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.

This revised Direct Phase II SBIR research project will improve the lives of people by providing researchers with novel and more physiologically-relevant tools and methods for in vitro testing. These tools will be used for testing potential toxicity of drugs and chemicals as well as for developing therapeutic drugs. This research project will use advanced products and methods to produce in vitro human tissue models of human respiratory and skin tissues. These products and tissue models will be utilized by researchers to test consumer products, cosmetics and chemicals for potential toxicity to the skin, and for testing chemicals and products such as tobacco smoke for potential toxicity to respiratory tissues.