Gelatin methacryloyl and its hydrogels with an exceptional degree of controllability and batch-to-batch consistency

Gelatin methacryloyl (GelMA) is a versatile material for a wide range of bioapplications. There is an intense interest in developing effective chemical strategies to prepare GelMA with a high degree of batch-to-batch consistency and controllability in terms of methacryloyl functionalization and physiochemical properties. Herein, we systematically investigated the batch-to-batch reproducibility and controllability of producing GelMA (target highly and lowly substituted versions) via a one-pot strategy. To assess the GelMA product, several parameters were evaluated, including the degree of methacryloylation, secondary structure, and enzymatic degradation, along with the mechanical properties and cell viability of GelMA hydrogels. The results showed that two types of target GelMA with five batches exhibited a high degree of controllability and reproducibility in compositional, structural, and functional properties owing to the highly controllable one-pot strategy.


Results and Discussion
Controllable preparation of highly and lowly substituted gelatin methacryloyl with five batches (DS100_1~5 and DS60_1~5). GelMA samples with five different batches were synthesized in the CB buffer system by a one-pot method as illustrated in Fig. 1. Modified synthesis parameters (10 (w/v)% gelatin, 0.25 M CB buffer, a reaction time of 1 h, and reaction temperature of 55 °C) were utilized according to the literature as seen in Table 1 20 . In this study, two types of GelMA samples (target degrees of substitution (DS): DS = 100% and 60%) with five batches were synthesized with feeding mole ratios of MAA to amino groups of gelatin at 1.859:1 and 0.628:1, respectively. GelMA samples (DS = 100% and 60%) with different batches were labeled as DS100_1~5 and DS60_1~5. The obtained products appeared white yellowish. The yields of all GelMA products were around 90% (92%, 93%, 88%, 90%, and 88% for DS100_1~5 and 92%, 94%, 92%, 91%, and 92% for DS60_1~5, respectively), indicating that the current one-pot GelMA batch process can produce consistent yields.
The main challenge of GelMA synthesis is to precisely control the DS and properties of GelMA in every batch because less controllable reaction systems can lead to less controllable outcomes of GelMA, as seen in Table S1 15 . There are many parameters involved in the reaction of gelatin and methacrylic anhydride (MAA) such as pH, temperature, reaction time, a gelatin concentration, a buffer system, a mole ratio of gelatin and MAA, and stirring speed. The crucial thing of GelMA synthesis is to maintain the pH of the reaction solution since the byproduct (methacrylic acid, MA) can decrease the pH of the solution during the reaction, hindering the forward reaction owing to the protonation of free amino groups. To this end, sequential or dropwise addition of MAA was The process of GelMA production including synthesis, paper filtration, tangential flow filtration (TFF), and lyophilization. employed to favor the forward reaction while the pH of the solution was adjusted simultaneously 21,22,25 . However, this method demands and depends on additional labor, which may be less controllable. Recently, Sewald et al. reported gelatin type A and type B methacryloyl with various degrees of substitution (DS) using a reaction system of PBS and pH adjustment. Even though they successfully prepared three batches of GelMA with various DS, GelMA materials exhibited relative high standard deviations in terms of methacryloylation and swelling/ mechanical properties 24 .
