Photopolymerizations for Creating Engineered Microenvironments: From 3D Culture Models to Biomanufacturing Facilitated by Interdisciplinary Collaborations

By April M. Kloxin, Ph.D., Chemical and Biomolecular Engineering and Materials Science and Engineering, University of Delaware

Photochemistry increasingly is used as a tool in both academic and industrial applications, largely owing to its scalability and precision in controlling when and where reactions happen under mild conditions through the application of light. ^1-3 Consequently, approaches and technologies that use photochemistry provide significant opportunity for collaboration between academic and industrial research, facilitating the translation of basic scientific discoveries into practical solutions that address outstanding challenges and societal needs. Examples of these opportunities for basic-to-applied research collaborations with photochemistry can be found in a number of fields, including in biomaterials and bioengineering. In this article, two examples will be highlighted from the author’s own collaborative research that demonstrate the potential for photochemistry to bridge the gap between fundamental and applied science. The article also will provide insights on how to foster stronger partnerships between researchers from academic institutions, national institutes and laboratories, and industry to drive innovation and create meaningful impact. This article builds off topics presented at the Photopolymerization Fundamentals Conference in 2023 and in a webinar hosted by RadTech and the Photopolymerization and Additive Manufacturing Alliance (PAMA) in 2024.

Photopolymerizations and cell-culture applications

Photopolymerizations have proven to be relevant in a variety of cell-culture applications, including well-defined three-dimensional (3D) cultures. ^4-7 The growing recognition of the importance of the human cell microenvironment’s role in regulating cellular function and fate has led to the creation of photopolymerized materials inspired by human tissues. Photo-initiation conditions compatible with live cells have been demonstrated, and design principles for forming soft materials from building blocks with photopolymerizable reactive handles have been established. Hydrophilic building blocks, such as polymers, peptides and proteins, and polysaccharides, have been functionalized with reactive groups, such as electron-poor (e.g., (meth)acrylates) or electron-rich (e.g., norbornenes, allyls) vinyl groups that can be photocrosslinked with themselves or with multi-functional thiols, respectively. These rapid reactions can be initiated with cytocompatible doses of long wavelength ultraviolet (UV) or visible light. The result is the formation of water-swollen networks, or hydrogels, suitable for both two-dimensional (2D) and 3D culture of cells.

When the author established her lab as a faculty member at the University of Delaware (UD), the author began utilizing photo-initiated thiol-ene reactions to form hydrogels inspired by the common metastatic sites for breast cancer. ^8-15 Informed by the work of and collaborations with biological, translational and clinical researchers, the author came to recognize the pressing need for well-defined 3D culture systems to study breast cancer late recurrence, one of several metastatic diseases that have high mortality rate owing, in part, to the lack of technologies for better understanding mechanism, early detection and treatment. Utilizing photopolymerized hydrogels as synthetic extracellular matrices (ECMs), reductionist 3D culture models were established, inspired by bone marrow and lung tissues, the two most common sites for breast cancer late recurrence, as tools for understanding aspects of this challenging disease. Informed by existing literature, different compositions of accessible building blocks were selected, including biologically-inert multi-arm poly(ethylene glycol) (PEG) functionalized with thiols for controlling network connectivity (and thereby modulus) and peptides functionalized with one or two allyloxycarbonyl groups for imparting bioactivity to the network (Figure 1). By varying the concentration and chemical identity of these building blocks, well-defined synthetic ECMs were created with relevant mechanical and biochemical properties inspired by the native ECM.

These photopolymerized materials were used to test hypotheses about how the composition of the cell microenvironment influenced breast cancer growth vs. dormancy. ^{14, 15} Leveraging the tunable properties afforded by photopolymerization, it was determined that the composition of the matrix surrounding the cells, in conjunction with the subtype of the breast cancer cell (e.g., associated with slow growth vs. rapid metastasis), plays a significant role. Additionally, other cells found in the metastatic niche, such as bone cells, also impacted the outcomes. These ‘inputs’ determined whether the cell entered a dormant state (‘asleep’), which is hypothesized to allow evasion of the primary tumor treatment and allow cells to later ‘wake up’ for a late recurrence, or continued growth (always ‘awake’), which is hypothesized to leave cells susceptible to the treatment of the primary tumor and less likely to lead to late recurrence. A range of molecular tools were used with these synthetic ECMs, enabled by the molecular design of the material. For example, cells could be imaged in three dimensions within the optically clear synthetic ECM, and transcriptomics and flow cytometry analyses could be performed on cells harvested from the synthetic ECM using enzymes. These 3D culture systems, benchmarked against preclinical models and clinical data, provide opportunities for evaluating therapeutic strategies to prevent late recurrence.

