This Small Business Innovation Research (SBIR) Phase I project is to apply novel bio-orthogonal tetrazine ligation technology to fluorescent labeling of DNA replication, and to ultimately produce a fluorescence imaging kit (FIK) for cell proliferation. The advantages are two-fold: 1) Because of the novel chemical mechanism, the FIK would allow for in vivo cell imaging, essentially providing continuous observation of cell proliferation with minimal interference from cell metabolism. Fluorescent microscopy could be done with live cells. 2) A single cell could be monitored through various stages of cell cycle. None of the existing methods allow for in vivo imaging of cell proliferation. Thus, conceptually, the difference between the capabilities of the existing assays and proposed FIK assay is analogous to the difference between a snapshot and a video recording.
The broader impact/commercial potential of this project is to develop an assay for cell proliferation compatible with high-throughput screening technology. The assay can be applied to differentiate between quiescent and actively cycling stem cells, and, eventually, to differentiate between cancer stem cells and cancer cells. The low cost of reagents, simplicity of sample handling, and readiness to be adapted to a multi-well format make the proposed methodology an ideal candidate for application to high-throughput screening assays that will find applications in the fields of cancer biology and neurogenesis, as well as developmental and molecular biology. These fields all share a common research need for close observation of cell processes.
The goal of the project has been to develop a commercial toolkit for fluorescence imaging of cell proliferation—to trace the growth of cell populations by observing how the "mother cell" grows and divides to produce two "daughter cells". The fluorescence imaging kit (FIK) would be used as a research tool to study cell proliferation in many fields of biology concerned with cell development such as cancer research, neurosciences, and stem cell research, to name a few. This toolkit would consist of two chemical reagents, as well as a simple biological protocol on how to use them for cell imaging. One of the main objectives of the project has been synthesis of the reagents of FIK. This goal has been accomplished in full. Several alternate versions of each reagent have been synthesized in order to achieve optimal cell imaging properties. Following completion of the synthesis, optimization of the biological protocols, the second major goal of the project, was initiated. In the first stage of this work we established a proper cell system for studying proliferation. This system has been tested using commercial reagents for its robustness and reproducibility. In the second stage, cell permeability, as well as toxicity of the FIK reagents has been tested. All of the reagents have been found to be cell permeable and non-toxic at the experimental concentrations. Optimization of the cell imaging protocols is still ongoing. For this project, MBMR Biolabs, Inc. has established an academic and research collaboration with the University of Delaware, as well as a strong advisory team ranging from experts in biology and chemistry to experts in business development and marketing. As part of its outreach activities, MBMR has established a web presence, highlighting the company’s technology, team, and press releases, and has taken every opportunity to disseminate information about MBMR’s project-related activities by word-of-mouth to lay members of the public and MBMR’s business and non-science network. Importantly, MBMR’s efforts have resulted also in the training for the under-served persons, namely scientific training for persons with disabilities. As part of the commercialization activities, MBMR has reached out to possible strategic collaborators to help take the product to the next level. Further, the company has reached out to commercialization advisors, including the New York Industrial and Technology Assistance Corporation, the Dartmouth Entrepreneurial Network meetings, and has participated in meetings with business leaders at the Astia Summit and Golden Seed Presentations.
