Name: reconstituting cytoarchitecture and function of human epithelial tissues on an open-top organ-chip
Uploaded: 2023-02-17T12:40:00
Description: Watch this Scientific Journal Video about Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip at JoVE.com

Human colonoids were obtained from intestinal resections in accordance with the guidelines of the Institutional Biosafety Committee of the Cincinnati Children&#39;s hospital (IBC 2017-2011).
1. Surface activation
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	<li>Preparation of activation buffer
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		<li>Place the crosslinker and solvent buffer reagents under the biosafety cabinet (BSC) and let them equilibrate at room temperature (RT) for 10 min before use.</li>
		<li>Reconstitute 5 mg of crosslinker in 5 mL of...

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	<li>Van Norman, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+animal+studies+for+predicting+toxicity+in+clinical+trials:+Is+it+time+to+rethink+our+current+approach.">Limitations of animal studies for predicting toxicity in clinical trials: Is it time to rethink our current approach.</a> JACC: Basic to Translational Science. 4 (7), 845-854 (2019).</li><li>Wange, R. L., Brown, P. C., Davis-Bruno, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+the+principles+of+the+3Rs+of+animal+testing+at+CDER:+Past,+present+and+future.">Implementation of the principles of the 3Rs of animal testing at CDER: Past, present and future.</a> Regulatory Toxicology and Pharmacology. 123, 104953 (2021).</li><li>Mosig, A. 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The Open-Top Chip represents an enabling platform for investigating the complex cellular interplay occurring between endothelium, stroma, and epithelium in a controlled microenvironment, in real time. This technology offers critical advantages over conventional organotypic and organoid cultures, such as the integration of physical and biochemical cues that are relevant to reconstitute the human tissue microenvironment, including fluidic shear (flow), cyclic stretching, and reconstruction of the epithelial surface topogra...

