Page 78 - GTM-4-1

Global Translational Medicine Gelatin-based cell carriers for tooth-germ organoids

dissolved at 10% w/v in 100 mL of 0.25 M, pH 9.4 carbonate- The mixture was transferred to a polydimethylsiloxane
bicarbonate buffer (Sigma Aldrich, United States) at 55°C. (PDMS)-coated 6-well plate. Light crosslinking (405 nm;
The pH of the solution was adjusted to 9.4, and 0.938 mL Sovol, China) was performed at 20 mW/cm for 2 min at
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methacrylic anhydride (Sigma Aldrich, United States) was a distance of 13 cm from the top of each well, followed by
added slowly under continuous magnetic stirring at 500 flipping the gelled discs and repeating the light crosslinking
revolutions per minute (rpm) to achieve a targeted 100% on the opposite side of the GelMA HG discs. The solidified
degree of substitution. The reaction was continued for GelMA HG was cut into approximately 3 mm × 3 mm pieces
1 h at 55°C and was stopped by lowering the pH to 7.4. and frozen at -80°C or in liquid nitrogen. Once frozen, the
After filtration, the solution was dialyzed for 2 days and GelMA pieces were transferred to a pre-chilled pestle and
lyophilized for use in subsequent experiments. mortar and ground into fine particles by repeated grinding
motions (Figure 1B). This process took about 1 h. The
2.2. Preparation of methacrylated gelatin particles were collected and lyophilized. The average size
microspheres
of rehydrated GelMA MP was determined by measuring
Methacrylated gelatin microspheres were prepared as the longest side of irregular shaped microparticles using an
previously described. Briefly, lyophilized GelMA was fully optical light microscope (Echo Revolve) and FIJI software
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dissolved at 10% w/w in 5 mL of deionized water (diH O) (NIH ImageJ2).
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at 37°C. Separately, 75 mL of mineral oil (Sigma Aldrich,
United States) was heated to 37°C in a two-neck, 250-mL 2.4. Preparation of methacrylate gelatin hydrogel-
round-bottomed flask. To this, 1 mL of Tween 20 (Thermo containing cells
Fisher Scientific, United States) was added and stirred at Methacrylate gelatin hydrogels are widely used as cell
600 rpm for 5 min. The 10% GelMA solution was mixed carriers. 21,22 In this study, GelMA HG-containing cells
with 14 mg (12.5 mM) ammonium persulfate (Sigma were used as the control. Briefly, 15 µL of 10 mg/mL
Aldrich, United States) and added dropwise to the oil LAP and 85 µL of culture media were added to 200 µL of
phase. The oil-water emulsion was stirred at 400 rpm for 15% w/v GelMA solution containing 1.5 × 10 hDPSCs.
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5 min, and nitrogen gas was bubbled through the emulsion After thorough mixing, 30 µL of this mixture (with a final
for 20 min at 45°C to purge oxygen. Under continuous GelMA concentration of 10%) was transferred to a PDMS
stirring, the temperature was increased to 100°C for 40 min mold (6 mm in diameter) and crosslinked for 40 s using
to allow for crosslinking. a 405 nm light source (Sovol, China). The gelled GelMA
After the reaction, the mixture was centrifuged HG discs contained 3 mg of GelMA (dry weight) per disc
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at 2,500 rpm at 4°C to collect the GelMA MS pellet. and 1.5 × 10 cells per disc. The GelMA HG discs were
The pellet was washed extensively with distilled water rinsed with PBS and cultured in a 48-well cell-repellent
(diH O; including one overnight wash to remove residual plate (Greiner Bio-One, Austria) in a cell culture medium.
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surfactant), followed by 50%, 70%, and 100% acetone The entire process of preparing GelMA HG with cells took
washes to remove oil residuals. Following the acetone approximately 30 min (Figure 1C).
washes, the GelMA MS was washed three times in diH O 2.5. Preparation of gelatin microspheres
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with a 15-min mixing before centrifugation to remove
residual acetone (Figure 1A). The entire process took Gelatin microspheres were synthesized using a modified
approximately 2 days. Finally, the pellet was lyophilized water-in-oil polymerization method as described
and stored at 4°C if not used immediately. The average size previously. Briefly, two grams of porcine type A gelatin
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of rehydrated GelMA MS was determined by measuring was dissolved in 10 mL of distilled water at 55°C to create
the microsphere sizes using an optical light microscope a clear solution. The gelatin solution was then added
(Echo Revolve, United States) and FIJI software (National dropwise to pre-heated olive oil (Ward’s Science, United
Institute of Health [NIH ImageJ2 version:2.14.0/1.54f, States) at 40°C, under constant stirring at 500 rpm. The
USA). water-in-oil emulsion was mixed for 30 min at 40°C, then
immediately cooled to 4°C. To crosslink, 20 mL of chilled
2.3. Preparation of methacrylated gelatin acetone containing 200 µL of 25% glutaraldehyde was
microparticles added slowly using a syringe, tubing, and an 18 G needle.
To prepare GelMA MP, a 10% (w/v) GelMA solution (10 mL) After fixing at 4°C for 1 h, the oil and acetone phases
was prepared. A 1% stock solution of the photoinitiator were removed. The Gel MS pellets were washed with
lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, acetone, followed by centrifugation at 3,400 rpm at 4°C for
Sigma Aldrich, United States) was added to the GelMA 5 min. After three washes with acetone, 20 mL of 10 mM
solution to achieve a final concentration of 0.05% (w/v). glycine was added to the Gel MS to neutralize residual

Volume 4 Issue 1 (2025) 70 doi: 10.36922/gtm.5897