Building Gut-on-a-Chip: PDMS, Cells & TEER

Learning Goal: Establish a fully functional, physiologically relevant gut-on-a-chip microphysiological system. This curriculum covers designing and fabricating a microfluidic device using polydimethylsiloxane (PDMS) soft lithography, establishing a viable co-culture of human intestinal epithelial cells (Caco-2) and gut microbiota, and validating the biological barrier integrity using on-chip Transepithelial Electrical Resistance (TEER) measurements.

• Prerequisites: Basic college-level biology/histology, introductory fluid mechanics, and standard laboratory biosafety training (BSL-2).
• Estimated Total Study Time: 26 Hours

Module 1: Foundations of Microfluidics and Gut Anatomy

This module introduces the mechanical and biological principles that govern organ-on-a-chip systems. You will study how fluid behaves at micro-scales—specifically, the transitions to low Reynolds number regimes, laminar flow, and wall shear stress. In parallel, you will analyze the histology of the human intestinal epithelium, focusing on its cellular composition, polarized structure, and the physiological significance of the extracellular matrix (ECM).

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Mod-01 Lec-01 Introduction to Microfluidics

@iit56:5337,141

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Microfluidics is the study of fluid flows at characteristic length scales of microns (approximately the thickness of a human hair), where surface effects dominate over volume effects due to the high surface-to-volume ratio. This interdisciplinary field combines microfabrication, chemistry, biology, mechanics, and control systems to create devices handling small fluid volumes (femtoliters to microliters) for applications including biomedical diagnostics, drug delivery, and precise chemical analys

@iit · 56:53

• Why this video: This academic lecture provides an in-depth mathematical and physical foundation of fluid dynamics at micro-scales. It is essential for understanding why fluids behave in a strictly laminar fashion within micrometer-sized channels, a concept crucial to designing microfluidic devices.

Cells and tissues: types and characteristics - Human histology | Kenhub

@Kenhub24:38242,907

Cells and tissues: types and characteristics - Human histology | Kenhub

The human body is composed of four primary tissue types that work together to maintain homeostasis: epithelial tissue (which covers and protects internal and external surfaces, classified by cell shape and arrangement into types like simple cuboidal, simple squamous, and stratified squamous), connective tissue (the most abundant tissue type, consisting of cells within an extracellular matrix containing collagen, elastic, and reticular fibers, including blood, loose connective tissue, dense conne

@Kenhub · 24:38

• Why this video: A comprehensive review of human histology is necessary to model the gut barrier. This video covers the primary characteristics of epithelial tissue, explaining cell shapes, polarization, and how these cells interface with the extracellular matrix to form protective, semi-permeable membranes.

Engineering human organs onto a microchip | Dan Huh | TEDxPenn

@TEDx16:1957,432

Engineering human organs onto a microchip | Dan Huh | TEDxPenn

Organ-on-a-chip technology uses microfabrication techniques to create microengineered devices that can grow human cells and mimic the structure and environment of living human organs, such as lungs and eyes, allowing researchers to test drug responses in a more realistic and predictive model than traditional cell culture or animal testing, thereby potentially reducing the time and cost of drug development.

@TEDx · 16:19

• Why this video: Delivered by one of the pioneer researchers in the field, this talk bridges the gap between engineering and biology. It explains the core philosophy of microphysiological systems (MPS)—why static Petri dish cultures fail to replicate human physiology and how dynamic microenvironments mimic organ-level functions.

Knowledge Checkpoint

• Understand the definition of Reynolds number ($Re$) and why it is typically $< 1$ in microfluidic channels.
• Explain how laminar flow differs from turbulent flow and why mixing in microfluidics depends primarily on diffusion.
• Identify the main structural layers of the intestinal barrier, focusing on the role of the mucosal lining and polarized epithelial cells.
• Articulate how mechanical strain (e.g., cyclic stretching or fluid shear stress) influences tissue differentiation on-chip.

Module 2: Cleanroom Fabrication & PDMS Soft Lithography

This module focuses on the micro-engineering and material fabrication phase. You will follow the step-by-step master mold fabrication using photolithography, learn to handle and cure polydimethylsiloxane (PDMS), and master the chemical principles of atmospheric plasma treatment to covalently bond microfluidic layers.

