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

摘要

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.

摘要

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.

引言

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 "3Rs" 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 organs^3,4.

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 process^5,6. 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 cells^10,11.

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 body^12,13. 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 functions^15,16. 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 "tailored" 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 conditions^19,20. 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 in vitro^22,23. 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).

方案

Human colonoids were obtained from intestinal resections in accordance with the guidelines of the Institutional Biosafety Committee of the Cincinnati Children's hospital (IBC 2017-2011).

1. Surface activation

1. Preparation of activation buffer
   1. 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.
   2. Reconstitute 5 mg of crosslinker in 5 mL of the solvent buffer using a sterile light-impervious container or a transparent 15 mL conical tube wrapped in aluminum foil to protect the crosslinker solution from direct light exposure.
   3. Vortex the solution for 1 min to remove all clumps, and then pipette 50 µL of the crosslinker solution directly into the bottom channel of the chip and 150 µL into the open-top chamber.
   4. Remove any excess of crosslinking solution from the surface of the chip using an aspirator. Then pipette additional 50 µL of the crosslinking solution directly into the bottom channel of the chip and 150 µL into the open-top chamber to remove any remaining air bubble.
2. Activation with UV crosslinking machine
   1. Gently remove the lid from the chip under the BSC and store it in a sterile container.
   2. Transfer the chips containing the crosslinker solution into a Petri dish, close the Petri dish to avoid contamination, and place the dish with the chips under the UV crosslinking machine.
      NOTE: Remove the Petri dish lid to maximize the UV exposure.
   3. Set the UV crosslinking machine with a peak wavelength of 365 nm at an intensity of 100 µJ/cm², and turn on the UV light for 20 min.
      NOTE: After 20 min of UV treatment, the crosslinker solution will look darker (brown).
   4. Bring the chips back under the BSC and aspirate the oxidized crosslinker solution. Then, rinse all the chips three times with the solvent buffer and let the chips dry under the BSC for 5-10 min to complete the chemical functionalization of the polydimethylsiloxane (PDMS) surface.

2. Preparation the stroma equivalent

1. Preparation of 10x reconstruction buffer (100 mL)
   1. Dissolve 2.2 g of sodium bicarbonate in 75 mL of 0.067 M NaOH in double-distilled water.
   2. Add 4.76 g of HEPES and bring the volume to 100 mL using double distilled water.
   3. Sterile filter the solution under the BSC using a disposable sterile bottle-top filter with a 0.22 µm membrane.
      NOTE: The solution is stable for about 6 months when stored at 4 °C.
2. Estimation of the pre-gel solution volume
   1. Multiply the number of chips needed for the experiment by 150 µL (inner volume of the central open-top chamber) to estimate the amount of pre-gel solution required for the experiment:
      ​Volume needed = (Number of chips x 150) µL
   2. Prepare the collagen pre-gel solution on ice by mixing: 1 volume of 10x EMEM containing the cells of choice, 1 volume of 10x reconstruction buffer (see step 2.1), 8 volumes of collagen I solution (10 mg/mL) and 1 µL of 1 N NaOH solution for each mg of collagen I.
      NOTE: It is recommended to prepare an extra +15% volume to account for experimental errors; the example described in section 2.2 provides a detailed step-by-step procedure for preparing enough pre-gel solution for 12 chips and an extra 15% volume.
3. Preparation of pre-gel solution for the stroma equivalent (for 12 chips)
   1. Bring the following solutions under the BSC on ice: 10x EMEM; 10x Reconstruction buffer (see step 2.1); Collagen I solution (10 mg/mL); and Sterile 1 N NaOH solution.
   2. Culture tissue-specific mesenchymal cells as directed by the providers until 80%-90% confluent, and then dissociate the cells using trypsin or other methods as recommended by the cell provider. Collect the cells in a pellet by centrifugation at 250 x g for 5 min at 24 ˚C.
   3. Resuspend the cell pellet in 225 µL of ice-cold 10x EMEM, and add 225 µL of ice-cold 10x reconstruction buffer (see step 2.1). Mix the solution by gently pipetting up and down, and then add 1,800 µL of ice-cold collagen I solution.
   4. Pipette up and down 5-6 times, avoiding bubbles to mix the pre-gel solution while on ice.
4. Incorporation of the stroma equivalent on chip (for 12 chips)
   1. Neutralize the pre-gel solution with 18 µL of 1 N NaOH. Mix gently by pipetting up and down 5-6 times, and then pipette 150 µL of cell-laden hydrogel into the central chamber of the Open-Top Chip avoiding bubbles.
      NOTE: If micropatterning is required, please move to the next step (section 3).
   2. Group the chips into separate Petri dishes, including a centrifuge tube cap filled with 2 mL of sterile ddH₂O in each Petri dish, and incubate the Petri dishes(s) in the incubator at 37 °C, 5% CO₂.
      ​NOTE: After 90 min, the cell-laden hydrogel will be completely polymerized.

