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International Journal of Bioprinting 3D-printed EVs for nasal septal defects

2.2. Isolation, identification, and staining of EVs 2.2.4. Western blot
from adipose-derived stem cells (ADSCs) The protein concentration of EVs was measured using a
BCA kit (Bvant, China) according to the manufacturer’s
2.2.1. Isolation of EVs method, and the number of EVs in each sample was
The acquisition of 3D EVs is based on the findings of Chen calculated based on the assay results. Sodium dodecyl
et al., which involved the use of coaxial printing to obtain sulfate (SDS) buffer (Servicebio, China) was added to
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3D adipose-derived stem cell (ADSC)-derived EVs. In the sample and mixed by vortexing. The proteins were
this study, 1.5% alginate solution was used for shell flow at then denatured in water at 95°C before electrophoresis.
20 mL/h; ADSC suspension (ScinencellTM Laboratories, After electrophoresis, the gels were separated, and the
USA) was used for core flow at 5 mL/h.; 3% CaCl solution positive EVs protein markers, CD63 and TSG101 (Abcam,
2
was used as the receiving medium. The alginate solution in England), and the negative protein marker calnexin
the shell flow rapidly gelled in the CaCl solution, forming were transferred to a polyvinylidene fluoride (PVDF)
2
microfibers that encapsulated the cells within approximately membrane (Millipore, Germany). After blocking with a 3%
10 min. The microfibers were then rinsed with a 0.9% bovine serum albumin (BSA) blocking solution (Solarbio,
NaCl solution for 5 min, before being transferred to a 150 China), the membrane was incubated with primary and
mm Petri dish (Zhennuo, China) containing 25 mL MSC secondary antibodies and visualized accordingly thereafter
complete medium (NC0103; Yocon, China). The dish (Chemiscope, China).
was then placed in a 5% CO incubator at a temperature
2
of 37°C. The cell culture medium was collected every 24 2.2.5. Cell uptake
h and replaced promptly, and the collected medium was To validate the uptake of 3D-derived EVs by nasal
frozen at −80°C for storage. septal chondrocytes, EVs were stained with PKH26 red
fluorescent dye (Uimbio Shanghai, China) for 15 min
Cell culture media uses continuous centrifugation at 37°C, and 3D-EV-loaded chondrocytes were treated
and filtration for EV collection. Briefly, the cells were for 24 h after centrifugation to remove excess dye. After
precipitated at 300 ×g for 10 min at 4°C using an Overspeed incubation, the cells were fixed with 4% paraformaldehyde
Centrifuge (Himac Japan) and subsequently at 2000 ×g (Biosharp, China) for 15 min, washed three times with
for 30 min to remove dead cells. The remaining purified PBS, and permeabilized with Triton-100 (Coolaber, China)
medium was centrifuged at 10000 ×g for 30 min, and the for 10 min. The membranes were stained using fluorescein
supernatant was filtered through a 0.22 μm filter (Sartorius, isothiocyanate (FITC; Aladdin, China) and washed with
Germany) to remove excess protein. Next, the supernatant PBS before staining the nuclei using DAPI. The images
was centrifuged at 100000 ×g for 70 min; the sediment was were captured under an inverted fluorescence microscope
washed with phosphate-buffered saline (PBS) (zqxzbio, (Nikon, Japan).
China); and the solution was transferred to a new tube and
centrifuged again at 100000 ×g for 70 min to obtain the 2.3. Characterization of molecular structure
3D-cultured EVs. and microstructure
Scanning electron microscopy (SEM) was used
The 2D EVs were synthesized according to the to characterize the electrospun biofilm and the
traditional preparation method. ADSCs (passage [P]3–6) microstructure of the composite scaffold; the fiber
30
were cultured in serum-free stem cell medium (Corning, diameter of the scaffold was calculated using Origin 2021
United States of America [USA]) for 48 h; the supernatant (OriginLab, United States). Briefly, a biological scaffold
was collected and purified via ultracentrifugation; and the of suitable size was prepared and its surface was evenly
2D-cultured EVs were obtained. covered with chondrocyte suspension and Dulbecco’s

2.2.2. Transmission electron microscopy Modified Eagle Medium (DMEM; Gibco, China)
The EVs were loaded onto Formvar carbon-coated EM supplemented with 10% FBS for 2 days. After complete
copper mesh (HEAD, China), stained with PKH26 kit cell adhesion and growth, the scaffold was washed with
(Sigma-Aldrich, USA), air-dried, and observed using PBS. The biofilm of the loaded cells and the composite
transmission electron microscopy (TEM; Hitachi, Japan). scaffold were freeze-dried, and the composite scaffold
was treated with liquid nitrogen embrittlement to expose
2.2.3. Nanoparticle tracking analysis the cross-section. The surface of the scaffold was sprayed
The EV suspension was diluted to 1 mL with stock with gold at 18 mA using an ion sputter coater (Technol,
solution and transferred to a 1 mL syringe. The particle China) for 60 s. SEM was used to observe the surface
size distribution of EVs was detected using nanoparticle morphology of the scaffold; the fiber diameter of the
tracking analysis (NTA; Malvern Panalytical, Netherlands). scaffold was calculated accordingly.

Volume 10 Issue 6 (2024) 177 doi: 10.36922/ijb.4118