3D Flipwell Engineering for Developing Asynchronous Systems for Toxicologic and Immunomodulatory Therapies in Bacterial, Gut, and Immune Cells

Summary

This article provides a detailed protocol with key steps to guide the 3D Flipwell engineering and utilization. We describe and show how to assemble the co-culture insert stacks and utilize them for co-culturing stratified layers of gut bacteria, gut epithelia, and macrophages to model the gut mucosal environment.

Abstract

To study the roles of bacterial secretome in immune cell polarization and gut mucosal homeostasis, we developed a 3-dimensional co-culture model using commercially available inserts in a back-to-back stacked format. We termed this system 3D Flipwells, and successfully co-cultured bacteria, colon epithelial, and immune cells in stratified layers to recapitulate the gut mucosal microenvironment. This co-culture system enables the seeding of colon epithelial cells on one side of the membrane and undifferentiated non-adherent monocytes on the other, allowing for the synchronous progression of colon epithelial polarization, mucus formation, and monocyte-macrophage differentiation and polarization within a single co-culture without the need for detaching and replating pre-differentiated macrophages. This system is easy to construct and only requires a few readily available materials and tools. We tested its utility by analyzing the responses of co-culture components to sepiapterin (SEP), the endogenous precursor of BH₄ (tetrahydrobiopterin) -- a cofactor of nitric oxide synthase (NOS). We demonstrated that SEP treatment of 3D Flipwell induced biofilm formation by gut bacteria, mucus production by colon epithelium, and pro-immunogenic polarization of macrophages. This indicated that SEP triggered activation of a mucosal defense mechanism through crosstalk between different cellular components. Further experiments are needed to investigate the role of mucosal communications that lead to immune cell reprogramming and to evaluate the utility of this co-culture system to screen for novel immunomodulatory therapies.

Introduction

Immune cell-gut microbiome crosstalk greatly influences the efficacy of different disease treatments. Such a crosstalk is markedly modified by external stimuli, including but not limited to host metabolic abnormalities, dietary changes, disease manifestation, and pharmacologic treatment. These changes could lead to an imbalance in the microflora composition, a condition termed dysbiosis^1,2,3.

To better understand the interactions between microbes and host immune cells in the gut, elaborate co-culture systems have been developed by different groups in an attempt to recapitulate the gut mucosal environment. From the gut-on-chip system developed by Kim et al.⁴ to a more complex assembly using a specialized absorbent substrate⁵, these co-culture systems are not simple to build and are unable to retain the full spectrum of secreted components due to their absorption by the co-culture substrate. Noel et al. thus developed an alternative co-culture system to model the gut mucosa that is relatively easier to fabricate⁶. This new system utilizes a single culture insert with colon epithelial cells seeded on one side of the membrane and pre-differentiated macrophages on the other side⁶. However, seeding pre-differentiated macrophages requires their pre-differentiation somewhere else and their dissociation and replating into a co-culture, potentially affecting their cellular integrity and viability.

By incorporating improvements to these previous systems, we developed a novel three-dimensional (3D) multi-layered co-culture system that we termed the 3D Flipwell system^7,8. This system is composed of cell culture inserts stacked back-to-back, allowing for the synchronous progression of colon epithelial polarization, mucus formation, macrophage differentiation, and polarization within a single co-culture. This system enables us to examine changes in the gut mucosal environment and interactions between gut bacteria, epithelial cells, and immune cells. Additionally, this system can be used to examine the effects of external stimuli, including bacterial metabolites, toxins, dietary nutrients, and pharmaceuticals, on the gut mucosa and the rest of the body^7,8.

By utilizing the novel co-culture insert system, we examined the responses of different cellular components to sepiapterin (SEP), the endogenous precursor of BH₄ (tetrahydrobiopterin)-a cofactor of nitric oxide synthase (NOS). We previously showed that SEP exerts pro-immunogenic activities on the breast tumor microenvironment by reprogramming tumor-associated macrophages to a pro-immunogenic type^9,10,11,12. We demonstrated that SEP induces synchronous events of biofilm formation by gut bacteria, mucus production by colon epithelium, and pro-immunogenic polarization of macrophages in co-cultures, indicating activation of a mucosal defense mechanism⁷. These findings support the utility of this new co-culture system in modeling the gut mucosal microenvironment⁷.