Recent studies reported that a carbonate-bicarbonate (0.25 M CB) buffer could be superior to phosphate buffer saline (0.01 M PBS) in terms of rendering free amino groups reactive via deprotonation and buffering capacity 20,22 . In this respect, a one-pot reaction strategy using the CB buffer at around pH 9 (above the isoelectric point of gelatin) could be ideal, and easy to control the reaction parameters 20 . On the other hand, the CB buffer at even higher pH (pH 11 and 12) can degrade quickly MAA as well as the formed methacrylate groups through hydrolysis, which is not so effective for GelMA synthesis 26 . The buffer capacity of the CB buffer was found to be optimal at around 0.25 M. Another important thing of GelMA synthesis is to improve the miscibility of two reactants (gelatin and MAA) because gelatin is soluble in warm water whereas MAA is insoluble in water. A high concentration of gelatin (above 10%) and a stirring rate of above 500 rpm can be conducive to the homogeneous mixing and reaction of gelatin and MAA because amphiphilic gelatin can serve also as a surfactant, and the high stirring speed may enlarge the reaction interface of gelatin and MAA via stabilizing MAA dispersion in the gelatin solution, subsequently leading to production of homogeneously reacted GelMA. Reaction temperature is also important to completely dissolve gelatin; a temperature between 30-60 °C is acceptable but a temperature above 60 °C might accelerate the backbone degradation of gelatin. A temperature at around 55 °C helps to rapidly dissolve gelatin. That is why the reaction temperature (55 °C) was chosen in this synthesis system. The reaction between gelatin and MAA normally can be complete within 1 hour 20 . Therefore, our one-pot method employing reaction parameters (10 (w/v)% gelatin, 0.25 M CB buffer, a reaction time of 1 h, reaction temperature of 55 °C, an initial pH of 9.4, and a reaction rate of 500 rpm) is exceptionally easy to control the parameters in every batch, resulting in good quality control of GelMA production. In addition, in our system, tangential flow filtration can reduce the dialysis time from several days to several hours through effectively removing the impurities of methacrylic acid and methacrylic anhydride.

Consistency of the degree of substitution of target GelMA batches (DS100_1~5 and DS60_1~5).
In GelMA production, reproducible methacryloyl functionalization of GelMA is a crucial factor for GelMA with different batches to display consistent hydrogel properties such as swelling behavior, mechanical properties, and degradation after photopolymerization. The amount of methacryloyl groups (AM) in GelMA was quantified by 1 H-NMR, TNBS, and Fe(III)-hydroxamic acid-based assays (AM NMR , AM TNBS , and AM Fe(III) , respectively). Basically, 1 H-NMR spectroscopy using TMSP as an internal reference can offer the quantification of both methacrylamide and methacrylate groups in GelMA simultaneously, whereas two different colorimetric methods (TNBS and Fe(III)-hydroxamic assays) provide the quantification of methacrylamide and methacrylate groups in GelMA, respectively [25][26][27] . The amount of methacryloyl groups (mmole g −1 ) in GelMA can be converted to the degree of substitution (DS; %) through normalization to the amount of the free amino group of original gelatin. Thus, AM NMR amounts to DS NMR whereas the sum of AM TNBS and AM Fe(III) leads to DS color . 1 H-NMR spectra were used for determining the amount of methacrylate and methacrylamide groups in GelMA products, as well as for identifying the presence of the byproduct (methacrylic acid) as presented in Fig. 2. In comparison with the 1 H-NMR spectra of gelatin (Fig. 2a,d), new proton peaks belonging to methacryloyl groups of GelMA appeared between 6.1-5.4 ppm and at 1.9 ppm, and apparently the free lysine signal (NH 2 CH 2 CH 2 CH 2 CH 2 -) of the unmodified gelatin at 3.0 ppm decreased markedly in DS60_1 and DS100_1 samples. DS100_1 displayed specific chemical shifts between 5.7-5.6 and 5.5-5.4 ppm for acrylic protons (CH 2 =C(CH 3 )CONH-) of methacrylamide groups and at 1.9 ppm for methyl protons (CH 2 =C(CH 3 )CO-) of www.nature.com/scientificreports www.nature.com/scientificreports/ methacryloyl groups, as well as additional small peaks at 6.1 and 5.7 ppm for acrylic protons (CH 2 =C(CH 3 ) COO-) of methacrylate groups, whereas DS60_1 appeared to show only some specific peaks at about 5.7, 5.5, and 1.9 ppm ascribing to methacrylamide groups (CH 2 =C(CH 3 )CONH-) in GelMA. Also, DS100_1 showed a higher peak intensity at 5.7, 5.5, and 1.9 ppm compared to DS60_1. In the 1 H-NMR spectra, GelMA samples (DS100_1~5 and DS60_1~5) with five different batches showed almost no batch-to-batch difference in terms of methacryloyl functionalization, as seen in Fig. 2b,c. Additionally, all the spectra demonstrated that in all GelMA products there remained little methacrylic acid (the byproduct) whose specific peaks normally appear at 5.7, 5.3 and 1.8 ppm.
