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

glutaraldehyde. After glycine treatment, the Gel MS was 2.9. Cell viability monitoring using a metabolic
washed three times with diH O. The Gel MS mixture activity assay
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was passed through a 100 µm cell strainer (Corning Inc., Cell viability on/in the cell carriers was assessed using the
United States) (Figure 1D). This process typically takes alamarBlue assay (Bio-Rad Laboratories, United States).
approximately 8 h. The filtered Gel MS (≤100 µm) was After 10 days of culture, 0.3 mL of alamarBlue solution
then lyophilized and stored at 4°C until use. (complete growth medium + 10% alamarBlue reagent) was
2.6. Culture of hDPSCs added to each well and incubated at 37°C for 30 min. After
incubation, 0.1 mL of the supernatant was transferred to
The hDPSCs were kindly provided by Dr. Pamela Yelick and a 96-well plate, and fluorescence intensity was measured
Dr. Weibo Zhang from Tufts University. Cells (passages using a multimode microplate reader (Spark , Tecan Life
7
®
5 – 9) were cultured in minimum essential medium Sciences, Switzerland) at excitation/emission wavelengths
(MEM)-alpha (HyClone, United States) supplemented with of 540 nm/590 nm. The fluorescent intensity was reported
10% fetal bovine serum (CPS Serum, United States) and in arbitrary units.
25 µg/mL of gentamicin (Sigma-Aldrich, USA) in tissue
culture-treated polystyrene plates (CELLTREAT, United 2.10. Deoxyribonucleic acid quantification
States). Cells were sub-cultured when they reached 80% Cells were released from aggregates or HG by enzymatic
confluence, if not immediately used for experiments.
digestion with 0.5 mg/mL collagenase in PBS for 2 h in
2.7. hDPSC seeding on different gelatin-based cell a 37°C incubator shaking at 200 rpm. After digestion,
carriers samples were treated with a cell lysis buffer (Cell Signaling
Technology, United States) containing 0.5% sodium
Three milligrams of lyophilized GelMA MS, GelMA HP, dodecyl sulfate (Fisher Scientific, United States). The
or GelMS were sterilized under ultraviolet light for 30 min released deoxyribonucleic acid (DNA) from cells cultured
(n=4 for each group). After sterilization, the samples were on/in cell carriers was quantified using the Helixyte Green
transferred to sterile Eppendorf tubes. Each tube was filled dsDNA Assay Kit (AAT Bioquest, Pleasanton, CA, USA)
with 1 mL of MEM-alpha complete medium and centrifuged following the manufacturer’s protocol.
at room temperature for 1 – 2 h to facilitate rehydration.
After rehydration, the medium was removed from each tube. 2.11. Osteogenic differentiation of hDPSCs on/in
hDPSCs were trypsinized and collected after reaching various cell carriers
80% confluence. One hundred µL of 1.5 × 10 /mL hDPSC After hDPSCs were cultured on GelMA MS, GelMA HP,
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suspension (1.5 × 10 cells/sample) was added to each tube. Gel MS, or in GelMA HG for 10 days, one set of samples
5
The cells were mixed with the cell carriers using pipette underwent osteogenic differentiation. Osteogenic medium
tips and incubated in the tubes in a tissue culture incubator (growth medium containing 10% fetal bovine serum,
for 30 min, with gentle mixing every 10 min. Following supplemented with 20 mM β-glycerophosphate, 50 µg/mL
the initial cell seeding, 200 µL of complete medium was ascorbic acid, and 100 nM dexamethasone; Sigma-Aldrich,
added to each tube. The mixed cells and carriers were then United States) was added to each well at a volume of 0.4 mL/
transferred to the wells of a 48-well cell-repellent plate and well. A control set was cultured in growth medium for
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cultured statically overnight. After 48 h, the culture was the same duration. The induction or growth medium was
continued with gentle shaking (200 rpm) on a BT4500 CO 2 changed every 3 days for a total of 10 days.
Orbi-Shaker (Benchmark Scientific, United States) in the
tissue culture incubator. The medium was changed every 2.12. Staining and quantification of osteogenic
3 days. differentiation
To evaluate osteogenic differentiation of hDPSCs on/in
2.8. Viable cell staining with calcein acetoxymethyl different carriers, samples were stained with 0.2 mL/well of
ester
alizarin red solution (EMD Millipore Corp., United States)
After 10 days of culture, cells on/in the carriers were stained for 5 min after 10 days of induction. Alizarin red stains the
with the live dye calcein, following the manufacturer’s calcium deposits formed during osteogenic differentiation
instructions. Briefly, the samples were treated with calcein of stem cells. The stained samples were washed in excess
acetoxymethyl ester (AM; 1 µM in growth medium) water and examined under a light microscope. Color (red,
(Corning Inc., United States) for 30 min in the cell culture green, blue [RGB]) images were captured using an Echo
incubator. After staining, the samples were washed with Revolve Microscope (Echo Revolve, United States) with
PBS and visualized using a fluorescence microscope (Echo a 10× objective. To quantify the intensity of red staining
Revolve, United States). in each image, at least 15 images from each group were

Volume 4 Issue 1 (2025) 71 doi: 10.36922/gtm.5897