Page 290 - IJB-10-2

International Journal of Bioprinting Kidney hydrogel print for renal cancer model

model with 8 mm inner diameter and 2 mm thickness for 2.13. Cubic ratio measurement
compressive and swelling tests. A mesh structure was printed using 10% GelMA-1%
Kidney, 10% GelMA-2% Kidney, and 10% GelMA-3%
2.9. Rheological analysis Kidney under 20°C (n = 50 per group). High-resolution
The rheological properties of GelMA solutions with or camera was used to capture the images for the pore
without dECM powders (n = 3) were measured with a structure of printed pattern immediately (n = 50 pores
rheometer (Thermo Scientific, USA). Rotational shear counted per group). Cubic ratio (Pr) was then calculated
thinning tests were then performed under increasing using Equation II:
shear rates (0.1 to 100 1/s) in 5 min at 20°C. The effect of L 2
a temperature change (i.e., 4–35°C) on GelMA precursor Pr = 16 A (II)
viscosity was determined under a constant shear rate where L and A represent the perimeter and area of the
of 1/s over 30 min. In addition, the storage (G’) and loss square pore shape, respectively, which were determined
(G’’) modules of GelMA-based precursors were further using NIH ImageJ. Ideally, the Pr value of a perfect square
evaluated to investigate the effect of a temperature change morphology should be 1, and the higher the Pr value is, the
(i.e., 4–35°C) under 1 Pa constant shear stress and 1 Hz better the bioink gelation degree is.
frequency.
2.14. Material spreading index measurement
2.10. Swelling test The material spreading index measurement was performed
All groups of disc-like GelMA samples (n = 5 per group) by printing with different nozzle sizes (i.e., 20G to 28G)
were soaked in DMEM medium and incubated at cell using the 10% GelMA-1% Kidney, 10% GelMA-2% Kidney,
culture condition (37°C and 5% CO ) for 5 days. The and 10% GelMA-3% Kidney samples (n = 10 images per
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diameter of disc hydrogels was then determined with group) at constant temperature of 20°C. The material
a digital micrometer (Deli, China) at multiple time spreading index was calculated using Equation III:
points, i.e., 0, 0.5, 1.5, 4, 6, 24, 48, 72, and 120 h, and D
normalized to the diameter at 0 h to calculate the scaled Materialspreadingindex = D a (III)
diameter values. where D indicates the nozzle inner diameter, and D
n
n
a
2.11. Compressive test indicates the diameter of the extrusion filament measured
Compressive test was performed to detect the compressive using NIH ImageJ.
modulus of all GelMA groups (n = 5 per group) with 2.15. Cell cultures
a universal tester (Shimadzu, Japan). All samples were ACHN cells were grown with complete DMEM medium
compressed at 0.5 mm/min rate to a strain of 50%. The containing 10% fetal bovine serum and 1% penicillin/
linear region slope on the beginning of the stress–strain streptomycin, and were maintained in 5% CO at 37℃.
curve was used to calculate the compressive modulus. Cells within passages of 10 were used for following
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2.12. Filament fusion test experiments.
The effect of kidney dECM powder on GelMA printability 2.16. Development of cell-laden RCC model
was evaluated in a filament fusion test, which was At 70–80% confluence, ACHN cells were detached
conducted in accordance with the procedures adapted using 0.25% trypsin-EDTA, followed by resuspending in
from a previously published method. 34,35 The test entails different groups of GelMA precursors. The final cellular
using a square pattern with increasing filament-to-filament concentration was 1 × 10 cells/mL. A nephron model for
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distance (1.5 to 4 mm with 0.5 mm increments). The 10% ACHN cell culture was printed using LB119 3D bioprinter
GelMA-1% Kidney, 10% GelMA-2% Kidney, and 10% (MEDPRIN, China). Cellular suspensions with different
GelMA-3% Kidney bioinks were printed at 20°C. High- groups of GelMA precursors were loaded to a 1 mL sterile
resolution camera (ZK4A08MTP, SANOTID, China) syringe, printed to a nephron structure, as shown in Figure
was used to capture the image of the printed patterns S1 (Supplementary File), with a nozzle of 24G at 20°C, and
immediately (n = 5 pattern printed per group). The pore photo-crosslinked with 405 nm UV light (10 W/cm ) for
2
area index (PAI) was calculated using Equation I: 1.5 min. ACHN cells with concentration of 1 × 10 cells/mL
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A − A
PAI = t a ×100% (1) were also mixed with Matrigel or 10% GelMA-2% Kidney
A a and added into PDMS models (8 mm inner diameter, 2
where A is the theoretical area of pores, and A is the mm thickness). The Matrigel samples were incubated for
a
t
actual area of pores. A was determined using NIH ImageJ. 30 min at 37°C to form Matrigels, and 10% GelMA-2%
a
For an ideal square pore, PAI should be 0, and A = A , Kidney was photo-crosslinked using 405 nm UV light (10
t
a
indicating that there has no material spreading. W/cm ) for 90 s. All cell-laden models were placed into
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Volume 10 Issue 2 (2024) 282 doi: 10.36922/ijb.1413