Historically, scientists have relied on preclinical animal testing for drug discovery, but a growing number of these methods have been questioned because of poor correlation with human outcome1. The implementation of the &#34;3Rs&#34; principles to Replace, Reduce, and Refine animal experimentation urges scientists to find new in vitro alternative methods to support preclinical drug and chemical toxicology risk assessment2. However, many in vitro models developed to date lack the biological architecture, cellular complexity, and mechanical environment necessary to recapitulate the dynamic nature of human living organs3,4.
Conventional in vitro preclinical systems typically employ 2D monocultures of human cells grown on a rigid plastic surface. These methods provide a tool for conducting simple mechanistic studies and enable rapid screening of drug candidates. Owing to their relatively low cost and high robustness, 2D models are often paired with automatic high-throughput systems and used for the rapid identification of potential drug candidates during the early stage of the drug development process5,6. However, such 2D models do not provide a translational approach for modeling tissue-level, organ-level, or systemic responses to therapeutic candidates, which is needed for accurate predictions of drug safety and efficacy during the preclinical stage of their development. Flat cell cultures do not recapitulate the native tissue microenvironment, including the complex multicellular interplay, biomechanical properties, and three-dimensional (3D) architecture of human tissues7. Cells growing on a flat surface often do not acquire a mature phenotype and, therefore, cannot respond to pharmacological stimuli as they would in the native tissue. For example, primary human alveolar epithelial cells grown in vitro exhibit a squamous phenotype and lose key phenotypic markers, including surfactant proteins C and B (SP-C and SP-B)8. In addition to insufficient differentiation, primary cells frequently become insensitive to biological stressors in vitro, as certain biochemical pathways associated with tissue inflammation become non-functional9. Such loss of cell function seems to be primarily associated with the use of stiff substrates as well as the lack of soluble factors naturally released by tissue-specific stromal cells such as lung fibroblasts and smooth muscle cells10,11.
Understanding that the lack of chemo-physical and biological complexity limits the physiological behavior of cells in vitro has fostered the development of more sophisticated multicellular models, which have proven to better capture the complexity of human tissues outside the body12,13. Since the creation of the first co-culture models in the early 1970s14, the introduction of synthetic and natural hydrogels has significantly improved the ability to mimic native tissue microenvironments and has become an invaluable tool for driving cellular differentiation, guiding the self-organization of cells into tissue-like structures, and restoration of native tissue functions15,16. For instance, when grown in the appropriate 3D scaffold, human cells can self-arrange into functional structures such as spheroids or organoids, expressing stem cell markers, and are capable of self-renewal17. In contrast, human cells (including stem cells), when grown on traditional 2D substrates, rapidly age and undergo senescence after a few passages18. In addition, hydrogels can be &#34;tailored&#34; to match specific tissue properties such as porosity, pore size, fiber thickness, viscoelasticity, topography, and stiffness or further engineered with tissue-derived cellular components and/or bioactive molecules enabling emulation of the physiological or pathological conditions19,20. Despite their enormous potential for drug testing, 3D hydrogel-based models used in pharmaceutical research do not fully recapitulate the complex cytoarchitecture of the in vivo tissues and lack important hemodynamic and mechanical stimuli normally present in the human body, including hydrostatic pressure, cyclic stretch, and fluid shear21.