Consistency of the secondary structure of GelMA batches. Gelatin exhibits partial triple helix formation at a low temperature in aqueous solutions and forms random coil structure upon heating. Its transition from triple helix to random coil is reversible. In comparison with gelatin, GelMA samples (DS100_1~5 and DS60_1~5) were expected to retain a certain degree of the secondary structure of gelatin even though the methacryloyl functionalization of GelMA can potentially interfere with helix formation 23,28 . Figure 4 shows the CD spectra of gelatin, highly substituted GelMA (DS100_1~5), and lowly substituted GelMA (DS60_1~5) that provide the information of their secondary structure at 4 °C and 37 °C. As presented in Fig. 4a,b, DS100_1~5 and DS60_1~5   Table 2. The amount of methacryloyl (AM, mmole g −1 ) and the degree of substitution (DS, %) of target DS100_1~5 and DS60_1~5. AM NMR , AM TNBS , and AM Fe(III) were measured by 1 H NMR spectra, TNBS assay, and Fe(III) assay, respectively. The degree of substitution (DS) of GelMA was calculated using either AM NMR or a combination of the colorimetric results (AM TNBS and AM Fe(III) ): DS NMR = AM NMR /0.3184 × 100 (%) and DS color = (AM TNBS + AM Fe(III) )/0.3184 × 100 (%). The amount of the free amino groups of gelatin was 0.3184 mmole g −1 . No methacrylate groups in DS60_1~5 were detected by Fe(III) assay. Data represent means ± standard deviations. www.nature.com/scientificreports www.nature.com/scientificreports/ displayed similarly a distinct rise in the intensity at 199 nm at 4 °C, compared with gelatin. The intensity of highly substituted GelMA (DS100_1~5) at 199 nm at 4 °C, ascribing to a portion of random coil formation, was slightly higher than that of lowly substituted GelMA (DS60_1~5), suggesting that higher methacryloyl functionalization of GelMA could further elicit random coil formation. On the other hand, the triple-helix contents of GelMA samples (DS100_1~5 and DS60_1~5) at 222 nm at 4 °C decreased markedly, compared with gelatin. DS100_1~5 exhibited a slightly lower intensity at 222 nm than DS60_1~5, indicating that lowly substituted GelMA could retain a more amount of the triple-helix formation at 4 °C than highly substituted GelMA. Additionally, GelMA with a higher DS (DS100_1) exhibited a less temperature-sensitive phase transition (helix-random coil transition) compared with GelMA with a lower DS (DS60_1), as seen in Fig. S1. It is speculated that the methacryloyation of free amino groups or hydroxyl groups in gelatin chains could reduce interchain or intrachain hydrogen bonding in the triple helix, leading to an increase in the random coil portion and a decrease in the triple helix formation 23 . Glycine-Proline-hydroxyproline tripeptides have been found to participate in the triple helix formation [29][30][31] . Hydroxyl groups of hydroxyproline can react with methacrylic anhydride (MAA) especially in a high feed of MAA 25,26 . Highly substituted GelMA (DS100_1~5) possessed the methacrylate group of around 0.01 mmole g −1 most likely from the reaction of hydroxyproline and MAA, which is presumed to obstruct partially the triple-helix formation. On the other hand, all GelMA as well as gelatin showed similar patterns in the CD spectra at 37 °C and exhibited a large increase in the intensity at 199 nm relative to the samples at 4 °C, as seen in Fig. 4c,d, indicating that GelMA materials including gelatin experience a helix-coil transition on heating. Highly substituted GelMA (DS100_1~5) showed a slightly higher intensity at 199 nm than lowly substituted GelMA (DS60_1~5). The triple-helix contents of all GelMA and gelatin at 222 nm at 37 °C decreased significantly compared with those at 4 °C, indicating that GelMA samples (DS100_1~5 and DS60_1~5) as well as gelatin appeared to completely lose the triple-helix formation and behaved like random coils at 37 °C. In addition, the CD spectra patterns of each GelMA group (DS100_1~5 or DS60_1~5) were almost the same even at different temperatures, meaning that each GelMA group with five batches was consistent in the secondary structure formation. GelMA (DS100_1~5 and DS60_1~5) displayed a higher degree of consistency not only in the composition of methacryloyl, but also in the protein secondary structure.