3. Surface micropatterning (optional)

1. Perform surface micropatterning of the stromal hydrogel after pipetting the neutralized collagen hydrogel (still in its liquid state) using 3D-printed stamps.
   ​NOTE: The 3D-printed stamps can be obtained in various customizable designs, as previously described elsewhere²⁴.
2. Pipette 20 µL of the neutralized collagen I pre-gel solution on the patterned surface of a sterile 3D-printed stamp and insert the stamp inside (on top) of the open-top chamber while the stromal hydrogel is still in a liquid form.
3. Remove any residue of the hydrogel that may spill from the top of the open-top chamber using an aspirator (or a pipette). Group all chips into separate Petri dishes and include a centrifuge 15 mL conical tube cap filled with 2 mL of sterile ddH₂O in each Petri dish.
4. Incubate all Petri dishes(s) at 37 °C, 5% CO₂ for 90 min, and then bring the chips back under the BSC and gently remove the stamps using precision tweezers to reduce the risk of damaging the hydrogel.

4. Coating the epithelial and vascular surface with tissue-specific ECM proteins

1. Coating the vascular microfluidic chamber with extracellular matrix proteins
   1. Multiply the number of chips needed for the experiment by 20 µL (volume of the vascular channel) to estimate the volume of vascular ECM coating solution required for the experiment:
      Volume needed = (Number of chips x 20) µL
      ​NOTE: It is recommended to prepare an extra 15% volume to account for experimental errors.
   2. Prepare the vascular ECM coating solution for all chips (for example, 300 µL per 12 chips) using ice-cold PBS or HBSS.
      NOTE: Refer to the specific organ-protocol table in the supplementary materials section (Supplementary Table 1 for the skin; Supplementary Table 2 for the alveolus; Supplementary Table 2 for the airway, and Supplementary Table 4 for the intestine) to identify the specific reagents and recommended ECM composition.
   3. Pipette 20 µL of vascular ECM coating solution into the vascular channel of each chip.
2. Coating the apical surface of the stromal equivalent with extracellular matrix proteins
   1. Multiply the number of chips needed for the experiment by 50 µL (volume of the vascular channel) to estimate the volume of epithelial ECM coating solution required for the experiment:
      Volume needed = (Number of chips x 50) µL
      NOTE: it is recommended to prepare an extra 15% volume to account for experimental errors.
   2. Prepare enough ECM coating solution for all chips (for example, 750 µL per 12 chips) in ice-cold PBS or HBSS and transfer 50 µL of epithelial ECM coating solution directly on top of the hydrogel surface.
      NOTE: Refer to the specific organ-protocol table in the supplementary materials section (Supplementary Table 1 for the skin; Supplementary Table 2 for the alveolus; Supplementary Table 2 for the airway, and Supplementary Table 4 for the intestine) to identify the specific reagents and recommended ECM composition.
   3. Group the chips into separate Petri dishes, including a centrifuge 15 mL conical tube cap filled with 2 mL of sterile ddH₂O in each Petri dish, and incubate the Petri dishes(s) in the incubator at 37 °C, 5% CO₂ for 2 h before proceeding with epithelial cell seeding.

