This Small Business Technology Transfer (STTR) Phase I project aims to use a newly discovered peptide (h9e) hydrogel technology to provide an affordable and easy-to-use 3D cell culture system with high throughput screen and accurate in vivo representative. To bridge the fundamental understanding of cellular characteristics and the extensive complexity of tissue and organs, hydrogel is the most promising material for 3D cell culture. Most of the existing hydrogel materials are limited under the physiological conditions, complex operating steps for cell encapsulation, difficulties for cell recovery from culture scaffold and high production cost. The objectives of this project are to 1) develop desirable h9e-medium hydrogel for cancer, normal and stem cell culture and 2) reduce hydrogel production cost. The success of this project will provide a superior 3D cell culture system with convenient cell encapsulation and recovery properties. Standard user-friendly protocols and successful examples of different cell lines growing in this system will be built. The optimal procedures of h9e chemical synthesis will be delivered for large scale synthesis with lower production cost. Furthermore, the proposed works will provide new insight about how drug discovery and tissue regenerative development be achieved based on an in vitro 3D cell culture system.
The broader impact/commercial potential of this project, if successful, will be to provide an advanced life science tool for cell/tissue culture industry with over $6.0 billion global market. Simplification of operating process of 3D cell culture and reduction of hydrogel synthesis cost will greatly advance this technology to be used routinely for both academic and industry. Drug discovery and regenerative medicine development will be benefited from this high throughput screening and better in vivo representative cell culture system and extent to affect the pharmaceuticals, biotechnology and life sciences industries, which possess over $1,001.5 billion global market. Furthermore, the injectable capability of h9e hydrogel system will introduce many potential biomedical applications such as drug delivery, wounds healing, which are beyond the proposed 3D cell culture. The proposed approach will be broadly disseminated through international meetings, peer-reviewed journals, company website, and other outreach activities. Fundamental knowledge derived from this proposed technology will bridge the gap between cellular information and in vivo system and benefit all-level students, scientific community and entire society. The more accurate data derived from this tool will reduce the research use of animal models, which will relief the extensive criticism within our society.
Life science research progress and effectiveness has been significantly limited by the two dimensional (2D) cell culture, however, mainly because cells grown in a flat environment cannot accurately predict what is expected in a real three dimensional (3D) living body (i.e. animal, human body). The limitations of 2D cell culture, a $6 billion growing market, have motivated scientists to search for a 3D cell culture technology solution. Currently available 3D cell cultures, an approximate $77 million market, have severe application and process limitations that reduces their uses for life science research, such as poor cell encapsulation environment, low reproducibility, difficult cell isolation, and complicated and time consuming 3D cell culture procedures. PepGel is a high potential, pre-clinical (research) product driven company, commercializing the proprietary structure-forming hydrogels to the research and industrial health care market place. The main goal of the STTR Phase I and IB project is to develop a peptide-medium hydrogel matrix for 3D cell cultures and optimize the hydrogel production for cost reduction, believing this will lead to the first commercial product, named "PGmatrix Kit" for research. In this STTR project, we have successfully developed peptide-medium hydrogel with four most commonly used cell media (DMEM, MEM, L-15, RPMI). The physical properties of these hydrogels have been characterized. The drug diffusion kinetics in the hydrogel was also preliminarily studied by using selected anticancer drugs. More importantly, we have identified the hydrogel system for 3D cell culture of breast cancer cells and rat stem cells. Those cells grew very well in our hydrogel system. We identified and optimized conditions to permit long-term expansion in an undifferentiated, apparently pluripotent stem cell state in the hydrogel. Particularly, we successfully obtained 30+ passages for both rat embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) grown in the gel. Furthermore, we modified the peptide synthesis and purify procedure to obtained higher synthesis yield rate and product purity, which can help to simplify hydrogel preparation and reduce the product cost. The prototype of PGmatrix research kit is ready for feasibility trials. In addition, we established and characterized 3D cultures of human embryonic stem cells (HESCs) by using our PGmatrix hydrogel system. We have successfully grown HESCs both in 2D and 3D. We demonstrate that HESCs were able to grow in different concentrations of PGmatrix hydrogel ranging from 0.5% to 4% and were further passaged in PGmatrix hydrogel system. It is critical and great interest to demonstrate the feasibility for expanding human pluripotent stem cells in 3D hydrogel system. A viable commercial product would attract great attention for current stem cell culture market.
