Page 12 - IJB-9-5

International Journal of Bioprinting 3D printed edible bird’s nests

the medium. After a 1-h culture, the cells were observed 150 kDa; Aladdin, Shanghai, China) were prepared in
under an inverted fluorescence microscope (Leica Dmi 8, three concentrations as 1.25 mg/mL, 2.5 mg/mL, and
Morrisville, USA). 5 mg/mL, respectively. Recombinant proteins of meiyo

The cell-loading rate in different models was compared. epidermal growth factor (EGF; Boster, Shanghai, China)
In the 2D surface group, 500 μL of GelMA was added to were prepared in 25 µg/mL, 50 µg/mL, and 100 µg/mL.
2 cm (24 well plate) and photo-crosslinked, forming a 2D The HA samples were added to the polyvinylidene
2
surface available for cell culture. In 3D encapsulation group, difluoride (PVDF) membrane by dropping 50 µL in the
[16]
500 μL GelMA loaded with cells was crosslinked, whereas order of concentration . After drying, the membrane
in 3D printing group, 500 μL GelMA was loaded with cells was immersed in 5% bovine serum albumin (BSA) for
and grid printed before crosslinking. The cell density was 1 h. Then, the PVDF membrane was incubated with EGF
controlled across different experimental groups to obtain a at 37°C for 3 h, washed three times with PBS, blocked
70% field of view under the microscope to mimic the cell in 5% BSA for 1 h, and subsequently incubated with the
density in the logarithmic growth phase. Finally, the cells primary antibody (anti-EGF; Boster, Shanghai, China)
were collected and the total number of cells was calculated in 1% BSA overnight at 4°C. Secondary antibodies in 1%
to assess the cell loading rate. BSA were incubated for another 1 h at room temperature.
After rinsing with PBS, the HA-EGF binding was detected
EdU assay was employed to assess epithelial cell with SuperSignal West Pico Chemiluminescent Substrate
proliferation (BeyoClick™ Cell Proliferation Kit with (Thermos Scientific, Waltham, USA).
Alexa Fluor 594, Beyotime, Shanghai, China) . Briefly,
[17]
the different feeding layers were cultured with DMEM 2.8. Preparation of receiving layer
containing 10 µM EdU for 12 h. Following cell fixation H-HA and gelatin were the main components of the
using 4% (w/v) paraformaldehyde, cells were incubated receiving layer of TeeBN. To simulate the functional
with staining working buffer for 1 h before being co-stained components of natural EBN [11,19] , we constituted
with DAPI for 10 min. ImageJ (NIH, Bethesda, USA) was carbohydrate ingredients to adjust the proportion of
used to count the total number of cells and the number of the final composing monosaccharides, as illustrated in
positive cells separately. composition table of Figure 3A. For the receiving layer, all
the materials used are of food-grade quality.
In gene expression studies, RNA from cells cultured
in different feeding layer models were obtained via Trizol 2.9. Scanning electron microscope (SEM)
(Beyotime, Shanghai, China) extraction. A total of 2 μg The morphology of the receiving layer was characterized by
of cDNA was collected by All-in-One MasterMix (abm, a high-resolution SEM JSM-6700F (JEOL, Tokyo, Japan).
Vancouver, Canada). Then, real-time polymerase chain After freeze-drying, the sample was fixed on the specimen
reaction (PCR) was performed on CFX-96 Touch platform and coated with gold for SEM observation .
[18]
TM
(BioRad, Hercules, USA) using the GoTaq qPCR Master
®
Mix kit (Promega, Madison, USA) . Glyceraldehyde 2.10. Heavy metal assay
[18]
3-phosphate dehydrogenase (GAPDH) was used as the Heavy metals (As, Pb, Cu) in EBN and TeeBN (receiving
housekeeping gene and the relative expression of genes layer) were measured by inductively coupled plasma mass
was calculated by 2 -ΔΔCt . The primers are listed in Table S1. spectrometry (ICP-MS) (Thermo Scientific, Waltham
USA) by referring to the previously reported method .
[20]
Cell morphology was observed by the Alexa Fluor The samples (0.2 g) were weighed and placed in a
488 Phalloidin Cytoskeleton staining kit (ThermoFisher, polytetrafluoroethylene (PTFE) digestion tank, and 8 mL
Waltham, USA). Briefly, after being cultured for 24 h, the of 65% nitric acid was added as a digestive solution. At
feeding layer was washed once with PBS. Fixation and first, the samples were placed in the microwave digestion
permeation were performed using 4% paraformaldehyde apparatus for a 20-min pre-digestion. Next, the samples
for 30 min and Triton X-100 (Beyotime, Shanghai, were subjected to acidic gas evaporation on the heating
China) for 10 min. Phalloidin (1:500) was added for 1 h plate at 120°C for 3 min and the remaining sample solution
before the cells were stained with DAPI for 10 min at was diluted to 25 mL with deionized water. A standard
room temperature. Finally, the feeding layer was washed curve was drawn for each ICP-MS assay. Finally, the nitrite
three times with PBS and observed under an inverted content in the sample was calculated and obtained.
fluorescence microscope (Leica Dmi 8, Morrisville, USA).
2.11 Nitrite quantification
2.7. Dot blotting of EGF and hyaluronic acid (HA) Nitrite quantification was determined by iodometry
Three kinds of HA with different molecular weights (L- titration . The 100 mg sample dissolved in 50 mL of PBS
[21]
HA: 10–20 kDa, M-HA: 40–80 kDa, and H-HA: 100– and the pH value was adjusted to 4, an excess of 0.01 mg/mL

Volume 9 Issue 5 (2023) 4 https://doi.org/10.18063/ijb.691