5. Seeding epithelial cells on the stromal equivalent

1. Epithelial cell culture
   1. Culture tissue-specific epithelial cells as directed by the providers until 80%-90% confluent.
   2. Dissociate the cells using proteolytic enzyme procedures as recommended by the cell provider.
      NOTE: For best results, harvest epithelial cells at low passage (P1-P2) during the active growth phase when they reach between 70%-90% confluency.
   3. Once dissociated, centrifuge the cells and collect them as a pellet.
   4. Resuspend the epithelial cells to the appropriate cell/fragment density as indicated in the specific organ protocol table.
      NOTE: In this study, cell solution was used at a density of 3 x 10⁶ cells/mL for the skin, 1 x 10⁶ cells/mL for the alveolus, 6 x 10⁶ cells/mL for the airway and 8 x 10⁶ fragments/mL for the intestine.
2. Epithelial cell seeding
   1. Transfer the chips from the incubator into the BSC. Aspirate the coating solution from the vascular channel, and rinse the vascular microfluidic channel three times with 50 µL of fresh endothelial cell culture medium.
   2. Aspirate the coating solution from the hydrogel surface and rinse the stromal surface three times with 100 µL of fresh epithelial cell culture medium to remove any excess coating solution.
   3. Aspirate the rising medium and seed the hydrogel surface with 50 µL of the epithelial cell suspension using appropriate cell density, as indicated in the supplementary tables, and then transfer the chips back into the incubator for 2 h (or overnight for colonoids).
   4. Gently rinse the hydrogel surface with the cell culture medium twice to remove cellular debris. Finally, refresh the medium by autoclaving in sealed autoclavable containers, and connect the chips to the peristaltic pump.

6. Connecting chips to flow

1. Preparing the fluidic parts
   1. Cut 2 inches of biocompatible polypropylene-based thermoplastic elastomer (TPE) transfer tubing (Table of Materials) to prepare the short microfluidic tubing that is required for connecting the chips to the media reservoirs.
   2. Cut 7.5 inches of biocompatible TPE transfer tubing to produce the long microfluidic tubing.
   3. Prepare enough 18 G and 19 G metal connectors (Table of Materials).
      NOTE: It is recommended to prepare and sterilize the tubing and connectors described in steps 6.1.1 and 6.1.2 at least 1 day before connecting the Open-Top Chips to the peristaltic pumps.
   4. Pierce the lid of each medium reservoirs with a 4-inch hypodermic needle (Table of Materials).
2. Medium degassing
   1. Allow the cell culture medium to equilibrate to room temperature (RT).
   2. Transfer the volume of medium needed into a conical filtering tube.
   3. Apply a negative vacuum pressure of -20 PSI to degas the medium (vacuum-driven filtration).
      NOTE: If a vacuum is not available, the cell culture medium can be left to equilibrate in the incubator overnight to achieve similar results.
3. Prepare the Open-Top Chip for fluid flow
   1. Bring the chips and the sterile fluidic parts under the BSC and align the lid (top portion) of the Open-Top Chip for sealing the Open-Top Chip prototype before starting fluid flow.
   2. Pipette 200 µL of the degassed cell culture medium (see organ-specific tables) into the inlet port of both the top and bottom channels of the chip while paying attention to avoid bubbles.
   3. Pipette 300 µL of the culture medium into the short microfluidic tubing to prime the inner surface of the tubes and connect the short tubing to the inlets of the top and bottom channels of the chip.
4. Connect and prime the microfluidic surfaces
   1. Position the medium reservoirs into the farm rack, connect the hypodermic needle to the bottom inlet of the chips, and finally, accommodate all the chips in the housing carrier(s) inside the incubator.
   2. Connect the chips to the peristaltic pump, and then inspect all the connectors to make sure that all the chips are properly connected and that there is no visible leakage of cell culture media.
   3. Use the Purge button on the pump and hold it for about 15 s or until the droplets of the cell culture medium appear at the end of the outflow tubing.
   4. Use the control system on the pump to set the appropriate organ-chip flow rate (Figure 2, Figure 3, Figure 4, and Figure 5).

