Yes, I know what you are thinking: Jell-O? Why would Jell-O be used in a materials lab for growing cells? Well, it isn’t the taste of this childhood snack that makes it perfect for tissue engineering; it is its composition. The most abundant protein in our body is a compound called collagen, which makes up most of the extracellular matrix that cells live in. If you take collagen and break it up into smaller pieces, you get gelatin, the material that makes up Jell-O. Tissue engineers, who work to maintain, improve, and/or restore biological tissues in our bodies, can use gelatin to simulate an in vivo environment, meaning the natural environment of a living organism. This not only opens applications in tissue culture but also has innumerable applications for injectable therapeutics, which are injectable solutions that can regrow or enhance existing tissue. While the field of tissue engineering is in its infancy, promising therapeutics with tissue culture using scaffolds like gelatin are already being applied to synthetic skin and organs for injured patients.
Injuries and degenerative diseases, such as Alzheimer’s and multiple sclerosis, can lead to long recoveries and/or irreversible damage. To alleviate these effects, one could inject specific stem cells into the damaged area to grow themselves, trigger new growth within your body, and limit inflammation. However, direct injection of cells into the body results in low viability and significant dispersion of the cells throughout the body, moving them away from where they are needed most. Here is where our “scientific Jell-O” comes in. Gelatin, which is a hydrated complex of polymers, can be used to surround encapsulated cells, thereby increasing viability and stabilizing cell location following their injection. My research in Dr. Kyung Jae Jeong’s lab at the University of New Hampshire, which was funded by the Research Experience and Apprenticeship Program (REAP) through the Hamel Center for Undergraduate Research, looked at ways to alter hydrogels made of gelatin to make them more efficient when used in injectable therapeutics.
Figure 1. A microscope image of Jeong lab's novel 10% gelatin microgels.
Microporous Hydrogel Methodology and Characterization
Conventional hydrogels are a complex of polymer chains that hold large amounts of water in comparison to their mass. However, these hydrogels lack porosity, mitigating cell growth and penetration of host biology. To address this, my faculty mentor, Dr. Jeong, and my graduate mentor, Dr. Seth Edwards, use novel injectable microporous hydrogels made of gelatin to promote cell spreading and proliferation. It may be easier to think of conventional hydrogels as one solid block of Jell-O with cells stuck inside, while a microgel structure is composed of many small beads of Jell-O with cells growing in the space between. My summer research focused on the alteration of microgel diameter. Controlling microgel diameter can influence hydrogel properties such as nutrient transfer and available surface area, and cellular responses such as cell morphology, spreading, and differentiation. These variables help characterize cell behavior for predictability and safety of therapeutics in vivo.
Figure 2. A microscope image of 5% gelatin microgels with a TWEEN 20 emulsifier developed by the author.
I formed the microgels by dissolving gelatin in water and dropping the solution into an oil bath, creating an emulsion. The gelatin solution breaks up into small droplets in the oil that can be collected and cured together. The small spheres of gelatin are connected by microbial transglutaminase (mTG), a bacterial enzyme that connects the glutamine and lysine substituents of gelatin. After curing, a bulk hydrogel is left with space for cells to grow in between the microspheres. (Figure 1)
I tried lower concentrations of gelatin during the emulsion process, varying the mixing speeds and microgel curing times. Each variable had its own effect, some greater than others. I also integrated an emulsifier (TWEEN 20) of varying concentrations to reduce polarity on the hydrated gelatin’s surface. By integrating an emulsifier into the hydrogel emulsion protocol and decreasing gelatin concentration to 5% (from the original 10%), the diameter of the microgels were reduced to an eighth of their original size (around 40 µm). (Figure 2)
With this new manipulation of microgels I further researched cytotoxicity (toxicity of the environment to the cells). To do this, I cultured 3T3 cells (a fast-growing cell line of mouse fibroblasts) and formed microgels with small diameters and the control diameters. After three days of culture, I compared the control of pure gelatin microgels with the TWEEN 20 with a lowered gelatin concentration.
Figure 3. A confocal microscopy stack image (left) and slice image (right) of living cells (green) and dead cells (red) growing within a microporous hydrogel structure.
I measured viability by performing a live/dead assay with confocal imaging. This entails using a high-definition microscope and adding dye to stain living cells green and dead cells red. I detected no difference in cell viability between the two hydrogels. (Figure 3)
For further analysis I conducted a rheology test on the bulk hydrogel I had created with smaller microgels to measure and characterize its elasticity. This showed that the smaller hydrogels formed a stiffer bulk hydrogel than the one formed with larger control microgels at all points in the curing process. Rheology was performed only once, making the results less reliable. However, the stiffness is most likely caused by a larger surface area, which makes more interactions through mTG cross-links/bonds.