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Photolithography Walkthrough: SU-8 2035 with a Mylar Mask

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Photolithography Walkthrough: SU-8 2035 with a Mylar Mask

This video demonstrates the complete SU-8 photolithography workflow for creating microstructures: wafer cleaning with acetone and IPA, spin coating SU-8 2035 at specified speeds and times, soft baking, mask alignment with water contact layer, UV exposure with calculated dose, post-exposure bake, and development in SU-8 developer with IPA rinse. Key parameters include spin speeds of 500 rpm (15 sec) and 3,000 rpm (38 sec) for 35-micron layers, soft bake at 65°C (2 min) and 95°C (5 min), and post-

@calcommunitycontent · 10:36

• Why this video: An exceptionally clear, step-by-step walk-through of the photolithography process in a cleanroom. It details wafer preparation, SU-8 spin coating, soft baking, alignment with photomasks, UV exposure, and development to yield a high-fidelity master mold.

PDMS Microfluidic Device Fabrication: SU-8 Soft Lithography Tutorial

@microfluidicsandmultiphase843410:4466,860

PDMS Microfluidic Device Fabrication: SU-8 Soft Lithography Tutorial

This video demonstrates the complete process of fabricating PDMS microfluidic devices through soft lithography, which involves creating a master mold by patterning SU-8 photoresist on a silicon wafer using UV lithography, then casting PDMS (prepolymer:curing agent ratio of 10:1) onto the mold, curing it, punching inlet/outlet holes, treating the PDMS surface with oxygen plasma to make it hydrophilic, and finally bonding it to a glass substrate to create a sealed microfluidic device.

@microfluidicsandmultiphase8434 · 10:44

• Why this video: This tutorial outlines the practical replica molding process. It demonstrates how to mix PDMS polymer base with curing agent at a 10:1 ratio, degas the mixture in a vacuum chamber, cure it, peel the elastomeric structures, and punch fluidic ports.

PDMS Replica Molding and Bonding

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PDMS Replica Molding and Bonding

This video demonstrates the complete process of creating PDMS (Polydimethylsiloxane) replicas through replica molding and bonding. The process involves mixing PDMS base with curing agent in a 10:1 weight ratio, thoroughly mixing for one minute, degassing the mixture under vacuum to remove bubbles, casting the degassed PDMS into a 3D-printed mold, performing a second degassing after filling, curing at 65°C for two hours, demolding using a spatula technique, creating inlet/outlet holes with a punc

@SoftFluidics · 13:00

• Why this video: Focused intensely on the physical replica molding process, this video provides excellent close-up details on cutting cured PDMS slabs, aligning them, and prepping surfaces for permanent bonding.

Microfluidics by soft lithography - Part 9 - Bonding

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Microfluidics by soft lithography - Part 9 - Bonding

Atmospheric plasma bonding is a technique used to permanently attach PDMS microfluidic chips to glass substrates by exposing both surfaces to plasma, which creates covalent silicon-oxygen-silicon bonds; the process requires evacuating the chamber, introducing controlled gas flow, generating plasma for approximately 15 seconds, and then carefully aligning and pressing the PDMS chip onto the substrate to form a permanent bond.

@dk-hu3er · 09:56

• Why this video: This video focuses on atmospheric oxygen plasma treatment, explaining the molecular mechanisms (e.g., exposing silanol groups $-SiOH$) that enable irreversible covalent bonding between PDMS and glass slides.

Knowledge Checkpoint

• Describe the difference between positive and negative photoresists (such as SU-8).
• State the correct mixing ratio for SYLGARD 184 (elastomer base to curing agent) and explain why degassing is required.
• Understand the chemical surface activation process that occurs during oxygen plasma bonding.
• Troubleshoot common bonding failures (e.g., dust contamination, incomplete contact, or over-exposure to plasma).

Module 3: Epithelial Cell Culture and On-Chip Seeding

In this module, you will master biological preparations for gut-on-a-chip culture. This includes maintaining Caco-2 human intestinal epithelial cells, coating the hydrophobic PDMS microchannels with an extracellular matrix (ECM) scaffold, calculating physiologic shear stresses, and introducing cells into microfluidic channels under flow.

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Caco-2 cells, derived from human colorectal carcinoma in 1977, are the most popular intestinal cell line for studying gut barrier function. When grown in transwell plates for 21 days, they form polarized barriers with tight junctions, secretory vesicles, microvilli, and brush border. They express a full complement of peptide and amino acid transporters. The protocol involves: resuscitating cells from liquid nitrogen, passing 3 times in flasks, seeding at low density (6×10^4 cells per well), cult

@fooddigestion · 06:53

• Why this video: This masterclass highlights Caco-2 cell line biology. It details why Caco-2 is the standard cell line used for modeling the intestinal barrier, their cancer-derived origin, and their ability to spontaneously differentiate into polarized monolayers.

