This Small Business Innovation Research (SBIR) Phase I project is the development of a lung tissue model based on a new cell culturing platform utilizing nanoparticle-based reagents and magnetic fields to levitate tissue samples and allow three-dimensional (3D) growth. This method naturally grows tissue at the air-liquid interface and is well suited for co-culturing, which are crucial for an accurate in vitro lung model. The application and market for this product will be drug discovery and toxicity screening. In the early stages of drug development, candidate compounds are screened for efficacy and toxicity in in vitro model systems. Currently, these tests are performed in high-throughput assays using two-dimensional (2D) tissue, but 2D culturing alters cellular responses and often leads to misleading results. Subsequent tests in animal models are costly, ethically contentious, and sometimes inaccurate since model organisms may not faithfully reproduce human responses.
The broader impact of this proposal follows from the potential to use the tissue model for improved in vitro efficacy and toxicity assays to reduce the cost of drug development. The reduction of animal testing is also an important goal for ethical reasons. In addition, a lung tissue model that recapitulates natural tissue properties and responses can also find application in toxicity screening of environmental, cosmetic, and industrial factors. The current worldwide expenditure for animal toxicity testing is over $10 billion/yr, and replacement in vitro methods are already over $200 million/yr and growing rapidly. An improved lung model will have significant impact in these areas.
This SBIR was directed at developing a lung tissue model based on a new cell culturing platform utilizing nanoparticle-based reagents and magnetic fields to levitate cells and enables three-dimensional (3D) tissue growth. This method grows tissue at the air-liquid interface and allows the co-culturing of different cell types, which are unmet and necessary conditions for generating an accurate in vitro model of human lung tissue. The application and market for this product is drug discovery and toxicity screening. In the early stages of drug development, candidate compounds are screened for efficacy and toxicity in in vitro model systems. Currently, these tests are performed in high-throughput assays using two-dimensional (2D) tissue, but 2D culturing alters cellular responses and leads to misleading results. Subsequent tests in animal models are costly, ethically contentious, and often inaccurate since model organisms are known to poorly reproduce human responses. The broader impact of this proposal follows from the potential to use the tissue model for improved in vitro efficacy and toxicity assays to reduce the cost of drug development. The reduction of animal testing is also important for ethical reasons. In addition, lung tissue models that recapitulate natural tissue response can also find application in toxicity screening of environmental, cosmetic, and industrial factors. The worldwide expenditure for animal toxicity testing is over $10 billion/yr, and replacement of in vitro methods is already over $200 million/yr and growing rapidly. An improved in vitro lung tissue model will have significant impact in these areas. Here, we developed a multi-cellular lung tissue model by first culturing human pulmonary endothelial (PEC), small airway epithelial (SAEpiC), pulmonary fibroblast (HPF), and human tracheal smooth muscle cells (SMC). These are the four major lung cell types, and they play roles in many lung disorders. Importantly, these represent the first primary cell types cultured with n3D’s magnetic levitation method (MLM). We achieved our objectives, and Fig. 1 displays the results of levitated cell culture of these four major cell types, which were generated from human primary cells. All cell types were found to grow robustly under a broad range of conditions. We found only small differences in the optimization of different cell types, and thus, give a combined protocol that notes any specific alternate methods to follow for a particular cell type. Furthermore, finding the optimal procedures for levitating a certain cell type involves making tradeoffs between competing metrics (Fig. 2). Typically, one desires the highest yield of levitated cells so that fewer cells are wasted by remaining on the Petri dish bottom. One important outcome of this optimization phase was obtained by comparative genomic hybridization (CGH) screening, where HPF cells showed no chromosomal abnormalities in cells treated with nanoparticles and/or levitated. This result indicates that perturbations caused by nanoparticles and levitation forces are negligible compared to the improved in vivo similarity offered by 3D culturing. Another metric that was established/confirmed with lung primary cells was the rapid formation of the 3D structures. The concentrating effect of the magnetic field induces cell-cell interactions that lead to unprecedented rapid formation of 3D tissue (Fig. 3). Next, there is a shortage of co-culture techniques that effectively recreate native tissue architecture. Tissues can be generalized to have a layered structure. For alveoli, there are four layers made up of epithelial cells, SMC, HPF, and endothelial cells. This is nearly impossible to reproduce in a 2D cell culture environment, and no 3D cell culture system supports the layering of multiple cell types. Thus, there exists a need for a 3D co-culture system that can assemble a cellular environment similar of native tissue. Therefore, we developed a simple device that works with the basic MLM to accomplish this goal. This tool, which is called a magnetic pen, is now part of n3D’s intellectual property portfolio (Fig. 4). One of the pen designs includes a high-throughput version for handling multiple cultures. Next, we adapted the MLM and magnetic pen for culturing cells in 3D and can produce multicellular structures with in vivo-like properties within 24 hours. One of our proof-of-concept tests shows that we successfully assembled a layered structure by co-culturing HPFs and aortic valve endothelial cells (PAVECs) (Fig. 5). Recently, we have generated a single tissue construct with all four cell types. Finally, we compare and show the similarity between fibroblasts cultured with the MLM and the connective tissue of an in vivo sample (Fig.6A and B). Here, we compare HPF 3D structure with previously published data from the human trachea (the in vivo image from B and C were obtained respectively from the Interactive Histology Atlas of the University of Oklahoma). This tissue is composed mostly of fibroblasts. HPF cells are usually bipolar or star-like in shape in vivo, and the 3D structure (Fig. 6A) showed these types of morphologies while the 2D cultures (Fig. 6D) did not.
