Elucidation of the liver pathophysiology of COVID-19 patients using liver-on-a-chips

Acknowledgments

Supplementary material

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Supplementary material is available at PNAS Nexus online.

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Funding

This research was supported by the iPS Cell Research Fund, the

COVID-19 Private Fund (to the Dr. Shinya Yamanaka laboratory,

CiRA, Kyoto University), the Mitsubishi Foundation, the Joint

Usage/Research Center program of Institute for Frontier Life and

Medical Sciences Kyoto University, the Japan Agency for Medical

Research

and

Development

(AMED)

(Grant

number:

JP20fk0108533, JP21fk0108492, and JP21gm1610005), and Japan

Science and Technology Agency (JST), (ACT-X, Grant number:

JPMJAX222A). S.D. was supported by a Grant-in-Aid for JSPS

Fellows.

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Author contributions

S.D. research design, generation and characterization of LoCs,

data analyses, statistical analysis, and manuscript writing. K.K.

fabrication of microfluidic devices.

R.H. SARS-CoV-2 infection experiments. A.S. qPCR analyses.

M.Y. collection and analysis of clinical samples. R.K. cell culture

experiments. P.G. cell culture experiments. R.N. HPLC analysis.

T.N. SARS-CoV-2 infection experiments. T.Y. RNA-seq analysis.

Y.T. fabrication of microfluidic devices. M.N. collection and ana­

lysis of clinical samples. K.T. research design, SARS-CoV-2 infec­

tion experiments, data analyses, manuscript writing, funding

acquisition, and final approval.

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Medium containing 10 μM of RDV, MPV, or BARI was injected into

the PDMS device (200 μL medium/channel). Half of the medium

was collected at 1, 2, or 4 h after the drug treatment. When collect­

ing the supernatant, the same amount of culture medium con­

taining the substrate was added. The collected supernatant was

mixed with the same volume of acetonitrile. Samples were fil­

trated with Cosmonice Filter W of a pore size of 0.45 µm and

then analyzed by HPLC to measure the concentration of RDV,

MPV, or BARI according to a standard curve. HPLC analysis was

performed using a LO-20AD SPD + RF (DGU-20A, LC-20AD,

RF-20A xs, SIL-20AC, CBM-20A, SPD-20A, CTO-20AC; Shimadzu).

The HPLC methods are summarized in Table S4.

We thank Dr Misaki Ouchida (Kyoto University) for creating fig­

ures, Dr Peter Karagiannis (Sofia Science Writing) for critical read­

ing of the manuscript, Dr Yoshio Koyanagi, Ms. Naoko Misawa,

and Dr Kazuya Shimura (Kyoto University) for the setup and oper­

ation of the BSL-3 laboratory at Kyoto University, Ms. Masami

Yamashita (Kyoto University) for the cell culture, Ms. Kazusa

Okita and Ms. Satoko Sakurai (Kyoto University) for technical as­

sistance with the RNA-seq experiments, and Ms. Natsumi

Mimura, Ms. Emi Sano, Ms. Naoko Yasuhara, and Mr. Takuro

Nobe (Kyoto University) for technical assistance.

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