Here you will know , how to write a critical review & what sections are suppose to analyse why reading a paper. Here is a critical review of a paper titled ' Repeated dose multi-drug testing using a microfluidic chip-based coculture of human liver and kidney proximal tubules equivalents' published recently in 2020 in reputed journal nature.

1. 1
Repeated dose multi-drug testing using a microfluidic chip-based
coculture of human liver and kidney proximal tubules equivalents
Report of critical review submitted in
partial fulfilment of the requirements
for the award of the degree
of
Master of Technology
(Bioprocess technology)
by
Sourabh Santosh Gurav
(20BPT218)
Institute of Chemical Technology
Mumbai – 400 019
Under the guidance of
Dr. Sadhana Sathaye

2. 2
TABLE OF CONTENT
 LIST OF ABBREVIATIONS……………………………………………………...….3
 LIST OF FIGURES……………………………………………………………...……4
 LIST OF TABLES……………………………………………………………….……4
1. INTRODUCTION ……………………………………………………………………5
2. RESEARCH PAPER FOR CRITICAL REVIEW……………………………………7
3. Overview of the paper…………………………………………………………………9
3.1.Objective…………………………………………………………………………10
3.2.Material and methods ……………………………………………………………10
3.2.1. Design and fabrication of micro-physiological 2-OC……………………10
3.2.2. Human Cell sources………………………………..…………………….10
3.2.3. De-novo formation of liver equivalents………………………………….10
3.2.4. Formation of renal proximal tubule barrier model………………………10
3.2.5. Chip based co-cultures…………………………………………………...11
3.2.6. Model compounds used…………………………………………………..11
3.2.7. Procedure for administration of drug…………………………………….11
3.3.Analysis and observations………………………………………………………..12
3.3.1. Cell viability and metabolic activity……………………………………..12
3.3.2. Protein Expression in surrogate blood circuit……………………………12
3.3.3. Protein expression in excretory lumen…………………………………...13
3.4.Results……………………………………………………………………………15
4. CRITICAL ANALYSIS………………………………………………………………16
4.1.Objectives………………………………………………………………………...16
4.2.Title of the paper…………………………………………………………………16
4.3.Originality of the paper…………………………………………………………..16
4.4.Grammatical errors or typographical mistakes…………………………………..16
4.5.Technical correctness and missing data………………………………………….17
4.6.Clarity…………………………………………………………………………….17
4.7.Typographical and grammatical errors…………………………………………...17
4.8.Limitations of the technique……………………………………………………...18
5. DISCUSSION…………………………………………………………………………19
6. REFERENCES ……………………………………………………………………….21

3. 3
LIST OF ABBREVIATIONS
ABBREVIATION DESCRIPTION
CsA Cyclosporin A
RFP Rifamcipin
2-OC Two Organ Chip
RPTEC/TERTI Renal proximal Tubule Epithelial Cells
MOC Multiple Organ Chip
LDH Lactate Dehydrogenase
DMSO Dimethyl SulFoxide
MPS Micro-Physiological System
CYP3A4 cytochrome P450 3A4
P-gp p-glycoprotein
TBiL total bilirubin
α1-MG α1-microglobulin
Col IV collagen IV
BSEP bile salt export pump
FABP-1 fatty acid binding protein-1

4. 4
LIST OF FIGURES
Fig 1 : Microfluidic chip………………………………………………………………………5
Fig 2 : A.) Organ on a chip device
B.) Picture of the organ on a chip device (directions indicated by arrows visualize
perfusion of the red and blue dyes)…………………………………………………..6
Fig 3 : Schematic overview of the 16-day coculture………………………………………….9
Fig 4 : Corning® Transwell® Permeable Supports………………………………………….11
Fig 5 : Production of LDH & lactate and consumption of Glucose…………………………12
Fig 6 : Expression of aminotransferase, Alkaline phosphatase & total bilirubin……………12
Fig 7 : Expression of Glucose, creatine & urea……………………………………………...13
Fig 8 : Expression of Albumin, gamma-glutamyl transpeptidase and Lactate……………....13
Fig 9 : Expression of (A) GGT (B) ALP (C) NAG (D) Glucose (E) Lactate (F) KIM-1
(G) Cystatin C (H) Collagen IV (I) Clusterin (J) NGAL (K) Osteoactivin (L) IP-10
(M) GST-α (N) albumin (O) FABP-1 (P) Renin (Q) α1-microglobulin (R)
Osteopontin (S) TIMP-1 (T) EGF in excretory media on days 1, 7 and 14………….14
LIST OF TABLES
Table 1 : Changes in levels of all biomarkers………………………………………………19

