#2P-245

NEXT LEVEL DRUG RESEARCH IN AN EX
VIVO TISSUE GUT-ON-A-CHIP MODEL:ADVANCED APPLICATIONS OF THE
INTESTINAL EXPLANT BARRIER CHIP

Joanne M. Donkers1
Esmée Wierenga1
Steven Erpelinck2
Kazuhiro Ariga3
Evita van de Steeg1
1TNO, Metabolic Health Research, Leiden, The Netherlands
2TNO, Biomedical Health, Leiden, The Netherlands
3TNO, Pharma, Japanjoanne.donkers@tno.nl

Papp x 10-6 cm/s (3-24 h)n.a.
1.9 ± 0.6
41.1 ± 5.0
24.6 ± 3.8

Metabolism
Phase I

Parent compoundMidazolam
DiclofenacTestosterone
Coumarin
Irinotecan
7-OH coumarinPhase IIMETHODS Human jejunum, ileum or colon tissue explants werefixed in the IEBC and apically exposed to drugs
dissolved in standard cell culture medium or in fecal
water containing microbial excretion products (iscreentechnology [2]) for 24 hours.
o Phase I and phase II metabolism using coumarinand 7-OH coumarino SCFA enriched iscreen spent medium Addition of aerobic-anaerobic interface to study hostmicrobe interactions in a more physiologicalenvironment.
Human or porcine colon

IEBCMicrobiome

RESULTS
Host-microbe interactions

 Inulin-treatment of microbiome stimulated short chain fatty acid (SCFA) production.
 Increased SCFA levels in i-screen spent medium improved tissue barrier integrity:
 1.7-3.5 fold reduction in FITC-dextran 4000 (FD4) leakage.
 1.4-2.1 lower transcellular and paracellular transport of antipyrine and atenolol,respectively.
 Furthermore, the release of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8 was(significantly) reduced.
APapp value (x 10-6 cm/s)

Figure 2. Schematic representation of the microfluidic setup andsampling possibilities in the IEBC. Recirculating flow rate: 2 mL/hr.
Apical administration of drugs or fecal water containing microbialexcretion products [2].

Measured metabolites
Enzymes involved
1-OH midazolam, 4-OH midazolam CYP3A44-OH diclofenac
CYP2C96β-OH testosterone,
CYP3A4/17β hydroxysteroid
androstenedione
dehydrogenase
7-OH coumarin
CYP2A6SN38
CES27-OH coumarin glucuronide, 7-OH UGTs, SULTs
coumarin sulfate

B806040*200Atenolol

AntipyrineC1.0400.8

300.60.40.20.0

REFERENCES

1 Eslami Amirabadi H. & Donkers J.M. et al., Lab Chip(2022) 22: 326-342
2 Fehlbaum et al., Int. J. Mol. Sci. (2018) 19(10): 3097

100**E

500
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F

2000
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20000
15000
10000
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000Figure 5. Human colon tissue explants in the IEBC were exposed to spent medium from i-screen (untreated control,inulin-treated, and untreated control with added SCFA) for 24 hours (n = 5–13/group. A) The average apparent
permeability (Papp) of atenolol (10 μM) and antipyrine (10 μM) was calculated between 20-24 hours. B) Average
FD4 permeability between 20-24 hours. C-F) TNF-α, IL-1β, IL-6, and IL-8 secretion into the basolateral compartment
after 24h

I-screen ctrl
I-screen superhealthy
I-screen ctrl + SCFA An aerobic-anaerobic interface was developed to co-culture living microbiome with ex vivo tissue*explants (patent WO2022055345 filed)
1.5*
Tissue T=0h

Figure 6. Aerobic-anaerobic interface of the IEBC. Strict
anaerobic Bifidobacterium Animalis was co-cultured
with porcine colon tissue. Attachment of B. animals tothe tissue was demonstrated by qPCR.

Figure 3. Colon tissue of human or porcine origin is added in the IEBC.
In host-microbe interaction studies microbiome is added at the apicalside of the tissue.

20DIL-8 (pg/mL)≥ 24 hours

Table 2. Overview of metabolizing enzymes assessed in IEBC
(and in InTESTine™)

IL-6 (pg/mL)

 To study advanced research questions in the field of:
1. Drug absorption & metabolism.
2. Host-microbe (immune) interaction

CompoundCoumarin
7-OH coumarin7-OH coumarin glucuronide
7-OH coumarin sulfate

CONCLUSIONSFold change

AIM

Figure 4. Human jejunum tissue explants were mounted in the IEBC
and exposed to coumarin (3 µM) or 7-OH coumarin (333 µM) for 24hours (n = 4). Parent compound and metabolites were measured by
LC-MS/MS. Data are presented as mean + SD.

Table 1. Transport rates of coumarin and its metabolites fromthe apical to basolateral side of the tissue

IL-1b (pg/mL)

Figure 1. Intestinal Explant Barrier Chip (IEBC) and laboratory set-up
for multiple IEBCs.

 Coumarinwaspredominantly
hydroxylated to 7-OH coumarin (viaCYP2A6) at the apical side of theintestinal tissue.
 7-OH coumarin was metabolized further
into 7-OH coumarin glucuronide and 7OH coumarin sulfate via phase II
conjugating enzymes and appearedbasolateral at high rates.
 Effective and preserved phase 1 and 2
metabolism was also shown for
midazolam, diclofenac, testosterone, andirinotecan.

TNF-a (pg/mL)

 The Intestinal Explant Barrier Chip (IEBC) is a gut-on-achip model with the unique property of integratingsmall ex vivo intestinal tissue explants between twomicrochannels.
 Intestinal permeability of small molecule drugs can bestudied in the IEBC for 24 hours. Showing good rankorder relationship with the in vivo fraction absorbed [1].

RESULTS
Phase I and Phase II metabolism

FD4 permeability (avg. %/h)

INTRODUCTION

Tissue T=24h
1.0

Tissue T=24h co-incubated with B. animalis

0.50.0 The IEBC can successfully answer advanced drug absorption and metabolism
questions.
 The IEBC is and advanced physiological relevant in vitro model to study andunderstand host-microbe effects on tissue functionality and local inflammation in
the human intestine.