The overall goal of this protocol is to provide step-by-step instructions for the construction of this new co-culture of gut bacteria, gut epithelia, mucus, and immune cells to model the gut mucosal environment. We also demonstrate how we could visualize the crosstalk of co-cultured cells under the influence of an external stimulus, for example, SEP treatment. Further research utilizing this co-culture system is needed to determine secreted cellular components that facilitate the interactions between different cells in co-culture, as well as to test the utility of this co-culture model for high-throughput screening of new therapeutics. Furthermore, to attain the physiological condition of the gut mucosal environment, this co-culture system would require further modifications. For example, to co-culture aerobic mammalian cells with anaerobic bacteria, these bacteria would need to be separately cultured in anaerobic media that remove oxygen or overlain with liquid paraffin that prevents oxygen entry, as reported elsewhere. Such advanced utility and application of this co-culture system would warrant further investigation.

Protocol

1. 3D Flipwell engineering

1. All work should be done inside a cell culture cabinet. Place all supplies inside the cabinet.
2. Open the Petri dish cover and set aside. Open the scalpel blade from its protective casing and place it on the edge of the Petri dish bottom. Ensure sterility.
   CAUTION: Careful handling of the scalpel blade is required. Use cut resistant, fiber reinforced gloves to protect hands if needed.
3. Open the sterile protective packaging of the 12-well commercial insert. Ensure sterility. Take a set of sterile tweezers. Gently grab the insert and remove it from the packaging.
4. Hold the insert so its bottom is facing up. With the other hand, take the sterile scalpel blade and bring it up to the insert.
5. Using a C-shaped motion, pierce the membrane at the edge and gently slide the scalpel blade around the insert bottom, staying close to the plastic wall.
6. Cut the PET membrane out and use the second set of tweezers to remove the membrane. Discard the membrane.
7. Use the scalpel blade to scrape any white membrane shavings to clean the edge and rim of the insert. Carefully set the newly prepared insert back into its original sterile pack.
8. Next take the silicone adhesive tube and cut off the nozzle tip with the scalpel blade to make an opening. This opening should be 3 mm wide.
9. Test the bead size by squeezing the glue out on the paper towel. The bead should be 2-3 mm thick. Adjust the nozzle if the bead is less than 2 mm and is hard to squeeze out of the tube.
   1. Another way to apply and spread the glue bead is to use a 200 µL pipette tip with its tip cut off. Trim off 3-4 mm off with scissors and use a spatula to spread the glue around the rim of the insert.
10. Peel the protective cover from the second insert and set aside. Pick up sterile forceps, grab the second insert from its packaging with the membrane facing up.
11. Gently spread a thin 2-3 mm bead of silicone all the way around the insert bottom, being careful not to touch the membrane.
12. Pick up the second set of sterile forceps and carefully lift the first insert (without the membrane) out of its protective pack.
13. Bring the two inserts together bottom-to-bottom so that the bottom rims align. Excess glue may be forced out of the joint. Leave the glue to dry. It can be cut off after the glue is completely dried (24-72 h). This is optional.
    NOTE: The final Flipwell Co-culture Insert (FCI) stack should only have one PET membrane in the middle. It is best to have the L-shaped handles of the commercial inserts positioned such that they are located 180° away from each other (at 3 and 6 o'clock). Follow silicone glue manufacturer's recommendations for drying time, which may be 24-72 h depending on the brand. For most silicone-based glues, a 72 h drying time is preferred. The glue can also be dried for only 24 h with UV sterilization.
14. When the FCI stack is glued, place it inside the original sterile pack to dry or inside a deep Petri dish. Use the lid from the specified Petri dishes to cover the stack assembly.
15. To sterilize the stack assemblies, open the Petri dish lid. Use sterile forceps to hand the FCI stack on the rim of the specified deep Petri dish rim.
16. Close the glass sash of the BSC cabinet. Turn on the UV light. Step away from the cabinet for safety and sterilize each side of the membrane 30 min-1 h. Turn off the UV light when each sterilization cycle is complete.
17. Place the Petri dish lid (as specified) over the Petri dish with the insert stacks and set aside.