5. 5
1. INTRODUCTION
Microfluidics is the technology that uses and manipulates fluids at a microscale usually of
volume 10-9
to 10-18
L in channels that range in size from tens to hundreds of microns. Using
the technology cells can be grown in a 3D environment that simulates the physiological
environment of human organs. This chip set up has micro-channels mimicking the
physiological conditions and regulating key parameters like concentration gradients , shear
force , cell patterning , tissue-boundaries, and tissue–organ interactions . The 3D arrangements
are typically created by the addition of biocompatible materials such as hydrogels. These
materials can prevent mechanical damage and shape three-dimensional arrangements. For
decades, traditional two-dimensional (2D) cell culture systems formed an important platform
for life science research. Using 2D systems, the functions of various cells are studied by
culturing cells or cell products. However, 2D systems fail to accurately simulate the
physiological conditions of living tissues/organs, intra-organ interactions and
microenvironmental factors and often require verification in in vivo animal models. Moreover
two or more different cells (non-parenchyma cells) can be simultaneously cultivated for better
representation of organ system rather than just using cells in a petri dish or a suspension culture.
Each organ chip is composed of a flexible and clear polymer that contains a hollow microfluidic
channels lined by living human organ specific interfaced with a human endothelial cell lined
artificial vasculature, Mechanical forces are also applied which recreates physical environment
of living organs like breathing motion and peristalsis in intestine. The set-up is translucent
providing a window into the inner working of human cells in context of real organ.
Fig 1 : Microfluidic chip

6. 6
Although animal experiments are indispensable for preclinical screening in the drug discovery
process, various issues such as ethical considerations and species differences remain. To solve
these issues, cell-based assays using human-derived cells have been actively pursued.
However, it remains difficult to accurately predict drug efficacy, toxicity, and organs
interactions, because cultivated cells often do not retain their original organ functions and
morphologies in conventional in vitro cell culture systems.
Fig : A.) Organ on a chip device B.) Picture of the organ on a chip device (directions
indicated by arrows visualize perfusion of the red and blue dyes).

7. 7
2. RESEARCH PAPER FOR CRITICAL REVIEW
2.1. Name : Repeated dose multi-drug testing using a microfluidic chip-based coculture of
human liver and kidney proximal tubules equivalents.
2.2. Name of the Journal : Nature
2.3.Impact factor of journal: 42.778 dated 2019
2.4. Authors :
Lin N, Zhou X, Geng X, Drewell C, Hübner J, Li Z, Zhang Y, Xue M, Marx U, Li B.
2.5. Dates :
 Received - 2019 Nov 18
 Accepted- 2020 Apr 15
 Published- 2020 Jun 1
2.6. Digital Object Identifier : 10.1038/s41598-020-65817-0
2.7. PMID (unique identifier number used in PubMed) : 32483208
2.8. Cited by following three articles :
 Jin, M., Yi, X., Liao, W., Chen, Q., Yang, W., Li, Y., Li, S., Gao, Y., Peng, Q., &
Zhou, S. (2021). Advancements in stem cell-derived hepatocyte-like cell models for
hepatotoxicity testing. Stem cell research & therapy, 12(1), 84.
https://doi.org/10.1186/s13287-021-02152-9