Microphysiological systems (MPSs) such as Organs-on-chips (OOCs) have recently emerged as tools that are capable of capturing complex physiological responses&#8239;in vitro22,23. These models often employ the use of microfluidic platforms, which enable the modelling of the dynamic microenvironment of living organs.
We have combined the principles of 3D tissue bioengineering and mechanobiology to create an Open-Top Chip model of complex human epithelial tissue. This allowed us to closely recapitulate the multicellular and dynamic microenvironment of epithelial tissues. This includes tissue-specific biochemical and biomechanical cues naturally present in living organs but often neglected by traditional in vitro models24. The Open-Top Chip incorporates two compartments: a vascular compartment (Figure 1A) and a stromal compartment (Figure 1B) separated by a porous membrane, allowing for the diffusion of nutrients between the two chambers (Figure 1C). The vascular compartment is exposed to continuous fluid flow to recapitulate physiological shear stress, while the stretchable design of the stromal chamber allows for the modeling of the mechanical strain associated with breathing motions or intestinal peristalsis. The stromal compartment houses the tunable 3D hydrogel scaffold designed to support the physiological growth of tissue-specific fibroblasts. It possesses a removable lid that facilitates the establishment of an air-liquid interface, a condition that allows greater emulation of human physiology of mucosal tissues as well as direct access to the tissue for administrating drugs directly onto the epithelial layer. Supplementary Figure 1 captures some of the key components of the Open-Top Chip design including dimensions and biological compartments (Supplementary Figure 1A-D) as well as the main technical steps described in this protocol (Supplementary Figure 1E).
Perfusion of the Open-Top Chip is achieved with a programmable peristaltic pump (Figure 1D). The peristaltic pump setup allows 12 Open-Top Chips to be perfused simultaneously. Most incubators can house two setups enabling the culture of up to 24 chips per incubator. Mechanical stretching is achieved using a custom-made programmable vacuum pressure regulator (Figure 1E). It consists of an electro-pneumatic vacuum regulator controlled electronically by a digital-to-analog converter. In other words, the electro-pneumatic vacuum regulator generates a sinusoidal vacuum profile with an amplitude and frequency that is determined by the user. Cyclic strain ranging from 0% to 15% is generated by applying negative pressure to the vacuum channel of the Open-Top Chip at an amplitude ranging from 0 to -90 kPa and a frequency of 0.2 Hz. It is a custom-made system equivalent to the commercially available Flexcell Strain Unit previously adopted and described in other papers25. To mimic the mechanical tissue deformation associated, for example, with the breathing motion of the lung or the peristalsis of the intestine, the pneumatic actuator applies sinusoidal vacuum/strain waves whose magnitude and amplitude can be adjusted to match the physiological level of strain and frequency that human cells experience in their native tissue.
Here, we describe an efficient and reproducible method for engineering and culturing organotypic epithelium equivalents on a prototype Open-Top Chip platform. It allows the generation of complex organ models such as skin, alveolus, airway, and colon while integrating a vascular fluid flow and mechanical stretching. We will outline key technical aspects that must be considered while implementing principles of tissue engineering for generating complex epithelial models. We will discuss the advantages and possible limitations of the current design.
An overview of the main steps used to achieve tissue and organ maturation, including flow and stretch parameters, is reported in: Figure 2 for the skin, Figure 3 for the alveolus, Figure 4 for the airway, and Figure 5 for the intestine. Additional information concerning media composition and reagents used for culturing the different organ models are included in the supplementary tables (Supplementary Table 1 for the skin; Supplementary Table 2 for the alveolus; Supplementary Table 3 for the airway, and Supplementary Table 4 for the intestine).