Caco 2 Cell Permeability Assay

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Caco 2 Cell Permeability Assay

The Caco-2 cell permeability assay uses human colon carcinoma cells cultured on permeable supports to model intestinal epithelium, where differentiated cells form tight junctions and express transporter proteins to predict drug absorption; the method involves cultivating cells for 21 days until polarization is achieved, measuring transepithelial electrical resistance (TEER > 250 Ω·cm²) to confirm monolayer integrity, then performing drug transport experiments with test compounds (10-200 μM) acro

@creativebioarray9401 · 04:40

• Why this video: This video demonstrates how to run permeability assays using differentiated Caco-2 cells. It introduces the functional behavior of intestinal monolayers, highlighting brush border formation and cellular tight junctions.

Microfluidic Shear stress calculator | Tutorial video

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Microfluidic Shear stress calculator | Tutorial video

This tutorial demonstrates how to use a microfluidic shear stress calculator tool that predicts shear stress levels in experimental setups by inputting parameters such as pressure, flow rate, fluid properties (dynamic viscosity and density), tubing dimensions (inner diameter and length), and chip geometry; the tool calculates shear stress, flow rate, pressure, Reynolds number, and velocity to help researchers design optimal experimental conditions for cell culture and organ-on-a-chip studies by

@Fluigent · 03:05

• Why this video: Fluid flow generates shear forces that are biologically critical for gut maturation. This tutorial explains how to calculate fluid flow rates, channel geometry, and viscosity to target physiological wall shear stress levels.

Curriculum Gap Acknowledgment & Self-Study

⚠️ Curriculum Gap: The existing video library has limited coverage of the physical, step-by-step setup of microfluidic tubing, syringe pumps, bubble traps, and cell suspension injection directly inside a microchannel under a cell culture hood.

To close this gap, search YouTube or academic literature for:

• "Caco-2 cell seeding microfluidic channel perfusion protocol"
• "How to connect microfluidic tubing and syringe pumps without bubbles"

Knowledge Checkpoint

• Explain why Caco-2 cells require up to 21 days to fully differentiate in static cultures, and how microfluidic flow accelerates this maturation timeline.
• List common ECM proteins (e.g., Collagen I, Fibronectin, Matrigel) used to promote epithelial attachment inside microfluidic channels.
• Calculate the target flow rate ($\mu L/min$) required to produce a physiological wall shear stress of $0.02\text{ Pa}$ ($0.2\text{ dyn/cm}^2$) inside a microchannel with known dimensions.
• Describe the structural features of polarized epithelial cells (i.e., apical vs. basolateral domains).

Module 4: TEER Measurement & Barrier Function Validation

To ensure the engineered tissue acts as an effective physical barrier, you must validate its junctional integrity. You will learn the electronic and biophysical principles of Transepithelial Electrical Resistance (TEER), construct or integrate electrodes within microchannels, and measure the electrical resistance of the cell layer under flow.

Note: In practical research workflows, you must first validate the base epithelial monolayer's barrier integrity using TEER before exposing it to microbiota (as presented in Module 5).

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What is TEER

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What is TEER

Transepithelial/Endothelial Electrical Resistance (TEER) is a quantitative technique that measures the electrical resistance across a cell monolayer grown on a porous membrane, reflecting the tightness of cell junctions and barrier function; it qualitatively assesses cell monolayer health and quantitatively evaluates cellular confluence by detecting changes in ion flow through transcellular and paracellular pathways, with values typically increasing from low levels immediately after cell seeding

@WPIInc · 03:54

• Why this video: This video introduces the physical and mathematical concepts behind TEER. It explains how applying an AC current across an epithelial monolayer allows researchers to quantify the resistance of paracellular pathways (tight junctions).

Real-Time TEER Monitoring for Organ-on-Chip Barrier Integrity

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Real-Time TEER Monitoring for Organ-on-Chip Barrier Integrity

Achieving stable TEER measurements requires fixed planar electrodes eliminating positioning variability and maintaining controlled incubator environments since TEER is temperature-sensitive. Wireless contactless readout prevents disturbance during measurement. Gaps or holes in cell layers cause current leakage, producing artificially low TEER values that misrepresent true barrier function—requiring fully confluent monolayers before measurement. Perfusion initially increases TEER due to shear-ind

@Dynamic42 · 04:10

• Why this video: Transitioning from Transwells to microfluidic chips introduces unique challenges like electrode positioning and temperature fluctuations. This video shows how to integrate stable, planar electrodes directly within microfluidic systems to achieve reproducible measurements.