8. 8
 Thompson, C. L., Fu, S., Knight, M. M., & Thorpe, S. D. (2020). Mechanical
Stimulation: A Crucial Element of Organ-on-Chip Models. Frontiers in
bioengineering and biotechnology, 8, 602646.
https://doi.org/10.3389/fbioe.2020.602646
 Klak, M., Bryniarski, T., Kowalska, P., Gomolka, M., Tymicki, G., Kosowska, K.,
Cywoniuk, P., Dobrzanski, T., Turowski, P., & Wszola, M. (2020). Novel Strategies
in Artificial Organ Development: What Is the Future of
Medicine?. Micromachines, 11(7), 646. https://doi.org/10.3390/mi11070646

9. 9
3. OVERVEIW OF THE PAPER
3.1. Objective : Drug toxicity is one of the major reasons for post-market drug withdrawal;
therefore it is crucial to perform preclinical toxicity testing on candidate drugs. Owing to
the existence of species differences, the results obtained from animal models cannot be
fully adapted to humans. This paper describes the drug-induced nephrotoxicity and
hepatotoxicity experimental procedure conducted on microfluidic chip based model of liver
spheroids (human HepaRG cell line, non-parenchyma cells- HHSteC cell line) and renal
proximal tubule barriers (human proximal tubule cell line RPTEC/TERT1) in a connecting
media over 16 days. Toxicity profiles of the two different doses of Cyclosporin A on
different target organs were discriminated and showed that concomitant treatment with
rifampicin from day 6 onwards decreased the CyclosporinA (CsA) concentration and
attenuated the toxicity compared with that after treatment with CsA for 14 consecutive
days.
Fig 3 : Schematic overview of the 16-day coculture
As shown in the above timeline figure, Cyclosporin A an immunosuppressant drug was used
to test its toxicity using a chip based liver-proximal tubule coculture system. A repeated
treatment with a combination of CsA and rifampicin (RFP), a well-known inducer of hepatic
enzymes and transporters, was applied to further evaluate drug metabolism and toxicity on the
two-organ-chip (2-OC). The objective of the paper is to assess microfluidic multi-organ chip
platform for co-cultivation reproducible and well-defined liver spheroids and renal proximal

10. 10
tubule barriers, check inter organ communication from designed fluid-to-tissue ratio in the 2-
OC model and eligibility of cocultured tissue equivalents for drug toxicity screening.
3.2. Material and Methods :
3.2.1. Design and fabrication of the microphysiological 2-OC : Fabrication of the 2-OC
was performed by TissUse GmbH as described by Wagner et al. . Fluid fow in the
circuit was established by an on-chip pulsatile micropump modifed afer Wu et al. Te
on-chip micropump was actuated by pressured air through an external control unit
provided by TissUse GmbH.
3.2.2. Human Cell sources :The human HepaRG cell line and the human proximal tubule
cell line RPTEC/TERT1 (CRL-4031) were purchased from ATCC (ATCC, Manassas,
VA, USA). HHSteC cell line was purchased from ScienCell Research Laboratories
(ScienCell, Carlsbad, CA, USA).
3.2.3. De novo formation of liver equivalents : HepaRG cells were cultured in HepaRG
medium from Life Technologies (Life Technologies; Carlsbad, CA, USA) at 370
C and
5% CO2 with medium exchange every other day for 2 weeks till full confluence. Later
medium containing 2% DMSO was added for differentiation for next 2 weeks. HHSteC
cells were expanded in Stellate Cell Media purchased from ScienCell Research
Laboratories (ScienCell, Carlsbad, CA, USA) till 80% confluence. The formation of
human liver spheroids was performed as previously described by Wagner et al. Both
the cells are harvested and grown in a 384 well spheroid plate on a shaker for 3 days
and to form round and compact spheroids. Forty spheroids were collected to form a
single liver equivalent for one circuit of the 2-OC and assessed using EVOS XL Core
digital inverted microscope (Life Technologies, Carlsbad, CA).
3.2.4. Formation of renal proximal tubule barrier model : RPTEC/TERT1 cells were
cultured in DMEM and Ham’s F-12 (DMEM/F12) medium with some additional
supplements. 105
cells were seeded on each transwell permeable support for 4 days
under static conditions followed by 3 days under perfused conditions in 2-OC. The
insert membranes were positioned at 100 μm above the bottom of the culture
compartment to ensure free media passage below the proximal tubule barriers. Medium
and supplements were purchased from Life Technologies (Life Technologies; Carlsbad,