reconstituting cytoarchitecture and function of human epithelial tissues on an open-top organ-chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

Surface micropatterning 
Micropatterning of the extracellular matrix (ECM) can be used to replicate the spatial configuration of the intestinal crypt interface. The Open-Top Chip configuration can be modified to integrate micropatterned stamps specifically designed to mimic the natural topography of the colonic epithelium-stroma interface (Figure 6A,B) and the intestinal crypts at micrometer scale (Figure 6C-</stron...

Watch this Scientific Journal Video about Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip at JoVE.com

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...

Traditional diffusal culture methods often fail to recapitulate the complex environment of living organs, leading to the loss of tissue-specific markers and functions. We have developed an open-top chip that combines diffusal culture and microfluidic principle to mimic the microenvironment of epithelial tissues closely. By including biochemical and biomechanical cues that are naturally present in living organs but often neglected by traditional in vitro models, the open-top chip represent a better alternative between closed systems.Demonstrating the procedure will be Adya Panchal today, a research student in my laboratory. To begin, bring the required reagents under the biosafety cabinet on ice. Culture tissue-specific mesenchymal cells to obtain 80%to 90%confluency and then dissociate the cells using trypsin or other methods as per the manufacturer's recommendations.Pellet the cells at 250g for five minutes at 24 degrees Celsius. Resuspend the cell pellet in 225 microliters each of ice-cold 10X EMEM and reconstruction buffer. Mix the cells by pipetting and add 1, 800 microliters of ice-cold Collagen 1 solution.Next, mix the pre-gel solution on ice by pipetting up and down five to six times. Neutralize the pre-gel solution with 18 microliters of one normal sodium hydroxide, mix gently by pipetting, and then pipette 150 microliters of cell-laden hydrogel into the central chamber of the open-top chip while avoiding bubbles. Group the chips into separate Petri dishes, including a centrifuge tube cap filled with two milliliters of sterile double-distilled water in each Petri dish, and incubate the Petri dishes at 37 degrees Celsius and 5%carbon dioxide.To perform surface micropatterning of the stromal hydrogel, pipette 20 microliters of the neutralized Collagen 1 pre-gel solution on the patterned surface of a sterile 3D-printed stamp and insert the stamp inside of the open-top chamber while the stromal hydrogel is still in a liquid form. Remove any hydrogel residue that may spill from the top of the open-top chamber using an aspirator. Group all chips into separate Petri dishes with a conical tube cap filled with water as demonstrated earlier.And incubate all Petri dishes at 37 degrees Celsius and 5%carbon dioxide for 90 minutes. At the end of the incubation, bring the chips back under the biosafety cabinet and gently remove the stamps using precision tweezers to reduce the risk of damaging the hydrogel. Once the cultured cells attain 80%to 90%confluency, dissociate the cells using proteolytic enzyme procedures as recommended by the cell provider.After dissociation, pellet the cells by centrifugation and resuspend the epithelial cells to the appropriate cell fragment density as described in the manuscript. To seed the epithelial cell, transfer the chips from the incubator into the biosafety cabinet and rinse the stromal surface three times with 100 microliters of fresh epithelial cell culture medium to remove any excess of coating solution. Once the rinsing medium is aspirated, seed the hydrogel surface with 50 microliters of the epithelial cell suspension using appropriate cell density, and then transfer the chips back into the incubator for two hours.To remove cellular debris, gently rinse the hydrogel surface with the cell culture medium twice, refresh the medium by autoclaving in sealed autoclavable containers, and connect the chips to the peristaltic pump. Pause the peristaltic pump, carefully disconnect the chips from the pump, and extract the chip housing carrier. After transferring the chips from the incubator to the biosafety cabinet, remove the volume of the medium left into the top reservoir.Then, aspirate all the medium from the top microfluidic channel gently and clamp the short microfluidic tubing connected to the top inlets using binder clips to reduce media evaporation and maintenance of air-liquid interface, or ALI. Place the open-top chip on the housing carrier back into the incubator. Reconnect the chips to the peristaltic pump and resume the flow by starting the peristaltic pump.Rinse the vascular chamber with endothelial cell culture medium and then seed 25 microliters of endothelial cell suspension. Flip the chips upside down to allow endothelial cells to attach to the upper surface of the microfluidic chamber. Group the chips into Petri dishes.Place them back into the incubator as demonstrated earlier and let the endothelial cells attach for one hour. At the end of the incubation, transfer the chips from the incubator to the biosafety cabinet and rinse the vascular channel with endothelial cell culture medium twice to remove cellular debris. Lay the chips flat to facilitate the adhesion of the endothelial cells to the bottom surface of the vascular channel.Fill the vascular medium reservoir with the degassed vascular cell culture medium under the biosafety cabinet. Place the chips back inside the chip housing carrier and reconnect the chips to the medium reservoirs on one end and to the peristaltic pump on the other. Inspect the microfluidic connections to ensure all the chips are correctly connected and no visible droplets of medium dripping.Then, resume fluid flow by starting the peristaltic pump. The micropatterned gel surface with and without cells is visualized. Histological analysis revealed mature multilayered stratified epidermis differentiated on chip.The top view picture of the skin chip showed the presence of the fibroblasts inside the dermal layer. PECAM-1, VE-cadherin, and von Willebrand showed the differentiation of human microvascular endothelial cells co-cultured in the open-top skin chip. MUC5AC, alpha and beta tubulin, chloro cell protein 16, p63, and ZO-1 fluorescent staining showed mature airway epithelium.The beating Cilia and differentiated mature pseudo-stratified epithelium were observed on open-top airway chip. Type I, Type II, and E-cadherin fluorescent staining showed the presence of mature pneumocytes on chip. Electron microscopy revealed the presence of microvilli and lysosomal vesicles, evidence of mature alveolar phenotype.The histological analysis showed the presence of flat squamous cells consistent with the Type I phenotype, and cuboidal cobblestone-like cells coherent with the Type II phenotype, and fibroblasts inside the dermal layer. The presence of enterocytes and the mature goblet was confirmed by mucin 2 and E-cadherin staining. The fibroblasts inside the dermal layer confirmed the presence of a simple columnar epithelium.All reagents must be cold and the surfaces of the gel should be adequately rinsed to obtain an even surface. It's important to remove any non adhering cells and debris to obtain even cell adhesion and a compact foul monolayer and vascular wall. A similar approach can be employed to generate other types of epithelial organ models, such as the intestine and the lung, to assess how specific drugs or environmental factors may affect the homeostasis of human tissues, with a particular emphasis on epithelial and vascular barrier function.With easy access to the efficacy phase of the stroma compartment, researching can now generate specific face patterns and reproduce functional tissue architecture, such as the cryptviveoli, which is difficult to achieve with other devices.

Research

JoVE Journal

Bioengineering

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...