Results and Avenues for Application within Tissue Engineering
In the end I was able to decrease the diameter of the gelatin microgels, but I have yet to show their relation to cell growth. In theory, the differing concentrations of gelatin and emulsifier used in the microgels I created should induce different cell proliferation and differentiation. It has been shown through conventional nonporous hydrogels that stiffness does change cell differentiation trends. However, it is unclear if the cells are affected by the stiffness change in the porous environment. While the bulk hydrogel may have more mTG cross-linking, and thus be stiffer, there is still a large interior network that could limit the cells from experiencing overall stiffness. If that is the case, then a variation in hydrogel stiffness is important for different applications within the body. Depending on the biological environment this technology is applied to, less stiff hydrogel that degrades more quickly may be required, while others require the opposite. For example, neurons might like a less rigid structure with a smaller microgel for increased surface area, while bone cells may like a stiffer gel with larger microgels for better penetration of host biology.
Having control over all variables in the hydrogel leads to greater specificity and customization, depending on the intended use. As of now, conventional hydrogels are nonporous, and therefore restrict cell growth, penetration of host biology, nutrient transfer, and cell viability. The novel microporous hydrogels fix these problems but are not fully understood. Every cell location and cell type will need different conditions for efficient use in injectable therapies. These conditions can theoretically be accommodated with this technology, but these possibilities must be researched. If my research could be extrapolated, a full understanding of microgel cross-linking and diameter could lead to a new field of injectable therapeutics. Personally, I hope to continue my work in the Jeong lab by applying this technology to the culture of red blood cells. And to think, all of this possibility was sitting right in front of our faces in Jell-O’s composition.
I would like to acknowledge my faculty research mentor Dr. Kyung Jae Jeong for not only taking me on as a first-year researcher but accelerating my learning and cultivating an environment of opportunity. Further, I am grateful to Dr. Seth Edwards, my graduate mentor, for being alongside me every step of the way and for always being in my corner—truly the guiding voice in my process. None of this would be possible without the amazing staff and donors (Mr. Dana Hamel, Dr. George Wildman, Mr. Nicholas Bencivenga) at the Hamel Center for Undergraduate Research at UNH; without the Research, Experience, and Apprenticeship Program, I would not be writing today. I owe my future to the work and charity of these people, and I am forever grateful to the numerous efforts that have been made to benefit me.
Author and Mentor Bios
Originally from Concord, New Hampshire, Jack Fenway Reynolds will graduate in May 2026 with a bachelor of science degree in bioengineering. He conducted his research on microgels through a Research Experience and Apprenticeship Program (REAP) grant funded by the Hamel Center for Undergraduate Research. Jack was looking for a project involving neurons and therapeutics, specifically in neurodegenerative diseases due to his mother's diagnosis with multiple sclerosis. He is very interested in the limited regenerative capacity of neurons and methods of circumnavigating this limitation. In talking with Dr. Jeong about possible topics relating to Jack’s interests, Dr. Joeng proposed the microgels his lab had been working on. Jack was pleasantly surprised at how accessible research was. As a new researcher, Jack describes having a steep learning curve, but he was mostly independent and was involved in scientific conversation with Ph.D. students and professors within a few weeks. He decided to submit to Inquiry because he is passionate about the public understanding of science. While using gelatin in this project, he wanted to convey the simplicity of this technology to excite people about the future, highlight the possibilities, and maybe inspire someone else. Jack would like to pursue a Ph.D. in a related scientific field and work in pharmaceutical/therapeutic development. While the research was very enlightening for the content and methodology, writing for Inquiry has shown him where science meets the public. Jack now wants to be on the cutting edge of science and educate the public about scientific developments.
Dr. Kyung Jae Jeong is an associate professor of chemical engineering and bioengineering at the University of New Hampshire, beginning in 2013. His research interests revolve around biomaterials, drug delivery systems, medical devices, and tissue engineering. He mentored author, Jack Reynolds, for a Research Experience Apprenticeship Program (REAP) during the summer of 2023. Dr. Jeong has been interested in creating functional injectable hydrogels for medicine. However, the injectable hydrogels were nonporous and not optimal for cell delivery. His lab worked to develop the approach of assembling gelatin microgels using an enzymatic reaction in 2018 and applied the approach to neural stem cell encapsulation last year. Dr. Jeong has mentored Inquiry author, Ryan Boudreau previously. He describes Jack as “pleasant to work with” as well as being “highly motivated to learn new things, intellectually bright, and hard-working.”
Copyright 2024, Jack Reynolds