11. 11
CA, USA). EVOS XL Core digital inverted microscope (Life Technologies, Carlsbad,
CA) was used for visualizing cell differentiation & morphology.
Fig 4 : Corning® Transwell® Permeable Supports
3.2.5. Chip based Cocultures: 14 microchips were loaded. A circulating medium of 500 μl
(half of volume replaced & collected) and a medium of 100 μl on the top of the barrier
insert (exchanged daily) were employed with a micropump frequency of 0.8 Hz at a
rate lower than 9 μl/min. At the end of the 16-day coculture organ-specific functional
markers of the liver or proximal tubule equivalent were analyzed by
immunofluorescence and qRT-PCR.
3.2.6. Model Compounds used :
 Cyclosporin A and Rifampicin from National Institutes for Food and Drug Control,
Beijing, China were dissolved in DMSO, stored in the dark at 4 °C, and then diluted
to a final concentration of 0.1% DMSO when used.
 CsA has been identified as a substrate as well as an inhibitor of CYP3A4 and P-gp,
which undergoes hepatic and intestinal metabolism in humans, while the renal
elimination of CsA mainly depends on intrarenal P-gp.
3.2.7. Procedure for administration of Drug :
 After setting up the chips with the coculture, the microfluidic chip was kept under
perfused condition for 3 days.
 Medium flow (circulating medium and medium above barrier) was on as per above
mentioned parameters

12. 12
 20 µM CsA were applied daily until day 6
 Next 20 µM CsA and 25 µM RFP were administered daily until day 13.
 Control culture : Medium containing 0.1% DMSO
3.3. Analysis and observations :
3.3.1. Cell viability and Metabolic activity:
Cell viability was monitored by measurement of lactate dehydrogenase and metabolic activity
was detected daily by measurement of glucose and lactate levels in supernatants of two
compartments using the Lactate Colorimetric Assay Kit (Biovision, Milpitas, CA, USA)
Fig 5 : Production of LDH and lactate & consumption of glucose
3.3.2. Protein expression in surrogate blood circuit:
Protein expression of non-invasive toxicity biomarkers in surrogate blood after daily
administration of CsA alone or with concomitant use of RFP in liver-proximal tubule coculture
chips for 14 days with LC-MS/MS analysis
Fig 6 : Expression of aspartate aminotransferase, Alkaline phosphatase and Total bilirubin

13. 13
Fig 7 : Expression of Glucose, Creatine and Urea
Fig 8 : Expression of Albumin, gamma-glutamyl transpeptidase and Lactate
3.3.3. Protein expression in the excretory lumen :
Protein expression of noninvasive toxicity biomarkers in the excretory lumen afer daily
administration of CsA alone or with concomitant use of RFP in liver-proximal tubule coculture
chips for 14 days using a 7180 automatic biochemistry analyzer (Hitachi, Japan).

14. 14
Fig 9 : Expression of (A) GGT (B) ALP (C) NAG (D) Glucose (E) Lactate (F) KIM-1
(G) Cystatin C (H) Collagen IV (I) Clusterin (J) NGAL (K) Osteoactivin (L) IP-10 (M)
GST-α (N) albumin (O) FABP-1 (P) Renin (Q) α1-microglobulin (R) Osteopontin (S)
TIMP-1 (T) EGF in excretory media on days 1, 7 and 14.

15. 15
3.4. Results :
The tissue equivalents liver spheroids and renal proximal tubule barriers were cocultured in the
microfluidic multi-organ chip platform. The process was reproducible and well defined. The
cocultured tissue equivalents expressed tissue-specific markers, enzymes and transporters and
could be challenged by CsA with hepatotoxicity and nephrotoxicity which could be minimized
by the hepatic drug-metabolizing inducer RFP. The present microfluidic chip-based platform
fits standardized sizes of microtissues and formats of tissue culture, which can be widely
applied in drug screening and is being increasingly accepted by regulatory agencies.