Organ-on-Chip Fabrication with Integrated Electrodes for TEER | Protocol Preview

@JoVEJournal00:40377

Organ-on-Chip Fabrication with Integrated Electrodes for TEER | Protocol Preview

Microfluidic chips with integrated electrodes enable direct measurement of trans-endothelial or trans-epithelial electrical resistance (TEER) in organ-on-chip systems. This technology allows researchers to measure the barrier function of tissues such as the blood-brain barrier directly within the chip. The main advantages include easy integration of electrodes into organ-on-chip systems and the ability to compare TEER values across different systems, facilitating standardized comparisons in barr

@JoVEJournal · 00:40

• Why this video: This protocol preview shows how researchers physically align metal wire or sputtered planar electrodes directly with PDMS microchannels to enable real-time electronic monitoring without taking the chip out of the incubator.

Fabrication of PDMS Microfluidic Devices Integrated with an Electrical Circuit

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Fabrication of PDMS Microfluidic Devices Integrated with an Electrical Circuit

This video demonstrates the fabrication process of a PDMS microfluidic device integrated with an electrical circuit, involving photolithography on an aluminum-coated glass slide to pattern electrodes, followed by spin coating PDMS and irreversible bonding to create a device capable of generating electric fields at specific points within microchannels for applications in electrokinetic manipulation and sensing.

@microfluidicsandmultiphase8434 · 11:45

• Why this video: A highly technical fabrication guide showing how microfluidic channels are aligned and permanently bonded over patterned glass electrodes or electrical components to produce integrated bio-sensors.

Curriculum Gap Acknowledgment & Self-Study

⚠️ Curriculum Gap: The existing video pool demonstrates general TEER theory and commercial Transwell insert measurements (such as Chopstick electrodes), but lacks hands-on lab footage showing a researcher manually threading platinum wire electrodes into microfluidic PDMS ports or soldering leads under flow.

To close this gap, search YouTube or academic literature for:

• "How to integrate electrodes in PDMS microfluidic chip for TEER"
• "On-chip TEER measurement tutorial microfluidics"

Knowledge Checkpoint

• Define Transepithelial Electrical Resistance (TEER) and state its standard SI unit of measurement ($\Omega \cdot \text{cm}^2$).
• Explain why AC current (alternating current) is used instead of DC current (direct current) to measure biological resistance.
• Calculate the final normalized TEER value given raw resistance readings, blank chip values, and the active membrane surface area: $\text{Normalized TEER} = (R_{\text{tissue}} - R_{\text{blank}}) \times \text{Area}$
• Identify how external variables (such as cell culture media temperature and pH) distort TEER resistance values.

Module 5: Gut Microbiota Co-Culture & Microenvironment Control

The final module covers co-culturing obligate/facultative anaerobes alongside human epithelial cells. You will study how researchers design dual-channel devices that support human cells on an oxygen-rich top channel and gut microbiota on an oxygen-depleted (anoxic) bottom channel, mimicking the natural oxygen gradient of the intestinal tract.

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Intestinal chips enable direct culture of human gut microbiomes with anaerobes and aerobes in contact with epithelium and mucous layers. Hypoxia gradients maintain oxygen levels below 0.5% for anaerobes while keeping epithelial cells viable. Barrier function improves with healthy microbiome presence, and complex microbiomes from human stool specimens sustain over five days with over 200 operational taxonomic units. Kidney glomerulus chips using induced pluripotent stem cells require one beat-per

@axialxyz · 04:27

• Why this video: Dr. Donald Ingber explains how the Wyss Institute developed human intestine chips. He discusses the critical physiological need to co-culture complex, living microbiomes with human cells by establishing steady oxygen gradients across the membrane.

Culture techniques for Anaerobic bacteria | microbiology | Notes by microbial concepts

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Culture techniques for Anaerobic bacteria | microbiology | Notes by microbial concepts

Anaerobic bacteria require specialized culture techniques to grow because they cannot survive in oxygen-rich environments; the main methods include pre-reduced media (boiled and flushed with nitrogen gas), anaerobic chambers (controlled glove boxes with H2, CO2, and N2 atmospheres), gas packs (chemical generators producing CO2 and H2), anaerobic jars (similar to gas packs with sodium bicarbonate and borohydride), candle jars (using burning candles to create CO2-rich environments), and the biolog

@microbialconcepts · 16:51

• Why this video: Before seeding anaerobes on-chip, they must be cultured off-chip. This lecture details standard laboratory methods for anaerobic growth, including anaerobic jars, gas packs, thioglycolate reduction media, and nitrogen flushing.

