Silk Fibers-Assisted 3D System for Large-Scale Culture of Human Urine-Derived Stem Cells Suitable for Late Mitotoxicity Testing

项目摘要 体外三维培养是评估抗逆转录病毒引起的迟发性线粒体毒性（MtT）的一种有前途的方法。 疗法标准MtT测试需要大量的细胞进行系列评估。但没有 3D平台可用于大规模生产长期培养的原代人类细胞。在目前可访问的 3D球体模型，控制结构几何形状的能力有限，不支持可靠的 和扩大细胞生产。此外，在3D球体中创建大量的原代人类细胞 系统是费时，劳动密集型，昂贵的MtT评估。因此，一种新的方法， 迫切需要解决其中每一个问题。组织修复用多孔生物材料中的细胞接种 可能会为这些目前阻碍进步的障碍提供一个解决方案。具有多孔微观结构的各种生物材料 已经用于组织工程的3D培养，例如天然材料（蜘蛛丝，壳聚糖， 由胶原、明胶、纤维蛋白原、透明质酸、藻酸盐制成的微球）和合成材料（PGA， PLGA、PLLA）。然而，丝素蛋白作为天然生物聚合物具有突出的特性， 生物相容性、生物降解性、耐久性和再生医学的灵活性。纤维生物基质， 高孔隙率具有互连的孔隙网络，其提供锚定位点以将细胞保持在一起， 促进营养和氧气扩散以及废物去除，以在长期3D培养中实现有效的细胞生长。我们 最近的研究表明，人尿源性干细胞（USCs）的体外3D球体模型可以 用于肾毒性测定。为了扩展我们正在进行的研究，并弥合当前3D与3D之间的差距， 球体培养和MtT测试，我们将探索一种新的技术，以提供大量的初级 人体细胞因此，本R 03研究的总体目标是制定大规模生产 适合于晚期MtT测试的3D培养中的人USC。我们假设体外3D培养系统 多孔丝纤维基质（SFM）将支持足够的USCs（≥ 3 x 106/样本）用于长期生长（≥ 8周）， 具有稳定的线粒体拷贝数和功能，最终用于后期MtT测试。为了验证这一 假设，我们提出这些目标：目标1。蚕丝纤维三维培养体系实验策略的优化 大规模生产人类USCs的网络;目标2。USC-SFM的3D培养系统， 与3D球体培养进行比较。这将是第一项用人类USCs测试丝纤维网络的研究， MtT测试。我们将在我们现有的研究中使用这个新开发的3D系统（USCs的3D培养系统 用于NTRI诱导的有丝分裂毒性; R21 AI 152832）。我们希望USC-SFM的3D文化作为一个更少的劳动- 密集、更有效和成本效益高方法将能够维持大量的人类干细胞 丝纤维内的细胞随着时间的推移具有稳定的线粒体数量和功能。这个提议的3D文化 与3D球状体相比，USC-SFM可能产生适用于抗病毒药物中晚期MtT测定的可靠平台。 药检

PROJECT SUMMARY In vitro 3-D cultures are a promising way to assess delayed mitochondrial toxicity (MtT) caused by antiretroviral therapy. Standard MtT testing requires large numbers of cells for serial assessments. However, there are no 3D platforms available for mass production of primary human cells for long-term culture. In currently accessible 3D spheroid models, ability to control the geometry of the structures is limited, which does not support reliable and scaled-up cell production. Furthermore, creating large numbers of primary human cells in 3D spheroid systems is time-consuming, labor-intensive, and expensive for MtT assessment. Thus, a novel approach to address each of these issues is urgently demanded. Cells seeding in porous biomaterials used for tissue repair might provide a solution to these current barriers to progress. Various biomaterials with porous microstructure have been used in 3D culture for tissue engineering, such as natural materials (spider silk, chitosan, microspheres made from collagen, gelatin, fibrinogen, hyaluronic acid, alginate) and synthetic materials (PGA, PLGA, PLLA). However, silk fibroin as a natural biopolymer possesses outstanding characteristics with biocompatibility, biodegradability, durability, and flexibility for regenerative medicine. Fiber biomatrices with high porosity have interconnected pore networks, which provide anchoring sites to hold the cells together and facilitate nutrient and oxygen diffusion and waste removal for efficient cell growth in long-term 3D culture. Our recent study demonstrated that an in vitro 3D spheroid model of human urine-derived stem cells (USCs) can be used for nephrotoxicity assays. To extend our ongoing study and bridge the gap between current 3D spheroid cultures and MtT testing, we will explore a novel technology to provide large numbers of primary human cells. Thus, the overall goal of this R03 study is to develop a strategy for large-scale production of human USC in 3D culture suitable for late MtT testing. We hypothesize that an in vitro 3D culture system with porous silk fiber matrix (SFM) will support adequate USCs (≥ 3 x 106/sample) for long-term growth (≥ 8 weeks), with stable mitochondrial copy number and function, to eventually be used in late MtT testing. To test this hypothesis, we propose these aims: Aim 1. Optimize experimental strategies for 3D culture system of silk fiber network for large-scale production of human USCs; Aim 2. Validate a 3D culture system of USC-SFM and compare it to 3D spheroid culture. This will be the first study to test a silk fiber network with human USCs for MtT testing. We will use this newly developed 3D system in our existing study (3D Culture Systems of USCs for NTRI-Induced Mitotoxicity; R21 AI152832). We expect that 3D culture of USC-SFM as a less labor- intensive, more efficient, and cost-effective approach will be able to maintain large amounts of human stem cells within silk fibers with stable mitochondrial quantities and function over time. This proposed 3D cultures of USC-SFM, in contrast to 3D spheroids, may yield a reliable platform suitable for late MtT assays in antiviral drug testing.