16. 16
4. CRITICAL ANALYSIS
4.1. Objective
 The objective of the research is met by showing the differences in expression of toxicity
biomarkers with the administration of two drugs.
 The coculture is established on a microfluidic chip properly for 16 days.
 Two tissue equivalents (liver spheroids and human proximal tubule cell line barrier)
were cocultured as given in the title of the paper.
 Multidrug testing was done. Two drugs CsA and RFP were employed in the study.
4.2. Title of the paper :
The title for the paper exceeds two lines and has a character count of 129 (with spaces).
The formatting guidelines for the multidisciplinary science journal Nature states that titles
should not exceed two lines in print. This equates to 75 characters (including spaces).
4.3. Originality of paper :
 The organ-on-chip has already been in use for 9 years for drug testing because the
microfluidic chip tries to accurately represent the in-vivo environment of the organ with
parameters like ECM, stress, growth in space, etc.
 Drug metabolism and toxicity assessment integrated into a single chip have been
successfully attempted previously.
 Though the administration of two drugs concomitantly in a two organ equivalent set-up
on the chip is the additional improv done here. Also, Liver-renal proximal tubule
coculture is attempted for the first time.
4.4. Technical correctness & missing Data:
 The changes in pH of the media circulating could have been studied as it would alter the
charge on the drug. Rifamcipin’s ester functional group is quickly hydrolysed in bile,
and it is catalysed by a high pH and substrate-specific esterases. Moreover perturbations
in pH can be indicative of metabolic distress, loss of kidney/liver organoid function, or
tumor activity.

17. 17
 The full form of MOC should have been mentioned in the beginning as it creates
confusion with ‘Material of construction’ in context to design of chip. MOC in the
research paper stands for Multiple Organ Chip.
 The fluid-to-tissue ratio upon which the 2-OC model is designed was not revealed.
 The data for the time interval at which the circulating medium was collected & replaced
is missing, otherwise the components of both media, the figures for the flow rate of the
media, and intervals at which replacement of excretory pool is done is given.
 The chemical composition of circulating medium for two types of tissue equivalents
used here was not revealed.
4.5. Clarity :
 The format of the paper is as per guidelines.
 References are hyperlinked .
 There are figures, Graphs, and illustrations for easy understanding for every
observation mentioned.
 The supplementary information is provided which can be accessed through the link
which contains additional images, table for primer sequences, list of acronyms in
details.
 The title of the paper is appropriate and correlates to the research done.
4.6. Typographical and grammatical errors :
 Under the paragraph ‘De-novo formation of liver equivalents’, the exponential part of
three numerical figures is given incorrectly (not as superscript). The figures should be
5×10-5
M for hydrocortisone hemisuccinate concentration, 0.1×104
for HHStec C cells
and 2.4×104
HepaRG cells.

18. 18
 In the paper downloaded from the link https://www.nature.com/articles/s41598-020-
65817-0, there is no space between two words ‘blood’ and ‘in’ , and reads as Biomarkers
in the surrogate bloodIn the surrogate blood media .
 Under the paragraph - Biomarkers in the surrogate blood, the sentence reads as AST
level in the coadministration group revealed a uction at the endpoint which seems
incorrect.
 Apart from few definite articles missing at few places , no other grammatical errors can
be seen.
4.7. Limitations :
 Development of single common medium for two different tissue equivalents is challenge
whenever cocultures or multi-organ chips are involved.
 Similarly choosing an appropriate biomaterial for MPS devices is a challenge since
different protein molecules can get absorbed or adsorbed on the inner surface. Since
different cells and materials have widely varying degrees of interaction with different
molecules, choice of materials in developing MPS devices should be made with caution.
 The fluid dynamic parameters like flow rate, the shear stress, friction, etc. also have
implications in circulating medium & cells, effectively maintaining physiological
pressure. Hence appropriate parameters have to be set.