Partnering for accessibility

How can these well-defined 3D model systems be made more accessible to the broader community for biological research and societal impact? One approach being taken is to partner with an industrial collaborator, Inventia Life Science, which has established a commercial bioprinter based on inkjet printing technology (RASTRUMÔ). This system enables the creation of PEG-peptide hydrogels in multi-well plates for high-throughput 3D cultures (Figure 2). ^16-18 By using the company’s commercially available reactive polymer and peptide ‘bioinks’, the lab can print similar formulations to the photopolymerized synthetic ECMs into 96-well plates, allowing mechanistic and therapeutic studies on cell types of interest.

The collaboration aims to create 3D culture models with increasing complexity for a range of biological applications. Other partnerships have been established, including those with researchers at institutes and national labs, such as the Helen F. Graham Cancer Center and Research Institute and the Army Research Laboratory, to apply these well-defined, high-throughput 3D cultures in studies that offer new insights toward improving human health. More broadly, additive manufacturing approaches like this provide exciting opportunities to bridge fundamental and applied materials research in bioengineering, including the development of tissue and organ mimics through academic-industrial partnerships.

Integrating bioinspired soft materials into scalable systems presents new opportunities for controlling the cell microenvironment during biomanufacturing processes. The National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL), part of the Manufacturing USA network [which is supported the US Department of Commerce, National Institute of Standards and Technology (NIST)], is facilitating collaborations between academic and institute researchers and industrial partners to drive technological innovations that make biopharmaceutical manufacturing of products, such as therapeutic cells and proteins, more efficient and flexible. Through NIIMBL, the author’s group is collaborating with academic and industrial partners to establish scalable approaches for manufacturing cell therapies. The goal is to reduce costs and increase accessibility, while ensuring consistency and efficacy. ²⁰ Specifically, an approach has been established to create photopolymerized, bioinspired hydrogel coatings on membranes that can be integrated within flow-based devices to produce engineered immune cells, such as CAR T cells, for cancer treatment (Figure 3). This combination of bottom-up and top-down engineering approaches uniformly enhances cell activation and proliferation and allows for cell modification with less virus than current industry standards.

Future opportunities

The collaborations mentioned above illustrate how academic and industrial researchers can work together to drive innovation and create meaningful impact. Engaging with institutes like NIST and NIIMBL, along with other local and national centers and consortia [e.g., Delaware Biotechnology Institute (DBI), Industry-University Cooperative Research Centers (IUCRCs)], and professional societies and trade groups (e.g., RadTech, PAMA, DelawareBio) provides opportunities to connect with potential partners, identifying shared goals and strategically planning collaborations of mutual and broad interest from project conception through completion. Additionally, working through institutes or centers offers benefits, such as access to seed funding, core facilities and streamlining agreements between different entities. These collaborations foster vibrant training environments for graduate students by connecting them with end users and applications, which can be both motivating and valuable for networking and mentoring. Overall, collaborative partnerships help bridge the gap between basic and applied research, support technology commercialization and contribute to workforce development, ensuring research has a lasting impact.

Acknowledgements

Thank you to the researchers with whom the author has have had the pleasure of doing this work, to the funding agencies that have supported these research endeavors, and to the organizations that have provided structures and support to facilitate this interdisciplinary research! Specific contributions are noted in the referenced peer-review publications.

April M. Kloxin, Ph.D., is the associate department chair and joint professor in Chemical and Biomolecular Engineering and Materials Science and Engineering at the University of Delaware. She received her Bachelor’s and Master’s degrees from North Carolina State University and her Doctorate from the University of Colorado at Boulder. Kloxin can be reached at akloxin@udel.edu.

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