Page 526 - IJB-10-6

International Journal of Bioprinting Bacteriorhodopsin-embedded hydrogel device

fabricated structure preserves the photosensitive nature of br Germany). The surface of the freeze-dried construct was
and exhibits desirable photoelectrical properties, such as light carefully trimmed to reveal part of the internal structure.
intensity sensitivity, concentration sensitivity, and advanced The UV-VIS absorption spectrum of printing material
functions such as pattern recognition. These results lay the containing br was measured using a spectrometer (U-
groundwork for further innovative applications of br through 3900; HITACHI, Japan) with hydrogel containing no br as
biocompatible material, versatile fabrication techniques, and a reference. The scan range was set to 250–700 nm. The
comprehensive photoelectrical characterization. material to be measured was heated up to 45° in a metal bath
to increase its liquidity. Subsequently, it was transferred to
2. Methods an Eppendorf UVett and cooled to room temperature
TM
before conducting the absorption spectrum test.
2.1. Fabrication of br-embedded hydrogel construct
Figure 1A illustrates the fabrication process of the Rheological measurement was performed using a
hydrogel construct. Bacteriorhodopsin (br; lyophilized rotational rheometer (MCR302; Anton Paar, Austria) in
powder; Sigma-Aldrich, USA) was dissolved in Dulbecco’s oscillatory mode. The strain and frequency were set to 1%
phosphate-buffered saline (DPBS; Sigma-Aldrich, USA) at and 1 Hz, respectively. Cone-plate geometry with a 25 mm
a concentration of 1 mg/mL, resulting in a uniformly pale diameter, 2’ cone, and a 99 µm truncation gap was used. Gʹ is
purple solution after ultrasonic oscillation. The printing defined as the storage modulus, and Gʹ is defined as the loss
material containing br was prepared by combining 400 modulus. The printing material was heated up to 30° using
µLof the solution with 200 µL of gelatin solution (20 wt% a metal bath before being transferred to the rheometer.
in DPBS) and 100 µL of sodium alginate solution (1 wt% The temperature sweep was performed at 20–30°, and 70
in DPBS). The mixture was heated to 45°C and thoroughly sample points were recorded. A shear rate-viscosity test was
mixed for 20 s using a vortex mixer. An ITO glass then performed at 21°. The shear rate ranges from 0.1 to
measuring 30 × 20 mm with a surface resistance of 8 Ω/ 1000 1/s, and 100 sample points were recorded.
2
m was sonicated in ethanol, acetone, and deionized (DI) Biocompatibility was assessed by loading neural
water to prepare the printing substrate. The well-mixed progenitor cells (NPCs) obtained through the method
printing system was drawn into a 1 mL syringe, pre-chilled described in previous research onto the br-embedded
65
in a 4°C refrigerator for 10 min, and then loaded into a hydrogel. A cell suspension was mixed with gelatin/sodium
printer (Biomakers; SunP Biotech, China). The nozzle alginate hydrogel containing br, and fibrinogen was added
and the ITO glass substrate were cooled to 13 and 10°C, to enhance NPC adaptability. To evaluate cell viability
respectively, before printing. The printing speed was set using a live-dead assay, Calcein AM and propidium iodide
to 5 mm/s, and the extrusion speed was set to 0.8 mm / (PI) (DOJINDO, Japan) were applied to the construct,
3
min. Following printing, 500 µL of CaCl solution (2% wt and images were captured using laser scanning confocal
2
in DI water) was applied to immerse the printed structures microscopy (TI-FL; Nikon, Japan). A calcium assay was
to facilitate the crosslinking of sodium alginate. After 3 subsequently conducted to assess neural function. Fluo
min, the CaCl solution was aspirated, and the ITO glass 4-AM (DOJINDO, Japan) was applied to the construct,
2
with the printed structures was placed in a 65 mm Petri and time-lapse images were acquired using a laser confocal
dish. DI water was added to prevent the structures from microscope (FV3000; Olympus, Japan) under 488 nm laser
dehydrating. The Petri dish was sealed with Parafilm and excitation. Further details regarding the biocompatibility
stored in the dark at 4°C for future use. assessment methods can be found in Gai’s work. 65

2.2. Characterization of printing material 2.3. Photoelectrochemical characterization
and structure The br-embedded hydrogel construct on the ITO glass
The printed br-embedded hydrogel structure was observed substrate was positioned on the sample stage of a probe
under an optical microscope (SMZ800N; Nikon, Japan) workstation (Chuangpu Instrument, China) and examined
to determine if it was crosslinked properly. To distinguish using a microscope. A droplet of electrolyte (DPBS, pH 8)
hydrogel structure with varying br concentration, was dispersed onto the br-embedded hydrogel and covered
hydrogel with high br concentration was dyed with with another ITO glass, constructing a photovoltaic cell with
Coomassie brilliant blue G-250, while hydrogel with low a layered structure. The photovoltaic cell was then connected
br concentration was dyed with grape skin anthocyanin. to an electrochemical workstation (CHI660E; Shanghai
The printed construct was freeze-dried at −60° overnight ChenHua, China), with the br-embedded hydrogel-attached
in a lyophilizer (LGJ-12; SONGYUANHUAXING ITO glass connected to the working electrode, and the other
Technology, China) and then observed using a scanning ITO glass connected to the counter and reference electrode.
electron microscope (SEM; Zeiss GeminiSEM 300; Zeiss, A white light-emitting diode (LED) with adjustable light

Volume 10 Issue 6 (2024) 518 doi: 10.36922/ijb.4454