Page 390 - IJB-9-3

International Journal of Bioprinting Bioprinting of a multicellular model

2.4. Construction of sandwich culture time was determined based on the staining effect. After

The final concentration of tumor cells in the SW480 bio- dehydration with absolute ethanol and xylene, frozen
ink was 4.8×10 /ml. The bio-ink prepared for tumor cells pathological sections were sealed with neutral gum. The
6
was tiled into a 24-well plate using a micropipette and then stained tumor and stroma in 3D bioprinted multicellular
incubated in a cell incubator (37°C, 5% CO ) after adding tissues were observed under a light microscope.
2
the same amount of fresh medium. The culture medium 2.9. Immunofluorescence assay
was changed every 2 days.
The 3D bioprinted tissue was transferred to the operating
2.5. Cell morphology photography table 10 days later and fixed in 3% CaCl solution for
2
The morphology of colorectal cancer cells in 3D bioprinted 1 min. The supernatant was discarded, and the cells were
models from days 1 to 10 was examined using light washed with PBS for five times. Afterward, the tissue was
microscopy. fixed in 4% paraformaldehyde for 20 min; after discarding
the supernatant, it was washed with PBS for 5 times. Three
2.6. Cell viability groups of 0.2% Triton X-100 models were incubated for
30 min. The tissue was then blocked in 3% bovine serum
The 3D printing-M, 3D printing-S, and sandwich culture albumin (BSA) at room temperature for 60 min. The
group (3D culture) were labeled with fluorescence. Before primary antibody was diluted in PBS containing 1.5% BSA
cell viability staining, 3D bioprinted models and sandwich (Table 1), and the tissue was incubated with the antibody at
culture models were washed with phosphate-buffered 4°C overnight (16 – 20 h).
saline and then stained with calcein-AM (1 µmol/L; Sigma)
and propidium iodide (PI, 2 µmol/L; Sigma), which were Caudal-related homeobox transcription factor 2
kept out of the light for 15 min. After washing with PBS, (CDX2) plays a key role in the normal development
cells were observed under a confocal microscope. Green and differentiation of intestinal epithelial cells and the
indicates live cell and red indicates cell death. development of cancer tissues, and its expression is rarely
lost in colorectal cancer . Ki67 is an antigen associated
[27]
2.7. Cell proliferation assay with cell proliferation, whose function is closely related to
[28]
The Cell Counting KIT-8 Kit (CCK-8) was mainly used mitosis and is indispensable in cell proliferation . Ki67
to detect the proliferation status of 3D printing-M, 3D is often used to reflect the proliferation of tumors. CD68
printing-S, and 3D culture groups. 3D bioprinted models is the most reliable marker of macrophages and is often
[29]
and sandwich models were cleaned three times with PBS. used for immunofluorescence staining of macrophages .
Then, 1 mL of complete medium was added to the petri CD31, also known as platelet-endothelial molecule-1
dish along with 0.1 mL drop of CCK-8 reagent. Cells were (PECAM-1), is often present in blood vessel endothelial
[30]
incubated in an incubator for 2 h. Each proliferation assay cells .
was performed in triplicates. Thereafter, 0.1 mL of culture The three groups of specimens were removed after
medium was absorbed from each group and added to a incubation, left at room temperature for 30 min, and
96-well plate. The 96-well plate was placed in a microplate then cleaned 6 times with PBS for 6 times. Secondary
reader for detection at 450 nm/620 nm, respectively. Cell antibodies were diluted in PBS containing 1.5% BSA
th
rd
st
proliferation was measured on 1 , 3 , 5 , 7 , and 10 days, (Table 1), and were used to incubate with the tissues for
th
th
st
respectively. The measurement value on the 1 day was the 1 h at room temperature in the dark. After discarding the
baseline value, and the measurement value of the following
4 days was standardized according to the measurement
value of the 1 day. Table 1. Information on immunofluorescent antibodies
st
2.8. Hematoxylin and eosin staining of frozen antibody species antibody brand
pathological sections Ki67 rabbit Abcam; 1:200
The 3D bioprinted multicellular tissue was embedded in CDX2 rabbit Abcam; 1:200
warm 3% agarose solution and placed on ice for 3 min to CD68 mouse Abcam; 1:100
solidify. Frozen tissues were sectioned with a thickness CD31 mouse Abcam; 1;100
of 5 µm. The sections were fixed in xylene and absolute IgG Alexa Fluor 488 Goat Anti-Rabbit IgG Abcam; 1:500
alcohol and cleaned with distilled water after fixation. IgG Alexa Fluor 488 Goat Anti-Mouse IgG Abcam; 1:500
The frozen tissue sections were stained with hematoxylin
solution for 2 – 3 min, washed with running water, and IgG Alexa Fluor 594 Goat Anti-Rabbit IgG Abcam; 1:500
then counterstained with eosin solution. The flushing IgG Alexa Fluor 594 Goat Anti-Mouse IgG Abcam; 1:500

Volume 9 Issue 3 (2023) 382 https://doi.org/10.18063/ijb.694