7. Maintenance of chips

1. Organ-chip maintenance
   1. Prepare fresh cell culture medium for the epithelium and/or endothelium and perform the degassing steps (as previously described in step 6.2).
   2. Pause the peristaltic pump, carefully disconnect the chips from the pump, and extract the chip housing carrier(s). Transfer the chips from the incubator to the BSC and remove the volume of media left into the reservoirs.
   3. Replace the cell culture media to the top and bottom inlet reservoirs with 5 mL of fresh cell culture media and place the chip housing carrier(s) back into the incubator. Connect the chips to the peristaltic pump and restart the flow.
      NOTE: It is recommended to use the Purge function to rapidly refresh the medium to the vascular compartment and reduce the risk of air bubbles.
   4. Repeat steps 7.1.1-7.1.3 every other day, as per Figure 2, Figure 3, Figure 4, and Figure 5.
2. Establishing air-liquid interface (ALI)
   1. Pause the peristaltic pump, carefully disconnect the chips from the pump, and extract the chip housing carrier(s). Transfer the chips from the incubator to the biosafety cabinet, and remove the volume of medium left into the top reservoir.
   2. Gently aspirate all the medium from the top microfluidic channel and clamp the short microfluidic tubing connected to the top inlets using binder clips to reduce media evaporation and maintenance of ALI.
   3. Place the Open-Top Chip on the housing carrier(s) back into the incubator and reconnect the chips to the peristaltic pump.
      NOTE: It is recommended to use the Purge function to rapidly refresh the medium to the vascular compartment and reduce the risk of air bubbles.
   4. Resume the flow by starting the peristaltic pump.
3. Stretching (Optional)
   1. Pause the peristaltic pump and connect the vacuum ports of the chips to the vacuum module using two long microfluidic tubes per chip.
   2. Use the vacuum module to adjust the stretch setting to the condition recommended for each organ as specified inside the organ protocol tables: Supplementary Table 1 for the skin; Supplementary Table 2 for the alveolus; Supplementary Table 2 for the airway; and Supplementary Table 4 for the intestine.
   3. Visually inspect the tube connections to make sure all the chips are properly connected and there are no visible droplets of medium dripping.
   4. Resume the flow by starting the peristaltic pump.

8. Seeding endothelial cells in the vascular compartment

1. Prepare cells and chips for vascular cell seeding
   1. Culture the tissue-specific endothelial cells as directed by the providers until 80%-90% confluent. Dissociate the cells using a proteolytic enzyme procedure (as recommended by the provider) and, finally, resuspend the endothelial cells in a solution of 3 x 10⁶ cells/mL.
      NOTE: For best results, harvest the endothelial cells at low passage (P2-P4) during the active growth phase when they reach between 70%-90% confluency.
   2. Pause the peristaltic pump. Transfer the chips from the incubator to the BSC, disconnect the chips from the media reservoirs and any connected tubing, and then group the chips into separate Petri dishes.
   3. Refresh the cell culture medium of the epithelial compartment with fresh epithelial cell culture medium. Rinse the vascular channel with fresh endothelium cell culture medium twice, and then aspirate the medium from the vascular compartment.
2. Endothelial cell seeding
   1. Seed the bottom (vascular) channel with 25 µL of endothelial cell suspension (3 x 10⁶ cells/mL), flip the chips upside down to allow endothelial cells to attach to the upper surface of the microfluidic chamber.
      NOTE: Add 50 µL of the cell suspension per chip (600 µL per 12 chips).
   2. Group the chips into Petri dishes. Place them back into the incubator at 37 °C, 5% CO_2, and let the endothelial cells attach for 1 h.
   3. After 1 h, transfer the chips from the incubator to the BSC. Rinse the vascular channel with endothelial cell culture medium twice to remove cellular debris.
   4. Repeat steps 8.2.1-8.2.2 to seed the vascular channel once again with endothelial cells and lay the chips flat to facilitate the adhesion of the endothelial cells to the bottom surface of the vascular channel.
3. Reconnect the chip to flow
   1. Fill up the vascular medium reservoir with the degassed vascular cell culture medium under the BSC.
   2. Place the chips back inside the chip housing carrier(s) and reconnect the chips to the medium reservoirs on one end to the peristaltic pump on the other end.
      ​NOTE: It is recommended to use the Purge function to rapidly refresh the medium to the vascular compartment and reduce the risk of air bubbles.
   3. Visually inspect the microfluidic connections to make sure all the chips are properly connected and there are no visible droplets of medium dripping. Then, resume fluid flow by starting the peristaltic pump.

