Ultra-low content physio-chemically crosslinked gelatin hydrogel improves encapsulated 3D cell culture.

Introduction:: Gelatin hydrogels such as photo-crosslinked gelatin methacrylate (GelMA) are widely used to fabricate scaffolds for 3D cell culture, bioprinting, and tissue engineering due to cell-adhesive nature and tunable physiochemical properties. Yet, the current GelMA hydrogels require high gelatin concentration (5 - 15% w/v) to achieve desired stability and mechanical strength. Unfortunately, the high-concentration, dense matrix surrounding the cells - while highly stable – restricts the growth and viability of encapsulated cells. Current GelMA hydrogels at concentrations below 5% are generally unstable for 3D cell culture. The objective of this study was to evaluate the effect of combined physio-chemical crosslinking on the formation of ultra-low concentration (ULC) GelMA hydrogels and study 3D cell growth and organoid formation in these new matrices. We demonstrated that combined physical and chemical crosslinking enables the formation of stable gelatin hydrogel with a remarkably low gelatin concentration (1 % w/v). This ULC gelatin hydrogel enabled a stable 3D distribution of cells in the matrix, allowed the spreading of encapsulated 3T3 fibroblast cells, and induced the organoid formation of encapsulated human cholangiocarcinoma cells (HuCCT-1) compared to traditional 5% and 10% GelMA hydrogels.


Materials and Methods:: Gelatin was modified with methacrylate groups to prepare GelMA precursor. Redissolved GelMA powder in photoinitiator solution was either chemically crosslinked at 37°C using UV light to prepare traditional GelMA hydrogel or was allowed to form a physical gel at 4°C for one hour followed by immediate exposure to UV light for 5 minutes to chemically crosslink the hydrogel (Gel^Phy/photo). The mechanical properties of hydrogels were quantified using a rheometer. The 3D distribution of cells was analyzed by using a confocal microscope. Pore size was analyzed using field emission scanning electron microscopy (FESEM). For 3D culture, 3T3 fibroblast and HuCCT-1 cells were encapsulated in this ultra-low (1% Gel^Phy/photo) and traditional high-concentration (5% and 10% GelMA) hydrogel. To quantitatively analyze 3T3 cell spreading, cell area, and aspect ratio were analyzed by image analysis of calcein-AM stained cells. HuCCT-1 cells were stained for nuclei and F-actin to study morphology after 7 days in culture. All experiments were run in triplicates and all the data is presented as mean ± SD.


Results, Conclusions, and Discussions:: Results and Discussion: ULC gelatin hydrogels were successfully fabricated by combining physical and chemical crosslinking which was confirmed by an inverted vial test. 1% Gel^Phy/photo held its shape as compared to 1% GelMA which did not gel (Fig.1a(i)). This stability was presumably due to the formation of triple helices upon cooling, resulting in additional hydrogen bonding which contributed towards the improved mechanical properties as no increase in storage modulus was observed when 1% GelMA was subjected to UV exposure for 30 mins (Fig.1 a(ii)). The 1% Gel^Phy/photo enabled 3D encapsulation/distribution of cells in matrix, which could not be achieved in 1% GelMA with only chemical crosslinking (Fig.1b). SEM imaging revealed that the pore size was significantly larger in 1% Gel^Phy/photo compared to 5% gelatin hydrogels (Fig. 1c). Cell morphology analysis indicated significantly higher cell area and aspect ratio in 1% Gel^Phy/photo as compared to control 10% GelMA hydrogel that has been previously used for 3D cell culture (Fig.1d (i & ii)). This was likely due to large pore size of 1% Gel^Phy/photo which provided ample space for cellular growth, allowing efficient diffusion of oxygen and nutrients for cell growth, thus providing a better invitro-microenvironment for cells to grow. Furthermore, the encapsulated HuCCT-1 cells displayed a compact morphology and exhibited pseudo-glandular rosettes representing the corresponding tumor of origin (Fig.1e). While, in traditional 5% GelMA, they stayed as single cells indicating that low polymer concentration favors matrix remodeling which allowed HuCCT-1 cells to self-assemble into organoids.



Conclusion: Physio-chemical crosslinking enabled fabrication of low polymer content, stable gelatin scaffolds, which were elusive thus far. These new gelatin hydrogels favored cell spreading and morphogenesis of encapsulated 3T3 fibroblast and HuCCT-1 cells. Together, this study highlights the importance of the biopolymer content and its critical role in improving in-vitro 3D cell culture and to provide a more conducive, yet stable environment for 3D cell culture.


Acknowledgements (Optional): :

References (Optional): :