2. Testing for leakage prior to collagen coating

1. Remove the Petri dish lid. Add 500 µL of sterile PBS or sterile deionized water.
   NOTE: For visual aid, DMEM was used to aid in visualizing the FCI stacks' integrity.
2. Cover the Petri dish. Let it rest overnight. Check the FCI integrity in the morning. Discard the FCIs if leakage is detected. Make new insert stacks as needed.
3. Aspirate the liquid (PBS or water) used for testing. Cover the Petri dish with a lid.
4. Allow the stacks to dry (1-2 h) inside the BSC. Store the FCIs in a BSC until ready to use or proceed to coat them with collagen for seeding the adherent cells.

3. Collagen coating of the Flipwell membrane

1. Prepare a working stock of the collagen solution by diluting collagen solution supplied at 3 mg/mL to 1:30 dilution at 100 µg/mL. Add 33 µL of commercially made collagen solution to 1 mL of sterile deionized water in a microcentrifuge tube. Mix by vortexing.
   NOTE: For a larger volume, scale up and use 15 mL conical tube depending on the number of insert stacks to be coated on both sides of the membrane. Make this fresh each time. Do not reuse the working stock.
2. Open the Petri dish lid. Let the Flipwell hang off the Petri dish rim. Carefully add 200 µL of collagen solution to one side of the insert stack.
3. Let it sit 1 h. Aspirate the collagen solution. Add 200 µL of sterile PBS. Aspirate PBS.
4. Cover the Petri dish with lid and let the membrane dry for 60 min inside the cabinet. When the membrane is dry, take the sterile forceps/tweezers and flip the Flipwell to the opposite side and let it hang off the Petri dish rim.
5. Add 200 µL of collagen solution to the uncoated (now top) side of the membrane. Let it sit 1 h.
6. Aspirate the collagen solution. Add 200 µL of sterile PBS. Aspirate PBS. Cover the Petri dish with a lid and let the membrane dry for 60 min inside the cabinet. Allow the FCI stack to dry overnight or seed the same day.
   NOTE: The integrity of the collagen coating was not tested under long storage conditions. Once the Flipwells are precoated, they must be used within 24 h.

4. Bacterial insert construction for bacterial cultures

1. Place two sets of sterile forceps and the 24-well inserts inside the BSC. Carefully open the inserts and leave them inside their sterile packaging.
2. With sterile tweezers, lift the insert from its sterile pack. With the second set of tweezers/forceps, break off the small plastic feet at the bottom of the insert.
3. Test the 24-well insert by fitting it inside one of the sterile FCI stacks or original 12-well inserts.
4. If the insert is not sliding into the well, use tweezers again to break off more plastic material from the rim where the rim and feet meet.
5. Test the fit again until the 24-well insert is able to slide inside the 12-well insert well. Place the insert either inside its sterile pack or inside a sterile Petri dish.
6. UV sterilize or ethanol the insert (optional). Cover the pack back up or cover with the Petri dish lid until ready to use.

5. Needle construction

1. Bring the tools and all supplies inside a BSC. Hold a needle carefully with the nose pliers.
2. Measure the location of each bend on the needle using a ruler. With the second set of nose pliers, grip the needle at the selected location and bend the needle.
3. Create the second bend. To twist the needle, grip the needle with pliers at the base and at the U-shaped bend and twist in the opposite direction to create a 4-5 mm twist. This step is critical to easily insert the needle inside the Flipwell for the air bubble removal.
4. Affix the needle to a sterile 3- or 5-mm syringe. Sterilize the needle and the syringe base in 20 mL of 70%-95% ethanol inside a 50 mL conical tube.
5. Pull ethanol through the needle and syringe to ensure the needle and base of syringe are sterilized.
6. Remove the syringe and needle from the conical tube with ethanol and let air dry by resting on a Petri dish. Purge the needle with air to dry and leave it to dry for a few minutes.