19. 19
5. DISCUSSION
Table 1 : Changes in levels of all biomarkers
Day 1 Day 7 Day 14
Biomarkers in
surrogate
blood
Co
ntr
ol
CsA
5
μM
CsA
20
μM
Co
ntr
ol
CsA
5
μM
CsA
20
μM
CsA 20
μM+RFP
25 μM
Co
ntr
ol
CsA
5
μM
CsA
20
μM
CsA 20
μM+RFP
25 μM
AST — ↑ ↑ — ↓ ↑/— ↑*/↑ — ↓ ↑*/— —/↓#
ALP — ↑ ↑ — ↑ ↑/— ↑/↓ — ↑* ↑*/— ↑/↓#
Tbil — ↓ ↓ — — —/— ↑**/↑## — — —/— ↑**/↑##
Glucose — ↓ ↓ — ↓ ↓*/— —/↑ — ↓* ↓**/
—
↓/↑#
CRE — ↑ ↑* — — ↓/— ↓/— — ↓ ↓/— ↓/↑
UREA — ↓ ↑* — ↑ ↑/— ↑/↓ — ↓ —/— ↓/↓
ALB — ↑ — — — —/— ↓/↓ — — —/— ↓/↓
GGT — ↓ ↓ — ↑ ↑/— ↑/— — — ↓/— ↓/↓
Lactate — — ↑ — — —/— —/— — — —/— —/—
Day 1 Day 7 Day 14
Biomarkers in
excretory
lumen
Co
ntr
ol
CsA
5
μM
CsA
20
μM
Co
ntr
ol
CsA
5
μM
CsA
20
μM
CsA 20
μM+RFP
25 μM
Co
ntr
ol
CsA
5
μM
CsA
20
μM
CsA 20
μM+RFP
25 μM
GGT — — ↑ — — ↓/— ↑/↑ — ↓ ↓/— ↑/↑
ALP — — ↑ — — ↑/— ↑**/↑# — — ↑/— ↑*/↑#
NAG — ↑ ↑ — — ↑/— ↑/↑ — — ↑/— ↑/↑
Glucose — ↓ ↓* — — ↓*/— ↓**/↓ — ↓* ↓*/— ↓**/↓
Lactate — ↓ ↑* — ↑* ↑*/— ↑*/— — ↑* ↑*/— ↑/↓
KIM-1 — ↑ ↑** — ↑ ↑*/— ↑/↓ — ↑ ↑/— ↑/↓#
Cystatin — ↑* ↑* — ↑ ↑*/— ↑/↓ — ↑ ↑/— ↑/↓
Collagen IV — ↑ ↑** — — ↑*/— ↑*/↑ — ↑* ↑*/— ↑*/↑#
Clusterin — ↑* ↑* — ↑* ↑*/— ↓/↓## — — ↑**/
—
↑/↓#
NGAL — ↑ ↑* — — ↑**/
—
↑/↓## — ↑ ↑**/
—
↑**/—
Osteoactivin — ↑* ↑** — — ↑*/— ↑*/↑ — — ↑**/
—
↑/↓#
IP-10 — ↑ ↑ — — ↑/— ↑/↑ — — ↑*/— ↑/↓
GST-α — ↑* ↑* — — ↓/— ↓/— — ↓ —/— ↑/↑
albumin — ↑ ↑* — ↓ ↑/— ↓/↓ — ↓ ↓/— ↓/—
FABP-1 — ↑ ↓ — — —/— ↓/↓ — ↓ ↑**/
—
↑/↓#
Renin — — — — ↑* ↑*/— —/↓ — — ↑*/— ↑*/↓
α1-
microglobulin
— ↑ — — ↑ ↑/— ↓/↓# — ↑ ↑**/
—
↑/↓#
Osteopontin — ↑** ↑* — ↑ ↑*/— ↓/↓## — — —/— ↓/↓#
TIMP-1 — ↑ ↑* — ↑ ↓/— ↑/↑ — ↓ ↓**/
—
↓**/↑
EGF — — ↓ — ↑ ↓/— ↓/↓ — ↓ ↓/— ↓*/↓
Observations of the changes in levels of biomarkers in surrogate blood and excretory lumen
after daily administration of CsA alone or with concomitant use of RFP in liver-proximal tubule
coculture chips for 14 days. Experiments were performed in triplicates (control and low-dose
groups) or quadruplicate (high-dose and coadministration groups). * (#) or ** (##) indicates
significant difference p<0.05 or p< 0.01, respectively. * or # indicates differences (increase (↑)