Life Without Oxygen? Challenge Accepted

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Life Without Oxygen? Challenge Accepted

The oxic-anoxic interface is a thin boundary layer (approximately 100 micrometers thick) where oxygen and sulfur coexist. Bacteria like Achromatium oxaliferum thrive here, possessing both sulfur globules and the ability to roll upward in response to oxygen presence. These bacteria must sense and respond to shifting environmental conditions, demonstrating sophisticated behavioral adaptations to dynamic interfaces.

@journeytomicro · 00:46

• Why this video: A microscopic visualization of the oxic-anoxic interface—the highly precise, sub-millimeter boundary layer where oxygen concentrations transition from high to zero, serving as a biological analogy for the microenvironments designed inside dual-channel gut chips.

Curriculum Gap Acknowledgment & Self-Study

⚠️ Curriculum Gap: The existing video pool demonstrates general anaerobic biology and macroscopic culturing techniques but does not feature step-by-step videos of setting up nitrogen gas sweep lines or oxygen-scavenging membranes on dual-channel PDMS chips in the lab.

To close this gap, search YouTube or academic literature for:

• "Anoxic aerobic interface microfluidic gut chip co-culture setup"
• "Oxygen gradient control in microfluidic organs on chips"

Knowledge Checkpoint

• Explain why obligate anaerobes (e.g., Bacteroides or Clostridium species) are highly sensitive to oxygen and cannot survive in standard cell incubators.
• Describe the design of a dual-channel microfluidic gut-on-a-chip, explaining how the PDMS membrane separates the aerobic (human) and anaerobic (bacterial) microenvironments.
• Understand how nitrogen gas perfusion or chemical oxygen scavengers are utilized to establish an active oxygen gradient on-chip.
• Define the biological consequences of co-culturing bacteria under static conditions versus dynamic flow conditions.

Course Map

Key People Index

• Dr. Donald Ingber (Wyss Institute/Harvard/Emulate): Pioneer of organ-on-a-chip technology. His lab developed the primary mechanical lung-on-a-chip and gut-on-a-chip designs, demonstrating that mechanical force and microfluidic flow are vital for human cellular differentiation and co-culturing the human microbiome.
• Dr. Dan Huh (University of Pennsylvania): Leading bioengineer who worked closely with Dr. Ingber on the lung-on-a-chip. His laboratory at UPenn continues to push the boundaries of microphysiological systems (MPS) for drug screening and disease modeling.
• Dr. Geraldine Hamilton (Emulate/Wyss): Former researcher at the Wyss Institute and leading industry voice who helped translate organ-on-a-chip technology from laboratory benches to commercialized testing platforms.

Final Self-Assessment

Complete this comprehensive, practical self-assessment to verify your competence in designing, fabricating, and running a gut-on-a-chip experiment.

• Design and Lithography: I can draw a 2D photomask with fluid channels of known height and width, and successfully pattern an SU-8 master wafer using a mask aligner and developer chemistry.
• Soft Lithography Fabrication: I can scale base-to-curing agent ratios, degas PDMS, cure it in a convection oven, cut clean slabs, and punch fluidic inlets/outlets without tearing the elastomer.
• Surface Modification and Bonding: I can explain how oxygen plasma changes PDMS and glass surfaces to create permanent $Si-O-Si$ covalent bonds.
• ECM Functionalization: I can select and coat microchannels with an appropriate extracellular matrix mixture (e.g., Collagen I and Fibronectin) to promote epithelial cell attachment.
• Sterility and Seeding: I can cleanly culture, harvest, and inject a bubble-free suspension of Caco-2 cells into a sterile microfluidic device inside a biosafety cabinet.
• Dynamic Flow Calculations: I can calculate and set fluid flow parameters on syringe pumps to supply physiological wall shear stress to the cells.
• Electrode Integration: I can successfully build, integrate, and align platinum or silver wire electrodes into a microfluidic device to connect to a voltohmmeter.
• TEER Quantification: I can record raw AC resistance values, subtract system background resistance, and apply mathematical normalization ($\Omega \cdot \text{cm}^2$) to determine barrier confluence.
• Oxygen Gradient Establishment: I can explain the physical setup required to supply anaerobic media to a microbial channel while maintaining oxygen levels for human epithelial cells.
• Microbiome Co-Culture Execution: I can safely culture, isolate, and introduce anaerobic microbial strains alongside epithelial cells in an active, stable gut-on-a-chip device.