6. Seeding the colon epithelial Caco-2:HT29-MTX-E12 cell lines

1. Start culturing Caco-2 and HT29-MTX cell lines 2-3 weeks ahead of the experiment in separate 100 mm TC-coated dishes in their respective growth media.
   1. For HT-29-MTX media, use 440 mL of DMEM with high glucose (4.5 g/L) and 1% (v:v) Glutamax media with 50 mL of 10% (v:v) heat-inactivated FBS, 5 mL of 1% (v:v) Pen Strep, and 5 mL of HEPES 10 mM final concentration.
   2. For Caco-2 media, use 440 mL of DMEM with high glucose (4.5 g/L) and 1% (v:v) Glutamax media with 50 mL of 10% (v:v) heat-inactivated FBS, 5 mL of 1% (v:v) Pen Strep, and 5 mL of nonessential amino acids.
      NOTE: Determine the seeding density for the Caco-2 cell line using tissue culture coated 12-well plate wells. Calculate number of days to 100% confluency depending on the starting seeding density. We provide a seeding density as a guideline; however, that will vary with the growth media formulation, vendor, and FBS used. This step is critical to ensure a monolayer for the gut epithelial side of the Flipwell after THP-1 cells are plated and bacterial/SEP treatments are performed.
2. To passage Caco-2 and HT-29 cells, aspirate the culture media. Wash with 10 mL of PBS.
3. Trypsinize the cells by adding 2 mL of 0.25% Trypsin-EDTA. Incubate 5-10 min at 37 °C with 5% CO₂.
4. Add 7 mL of the proper-to-the-cell-type growth media to the plates. Transfer cells to separate sterile 15 mL conical tubes.
5. Pellet by centrifugation for 5 min at 500 x g at room temperature. Aspirate the supernatant without disturbing the cell pellet.
6. Resuspend the pellet in 1 mL of pre-warmed DMEM-based HT-29 media. Count cells with a hemacytometer or a cell counter.
7. Combine the two cell types Caco-2:HT-29 cells with a ratio of 9:1 in a new 15 mL or 50 mL conical tube, depending on the number of cells needed for the desired number of Flipwells.
8. Use collagen-precoated Flipwells for the steps below. Hang the collagen-precoated FCI stacks from the rim of the deep Petri dish.
9. Seed 500 µL of 7.5 x 10⁴ cells per cm² (2.6 x 10⁵ cells/12 well insert) per Flipwell's apical side. Calculate starting cell counts based on the number of stacks being used. This will include both cell types at a 9:1 ratio.
10. Cover the assemblies with a Petri dish lid. Incubate at 37 °C with 5% CO₂ overnight until the cells attach. The cells begin to produce mucus around 7-10 days in culture. Change media every other day to maintain cultures.
    NOTE: Total days in culture with treatments need to be calculated ahead of time. The flip and initial seeding density will depend on that duration.

7. Flipping the 3D Flipwell in preparation for seeding THP-1 cells

1. When Caco-2:HT-29 cells attach and begin to proliferate (the exact confluency needs to be estimated based on the media/FBS used and duration of culture) but are not at 100% confluency, flip the Flipwell to seed THP-1 cells on the other side of the membrane.
   NOTE: THP-1 cells need to have been expanded in a separate culture by this point, and the exact cell numbers needed will be estimated based on the number of FCI stacks used.
2. Open the Petri dish lid and aspirate the DMEM media from the apical (gut epithelial) side of the FCI stack.
3. Using sterile forceps, pick up the Flipwell carefully by the hook and rotate 180°. With the second set of sterile forceps, grab the FCI stack by the other hook that is now facing upward.
4. Lower the assembly back into the Petri dish and hang the hook over the Petri dish rim. If not ready to seed the THP-1 cells, add enough media to the deep Petri dish to bring the media level up and just to the top of the membrane.
   NOTE: The Flipwell should be stable in this position, and seeding with THP-1 cells and media addition should be considered right away now. These steps are done quickly. THP-1 cells need to be counted and ready to be plated. The THP1 cells were seeded first to weigh down the FCI stack for greater stability and final weight. DMEM media was then added to the deep Petri dish to bring the media level up to the underside of the membrane. The custom-made needle was then used to remove the air bubble, which further stabilized the FCI stack and allowed for the final volume adjustments.
5. Use the needle to remove the air bubble as described in the following step.