Consistency of hydrogel properties of GelMA batches (DS100_1~5 and DS60_1~5).
Gelatin undergoes only physical gelation at a low temperature whereas GelMA can form a physical gel at a low temperature and additionally form a chemical hydrogel via photopolymerization owing to photosensitive methacryloyl functionalization. GelMA hydrogels exhibit tailorable swelling behavior and mechanical properties, which depend on mainly their degree of substitution, their concentration and selected curing parameters (light intensity, Almost no batch-to-batch variation in CD curves was observed among DS100_1~5 or DS60_1~5, indicating that each gelMA group (DS100_1~5 or DS60_1~5) exhibited its own secondary structure. DS100_1~5 and DS60_1~5 exhibited a marked increase of the random coil portion in the intensity at 199 nm and a slight decrease of the triple helix formation in the intensity at 222 nm at 4 °C, compared with gelatin. An increase in the DS increased the random coil conformation of GelMA and decreased the triple-helix formation of GelMA. However, both DS100 and DS60 samples, like gelatin, lost the triple-helix formation at 37 °C and exhibited a similar amount of the random coil conformation at 222 nm.
www.nature.com/scientificreports www.nature.com/scientificreports/ exposure time of irradiation, and amounts of an initiator). Here, the batch-to-batch variation of hydrogel properties of GelMA (DS100_1~5 and DS60_1~5) was investigated in terms of swelling and mechanical stiffness.
First, GelMA hydrogels were fabricated through a simple method: Each 20 (w/v)% GelMA solution containing 0.5 (w/v)% I2959 was placed in a mold (8 mm in diameter and 1 mm in thickness) and then cured by 365 nm UV light (3.5 mW cm −2 and 5 minutes). As shown in Fig. 5a, GelMA (DS100_1~5 and DS60_1~5) bulk hydrogels exhibited structural integrity after photo-crosslinking. When GelMA hydrogels were soaked in DI water at an elevated temperature (50 °C) to expedite the swelling process, they began to swell and reached a swelling equilibrium within 60 min. In addition, any degradation of DS100 and DS60 hydrogels was not observed at the elevated temperature during the swelling test. DS100_1~5 hydrogels exhibited a lower swelling degree (%) compared with DS60_1~5 hydrogels. Swelling degrees of DS100_1~5 hydrogels were 1156 ± 12, 1148 ± 26, 1144 ± 23, 1155 ± 12, and 1152 ± 17%, respectively (n = 3, one-way ANOVA, p = 0.489) whereas those of DS60_1~5 were 2707 ± 39, 2726 ± 42, 2759 ± 36, 2733 ± 22, and 2726 ± 13%, respectively (n = 3, one-way ANOVA, p = 0.078). DS100_1~5 hydrogels should have a higher crosslinking density and a smaller mesh size compared to DS60_1~5, owing to a higher degree of methacryloyl functionalization, subsequently leading to less swelling. Additionally, the results demonstrated that each GelMA hydrogel group (DS100_1~5 or DS60_1~5) showed a higher degree of consistency in swelling behavior. The swelling of hydrogels is an important feature for the diffusion behavior of small molecules (nutrients and waste) in cell culture and drug delivery systems. Consistent swelling behavior of GelMA (DS100_1~5 or DS60_1~5) hydrogels could be used as a predictable basic reference for various bioapplications.
