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

项目总结 体外3-D培养是评估抗逆转录病毒引起的延迟性线粒体毒性(MTT)的一种有前途的方法 心理治疗。标准的四甲基偶氮唑蓝检测需要大量细胞进行系列评估。然而，没有 3D平台可用于大规模生产原代人类细胞，用于长期培养。在当前可访问的 3D椭球体模型，对结构的几何控制能力有限，这不支持可靠 扩大电池生产规模。此外，在3D球体中创建大量的原代人体细胞 系统是耗时、劳动密集型的，并且对于MTT评估来说是昂贵的。因此，一种新的方法来 解决这些问题都是当务之急。用于组织修复的多孔生物材料中的细胞种植 可能会为这些目前阻碍进展的障碍提供解决方案。具有多孔微结构的各种生物材料 已被用于组织工程的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、海藻酸盐制成的微球)和合成材料(PGA， PLGA、PLLA)。然而，丝素蛋白作为一种天然生物聚合物，具有突出的特性 再生医学的生物兼容性、生物降解性、耐用性和灵活性。纤维生物材料 高孔隙率具有相互连接的孔隙网络，这提供了将细胞连接在一起的锚定位置和 在长期的3D培养中，促进营养和氧气的扩散和废物的清除，以实现细胞的高效生长。我们的 最近的研究表明，体外培养的人尿源性干细胞(USCs)的三维球体模型可以 可用于肾毒性检测。以扩展我们正在进行的研究，并弥合当前3D 球体培养和四甲基偶氮唑蓝检测，我们将探索一种新的技术来提供大量的原代 人类细胞。因此，这项R03研究的总体目标是制定一项大规模生产 3D培养的人USC适用于后期的四甲基偶氮唑盐(MTT)检测。我们假设体外3D培养系统 多孔真丝纤维基质将支持足够的USC(≥3x106/样本)用于长期生长(≥8周)， 具有稳定的线粒体拷贝数和功能，最终用于后期的四甲基偶氮唑蓝检测。为了测试这一点 假设，我们提出了以下目标：目的1.优化蚕丝纤维三维培养系统的实验策略 用于大规模生产人USCs的网络；目标2.验证USC-SFM三维培养系统和 将其与3D球体培养进行比较。这将是第一项用人类干细胞测试丝素纤维网络的研究 四甲基偶氮唑盐试验。我们将在我们现有的研究中使用这个新开发的3D系统(USC的3D文化系统 对于ntri诱导的有丝分裂毒性；R21 AI152832)。我们预计南加州大学的3D文化-SFM作为一种更少的劳动力- 更密集、更高效、更具成本效益的方法将能够维持大量的人类干细胞 丝素纤维内的细胞具有稳定的线粒体数量和功能，随着时间的推移。这项提议的3D文化 与3D球体相比，USC-SFM可能会提供一个可靠的平台，适用于后期抗病毒的MTT法。 药物测试。

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.