Page 256 - IJB-10-3

International Journal of Bioprinting Bioprinting organoids for toxicity testing

2.3. Fabrication of DOs-laden microspheres by HE, unlabeled EPC, and LX-2; (ii) PKH67-labeled EPC,
electro-assisted inkjet printing unlabeled HE, and LX-2; and (iii) PKH67-labeled LX-2,
The printing procedure conducted in this experiment was unlabeled HE, and EPC. After 5 days of co-culture, the
similar to that reported in our previous study. The bioink three types of DOs-laden microspheres were obtained.
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was composed of 3.5% alginate (Sigma, 919373), 2 μg/ To allow clearer observation of the microplastic entry
mL ncLaminin 511 (Nuwacell Biotechnologies Co., Ltd., into cells within a short period, we used MPs in dosages
RP01025), and a cell mixture of hPSC-HE, hiPSC-EPCs, higher than the environmental concentrations. The DOs-
and LX-2 at 10:7:2 with a total density of 3 × 10 cells/mL. laden microspheres were exposed to 100 μg/mL red
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6
The electro-assisted inkjet printing device consisted of fluorescence-labeled MPs (for better visualization) (Wuxi
a static electricity power supply, a syringe pump (Longer Ruige Biotechnology Co., Ltd.) and MPTA at 100 μg/
Pump Ltd.), and a grounded collecting device. A disposable mL/100 nM. The DOs-laden microspheres were placed in
sterile syringe loaded with bioink was fixed to the syringe a sterile chamber at 5% CO and 37°C.
2
pump. Cell-laden microspheres were printed at a 10 The diffusion of MPs in the DOs was visualized and
mL/h propulsion speed, 2 cm electrode distance, and 12 recorded with a laser scanning confocal microscope
kV voltage. Microspheres were crosslinked with 300 mM (LSCM; Nikon, Z2). The fluorescence signal distribution
calcium chloride solution and washed with DMEM-F12 of MPs and PKH67 at the specified locations was analyzed
basal medium three times. and graphed with Nikon NIS-Elements Advanced
The post-printed cell-laden microspheres were cultured Research (AR). The fluorescence intensities and areas of
in organoid culture medium composed of DMEM-F12/ the more than 10 microspheres were analyzed by Nikon
M199/William (1:1:1), 500 μM monothioglycerol, 1× NIS-Elements AR. The fluorescence intensity of the MPs
B27 with insulin, 1% ITS, 50 μg/mL ascorbic acid, 0.1% (n > 10) was the average of the normalized fluorescence
BSA, 2% FBS, 1% v/v sodium pyruvate (Sigma, P2256), intensity of MPs, which was divided by the area of the
1% v/v NEAA (Sigma, M7145), 1% v/v GlutaMAX, 100 microsphere.
μM ascorbic acid, 20 ng/mL VEGF, 20 ng/mL hepatocyte
growth factor (HGF; R&D, 294-HG-025), and 5 ng/ 2.6. Cell live/dead staining
mL FGF2. The medium was refreshed daily. DOs-laden The cell viability in the post-printed microspheres, DOs
microspheres were formed after 5 days of culture. within microspheres, and DOs with pollutant treatments
was analyzed by in situ staining of microspheres for live/
2.4. Pollution exposure dead cells. The live/dead cells were stained with the
The spherical polystyrene MPs (Wuxi Ruige Biotechnology Calcein-AM/PI Kit (Merck, 04511-1KT-F) according
Co., Ltd.) were monodispersed particles with a diameter to the manufacturer’s instructions. The staining was
of 1 μm in water at a concentration of 25 mg/mL. The incubated for 10 min in the dark and observed using an
stock solutions were pasteurized before utilization. TBBPA LSCM (Nikon, Z2).The cell viabilities were the averages
(purity > 98.0%, TCI, Shanghai Development Co., Ltd., of the ratio of green fluorescence intensity to total cell
China) was dissolved in dimethyl sulfoxide (DMSO; fluorescence intensity (n > 3).
Amresco, USA, 0231). Polystyrene MPs and TBBPA were 2.7. Determination of liver function
diluted according to the manufacturer’s protocols. The Bile salt transport was examined with cholyl-lysyl-
MPTA complex was obtained by incubating 6 μg/mL PS- fluorescein (CLF) staining. DOs-laden microspheres were
MPs and 10 μM TBBPA in medium at 37°C overnight.
incubated with Williams’ E medium (BasalMedia, L660KJ)
The exposure dosage of PS-MPs was 600 ng/mL, supplemented with 5 µM cholyl-lysyl-fluorescein (CLF; BD
TBBPA was 100 nM, and MPTA was 600 ng/mL/100 Biosciences, BD-451041) for 15 min at 37°C and 5% CO .
2
nM. The vehicle control was 0.01% DMSO. DOs-laden After washing, CLF internalization was visualized with
microspheres were incubated with MPs, TBBPA, MPTA, confocal microscopy. The fluorescence intensity of CLF was
and vehicle control for 3 days. The media were changed analyzed by Nikon NIS-Elements AR. More than 20 DOs-
every day. laden microspheres from three independent experiments
were included in the calculation. The CLF intensities were
2.5. Tracing MPs in DOs calculated as the average fluorescence intensity of the CLF
The cell membranes of hiPSC-derived HE, EPC, and divided by the microsphere area.
LX-2 cells were labeled with a PKH67 Green Fluorescent
Cell Linker Kit (Merck, PKH67GL) according to the 2.7.1. Neutral lipid staining
manufacturer’s protocols. We printed three types of Lipid accumulation was detected using neutral lipid
microspheres with cell mixtures: (i) PKH67-labeled staining. DOs were harvested from microspheres, fixed

Volume 10 Issue 3 (2024) 248 doi: 10.36922/ijb.1403