As to mechanical properties of GelMA (DS100_1~5 and DS60_1~5) hydrogels at 20 (w/v)%, highly substituted GelMA materials (DS100_1~5) with an average of 30.20 ± 0.57 kPa were 1.9-fold stiffer than lowly substituted GelMA (DS60_1~5) with an average of 16.04 ± 0.93 kPa. As seen in Fig. 5b  www.nature.com/scientificreports www.nature.com/scientificreports/ statistically different from those of highly methacrylated GelMA with the methacrylation of around 0.6 mmole g −1 , assumingly because of the much influence of the physical crosslinking of lowly methacrylated GelMA. In our case, the mechanical and swelling properties of GelMA hydrogels showed a direct correlation with the degree of methacrylation. It is potentially because the hydrogels were prepared above 37 °C, which could rule out the physical gelation. The chemical gelation and crosslinking density of GelMA hydrogels formed by light could be a dominant factor of determining their mechanical properties and swelling behavior, resulting in distinct mechanical properties of each GelMA group and a low batch-to-batch difference within each GelMA group. GelMA hydrogels with a high degree of consistency and tailorability in mechanical properties could be a highly versatile tool for tissue engineering applications since tunable mechanical properties of soft hydrogels have been used to regulate cellular behavior such as proliferation, migration, and differentiation 32 .

Consistency of biodegradability and cell viability of GelMA (DS100_1~5 and DS 60_1~5) hydrogels.
Biodegradability has gained considerable attention in drug delivery and tissue engineering applications as a desirable feature of hydrogel materials. GelMA hydrogels exhibit enzymatic degradation properties; indeed, GelMA retains enzyme-sensitive sequences (proline-X-glycine-proline-, X: a neutral amino acid) as its parent gelatin and collagen do. Here, accelerated enzymatic degradation tests of GelMA (DS100_1~5 and DS60_1~5) hydrogels were conducted to investigate consistency of their degradation behavior. As displayed in Fig. 6a-c, the enzymatic degradation of bulk GelMA hydrogels appeared apparent, and their degradation speed was highly dependent on the DS of GelMA, which affects dominantly the crosslinking density of GelMA hydrogels. The higher the DS of GelMA hydrogels, the slower their degradation. DS100_1~5 hydrogels under the accelerated degradation conditions lost half of their masses at around 3.33 h, and DS60_1~5 hydrogels did at around 0.65 h. Actual half-lives of DS100_1~5 hydrogels were 3.23, 3.31, 3.60, 3.03, and 3.50 h, respectively whereas those of DS60_1~5 were 0.67, 0.64, 0.69, 0.61, and 0.62 h, respectively. Each GelMA hydrogel group followed a similar degradation pattern, which showed that there seemed to be little batch-to-batch difference in hydrogel structure and susceptibility to enzyme degradation within each hydrogel group. We presume that the crosslinking density caused by photocrosslinking of the methacryloyl group could be the main factor of making a difference in the degradation rate of GelMA (DS100_1~5 and DS60_1~5) hydrogels. GelMA DS100_1~5 hydrogels with a higher DS can slow down the enzymatic degradation owing to a higher crosslinking density in the hydrogels, compared with GelMA DS60_1~5 hydrogels with a lower DS 13 . www.nature.com/scientificreports www.nature.com/scientificreports/ Also, GelMA, like its parents (gelatin and collagen), maintains cell binding sites (e.g. RGD). Cell binding affinity of GelMA is an important feature that can promote cell viability and affect cell behavior such as cell proliferation and differentiation. We investigated cell viability of Huh7.5 cells cultured on and inside GelMA hydrogels (DS100_1~5 and DS60_1~5). As presented in Fig. 7a, GelMA hydrogels (DS100_1~5 and DS60_1~5) exhibited cell viability of above 87%. Cell viability of DS100_1~5 and DS60_1~5 hydrogels was not significantly different from one another (one-way ANOVA, p = 0.812). Cell viability values of DS100_1~5 hydrogels were 87.1 ± 7.1%, 92.4 ± 1.4%, 97.1 ± 2.4%, 91.6 ± 3.8%, and 96.9 ± 0.5%, respectively whereas those of DS60_1~5 hydrogels were 91.3 ± 2.5%, 88.6 ± 3.6%, 92.3 ± 1.5%, 90.7 ± 2.9%, and 89.7 ± 7.0%, respectively. Cells on GelMA (DS100_1~5 and DS60_1~5) hydrogels appeared as cell clusters possibly because GelMA substrates were soft and compliant. Huh 7.5 cells on soft hydrogels tended to form cell clusters or spheroids 13 . Cell clusters on DS100_1~5 hydrogels looked slightly more scattered than those on DS60_1~5. It was speculated that relatively stiffer DS100_1~5 hydrogel substrates could allow cells to be more spread and scattered, as compared to DS60_1~5 hydrogel substrates.