8. Air bubble removal

1. Presterilize the needle and syringe in ethanol and air dry prior to use (see step 5). Plunge the syringe plunger.
2. Hold the FCI stack by the arm with sterile forceps. Carefully lower the needle in and under the insert assembly and very gently place the gavage needle soft tip up to the air bubble.
3. Carefully and very slowly pull the syringe plunger up and watch the air bubble disappear slowly. Stop when the air bubble is about 2 mm in diameter, which is enough to retain good DMEM coverage for the colon epithelial cells.
4. Carefully remove the needle and syringe from the Flipwell. Proceed to the other insert assembly in the deep Petri dish.

9. THP-1 seeding and PMA treatment

1. Make THP1 media with 420 mL of Roswell Park Memorial Institute (RPMI)-1640 medium, 50 mL of 10% (v:v) heat-inactivated FBS, 5 mL of Glutamax 1% (v:v), 5 mL of HEPES (10 mM final concentration), 11 mL of Glucose (4.5 g/L final concentration), and 5 mL of Pen-Strep (1% v:v).
   NOTE: THP-1 cells need to be grown in RPMI-based culture medium for several days before seeding in the inserts. Make cell count calculations ahead of seeding and scale up based on the number of FCI stacks to be used.
2. Prepare the cell suspension based on 500 µL volume and at 0.5 x 10⁶ cells per 500 µL of THP-1 media. If PMA needs to be used for differentiation and polarization, add it to the RPMI media at 100 ng/µL.
   1. To make PMA, reconstitute 5 mg phorbol myristate acetate (PMA) in 1 mL of DMSO to make 5 mg/mL stock solution. Mix by vortexing. Aliquot into microcentrifuge tubes at 20 µL each and store at -20 °C.
   2. To use PMA, make a working stock at 50 µg/mL by diluting 1 µL of 5 mg/mL stock in 99 µL (1:100) of complete growth media. Add 1 µL of working stock to 500 µL of media, or 20 µL of working stock to 10 mL of media.
3. Centrifuge THP-1 cells at 94 x g for 5 min at RT to pellet. Resuspend in 5-10 mL of RPMI media. Count THP-1 cells with any preferred counting method.
4. Centrifuge again at 94 x g for 5 min at RT. Resuspend at 0.5 x 10⁶ cells per 500 µL of THP-1 media.
5. Add 500 µL of THP-1 cell suspension to the apical side of the co-culture insert stack. Adjust HT-29 media for the colon epithelial cells by adding media to the deep well Petri dish.
6. Remove the air bubble with a gavage needle as per step 8, if needed. Culture both cell types in the cell culture incubator at 37°C, 5% CO₂, 100% humidity until THP-1 cells attach (overnight) and proliferate for 1-2 days.
7. Prepare to either do treatments with SEP or other drugs or prepare for bacterial inserts (see step 4). Start culturing bacteria 1 day before the next FCI stack flip.

