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International Journal of Bioprinting Development and characterization of AAMP for hydrogel bioink preparation

were then taken, followed by measuring their red (R) and DMEM and HUVEC growth medium, respectively, and
blue (B) values in RGB fashion at 40 randomly sampled collected on days 0, 3, 7, 14, 21, and 28. At each time point,
pixels using image processing software (MATLab). The samples were collected into 1.5-mL centrifuge tubes and
standard deviation or percent variance of R and B values mechanically broken down with polypropylene pestles
was then calculated as an indication of the homogeneity. (Bio Plas, CA, USA) in 500 μL of 1% of Triton solution in
PBS. The resulted solution was then sonicated to lyse the
2.5. Multiphysics modeling and simulation with cell membrane. The amount of DNA was then measured
COMSOL with PicoGreen dsDNA assay kit (Invitrogen, MA, USA).
COMSOL Multiphysics simulation software was used to
simulate the mixing process in the AAMP at a frequency 3. Results and discussion
of 1 Hz for up to 50 cycles. Geometry of the dual-syringe 3.1. AAMP system setup
compartment was directly reconstructed in COMSOL.
Using moving walls together with a moving mesh, back Figure 1 shows the AAMP from the top view, including
and forth motion was coupled with fluid dynamics (CFD) the structural set-up and control electronics. The overall
device is lightweight and portable, establishing it as an
model to allow for comparison to the experimental data. efficient and low-cost option compared to that which exists
Viscosities of the fluid components were substituted based on the market today. The chassis sits on two rods that allow
on experimental value from the rheological measurement. for size manipulation of the device’s body. The AAMP is
Time response of fluid velocity, pressure, and concentrations controlled by Arduino which is easily customizable for
were plotted at selected time points after the computation.
speed and cycle settings. The stop sensors are located on
2.6. Cell viability study the side walls of the chassis and are adjustable, allowing the
user to regulate how far each syringe is pushed. This setting
A 4-day cell viability study was performed by mixing is especially important for the “one-size-fits-all” approach,
cell-laden hydrogel components with AAMP. Human in which our AAMP system is able to accommodate
mesenchymal stem cells (hMSCs) were cultured in DMEM syringes of different sizes and varying mixing volumes.
medium (Life Technologies, USA) supplemented with 10%
of fetal bovine serum (FBS, Life Technologies, USA) and 3.2. Colorimetric characterization of AAMP
1% of penicillin and streptomycin. For encapsulation of We have carried out characterization for the AAMP to
cells in the hydrogel, hMSCs were trypsinized and added study the effect of different mixing conditions on the mixing
to 2.5% alginate solution before mixing at 1:1 ratio with outcome. We first mixed 2.5% of sodium alginate and 6%
6% of xanthan gum. For dissolving alginate and xanthan of xanthan gum solutions as representative materials for
gum, DMEM medium was used as the solvent instead of characterizing the mixing. We also studied their rheological
deionized water. After 10, 30, and 50 cycles of mixing in properties before mixing using a rheometer (Figure 2). As
AAMP, hydrogels were extruded into a mold, forming shown in Figure 2A and B, both sodium alginate and xanthan
discs with a diameter of 6 mm and a thickness of 0.45 mm.
After physical crosslinking by submerging in 1% of CaCl
2
solution for 1 min, samples were then transferred into a A B
24-well plate, with 1 mL DMEM medium added to each well
for culturing. Samples were then analyzed on days 0, 1, and
4. On analyzing, samples were washed with medium. Live/
dead staining kit (Invitrogen™, Thermo Fisher, Waltham,
MA) was used to visualize live and dead cells. Results were
obtained with a fluorescence microscope (Zeiss, Germany)
and the viability was analyzed through MATLab scripts by C D
counting live and dead cells using image analysis.
2.7. Cell proliferation

Four-week cell proliferation was evaluated by DNA
quantification. Similar to the viability experiment, hMSC
– or human umbilical vascular endothelial cell (HUVEC)
– encapsulated hydrogels were molded into discs with
6 mm diameter and 0.45 mm thickness. After crosslinking, Figure 2. Rheological properties of (A) sodium alginate, (B) xanthan
hMSC- and HUVEC-laden samples were cultured in gum, (C) their mixture over time, and (D) alginate mixed with Ca .
2+

Volume 9 Issue 4 (2023) 402 https://doi.org/10.18063/ijb.705