In addition, cells were encapsulated inside GelMA hydrogels as presented in Fig. 2b. The average (93.7 ± 5.2%) of cell encapsulation efficiency of DS100_1~5 hydrogels was slightly higher than that (87.0 ± 7.3%) of DS60_1~5. The cell encapsulation efficiency of each GelMA group showed no statistical difference (one-way ANOVA, p > 0.05). Cells encapsulated inside DS100_1~5 hydrogels displayed an average cell viability of 87.7 ± 3.7% whereas those inside DS60_1~5 had an average cell viability of 87.8 ± 5.3%. In comparison to cells grown on GelMA hydrogels, cells inside GelMA hydrogels exhibited a round morphology possibly owing to the fact that cells were surrounded and packed by dense hydrogel matrices in a three-dimensional manner. Overall both DS100_1~5 and DS60_1~5 hydrogels were found to offer good cell viability with good batch-to-batch consistency.
Preparation of gelatin methacryloyl (GelMA, target DS = 100 and 60%) with five batches. Two kinds of gelatin methacryloyl (GelMA, target DS = 100 and 60%) materials with five different batches (DS100_1~5 and DS60_1~5) were prepared as illustrated in Fig. 1a,b. Details regarding reaction parameters are presented in Table 1. In brief, type B gelatin (10 g, 250 bloom, 3.18 mmole of free amino groups) was dissolved at 10 (w/v)% in carbonate-bicarbonate (CB) buffer (0.25 M, 100 mL) at 55 °C, and then the pH of the gelatin solutions was adjusted to 9.4. Two different amounts (0.938 mL for target DS = 100% and 0.317 mL for target DS = 60%) of methacrylic anhydride (MAA, 94%) were separately added to the gelatin solutions under magnetic stirring at 500 rpm. The reaction proceeded for 1 h at 55 °C, and the final pH of the reaction solutions was adjusted to 7.4 to stop the reaction. After being filtered, the solutions were dialyzed against water at 50 °C in a MasterFlex ® tangential flow filtration (TFF) system equipped with Pellicon ® 2 cassette (Darmstadt, Germany) containing a 10 k Da Biomax membrane, and lyophilized to obtain the final solid products. The average yield on GelMA products was around 90%. Finally, the degree of substitution (DS) of GelMA was calculated using either AM NMR or a combination of the colorimetric results (AM TNBS and AM Fe(III) ): www.nature.com/scientificreports www.nature.com/scientificreports/ helix structure or random coil). The acquisitions were performed at 4 or 37 °C after 300 μL of each solution was added in a quartz cell with an optical path length of 1 mm.

Degree of substitution of target
Swelling behavior of bulk GelMA DS100_1~5 and DS60_1~5 hydrogels. GelMA hydrogels (DS100_1~5 and DS60_1~5) for swelling, mechanical and enzymatic degradation testings were conducted as follows. Each GelMA solution (20 (w/v)% in deionized water) containing 0.5 (w/v)% I2959 (prepared at a concentration of 20% as a stock solution in 70% ethanol) was cured under UV light (365 nm with an intensity of 3.5 mW cm −2 ) for 5 min in a polytetrafluoroethylene (PTFE) mold with a diameter of 8 mm and a thickness of 1 mm. Prepared GelMA hydrogels were transferred into a small beaker containing 30 mL of deionized water, incubated for 120 min at an elevated temperature (50 °C) so as to expedite the equilibrium swelling and then weighed at appointed times (5,10,15,20,30,40, 50, 60, and 120 min). The swelling degree (Q t ) of GelMA hydrogels at different time points was calculated using the following formula, (w t : the weight of GelMA hydrogels at t min; w o : the dried weight of GelMA hydrogels after freeze-drying at 0 min).
