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 组织模型被用作动物试验的替代品。然而，插入支架技术还没有 近30年来取得了显著进步。目前可用的基于膜的插入支架仅是二维的 (2D)并且与天然细胞外基质相比过于刚性。这些2D支架并不适合 用于生产复杂的体外三维（3D）组织模型。静电纺丝技术可以生产 新的支架，更好地复制天然的3D细胞外基质，并克服了目前可用的限制， 脚手架在I期等效的初步工作中，我们生产了3D静电纺丝支架，并开发了 用于将这些支架连接到高通量筛选（HTS）Transwell®组织的制造工艺 文化嵌入我们还证明了利用3D静电纺丝支架Transwell®产品的可行性 用于产生全层人类皮肤和支气管组织的器官型体外模型。这些组织模型 与目前可用的模型相比，具有改进的生理相关性和功能，并且需要 作为动物实验的替代品。修订后的直接第二阶段SBIR提案的目标是进一步开发和 商业化这些新的静电纺丝支架插入物和器官型培养模型。目标1将使用3D 电纺支架插入物，以开发由人角质形成细胞组成的全层人皮肤模型， 人皮肤成纤维细胞目的2将利用三维静电纺丝支架插入物来开发全层人类 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。的 组织模型将在24孔和96孔HTS Transwell®支架板中产生，以及单独的6孔、12孔和14孔支架板。 和24孔Transwell®支架插入物形式。这些模型将在不使用动物源性 细胞外基质，并将是唯一的全厚度体外皮肤和气道组织模型商业化 以HTS 24孔和96孔形式提供。将对组织模型的屏障完整性进行表征， 形态学外观和功能，以及批内和批间重现性。模型的验证 几种监管认可的试验，包括皮肤刺激性和光毒性评估，以及 烟草制品的气道毒性，将为支架的直接商业应用提供关键机会。 产品和模型。该项目将产生的商业产品包括单个Transwell®支架 插入物以及24孔和96孔HTS Transwell®支架板，将作为独立产品销售 允许研究人员使用他们自己的细胞和培养基生产任何类型的组织模型。组织模型套件，用于 生产包括HTS Transwell®电纺支架板的人类皮肤和支气管模型 与预先合格的细胞和培养基一起， 伙伴关系。保守的市场渗透率为1%（全球植入物产品市场，体外组织模型 和非动物体外筛选测定> 15亿美元）将导致年收入超过1500万美元。