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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

pim.de.haan@rug.nl

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

pim.de.haan@rug.nl

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