9. Common endpoint assays

1. Disconnect chips for endpoint assays
   1. Pause the peristaltic pump, carefully disconnect the chips from the pump, and extract the chip housing carrier(s). Transfer the chip housing carrier(s) from the incubator to the BSC and set the chips free.
   2. Gently wash the central chamber of the Open-Top Chip with the epithelial cell culture medium and the vascular channel with the endothelium culture medium twice to remove any cellular debris.
   3. Remove the lid of the Open-Top Chip to access the apical compartment of the chip using tweezers.
2. Immunostaining for fluorescence microscopy
   1. Proceed with conventional sample fixation, permeabilization, and blocking.
      NOTE: In this study, the samples were fixed in 200 µL of 4% paraformaldehyde (PFA) for 1 h followed by rinsing with PBS, permeabilization in 0.1% Triton X-100 for 40 min and blocking in 1% bovine serum albumin for 1 h.
   2. Incubate the chips with primary antibodies (Table of Materials) at 4 °C overnight. and then wash both epithelial and endothelial compartments of the chips with 200 µL of PBS twice. Then, proceed with the appropriate secondary antibodies for 2 h.
      NOTE: Dilute the primary antibodies and secondary antibodies in PBS + 1% BSA at a dilution of 1:100 (primary antibody) and 1:200 (secondary antibody).
3. Immunohistochemistry
   1. Extract the stromal equivalents from the main chamber of the Open-Top Chip using tweezers, collect them into a 1.5 mL tube filled with 10% neutral buffered formalin and incubate for at least 24 h.
   2. Transfer the fixed stromal equivalent to the tissue processor and follow the below steps to achieve optimal sample dehydration.
      1. Submerge the hydrogel in 70% ethanol for 90 min.
      2. Remove the 70% ethanol and submerge the hydrogel in 80% ethanol for 90 min.
      3. Remove the 80% ethanol and submerge the hydrogel in 95% ethanol for 90 min.
      4. Remove the 95% ethanol and submerge the hydrogel in 100% ethanol for 90 min twice.
      5. Remove the 100% ethanol and submerge the hydrogel in a solution of xylene for 120 min twice.
      6. Remove the xylene solution and infiltrate the processed stromal equivalents in paraffin wax for 120 min (~2 h) twice.
   3. Embed the infiltrated stromal equivalents into sectioning paraffin blocks.
   4. At this point, the stromal equivalents can be sectioned as paraffin blocks using a microtome and processed according to conventional histological techniques²⁶.

结果

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-...

