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.

组织培养物插入利用基于膜的微孔膜支架是体外的关键组成部分 用作动物测试替代的组织模型。但是，插入脚手架技术尚未 在将近30年内显着进步。当前可用的基于电影的插入脚手架仅是二维 （2D），与天然细胞外基质相比非常刚性。这些2D脚手架不适合 用于生产复杂的体外3维（3D）组织模型。静电技术可以生产 更好地复制天然3D细胞外基质并克服当前可用的局限性的新型脚手架 脚手架。在第I阶段等效的初步工作中，我们生产了3D电纺支架并开发了 将这些脚手架附加到高通量筛选（HTS）Transwell®组织的制造过程 文化插入。我们还证明了使用3D电气传播支架Transwell®产品的可行性 用于产生人体皮肤和支气管组织的全厚度的有机体外模型。这些组织模型 与当前可用的模型相比，具有改善的身体相关性和功能，需要 作为动物测试的替代方案。这项修订后的II阶段SBIR提案的目标是进一步发展和 将这些新型的电纺支架插入物和有机培养模型进行商业化。 AIM 1将使用3D 电纺支架插入物以开发由人角质形成细胞和 人真皮成纤维细胞。 AIM 2将利用3D电气脚手架插入物来发展全厚的人 支气管气道模型由人支气管上皮细胞和人肺成纤维细胞组成。这 组织模型将在24和96孔HTSTranswell®支架板以及单个6-，12-- 和24-WellTranswell®脚手架插入格式。这些模型将在不使用动物衍生的情况下生产 细胞外基质将是商业上唯一的体外皮肤和气道组织模型 可作为HTS 24-和96孔格式可用。组织模型将以屏障完整性为特征， 形态学外观和功能，以及内部和相互重现性。验证模型的 几项监管的评估，包括评估皮肤刺激和光毒性，以及评估 烟道产品的气道毒性将为立即商业使用脚手架提供关键机会 产品和型号。该项目将导致的商业产品包括个人Transwell®脚手架 插入物以及24孔和96孔HTSTranswell®脚手架板将被销售为独立产品 允许研究人员使用自己的细胞和培养基生成任何类型的组织模型。组织模型试剂盒 产生人类皮肤和支气管模型，其中包括HTSTranswell®型电纺脚架板 还可以通过合格的细胞和培养基和生产方案以及通过 伙伴关系。 1％的保守市场渗透（全球插入产品市场，体外组织模型 非动物的体外筛查测定> 15亿美元）将导致年收入超过1500万美元。