Mechanical properties of bulk GelMA DS100_1~5 and DS60_1~5 hydrogels. Mechanical properties of GelMA hydrogels with a diameter of 8 mm and a thickness of 1 mm were characterized via sinusoidal shear rheometry. Frequency-sweep measurements were conducted using a rheometer (Discovery Hybrid Rheometer, Thermo, America), equipped with an 8 mm parallel plate (Peltier plate Steel). The storage modulus of each GelMA hydrogel sample was measured at 0.1% strain and 0.1-10 Hz within the viscoelastic range. The running temperature was maintained at 37 °C throughout the measurements.
Enzymatic degradation of bulk GelMA DS100_1~5 and DS60_1~5 hydrogels. GelMA hydrogels (20 (w/v)%) with a diameter of 8 mm and a thickness of 1 mm were tested for enzymatic degradation. After GelMA hydrogels were placed in deionized water for 3 h at 50 °C to rapidly reach the equilibrium swelling, they were transferred into a new 24-well plate with each well containing 1 mL of 0.05 (v/w)% collagenase (125 CDU mg −1 , solid) in Hank's Balanced Salt Solution including 3 mM CaCl 2 . Their enzyme degradation was conducted at 37 °C. Gross images of GelMA hydrogels were taken during the degradation, and mass loss of GelMA hydrogels was also measured.

Viability of Huh7.5 cells grown on and inside GelMA DS100_1~5 and DS60_1~5 hydrogels.
Human hepatocellular carcinoma cells (Huh7.5; ScienCell, Shanghai, China) were cultured in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere at 37 °C with 5% CO 2 . The medium was changed every 3 days. Bulk GelMA (DS100_1~5 and DS60_1~5) hydrogels were prepared in 48-well plates by curing 120 µL of each GelMA solution (20 (w/v)% in PBS) containing 0.5 (w/v)% I2959 in each well. After washing each hydrogel with PBS two times, 500 µL of a medium containing 1 × 10 5 cells was carefully added to each well coated with each GelMA hydrogel. For cell encapsulation, each GelMA solution (20 (w/v)% in PBS and 0.25 (w/v)% I2959) containing Huh 7.5 cells at a concentration of 3 × 10 6 cells mL −1 was cured in a mold (8 mm in diameter and 1 mm in depth) by exposure to light (365 nm with an intensity of 3.5 mW cm −2 for 5 min). Then unencapsulated cells were counted for cell encapsulation efficiency.
After 1 day, cell viability on and inside GelMA hydrogels was characterized using Live/Dead ® Cell Viability/ Cytotoxicity kit. Briefly, 4 µM calcein-acetoxymethyl (calcein-AM) and 8 µM ethidium homodimer-1 (EthD-1) in media were added to each well containing each cell-laden hydrogel, followed by incubation for 1 h at 37 °C. The cytoplasm of live cells and the nuclei of dead cells were stained by calcein-AM (green) and EthD-1 (red), respectively and were observed through a confocal microscope (C2 + , Nikon, Shanghai, China). Numbers of live and dead cells in three images of each hydrogel were counted using ImageJ. Cell viability was calculated using the following formula, = × . Cell viability (%) live cells the total number of cells 100 statistical analysis. Statistical analysis was carried out using the Microsoft Excel ® statistical analysis. A one-way ANOVA was used to test for differences among five groups. The standard deviation (s.d.) was calculated and presented for each treatment group. P values below 0.05 were considered statistically significant. The value of n denotes the number of performed samples or the number of independently performed attempts.