leipzig, Nic D.
中科院分区：
文献类型：
--
作者：
Patil, Pritam S.;Mansouri, Mona;Leipzig, Nic D.
氧气输送差是目前在球状体/类器官3D细胞培养平台领域中的主要障碍，此时其不能生长得足够大以真正生理相关并复制成人器官功能。为了克服内部局部氧运输不足，利用全氟化碳（PFC）改性壳聚糖和高度可扩展的油包水微乳液方法形成充氧微凝胶。形成细胞直径（约10-40微米）量级的微凝胶，使得它们在包含在3D球状体培养物中时直接与细胞缔合，而不被内化。这些微凝胶中固定的PFC的存在允许在掺入培养的微组织中时增强和调节氧转运。因此，证明了以50：1、150：1、400：1（细胞数量：微凝胶数量）的比例将充氧微凝胶掺入致密的基于人成纤维细胞的球状体中促进了更大的基于人细胞的球状体的生长，特别是在最高掺入比例（50：1）下，其缺乏限定的低氧核心。此外，与不含微凝胶的对照球状体相比，加入更多壳聚糖-PFC微凝胶后，缺氧染色逐渐减少。总双链（ds）-DNA的定量，总活细胞的指标，表现出类似的结果缺氧定量，显示更多的ds-DNA由于掺入氧化微凝胶。最后，直接测量球体内不同深度处的氧浓度，并证实由于壳聚糖-PFC微球的整合而导致的较高的氧分压。值得注意的是，掺入50：1壳聚糖-PFC微凝胶的球状体的中心比对照球状体提高了约30 mmHg的氧张力。这项工作证明了培养3D微组织的氧气运输的重要性，并提供了一种新的基于材料的方法来增强这些系统中的氧气运输。氧的可用性和运输是球状体/类器官研究的障碍。在这里，通过油包水细乳液产生用于维持体外微组织的氧合微凝胶。这些颗粒在接种时被掺入高密度的大细胞球体中，以局部增强氧合，从而在静态条件下的细胞培养期间维持细胞，减少缺氧和坏死-这提出了改善氧转运的新策略。
Poor oxygen transport is a major obstacle currently in the field of spheroid/organoid 3D cell culture platforms, which at this time cannot be grown large enough to be truly physiologically relevant and replicate adult human organ functions. To overcome internal local oxygen transport deficiencies, oxygenating microgels were formed utilizing perfluorocarbon (PFC) modified chitosan and a highly scalable water-in-oil mini-emulsion method. Microgels that are on the order of a cell dimeter (~10-40 microns) were formed allowing them to directly associate with cells when included in 3D spheroid culture, while not being internalized. The presence of immobilized PFCs in these microgels allows for enhancement and tuning of oxygen transport when incorporated into cultured microtissues. As such, it was demonstrated that incorporating oxygenating microgels from ratios of 50:1, 150:1, 400:1 (number of cells: number of microgels) into dense human fibroblast-based spheroids facilitated the growth of larger human cell-based spheroids, especially at the highest incorporation ratio (50:1), which lacked defined hypoxic cores. Further, hypoxia staining was increasingly reduced upon the incorporation of more chitosan-PFC microgels as compared to control spheroids with no microgels. Quantification of total double-stranded (ds)-DNA, an indicator of total live cells, demonstrated similar results to hypoxia quantification, showing more ds-DNA due to incorporation of oxygenating microgels. Finally, oxygen concentrations were measured at different depths within spheroids directly and confirmed higher oxygen partial pressures due to the integration of chitosan-PFC microspheres. Notably, the center of spheroids incorporating 50:1 chitosan-PFC microgels enhanced oxygen tensions by approximately 30 mmHg over control spheroids. This work demonstrates the importance of addressing oxygen transport for culturing 3D microtissues and offers a new materials-based approach to enhance oxygen transport in these systems. Oxygen availability and transport are a barrier to spheroid/organoid research. Here oxygenating microgels to sustain microtissues in vitro are created via water-in-oil miniemulsion. These particles are incorporated into high-density large cellular spheroids at seeding to locally enhance oxygenation to maintain cells, reduce hypoxia, and necrosis during cell culture under static conditions - presenting a new strategy for improving oxygen transport.
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