10. Second Flipwell Flip and air bubble removal for SEP and bacterial inserts

1. Open the Petri dish lid. Aspirate the RPMI media from the apical side. Hold the Flipwell by the arm with sterile tweezers and lift the stack.
2. Rotate the stack upside down. Using the second set of tweezers, hold the stack assembly the available arm.
3. Lower the stack into a new sterile deep well Petri dish and hang over the rim. Add 500 µL of DMEM media to the now epical/gut epithelial side.
4. Add enough RPMI media to the deep Petri dish until the media is just to the top of the membrane. The RPMI volume was estimated to be about 40-55 mL, but it will change depending on the number of FCI stacks used per deep Petri dish.
5. Remove the air bubble using step 8 leaving 2-3 mm bubble. Adjust RPMI media if needed. Cover the plate.
6. Culture until the colon epithelial cells begin to produce mucus. Change the media partially every other day.
   NOTE: If the colon epithelial cells were cultured long enough and have already produced mucus, they can proceed to the bacterial insert or treatments. Do not allow the media in the lower compartment to get below the insert's membrane underside. Add more DMEM media to keep the media level at the membrane. The ideal method for changing media is to remove half of the media from the basolateral compartment and leave a small volume of media in the apical compartment to prevent air infiltration.
7. For the SEP treatments, add SEP to the final concentration of 100 µM to wells at this time if the bacterial culture is not used. If bacterial culture and insert are used, add SEP to the bacterial culture. For the bacterial culture, see step 11.
   1. To make SEP, add 100 mg L-Sepiapterin to 84.3 mL of DMSO and mix by vortexing. Aliquot into microcentrifuge tubes at 100 µL each. Store at -20 °C. Use SEP aliquots at 1 µL per 500 µL media per insert assembly (final concentration of 100 µM).

11. Starting the bacterial culture (optional)

1. To study bacterial metabolites' effect on the immune cells, mucus and colon epithelium, add a bacterial insert to the apical side of the insert assembly. For bacterial insert construction follow step 4. We cultured Bacillus subtilis the day before the bacterial insert use.
2. Make and autoclave the Miller LB 2 days prior to bacterial insert use. To make LB, combine 6.25 g of Miller LB with 250 mL of deionized water in a 500 mL glass flask. Cover with aluminum foil. Autoclave, cool down to RT. Can store sterile LB at either 4 °C or RT.
3. Start growing Bacillus subtilis bacteria from glycerol stock the day prior to bacterial insert use.
4. To start the bacterial culture, inoculate 10 mL of Miller LB with Bacillus subtilis from a frozen glycerol stock inside a sterile 50 mL conical tube. Do not let the glycerol stock thaw.
5. Using a sterile 200 µL tip, pick a small amount of bacteria from the glycerol stock. Eject the tip into 10 mL of LB in the sterile 50 mL conical tube.
6. If using SEP, start treatment with 20 µL of SEP stock (the final concentration should be 100 µM). Add directly to the 10 mL bacterial culture in the 50 mL conical tube starter culture.
7. Culture Bacillus subtilis for 8 h in a shaking incubator at 220 rpm and 37 °C. Add the second treatment of SEP at the end of the day - add 20 µL of SEP to the final concentration of 100 µM to the 10 mL of LB with bacteria.
8. Culture overnight at 220 rpm, 37 °C. Add the third treatment of 20 µL of SEP to 10 mL of LB with Bacillus subtilis the following morning and continue shaking until ready to do the bacterial inserts.