讨论

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...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
10 倍 EMEM	Lonza	12-684F	中等;Stroma
18 规格针	MicroGroup	316H18RW	管不锈钢 316 焊接，18RW 全硬
19号针	MicroGroup	316H19RW	管不锈钢316焊接，19RW全硬
2站PharMed BPT	科尔-帕尔默	EW-95723-12	管，0.25 毫米，12 支/包
70% 乙醇和湿巾	-	-	用于表面灭菌
8-溴腺苷 3′，5′-环磷酸钠盐 （8-Br-cAMP）	Sigma	B7880	培养基补充剂
编号 A-83-01	托克里斯	2939
腺嘌	呤 Sigma	A9795
Advanced DMEM/F12	Thermo	12634010
气道上皮细胞	Lifeline Cell Technology	FC-0016
铝箔	-	-	-
肺泡细胞	Cell Biologics	H6621
抗 ABCA3	ABCAM 中文官网	ab24751	小鼠单克隆抗体 [3C9]
抗水通道蛋白 5 Alexa Fluor 647	ABCAM 中文官网	ab215225	兔单克隆抗体 [EPR3747]
抗水通道蛋白5	ABCAM 中文官网	ab92320	兔单克隆抗体 [EPR3747]
抗 β IV 微管蛋白	ABCAM 中文官网	ab11315	小鼠单克隆抗体 [ONS.1A6]
CD31 抗体 （PECAM-1）	ABCAM 中文官网	ab9498	小鼠单克隆抗体 [JC/70A]
抗 CK5	ABCAM 中文官网	ab75869	兔重组单克隆抗体 [AY1E6]
抗细胞角蛋白 10	赛默飞世尔	货号 MA5-13705	小鼠单克隆抗体 （DE-K10）
抗细胞角蛋白 14	ABCAM 中文官网	AB7800	小鼠单克隆抗体
抗 E-钙粘蛋白	ABCAM 中文官网	AB1416	小鼠单克隆抗体
抗丝聚蛋白	赛默飞世尔	PA5-79267	兔多克隆抗体
抗 HTI-56	台地生物	TB-29AHT1-56	小鼠单克隆抗体 （IgG1）
抗HTII-280	台地生物	TB-27AHT2-280	小鼠单克隆抗体 （IgM）
抗内卷	赛默飞世尔	货号 MA5-11803	小鼠单克隆抗体 （SY5）
抗亚型 TA p63-α， -β， -γ	生物伦根德	618902	兔多克隆抗体
抗 Ki67	ABCAM 中文官网	ab8191	小鼠单克隆抗体 [B126.1]
抗 LAMP3	ABCAM 中文官网	ab111090	兔多克隆抗体
抗成熟 SP-B	七山	WRAB-48604	兔多克隆抗体
抗 MUC5AC	赛默飞世尔	PA5-34612	兔多克隆抗体
抗粘蛋白-2	SantaCruz Biotechnology	sc-7314	小鼠单克隆抗体 （IgG1）
抗 p63	达科	GA662	小鼠单克隆抗体 p63 蛋白 （Dako Omnis） 克隆 DAK-p63
抗 PCNA	赛默飞世尔	PA5-32541	兔多克隆抗体
抗足臼素 （AT-1α）	ABCAM 中文官网	ab128994	兔多克隆抗体
抗 Pro + 成熟表面活性剂蛋白 B	ABCAM 中文官网	ab40876	兔多克隆抗体
抗表面活性剂 C	七山	WRAB-9337	兔多克隆抗体
抗子宫红蛋白/SCGB1A1	海特生物科技	HM2178	小鼠单克隆抗体 [AY1E6]
抗 VE-钙粘蛋白	ABCAM 中文官网	ab33168	兔多克隆抗体
抗 ZO-1	赛默飞世尔	33-9100	小鼠单克隆抗体 [1A12]
抗坏血酸	Sigma	A4544
吸液管	康宁 / 猎鹰	357558	2 mL，聚苯乙烯，独立包装
吸气小贴士	-	-	无菌（高压灭菌）
PBS 溶液中的 B27	热	17504044
阻滞剂 BSA （10X）	赛默飞世尔	37525	阻断剂
氯化钙	Sigma	C7902
CHIR 99021	Tocris	4423
