Bouwmeester, H.

de Haan, P.

Ianovska, M.A.

Mathwig, K.

Verpoorte, E

English

Gut-on-a-chip models have gained attention as replacements for other cell-based assays or animal studies in drug development or toxicological studies. These models aim to provide a more accurate representation of the in vivo situation in form and function; however, no digestive processes have been included in these systems so far. This work describes a miniaturized digestive system based on artificial digestive juices that digest liquid samples in a series of three microreactors. After optimization of the pH value of juices and mixtures, samples leading to fluorescent products were digested to demonstrate enzyme functionality and to determine kinetic parameters

ARTS repository - University of Groningen

   University of GroningenAn automated modular microsystem for enzymatic digestion with gut-on-a-chip applicationsde Haan, P.; Ianovska, M.A.; Mathwig, K.; Bouwmeester, H.; Verpoorte, EPublished in:21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, October 22-26, 2017,Savannah, Georgia, USAIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2017Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):de Haan, P., Ianovska, M. A., Mathwig, K., Bouwmeester, H., & Verpoorte, E. (2017). An automatedmodular microsystem for enzymatic digestion with gut-on-a-chip applications. In 21st InternationalConference on Miniaturized Systems for Chemistry and Life Sciences, October 22-26, 2017, Savannah,Georgia, USA (pp. 1593-1594)CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 13-02-2023AN AUTOMATED MODULAR MICROSYSTEM FOR  ENZYMATIC DIGESTION WITH GUT-ON-A-CHIP APPLICATIONS P. de Haan1,2, M.A. Ianovska1,2, K. Mathwig1, H. Bouwmeester3,4, and E. Verpoorte1 1University of Groningen, THE NETHERLANDS, 2TI-COAST, Amsterdam, THE NETHERLANDS, 3RIKILT – Wageningen University and Research, THE NETHERLANDS, and 4Wageningen University, THE NETHERLANDS.   ABSTRACT Gut-on-a-chip models have gained attention as replacements for other cell-based assays or animal studies in drug development or toxicological studies.  These models aim to provide a more accurate representation of the in vivo situation in form and function; however, no digestive processes have been included in these systems so far.  This work describes a miniaturized digestive system based on artificial digestive juices that digest liquid samples in a series of three microreactors.  After optimization of the pH value of juices and mixtures, samples leading to fluorescent products were digested to demonstrate enzyme functionality and to determine kinetic parameters.   KEYWORDS: Gut-on-a-chip, Digestion, Organ-on-a-chip, Enzyme kinetics  INTRODUCTION Several gut-on-a-chip models have been developed over the past few years, mimicking the barrier function of the human intestinal wall by growing intestinal cells on a porous membrane [1].  These models recreate the anatomy of the gastrointestinal (GI) tract for applications in drug or toxicology testing.  However, the digestive functions of the GI tract have not been taken into account in these systems, and test samples still require larger-scale, batch-wise in vitro digestion by a skilled person before introduction to the device.  This work describes progress in the development of an automated, miniaturized, digestive system based on artificial digestive juices [2].  Samples are continuously mixed with these juices (i.e. saliva, gastric juice and duodenal juice/bile – see Table 1) in physiologically relevant ratios, using three hybrid glass–poly(dimethylsiloxane) (PDMS) micromixers as bioreactors [3].  Enzymes in these mixtures digest compounds present in the test samples in a two-step process, in which the enzyme, E, first reversibly binds to a substrate, S, after which product, P, is formed:    𝐸𝐸 + 𝑆𝑆 ⇌ 𝐸𝐸 ∙ 𝑆𝑆 → 𝐸𝐸 + 𝑃𝑃 (1) The Michaelis-Menten equation shows that the reaction rate, v, depends on the substrate concentration, [S]:     𝑣𝑣 = 𝑑𝑑[𝑃𝑃]𝑑𝑑𝑑𝑑 =𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚 ∙ [𝑆𝑆]𝐾𝐾𝑚𝑚 + [𝑆𝑆] (2) In this equation, the maximum reaction rate, Vmax, and Michaelis constant, Km, are also included.  These two parameters can be estimated experimentally by measuring the formation of the reaction product over time at various [S].  In this work, these enzymatic parameters were determined on-chip using labeled substrates, which produce highly fluorescent reaction products after hydrolysis by digestive enzymes.  Table 1:  Optimization of the pH value of artificial digestive juices. Juice or Mixture Ratio Main species pH in original composition [2] pH in optimized composition    Calc. Meas. Calc. Meas. Sample 1 Pure H2O 7.00 6.96 7.00 6.96 Saliva 4 H2PO4–, OH– 6.71 6.45 7.00 6.75    Mixture in the Mouth 5    All of the above 6.71 6.47 7.00 6.76 Gastric Juice 8 HCl, H2PO4– 1.02 1.16 2.46 2.57    Mixture in the Stomach 13    All of the above 1.25 1.38 3.00 3.04 Duodenal Juice 8 HCO3–, HCl 7.58 8.82 7.12 8.32 Bile 4 HCO3–, HCl 7.76 9.03 7.35 8.81    Mixture in the Intestine 25    All of the above 2.12 2.53 7.00 7.31 978-0-692-94183-6/µTAS 2017/$20©17CBMS-0001 1593 21st International Conference on MiniaturizedSystems for Chemistry and Life SciencesOctober 22-26, 2017, Savannah, Georgia, USAEXPERIMENTAL The composition of artificial digestive juices [2] was optimized to obtain the physiological pH value for enzyme function in the respective compartments (Table 1).  Three hybrid glass–PDMS micromixers, containing grooves to generate chaotic flow profiles, were coupled in series to act as bioreactors [3,4], mixing the sample with digestive juices containing different enzymes (Figure 1, left).  Substrates with quenched fluorophores were digested in each stage of this system, producing highly fluorescent products.  Pictures were taken at the beginning and the end of each channel (50 mm length), representing a 40 s residence time in the channel.  The difference in fluorescence between these two points in the channel is directly proportional to [P], and can be used to determine the reaction rate in the initial 40 s.   RESULTS AND DISCUSSION Starch was digested as a model compound in the first compartment (mouth), and casein (a protein naturally occurring in milk) was digested by proteases in the stomach and intestine, at different values for [S].  The Michaelis-Menten plots (Figure 1, right) show a typical shape, with a clear saturation at higher [S].  Parameters Vmax and Km were estimated mathematically and are displayed in the plots.    CONCLUSION We demonstrate an automated, modular microsystem for enzymatic digestion, in which the different enzymes occurring in the human GI tract digest samples of model compounds.  The output of this system – or chyme – may be transferred to a gut-on-a-chip barrier model of the human intestine.    ACKNOWLEDGMENTS This research received funding from the Netherlands Organization for Scientific Research (NWO) in the framework of the Technology Area COAST.    REFERENCES [1] Huh et al. Nat Protoc 2013;8(11):2135-57.  [2] Walczak et al. Nanotoxicology 2013;7(7):1198-210.  [3] Ianovska et al. RSC Adv 2017;7(15):9090-9.  [4] Stroock et al. Science 2002;295(5555):647-51.   CONTACT   Pim de Haan: pim.de.haan@rug.nl     Figure 1:  Diagram of the three-stage enzymatic digestive system.  Chaotic micro-mixers digest samples using artificial digestive juices containing enzymes.  The Michaelis-Menten graphs on the right display the reaction rate, v, in each of these digestive stages versus the concentration of the substrate used.  1594

An automated modular microsystem for enzymatic digestion with gut-on-a-chip applications

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An automated modular microsystem for enzymatic digestion with gut-on-a-chip applications

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