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

用于大规模培养人尿液干细胞的丝纤维辅助 3D 系统，适用于晚期有丝分裂毒性测试

• 批准号: 10417268
• 负责人: YUANYUAN no ZHANG
• 金额: $ 7.75万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-03 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10417268
• 关键词: 3-Dimensional; Address; Alginates; Anti-HIV Agents; Antiviral Agents; Apoptosis; Biocompatible Materials; Biological; Biological Assay; Biopolymers; CASP3 gene; Caliber; Cell Adhesion; Cell Density; Cell Survival; Cells; Cellular Assay; Characteristics; Chitosan; Collagen; Complex; Consumption; Diffusion; Dose; Evaluation; Excision; Fiber; Fibrinogen; Fibroins; Gelatin; Geometry; Glutathione; Goals; Growth; HIV; Human; Hyaluronic Acid; In Vitro; Infiltration; Longevity; Metabolism; Microspheres; Mitochondria; Mitochondrial DNA; Modeling; Nucleosides; Nutrient; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen; Pharmaceutical Preparations; Ploidies; Porosity; Production; Reactive Nitrogen Species; Regenerative Medicine; Reverse Transcriptase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Scanning Electron Microscopy; Seeds; Silk; Site; Spiders; Structure; System; Testing; Therapeutic; Thick; Time; Tissue Engineering; Toxic effect; Toxicity Tests; Toxicology; Urine; Zalcitabine; antiretroviral therapy; biomaterial compatibility; cell growth; cost; cost effective; cost effectiveness; drug testing; flexibility; histological stains; human model; human stem cells; large scale production; nanosized; nephrotoxicity; new technology; novel; novel strategies; process optimization; scale up; stem cells; three dimensional cell culture; three dimensional structure; tissue repair; wasting

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.

项目概要 体外 3D 培养是评估抗逆转录病毒药物引起的延迟线粒体毒性 (MtT) 的一种有前途的方法 治疗。标准 MtT 测试需要大量细胞进行连续评估。然而，没有 3D 平台可用于大规模生产用于长期培养的原代人类细胞。在当前可访问的 3D球体模型，控制结构几何形状的能力有限，不支持可靠的 以及扩大细胞生产规模。此外，在 3D 球体中创建大量原代人类细胞 对于 MtT 评估来说，系统是耗时、劳动密集型且昂贵的。因此，一种新颖的方法 迫切需要解决这些问题。用于组织修复的多孔生物材料中的细胞接种 可能会为当前的这些进步障碍提供解决方案。各种具有多孔微结构的生物材料 已用于组织工程的 3D 培养，例如天然材料（蜘蛛丝、壳聚糖、 由胶原蛋白、明胶、纤维蛋白原、透明质酸、藻酸盐）和合成材料（PGA、 聚乳酸、聚乳酸）。然而，丝素蛋白作为一种天然生物聚合物具有突出的特性： 再生医学的生物相容性、生物降解性、耐久性和灵活性。纤维生物基质 高孔隙率具有互连的孔隙网络，提供了将细胞固定在一起的锚定位点 促进营养和氧气的扩散以及废物的去除，从而在长期 3D 培养中实现高效的细胞生长。我们的 最近的研究表明，人尿源性干细胞 (USC) 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 之间的差距 球体培养和 MtT 测试，我们将探索一种新技术来提供大量原代细胞 人体细胞。因此，这项 R03 研究的总体目标是制定大规模生产的策略 3D 培养中的人类 USC，适合后期 MtT 测试。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 USC（≥ 3 x 106/样本）以实现长期生长（≥ 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。为了测试这个 假设，我们提出以下目标： 目标 1. 优化丝纤维 3D 培养系统的实验策略 大规模生产人类 USC 的网络；目标 2. 验证 USC-SFM 的 3D 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 USC 测试丝纤维网络的研究 MtT 测试。我们将在现有研究中使用这个新开发的 3D 系统（USC 的 3D 培养系统） NTRI 诱导的细胞毒性； R21 AI152832）。我们预计 USC-SFM 的 3D 文化作为一种​​较少劳动力的 集约化、更高效、更具成本效益的方法将能够维持大量的人体干细胞 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 球体相比，USC-SFM 可能会产生适合抗病毒后期 MtT 测定的可靠平台 药物测试。