12. Addition of bacterial insert to the Flipwell (optional)

1. Perform bacterial insert construction as described in step 4. Perform bacterial culturing as described in step 11.
   NOTE: If a separate cell culture cabinet is available, that will work best. Otherwise, divide the work area inside a cell culture cabinet in half. Maintain bacterial and cell cultures apart. The cell cultures should not be returned to the cell culture incubator. The BSC will need to be sterilized and exposed to UV for 24 hours or longer after this method is performed.
2. Use two pairs of sterile forceps for the steps below. All steps below are to be done in the BSC.
3. With forceps, lift the pre-made sterile 24-well insert from its packaging. Set the insert inside a sterile Petri dish and cover the lid.
4. Repeat for the number of bacterial inserts needed. Transfer the Petri dish with the Flipwells from the incubator into the BSC and set aside on the opposite side of the dish with bacterial inserts.
5. Take out 300 µL of the DMEM media from the apical (top) side of the Flipwell to allow for the bacterial insert. Leave about 200 µL in the FCI stack's top side. Place the Petri dish lid over the insert assemblies.
6. Add 50-100 µL of B.subtilis culture to the 24-well bacterial inserts. Take care not to spill or contaminate the outer wall and rim of the bacterial insert. OD600 can be taken to monitor the culture's growth phase.
7. With sterile forceps, lift the bacterial insert with culture and carefully insert it inside the Flipwell top side. Rotate the arm and hang it from the rim of the FCI stack. Hold the stack with the second set of sterile forceps.
8. Repeat for the rest of the FCI stacks. Cover the Petri dish and rest inside the BSC 3 h. Do not bring inside the incubator.
9. After 3 h, use sterile forceps to remove the bacterial insert from the Flipwell. Repeat for the rest of the insert assemblies. Discard the bacterial inserts.
   NOTE: Discard bacteria and bacterial inserts per the institution's biohazardous waste disposal policies and safety and health guidelines. Bacterial components or bacteria itself were not examined.
10. To analyze changes to the mucosal, epithelial and immune cellular compartments, proceed to washing the membranes and preparing them for imaging.
11. Aspirate the DMEM media from the Flipwell top. Wash with 500 µL of sterile PBS. Using forceps, lift the FCI stack out of the Petri dish and transfer it into a new Petri dish.
12. Flip the Flipwell and hang it over the Petri dish rim. Wash the membrane with 500 µL of sterile PBS.
13. If storage is required, store the FCI stacks short term (30 min-overnight) in sterile PBS inside a 50 mL conical tube.
    NOTE: It is important to determine which imaging method will be used next and if the membrane will be cut in half for both electron microscopy and fluorescent antibody labeling.

13. Taking the Flipwell apart for scanning electron microscopy (SEM)

NOTE: To examine morphological changes in colon epithelial cells and macrophages after SEP/bacterial treatments, we fixed the membranes in a fixation buffer and then disassembled the Flipwells for scanning electron microscopy (SEM). PFA was used as a fixative; however, glutaraldehyde can also be used. It is important to consult with the Microscopy Core about their preferred fixation buffer, specific dehydration steps, and the initial fixation buffer concentrations. Membranes can be stored in the fixation buffer if storage is necessary. The work can be done outside the BSC. If the Imaging Core will be gluing the membrane down, it can be cut in half and positioned to image both sides separately.

1. Using a solvent-proof marker, label the FCI stack's plastic sides with a symbol coding for the cell type used. Alternatively, label the membrane directly with either a dot, a check mark, or an x to label the cell type used.
2. Place the Flipwell inside a 50 mL conical tube without disassembly. Add 4% PFA enough to submerge the stack. Keep in 4% PFA overnight.
   1. To make 4% PFA (methanol free), dissolve 40 g paraformaldehyde in 800 mL of 1x PBS. To make 0.4% PFA, dissolve 4 g paraformaldehyde in 800 mL of 1x PBS or dilute 4% PFA 1:10 with 1x PBS by adding 100 mL of 4% PFA to 900 mL of 1x PBS.
3. Exchange PFA for 0.4% for longer storage. Consult the Microscopy Core for storage duration and buffers.
4. To disassemble the Flipwell, hold it with both hands and twist off each glued part of the stack. The stack will disassemble into two parts: one part will have the membrane and the other will be the insert without the membrane. Discard the insert without the membrane.
5. Rotate the insert with the membrane and set it down with the membrane facing up. Can label the membrane with a small dot or x at the edge of the membrane to know which side has what cell type.
6. Hold the insert by the wall with forceps. Carefully cut out the membrane with a scalpel blade and place it inside a 1.7 mL tube or a small glass bottle with 500 µL-1 mL of 0.4%-4% PFA solution.
7. Submit to microscopy core for the dehydration steps and imaging. Dehydration steps are described in Beamer et al⁷.
8. Discard PFA or other fixatives per the organization's prescribed hazardous waste disposal policies and health and safety guidelines.