胶原蛋白 I	高级生物基质	5133	10 mg/mL（基质）
胶原蛋白 I	Advanced BioMatrix	5005	3 mg/mL（血管 ECM）
IV	Sigma	C5533
胶原蛋白-IV	Sigma	C5533-5MG	来自人胎盘的胶原蛋白，5 mg 粉末，复溶至 1 mg/mL
结肠成纤维细胞	细胞生物制剂	H6231
结肠微血管内皮细胞	细胞生物制剂	H6203
锥形管	-	-	15 mL 和 50 mL 聚丙烯， 无菌
交联剂 （ER-1）	模拟	10461	5 毫克粉末
DAPI（4'，6-二脒基-2-苯吲哚，二乳酸盐）	赛默飞世尔	D3571	DNA 探针
真皮成纤维细胞	ATCC	PCS-201-010
真皮微血管内皮细胞	ATCC	CRL-3243
地塞米松	Sigma	D4902
DMEM	ThermoFisher	11054020
DMEM/F-12	GIBCO
DMEM/F-12、GlutaMAX	GIBCO	10565-018	用于 ALI 培养基的基础培养基
驴抗小鼠 IgG H&L （Alexa Fluor 488）	ABCAM 中文官网	ab150105	驴抗小鼠二抗
驴抗小鼠 IgG H&L （Alexa Fluor 568）	ABCAM 中文官网	ab175472	驴抗小鼠二抗
驴抗小鼠 IgG H&L （Alexa Fluor 647）	ABCAM 中文官网	ab150107	驴抗小鼠二抗
驴抗兔 IgG H&L （Alexa Fluor 488）	ABCAM 中文官网	ab150073	驴抗小鼠二抗
驴抗兔 IgG H&L （Alexa Fluor 568）	ABCAM 中文官网	ab175470	驴抗小鼠二抗
驴抗兔 IgG H&L （Alexa Fluor 647）	ABCAM 中文官网	ab150075	驴抗小鼠二抗
杜尔贝科s PBS （DPBS-/-） （不含 Ca2+， Mg2+）	康宁	21-031-CV	1x
人表皮生长因子 （EGF），重组于大肠杆菌	PromoCell	C-60170	培养基补充剂中
F-12 火腿'的	Invitrogen	21700-108	用于血管 ECM
纤维色谱柱	高级生物基质	5133-20毫升	I型胶原溶液 （10 mg/mL）
康宁	纤连	356008
纤连蛋白，人，天然，	康宁	47743-654	人血浆纤连蛋白
细尖精密镊子	艾文	18056美国	Technik Style 5B-SA 精密不锈钢镊子
谷氨酰胺	Invitrogen	21700-108
谷氨酰胺	Invitrogen
山羊抗小鼠 IgG H&L （Alexa Fluor 594）	ABCAM 中文官网	ab150080	山羊抗小鼠二抗
山羊抗小鼠 IgG H&L （Alexa Fluor 647）	ABCAM 中文官网	ab150115	山羊抗小鼠二抗
山羊抗小鼠 IgG H&L （FITC）	ABCAM 中文官网	ab6785	山羊抗小鼠二抗
山羊抗小鼠 IgG1 Alexa Fluor 568	赛默飞世尔	编号 A-21124	山羊抗小鼠 IgG1 二抗
山羊抗小鼠 IgM Alexa Fluor 488	赛默飞世尔	A-21042	山羊抗小鼠 IgM 二抗
手持式真空吸尘器	康宁	4930	-
热灭活 HyClone FetalClone II 血清 （FCS）	GE Healthcare Life Sciences	SH30066.03
血细胞计数器	-	-	-
猪肠粘膜肝素钠盐	Sigma	H3149
HEPES	Thermo	15630080
人 [Leu15] - 胃泌素	Sigma	G9145
人结肠类	从临床切除		中获得 从临床切除获得
的人 EGF 重组蛋白	热	PHG0311L
人上皮生长因子	热
HyClone FetalClone II 血清 （美国）	GE 医疗	SH30066.02HI	无菌 FBS 热灭活
氢化可的松 21-半琥珀酸钠盐	Sigma	H4881
氢化可的松	PromoCell 的 PromoCell	C-64420	中等补充剂
冰桶	-	-	-
