Development of Cell Culture Inserts and 3D In Vitro Tissue Models Utilizing Novel Electrospun Scaffolds

利用新型静电纺丝支架开发细胞培养插入物和 3D 体外组织模型

• 批准号: 10697932
• 负责人: PATRICK J HAYDEN
• 金额: $ 88.47万
• 依托单位: BIOSURFACES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-17 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697932
• 关键词: 3-Dimensional; Animal Testing; Animals; Appearance; Biological Assay; Blood - brain barrier anatomy; Cell Culture Techniques; Cells; Chemicals; Coculture Techniques; Complex; Contracts; Cosmetics; Dermal; Development; Drug toxicity; Electrospinning; Epithelial Cells; Extracellular Matrix; Fibroblasts; Film; Funding; Goals; Government; Human; In Vitro; Individual; Industry; Intestines; Kidney; Laboratories; Letters; Lung; Manufacturer; Marketing; Membrane; Methodology; Methods; Modeling; Morphology; National Center for Advancing Translational Sciences; Penetration; Persons; Pharmaceutical Preparations; Phase; Phototoxicity; Physiological; Porosity; Production; Protocols documentation; Qualifying; Recovery; Reproducibility; Reproduction; Research; Research Contracts; Research Personnel; Research Project Grants; Skin; Skin Tissue; Small Business Innovation Research Grant; Technology; Testing; Therapeutic; Thick; Tissue Model; Tissues; Tobacco smoke; Toxic effect; Toxicology; Validation; Work; bronchial epithelium; commercialization; consumer product; efficacy testing; high throughput screening; human model; human tissue; improved; in vitro Model; in vitro testing; in vivo; keratinocyte; manufacturing process; novel; novel strategies; open source; protocol development; respiratory; response; scaffold; screening; skin irritation; tissue culture; tissue support frame; tobacco products; tool; two-dimensional

Tissue culture inserts that utilize film-based microporous membrane scaffolds are key components of in vitro tissue models that are used as alternatives to animal testing. However, insert scaffold technology has not significantly advanced in nearly 30 years. Currently available film-based insert scaffolds are only 2-dimensional (2D), and are excessively rigid compared to natural extracellular matrix. These 2D scaffolds are not well-suited for production of complex in vitro 3-dimensional (3D) tissue models. Electrospinning technology can produce novel scaffolds that better replicate natural 3D extracellular matrix and overcome limitations of currently available scaffolds. In Phase I-equivalent preliminary work, we produced 3D electrospun scaffolds and developed manufacturing processes for attaching these scaffolds to high-throughput screening (HTS) Transwell® tissue culture inserts. We also demonstrated the feasibility of utilizing the 3D electrospun scaffold Transwell® products for producing organotypic in vitro models of full-thickness human skin and bronchial tissues. These tissue models have improved physiological relevance and functionality compared to currently available models, and are needed as alternatives to animal testing. The goal of this revised Direct Phase II SBIR proposal is to further develop and commercialize these novel electrospun scaffold inserts and organotypic culture models. Aim 1 will utilize 3D electrospun scaffold inserts to develop full-thickness human skin models consisting of human keratinocytes and human dermal fibroblasts. Aim 2 will utilize the 3D electrospun scaffold inserts to develop full-thickness human bronchial airway models consisting of human bronchial epithelial cells and human pulmonary fibroblasts. The tissue models will be produced in 24- and 96-well HTS Transwell® scaffold plates, as well as individual 6-, 12- and 24-well Transwell® scaffold insert formats. The models will be produced without the use of animal-derived extracellular matrix, and will be the only full-thickness in vitro skin and airway tissue models commercially available as HTS 24- and 96-well formats. The tissue models will be characterized for barrier integrity, morphological appearance and function, and intra- and inter-lot reproducibility. Validation of the models for several regulatory accepted assays including assessment of skin irritation and phototoxicity, and assessment of airway toxicity of tobacco products, will provide key opportunities for immediate commercial use of the scaffold products and models. Commercial products that will result from this project include individual Transwell® scaffold inserts as well as 24- and 96-well HTS Transwell® scaffold plates that will be marketed as stand-alone products to allow researchers to produce any type of tissue models using their own cells and media. Tissue model kits for producing human skin and bronchial models that would include HTS Transwell® electrospun scaffold plates together with pre-qualified cells and culture medium and production protocols could also be offered through partnerships. A conservative market penetration of 1% (global market for insert products, in vitro tissue models and non-animal in vitro screening assays >$1.5 billion) would result in annual revenue exceeding $15 million.

利用基于薄膜的微孔膜支架的组织培养插入物是体外培养的关键组成部分 用作动物试验替代品的组织模型然而，插入支架技术还没有。 近 30 年来取得了显着进步，目前可用的基于薄膜的插入支架只是二维的。 (2D)，并且与天然细胞外基质相比过于刚性，这些 2D 支架不太适合。 用于制作复杂的体外 3 维 (3D) 组织模型 静电纺丝技术可以制作。 新型支架可以更好地复制天然 3D 细胞外基质并克服目前可用的局限性 在相当于第一阶段的前期工作中，我们生产并开发了 3D 静电纺丝支架。 将这些支架连接到高通量筛选 (HTS) Transwell® 组织的制造工艺 我们还展示了利用 3D 电纺支架 Transwell® 产品的可行性。 用于制作全层人体皮肤和支气管组织的器官型体外模型。 与当前可用的模型相比，具有改进的生理相关性和功能性，并且是需要的 作为动物试验的替代方案，修订后的直接第二阶段 SBIR 提案的目标是进一步发展和发展。 Aim 1 将利用 3D 技术将这些新型电纺支架插入物和器官培养模型商业化。 电纺支架插入物可开发由人类角质形成细胞和 Aim 2 将利用 3D 电纺支架插入物开发全层人类真皮成纤维细胞。 由人支气管上皮细胞和人肺成纤维细胞组成的支气管气道模型。 组织模型将在 24 孔和 96 孔 HTS Transwell® 支架板以及单独的 6 孔、12 孔支架板中制作 和 24 孔 Transwell® 支架插入格式将在不使用动物源性的情况下生产。 细胞外基质，并将成为唯一商业化的全层体外皮肤和气道组织模型 提供 HTS 24 孔和 96 孔格式。组织模型将表征屏障完整性、 形态外观和功能，以及批次内和批次间的重现性。 一些公认的监管测定，包括皮肤刺激和光毒性的评估，以及 烟草产品的气道毒性，将为支架的立即商业使用提供关键机会 该项目将产生的商业产品包括单独的 Transwell® 支架。 插入件以及 24 孔和 96 孔 HTS Transwell® 支架板将作为独立产品销售 允许研究人员使用自己的细胞和培养基制作任何类型的组织模型。 制作包含 HTS Transwell® 电纺支架板的人体皮肤和支气管模型 连同预先合格的细胞和培养基以及生产方案也可以通过 保守的市场渗透率为 1%（插入产品、体外组织模型的全球市场）。 和非动物体外筛选试验>15亿美元）将导致年收入超过1500万美元。