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faculty of mathematics and natural sciences
groningen research institute of pharmacy
Enzymatic digestion in chaotic mixers as microreactors for a fully integrated gut-on-a-chip
Pim de Haan1,2, Margaryta A. Ianovska1,2, Hans Bouwmeester3,4 & Elisabeth Verpoorte1
1 Pharmaceutical Analysis, Groningen Research Institute of Pharmacy, University of Groningen; 2 TI-COAST, Amsterdam; 3 RIKILT – Wageningen UR; 4 Division of Toxicology, Wageningen University; the Netherlands.
MicroNanoConference, December 14, 2016
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Contents
› Introduction
Drug and toxicology testing
Models for the human gastrointestinal tract
Laminar flow and chaotic micromixers
› Results
› Conclusion and future perspectives
Towards a gut-on-a-chip
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Introduction: Drug and toxicology testing
› Development of a drug takes >10 years [1]
› Safety and efficacy must be tested before drugs are allowed access to the market
Testing of drugs on
- Cell lines / tissue models
- Animals
- Humans
› Only 1 in 10,000 potential drugs makes it to the market
Elimination of ‘failing’ drugs: the sooner, the better
› Oral administration is preferred
[1] Esch et al. Annu Rev Biomed Eng, 2011.
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Introduction: The human gastrointestinal tract
› Digestive system
› Extraction of nutrients
› Absorption of orally administered drugs
Influence of digestive juices?
Mouth
Stomach
Intestine
Saliva
Gastric
juice Bile
Duodenal
juice
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Introduction: Models for the human gastrointestinal tract
› Larger-scale in vitro assay systems are used in academia and industry [2]
› Artificial digestive juices are available [3,4]
› Pro: Easy to use
› Con: Batch process, no continuous operation
› Is it possible to miniaturize this system, to save both time and chemicals?
[2] Egger et al. Food Res Int, 2016.
[3] Walczak et al. Nanotoxicol, 2013.
[4] Versantvoort et al. Food Chem Toxicol, 2005.
250 mL
Reagents
Sam
ple
s
37 °C
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Stomach Intestine
Sample in
Saliva
Gastric juice Bile
Chyme out
Duodenal juice
Mouth
Introduction: Our model
› Three-step digestion model, with three chaotic micromixers in series
› Mixing samples with digestive juices in relevant ratios [2,3]:
› Study both single-dose and continuous supplies of drugs/food/toxicants
Continuous processing, reminiscent of human gastrointestinal tract
[2] Egger et al. Food Res Int, 2016. [3] Walczak et al. Nanotoxicol, 2013.
1 µL/min
4 µL/min
8 µL/min
8 µL/min
4 µL/min 25 µL/min
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Introduction: Laminar flow and chaotic micromixers
› Digestion requires thorough mixing, to expose samples to digestive enzymes
› In microfluidic channels, flows are laminar
Mixing liquids is challenging!
› Chaotic micromixers have herringbone-shaped grooves to create a vortical flow, enhancing mixing [5,6]
[5] Stroock et al. Science, 2002.
[6] Ianovska et al. (submitted)
F
low
1 c
ycle
1 cycle = 2×6 grooves = 3.1 mm
60
50
100+60
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Results: Mouth
› Mixing 5 µM fluorescein in water with artificial saliva, at 37 °C
› Measured fluorescence intensity after each cycle of herringbones, and calculated the degree of mixing from the standard deviation of the signal [6]:
[6] Ianovska et al. (submitted)
Saliva
20 µL/min
Fluorescein
20 µL/min
100 µm
0 3.1 6.3 9.4 12.5 15.6 18.8 21.9 25
28.1 31.3 34.4 37.5 40.6 43.8 46.9 50
Distance (mm) from confluence
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Results: Mouth (II) – mixing in different ratios
› Mixing 5 µM fluorescein in water with artificial saliva, at 37 °C:
› Results in triplicate, ±SD
› At relevant 4:1 ratio saliva/sample:
Mixing of an aqueous sample with artificial saliva occurs within 10 mm in these chaotic micromixers, at lower flow rates
0
20
40
60
80
100
120
0,0 10,0 20,0 30,0 40,0 50,0
Deg
ree
of
mix
ing
(%)
Distance from confluence (mm)
20+20 µL/min
1+1 µL/min 0
20
40
60
80
100
120
0,0 10,0 20,0 30,0 40,0 50,0
Deg
ree
of
mix
ing
(%)
Distance from confluence (mm)
1+4 µL/min
0.4+1.6 µL/min
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Results: Stomach and Intestine
› Mixing of gastric juice with 5 µM fluorescein (in 4:1 saliva/water) in 8:5 ratio
Fluorescence is rapidly lost in gastric juice (pH 1.3)
› Mixing fluorescein (in a mixture of water/saliva/gastric juice 1:4:8) with duodenal juice and bile
Formation of solids and gas
- Possibly protein denaturation and CO2
100 µm
Sample (F)
5 µL/min
Gastric juice
8 µL/min
Sample (F)
13 µL/min
Duodenal juice
+ bile
12 µL/min
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Hyphenated Mouth, Stomach and Intestine
Sample
Saliva
Gastric juice
Bile + duodenal juice
Chyme out
Mouth
Stomach
Intestine
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Results: Hyphenated Mouth, Stomach and Intestine
› Serial mixing of 20 µM fluorescein with water, in appropriate ratios
› Fast mixing of liquids within a few cycles
1 µL/min
8 µL
/min
12 µL/min
4 µL
/min
Sample
Saliva
Gastric juice
Bile + duodenal juice
Chyme out
Mouth
Stomach
Intestine
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Future outlook: Towards a fully integrated gut-on-a-chip
› Present digested materials to ‘living’ gut-on-a-chip [7]
Ideally positioned to reduce the need for animal trials
› Partial in vitro model for pharmacokinetics of drugs
Digestion / metabolism
Absorption
› Future coupling to high-end analytical detection systems [7] Huh et al. Nat Protoc, 2013.
GUT LUMEN
PLASMA COMPARTMENTMedium in
Chyme in
To analysis
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Conclusion and future perspective
› Our micromixers in series can be used to efficiently mix artificial digestive juices
› These mixers will be used as a cell-free model for the gastrointestinal tract
› Relative scaling effects are to be studied
A miniaturized model could mean faster digestion
Benchmarking using a larger-scale in vitro system
› Working towards a fully integrated gut-on-a-chip, combining
On-chip digestion
Microfluidic barrier model
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Acknowledgments
› Funding
› GUTTEST consortium partners
› Pharmaceutical Analysis
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faculty of mathematics and natural sciences
groningen research institute of pharmacy
Enzymatic digestion in chaotic mixers as microreactors for a fully integrated gut-on-a-chip
Pim de Haan1,2, Margaryta A. Ianovska1,2, Hans Bouwmeester3,4 & Elisabeth Verpoorte1
1 Pharmaceutical Analysis, Groningen Research Institute of Pharmacy, University of Groningen; 2 TI-COAST, Amsterdam; 3 RIKILT – Wageningen UR; 4 Division of Toxicology, Wageningen University; the Netherlands.
MicroNanoConference, December 14, 2016