伊斯马特 IPC-N	Cole-Palmer	EW-78000-41	低速数字蠕动泵;q24通道（每12个芯片1个）
ITES	BioWhittaker	17-839Z
角质形成细胞生长因子（KGF），也称为碱性成纤维细胞生长因子7（FGF-7），人，在HEK	增殖细胞	C-63821
角质形成细胞ATCC		PCS-200-010
层粘连蛋白	生物层粘连	CT521-0501
层粘连蛋白，521 CTG （CT521）	生物层	CT521-0501	人重组层粘连蛋白 521
肺成纤维细胞	生物制剂	H6013
肺成纤维细胞	Lifeline Cell Technology	FC-0049
肺微血管内皮细胞	Lonza	CC-2527
肺平滑肌细胞	Lifeline Cell Technology	FC-0046
手动计数器	-	-	-
Masterflex （TPE） 传输管	Cole-Palmer	FV-96880-02	PharMed BPT，1/32" 内径 x 5/32" 外径
199 培养基，无酚红	热处理
微量离心管	-	-	1.5 mL，无菌
显微镜（带相机）	-	-	用于明场成像
N2	Sigma	17502001
N-乙酰半胱氨酸	Sigma	A5099
Noggin（HEK293T条件培养基）	Sigma	N17001
正常山羊血清	赛默飞世尔	50062Z	堵漏液
O-磷酸乙醇胺	Sigma	P0503
多聚甲醛 （4% wt/vol）	EMS	15710	固色剂
青霉素 链霉素	GIBCO	15140122
青霉素-链霉素	西格玛	P4333	10,000 U/mL;10 毫克/毫升
移液器吸头	-	-	P20、P200 和 P1000 无菌、低粘附
移液器	吉尔森	F167380	P20、P200 和 P1000
PluriQ 血清替代物（或 KO 血清替代物）	AMSBIO（或 Thermo）	N/A（或 C1910828010）
聚-L-赖氨酸涂层显微镜载玻片	西格玛	P0425	载玻片
Primocin	InvivoGen	ant-pm-1
黄体酮	Sigma	P8783
ProLong Gold	赛默飞世尔	P36931	含 DAPI 的抗淬灭封片剂
视黄酸	Sigma	R2625
ROCK 抑制剂 （Y27632）	Tocris	TB1254-GMP/10
R-spondin （HEK293T条件培养基）	Sigma	SCC111
SAGM SingleQuots 补充剂	Lonza	CC-4124
SAGMTM 小气道上皮细胞生长培养基 BulletKitTM	隆沙	CC-4124	中补充剂
SB2001190	托克里斯	1264/10
血清移液器	-	-	2 mL、5 mL、10 mL 和 25 mL 低内毒素，无菌
小气道上皮细胞生长培养基 （SAGM）	Lonza	CC-4124
溶剂缓冲液 （ER-2）	模拟	10462	25 mL 瓶
Steriflip-HV	Millipore	SE1M003M00	无菌过滤锥形管
灭菌 100 mm 方形培养皿	Thermo	103	无菌，每 6 片 1 片
T25 培养瓶	-	-	-
T75 培养瓶	-	-	-
三碘甲状腺原氨酸	Sigma	T5516
Triton X-100 （0.3% （vol/vol）	西格玛	T8787	透化剂
台盼蓝	西格玛	93595	0.4% 溶液
TrypEE 溶液	西格玛	12604013	细胞分离溶液
吐温-20	西格玛	P2287	透化剂
紫外光烘箱（峰频率 365nm，强度为 100 µJ/cm2）	VWR	21474-598	UVP，长距离UV，365 nm 60Hz型号CL-1000L
真空设置	-	-	最小压力：-70 kPa
人血管内皮生长因子 165 （VEGF-165），在大肠	PromoCell	C-64420
VEGF-165 中重组	PromoCell 的 PromoCell	C-64420	中等补充剂
血管性血友病因子偶联 FITC	ABCAM 中文官网	ab8822	绵羊多克隆抗体
水浴（或珠子）	-	-	设置为 37 °C
Wnt3A （L-Wnt3A 条件培养基）	ATCC	CRL-2647