Three years ago when I joined Jack Hu's Laboratory at the Institute of Biomedical Sciences, Academia Sinica, it was my first exposure to the multidisciplinary field of biomaterials research. Prior to that I received my graduate training as an immunologist from the National Taiwan University, and when I learned my project was to crosslink hydrogels inside a dendritic cell to preserve its T cell-activating function, I honestly didn't know what to make of the idea. On one hand the idea was so ludicrous that I couldn't imagine how hydrogel could possibly help preserve cellular function. On the other hand the idea made sense on paper and its feasibility seemed apparent. Despite my doubts, I went ahead with several other members in the lab to explore different hydrogel systems and crosslinking strategies to induce hydrogelation in cellular cytosol. The exploration process was far from smooth as biological cells are inherently fragile. Early gelation attempts frequently resulted in complete cellular rupture during the gelation process, but we continued to refine our protocol to facilitate intracellular gelation with minimal cellular disruption.
The first successful preparation of intracellularly gelated cell was made by Nina Chen, who is now a PhD candidate at UT Austin. Using HeLa cells and a UV-activated PEG-DA hydrogel system, she showed that a rapid freeze-thaw cycle followed immediately by UV crosslinking enabled intracellular hydrogelation with minimal cellular disruption. We observed that despite being biologically dead these intracellularly gelated cells preserved their cellular morphology for a long period of time, as if the cells could remain alive indefinitely. Within the lab, we came up with a lot of names for these cells, including zombie cells, ghost cells, undead cells, jello cells etc. Dr. Hu suggested that "cyborg cells" might be more appropriate as these cells are infused with synthetic implants for enhanced functions. Let's just say at the end of the day, we are referring to them as gelated cells for the sake of simplicity.
Figure 1. Preparation of intracellularly gelated cell using a UV-activated crosslinking system. (A) Schematic for the preparation of intracellularly gelated cells. (B) Fluorescence microscopy displaying distinctive plasma membrane and hydrogel components of fluorescein-DA-infused gelated cells. (C) Microscopy observation of gelated HeLa cells 30 days following storage in 4C.
Even though my colleagues and I performed numerous physicochemical characterizations to validate that the gelated cells and live cells possess the same lipid order, membrane fluidity, and protein mobility, I was truly blown away by gelated cells' capability when we first incubated CD8 T cells with gelated dendritic cells displaying cognate peptide antigens. Under fluorescence microscopy, we observed T cells dynamically engaging with the gelated cells, and it really looked as if the dendritic cells were alive to the T cell targets. The life-like interaction further led to dose- and time-dependent T cell expansions. More surprisingly, in an in vivo experiment, we showed that administration of gelated dendritic cells were equivalent to live dendritic cells in expanding adoptively transferred T cells. This result may find applicability in enhancing immunotherapies that can benefit from antigen-specific T cell expansion.
Video 1. Fluorescence microscopy observation of CD8+ T cells (OTI-specific) dynamically engaging with gelated dendritic cells displaying cognate peptide antigens.
To demonstrate the durability of the gelated cells, we further performed a study comparing gelated dendritic cells and non-gelated dendritic cells following 21 days of storage in the fridge. After 21 days, the gelated dendritic cells looked as good as new and the non-gelated controls ruptured and collapsed as expected. Upon injecting these different cells in mice with adoptively transferred T cells, we observed that the gelated dendritic cells remained capable in triggering T cell expansion. It is truly amazing to me as 21 day is a long enough period to even make milk spoil, yet the gelated dendritic cells remained functionally active. I was really impressed by how the simple incorporation of synthetic material can dramatically enhance the functionality of biological cells, and the study opens my eyes to the possibilities of bioengineering research.
Figure 2. Comparison of gelated dendritic cells and non-gelated dendritic cells.
(A) Morphology of gelated dendritic cells and non-gelated dendritic cells following 21 days of storage at 4C. Scale bars = 50 um. (B) In vivo expansion of adoptively transferred OTI-specific CD8 T cells by gelated dendritic cells, non-gelated dendritic cells, and gelated dendritic cells following homogenization.
Outside of bioengineering research for immunological applications, the intracellular gelation technique provides a highly versatile platform for biomembrane and cellular signaling studies. Our laboratory is currently collaborating with several laboratories to use the gelated cells to provide better insights into membrane protein dynamics and into signaling mechanisms via cell-cell contacts. It is interesting every time I explain the gelated cells to collaborators, I always see the same kind of confusion and amazement that reflect my very sentiment when I first heard of the idea. I believe the gelated cells can serve as a useful tool in multiple areas of biomedical research.