20. 20
or decrease (↓)) compared with the corresponding controls (—) or (/) when the
coadministration group was compared with the high-dose group(—), respectively.
1. The selection of the dosage of CsA applied in the coculture experiments was guided by
following general assumptions: the cell numbers of liver spheroids and proximal tubule
barriers represent approximately 1/100,000 of the size of the respective counterpart organ
in the human body.
2. 5 and 20 μM of CsA were used for the low- and high-dose groups, and 25 μM of RFP was
used for the concomitant treatment group.
3. Increase in LDH in excretory media, indicates the increase in the epithelial barrier
permeability induced by CsA-induced injury.
4. Increase in p53 gene expression in renal cells of the high-dose group revealed an induction
in response to cellular stress caused by a toxic dose of CsA.
5. Decreased dose of CsA in the concomitant treatment with RFP reduced the toxicity and
induced the proliferation of RPTEC/TERT1 cells and the reassembly of tightly packed
renal tubular epithelial barriers.
6. Hence the Ki67 mRNA expression in renal cells was significantly induced after repeated
coadministration of CsA and RFP.
7. A remarkable decrease in CsA concentration in surrogate blood and in the cytotoxic
indicators LDH and ALP could be observed in the RFP coadministration group on day 14.
8. The organ chip model suggested that CsA induces intrarenal P-gp but the slight inhibitory
effect on intrarenal P-gp after coadministration of CsA and RFP is not elucidated.
9. Upregulation of MRP-2 mRNA expression suggested enhanced drug clearance produced
by RFP in both the liver and the proximal tubule equivalents.
10. the hepatic enzyme AST and the non-organ specific cytotoxic enzyme ALP were decreased
by RFP.
11. TBiL, a serum biomarker of severe liver injury, was significantly elevated, although RFP
even slightly attenuated the inhibition of BSEP in the present experiment.
12. The cocultured proximal tubule barrier equivalents in chips expressed several biomarkers,
such as KIM-1, CLU, OA, and NGAL, for toxicity assessment over a long period of time,
mimicking the in vivo responses to nephrotoxic exposures.
13. A dose-dependent increase in OA supported the conclusion that OA can be categorized as
a sensitive biomarker for early proximal tubular damage.
14. The upregulation of Col IV in the coadministration group suggested the regeneration of
renal epithelial cells

21. 21
6. REFERENCES
1. Lin, N., Zhou, X., Geng, X., Drewell, C., Hübner, J., Li, Z., Zhang, Y., Xue, M., Marx, U.,
& Li, B. (2020). Repeated dose multi-drug testing using a microfluidic chip-based coculture
of human liver and kidney proximal tubules equivalents. Scientific reports, 10(1), 8879.
https://doi.org/10.1038/s41598-020-65817-0
2. Wu MH, Huang SB, Cui Z, Cui Z, Lee GB. Development of perfusion-based micro 3-D
cell culture platform and its application for high throughput drug testing. Sensors &
Actuators B Chemical. 2008;129:231–240. doi: 10.1016/j.snb.2007.07.145.
3. Corning® Transwell® Permeable Supports
https://b2b.vwrcanlab.com/assetsvc/asset/en_CA/id/9560361/contents
4. Sung, J. H., Wang, Y. I., Narasimhan Sriram, N., Jackson, M., Long, C., Hickman, J. J., &
Shuler, M. L. (2019). Recent Advances in Body-on-a-Chip Systems. Analytical
chemistry, 91(1), 330–351. https://doi.org/10.1021/acs.analchem.8b05293
5. https://www.nature.com/nature/for-authors/formatting-
guide#:~:text=within%20the%20text.-
Titles,%2C%20acronyms%2C%20abbreviations%20or%20punctuation.
6. Supplementary information, Repeated dose multi-drug testing using a microfluidic chip-
based coculture of human liver and kidney proximal tubules equivalents (2020) ,
https://static-content.springer.com/esm/art%3A10.1038%2Fs41598-020-65817-
0/MediaObjects/41598_2020_65817_MOESM1_ESM.docx