Page 14 - IJB-9-5

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

section, ENB vs. TeeBN were analyzed with paired samples affect the physiological function of the encapsulated cells
t-test. Significance was set to P < 0.05. (*P < 0.05, **P < or being too soft leading to collapse upon fabrication.
0.01, ***P < 0.001, and ****P < 0.0001)
Thus, we first explored this printability window by
3. Results and discussion adjusting temperature and pressure and evaluated the pore
factor (Pr) . A Pr close to 1 indicates an optimal printing
[27]
3.1. Preparation of the feeding-layer material condition. At the same time, its value <1 and >1 suggests
We aimed to fabricate a 3D matrix of GelMA to encapsulate the collapse of printing structure and closure of the circle;
epithelial cells as the feeding layer (Figure 2A). GelMA contrarily, the printing ink is the over concentrated
is renowned for superior biocompatibility. Despite its inducing irregular shape of the structure (Figure S1C) .
[15]
commercial availability, we chose to synthesize it in-house After testing 91 combinations, we eventually obtained four
in order to fine-tune the mechanical strength, which is categories of conditions: printable, shapeless, congested,
vital for printing and epithelial cell survival. We did it by and non-printable (Figure 2E); the samples with a Pr
adjusting the substitution ratio of MA to gelatin chains around 1 in red rectangles and printing sample shown in
(Figure S1A). H-NMR confirmed the incorporation of Figure 2F.
1
acrylamide double bonds (5.3 and 5.6 ppm) and suggested
the degree of methacrylate group at approximately 63% We then compared the different culturing types to
(Figure 2B). In addition, mixing LAP (0.25%, w/v) confirm that 3D-printed matrices are optimal for the
crosslinker induced rapid crosslinking (<10 s) under feeding layer in comparison with 2D surface culture and
blue light (Figure 2C), minimizing the unfavorable 3D gel encapsulation (without printing of the porous
environment caused by traditional UV crosslink method structure) (Figure 2G). The data showed that the epithelial
(>10 s) influences on cell viability. cells maintained high viability in both 2D surface and 3D
printing conditions (>80%) (Live/Dead staining, Figure 2H;
Rheological tests showed that the GelMA gel became quantification, Figure 2I). However, the 3D-printed matrix
increasingly viscous as the concentration increased from provided a high cell number per surface area (Figure 2J)
1.17 Pa.s of 5% (w/v) to 5.59 Pa.s of 15% (w/v), and the and a large proportion of actively proliferating cells (EdU
frequency dependence of the energy storage modulus staining; Figure 2K). In comparison, the 2D culture
increased with the modification degree (Figure 2D). A accommodated a limited number of cells while the 3D
10% concentration could provide adequate mechanical encapsulation model had both lower viability and fewer
support and avoid being too stiff for the in-gel growth of proliferating cells. More interestingly, after 7 days of culture,
epithelial cells, which are typical anchorage-dependent the epithelial cells expressed the highest level of AQP5
cells sensitive to mechanical cues . Measurements of the (acinar cell-specific marker) and the lowest level of αSMA
[25]
rheological properties, storage modulus, and loss modulus (myoepithelial marker) in the 3D-printed matrix [28,29] ,
through temperature scanning identified the window of among other cell cycle-related markers (Figure 2L) . This
[30]
the gelation state. At room temperature, the energy storage trend was in favor of cell secretory capacity.
modulus was greater than the loss modulus, and the gel
was robust; as the temperature rose to 33°C, the energy Before encapsulation, the cytotoxicity assay (CCK-8)
storage modulus gradually decreased to the loss modulus, showed that the material, having been rinsed to remove
and GelMA briefly presented a gel-state temperature any chemical agents used during the preparation
window (Figure S1B). The physical state of GelMA is in and crosslinking, had no toxicity to epithelial cells
gel state at this temperature window (22°C–30°C) around (Figure S1D) through co-culture with the GelMA soaking
room temperature, which is suitable and applicable for solution. After 24-h encapsulation, epithelial cells started
extrusion 3D printers and could not significantly affect cell to spread in hydrogel network, suggesting that these cells
[26]
viability . demonstrate their natural morphology in the soft feeding
layer (Figure 2M). To verify that the cells could secrete
3.2. Cell encapsulation in hydrogel through adequately, we chose EGF as the representative nutritional
3D printing ingredient of EBN and determined its amount by ELISA.
We fabricated GelMA into hydrogels to encapsulate the The cumulative release data showed increased EGF
epithelial cells through 3D printing. Compared with 2D secretion along the culture time, indicating the functioning
culture, 3D encapsulation enables a high and controllable of the feeding layer (Figure 2N).
cell number to produce sufficient nutritional factors, and
printing helps create a tissue-mimicking microenvironment 3.3. Preparation of receiving layer
for cell settlement. The GelMA ink for printing should be We employed edible hyaluronic acid (HA) as the main
in appropriate stiffness, avoiding being too hard that could structure of the receiving layer. It contains negatively

Volume 9 Issue 5 (2023) 6 https://doi.org/10.18063/ijb.691