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He, et al.
a 35 mm tissue culture plate was placed on the printing ki-67 (ab15580, Abcam) and rabbit anti-human albumin
platform. The nozzle tip was manually moved to a height (ab207327, Abcam) were used as the primary antibodies.
just touching the plate bottom. During printing, the nozzle The blocked samples were incubated in the primary
moved along an automatically planned path, depositing antibody solution overnight at 4°C and then treated with
bioink. After printing, the 3DP structures were ionically Alexa Fluor®488 (ab150077) - or Alexa Fluor®568
crosslinked for 3 min using a calcium dichloride (CaCl ) (ab175471) - conjugated goat anti-rabbit IgG (Abcam)
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solution. A schematic of the bioprinting process is shown as secondary antibodies. Cell nuclei were stained with
in Figure 1B. Moreover, non-printed sandwich cultures of 4′,6-diamidino-2-phenylindole. The fluorescence images
hiPSC-Heps were performed by pipetting the bioink into were captured using a confocal laser-scanning microscope
24-well culture plates, forming a hydrogel layer with the (LSM710META, Zeiss, Oberkochen, Germany) by layer-
same height (1.2 mm) as the 3DP structures. In addition, by-layer scanning. All images were stacked and rebuilt
hiPSC-Heps, at a density of 2 × 10 cells/cm , were to a single image. The mean fluorescence intensity of
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inoculated into 24-well culture plates for 2D cultures. these images was analyzed using ImageJ to quantitatively
An 8-day culture of the 2D, SW, and 3DP hepatic determine the expression level of the biomarkers.
tissue models was implemented with culture medium Samples of each group were prepared in triplicate. Four
refreshment occurring every other day. The experimental randomly captured images from each sample were used
timeline for this study is shown in Figure 1C. for the analysis.

2.3. Spheroid morphology analysis and 2.5. RNA extraction and quantitative real-time
measurement of cell viability and proliferation reverse transcription polymerase chain reaction

The morphology of the hiPSC-Heps spheroids was (qRT-PCR)
observed under a phase-contrast microscope (Eclipse Total RNA was extracted from hiPSC-Heps using
TS100, Nikon, Tokyo, Japan). Cell viability was TRIzol™ reagent (15596026, Invitrogen, Carlsbad, CA,
determined using the calcium acetoxymethyl ester USA). After RNA extraction, the concentration and purity
and propidium iodide double staining method (C542, of the RNA were measured using a spectrophotometer
Dojindo, Kumamoto, Japan). The SW and 3DP models (Nanodrop 2000, Thermo Scientific, Waltham, MA,
were incubated for 15 min in a staining solution USA). Reverse transcription of RNA was performed
containing 2 μg/mL calcium acetoxymethyl ester (green) using PrimeScript™ RT Master Mix (6210 A, TaKaRa,
and 3 μg/mL propidium iodide (red). The stained samples Kyoto, Japan), following the manufacturer’s protocol.
were observed under a confocal laser-scanning microscope The qRT-PCR reaction system was then prepared using
(LSM710META, Zeiss, Oberkochen, Germany). The Maxima SYBR Green qPCR Master Mix (RR420L,
number of living and dead cells was counted to calculate TaKaRa). Data collection was performed using a real-
the cell viability rates using ImageJ software. The cell time PCR detection system (7500, Applied Biosystems,
viability data were analyzed using four randomly captured Carlsbad, CA, USA). The relative gene expression levels
images from each sample. These fluorescent images were were calculated using the 2 –ΔΔCt method with β-actin
also used to measure the average diameter of hiPSC-Heps selected as the housekeeping gene. Primers used for qRT-
spheroids and the data were analyzed using ImageJ. Cell PCR are listed in Table A.1. Samples of each group were
proliferation was measured using a Cell Counting Kit-8 prepared in triplicate.
(CCK8) (C10350, Dojindo), and finally, a standard curve
was plotted using the known cell densities to convert the 2.6. Enzyme-linked immunosorbent assay
collected OD values to cell densities. All the experiments (ELISA) assay
mentioned above were performed in triplicate. The secretion of human albumin, α-1-antitrypsin (AAT),
2.4. Immunostaining and blood urea nitrogen (BUN) was measured using a
human albumin ELISA kit (EHALB, Invitrogen), a serpin
Samples from the 2D, SW, and 3DP models were A1 human ELISA kit (EHSERPINA1, Invitrogen), and a
collected for immunostaining after 8 days in culture. BUN colorimetric detection kit (EIABUN, Invitrogen),
The samples were fixed with 4% paraformaldehyde for respectively. Culture supernatants were collected 24 h
30 min and permeabilized with 0.1% Triton X-100 for after the medium change on days 2, 4, 6, and 8. The
20 min. Fluorescein isothiocyanate–conjugated phalloidin number of cells at each time point for sample collection
(ab176753, Abcam, Cambridge, UK) was used after was measured using the CCK8 kit. The amounts of
permeabilization for cytoskeletal staining. The samples albumin, AAT, and BUN were calculated according to
for ki-67 and albumin staining were subsequently blocked the standard curve, followed by data normalization per
in 2% bovine albumin for another 2 h. Rabbit anti-human 1 million cells per day. A total of 12 individual samples

International Journal of Bioprinting (2022)–Volume 8, Issue 3 179