14. Large droplet method for taking the Flipwell apart for confocal microscopy

NOTE: A new method was created to stain and image the membranes. The co-culture insert stack was kept intact with initial fixation, permeabilization and blocking steps done inside a 50 mL conical tube. Immunofluorescent labeling and confocal microscopy were used to image both sides of the membrane. The newly termed 12-well plate method was used for the overnight incubation steps, and a large droplet method was used to stain the bottom of the disassembled insert. Confocal Z-stack was used for imaging both sides with the membrane. The stained membranes were sandwiched between two circular glass coverslips, and the sandwiched assembly was placed in the middle between two metal rings of the commercially made round chamber to image. Multi-photon laser scanning confocal microscope was used with commercial imaging software.

1. To wash the membrane, place the co-culture insert stack directly into a 50 mL conical tube and add 15 mL of sterile PBS or wash each side separately (described below).
2. Aspirate the DMEM and add 300 µL of sterile PBS to the apical side. Aspirate PBS and carefully remove the stack from the Petri dish.
3. Flip the Flipwell and add 300 µL of PBS to the now facing upward RPMI of the FCI stack. Aspirate the PBS.
4. Using a solvent resistant marker label the stack's top and bottom side to know which side has which cell type.
5. Place the Flipwell inside a 50 mL sterile conical tube. Add 15 mL of sterile 4% PFA as the initial fixation buffer for 10 min. If overnight incubation is needed, exchange for 15 mL of 0.4% PFA in PBS, cover and leave overnight.
6. To go ahead with the immunostaining, make a bench space for the large droplet method.
   Tear off 10 x 14 inch pieces of plastic wrap and tape it to the bench at corners to create a smooth surface.
7. Using a 200 µL pipet create a large droplet with 200 µL of PBS. Twist off the insert assembly to separate into the inserts.
8. Using a solvent resistant marker, place a small dot close to the edge of one side of the membrane to know which cell type is used for staining and imaging.
9. Remove any residual silicone gasket from the insert-bottom if the insert with the membrane still has any residual glue. This is done to ensure that the insert will stay upright for the steps below.
10. Carefully position the FCI stack (with THP-1 cells) on top of the 200 µL large drop of PBS. Add 200 µL of permeabilization buffer to the top side with colon epithelial cells and keep for 10 min.
    1. To prepare the permeabilization buffer, use 0.1% v/v Triton-X100 in PBS.
11. Aspirate and add 200 µL of PBS. Repeat this wash 2x. Create a new droplet with 250 µL of blocking buffer.
    1. To make blocking buffer, add 250 µL of normal goat serum in 5 mL of 1x PBS (final 5% serum). Add 15 µL of Triton-X100 (final 0.3% v:v). Mix well and store at 4 °C for 1-2 days.
12. Place the insert on top of the blocking buffer. Block for 30 min at room temperature. Aspirate PBS from the apical side and add 200 µL of blocking buffer to the top of the insert. Both sides of the membrane are now ready for antibody staining overnight.

15. Immunofluorescent (IF) staining

NOTE: Staining is done overnight using a 12-well plate method or on bench using a large droplet method.

1. Prepare necessary antibodies to be used for staining both sides of the membrane overnight at 4 °C or on the bench at RT. Dilute all antibodies 1:100 in 1% BSA in PBS. Alternatively, follow the manufacturer's recommendations and optimize staining to determine ideal concentrations and antibody dilution buffer.
2. Large droplet method
   1. Create a large rectangle using a plastic wrap on top of the bench and tape the corners down. Pipet 200 µL of antibody diluted on top of the plastic to create a large droplet. Place the insert membrane down on top of the large droplet.
   2. Add 200 µL of primary Ab to the top of the insert. Incubate at RT for 2 h.
3. 12-well plate method
   1. If using a 12-well plate method overnight, add 300 µL of primary antibody to the top of the insert.
   2. Add 300 µL of primary antibody to the well of the 12-well plate. Place the insert inside the 12-well plate well on top of the droplet. Cover the plate with a lid and place it at 4 °C overnight.
4. When staining is completed, wash both sides of the membrane with PBS. Using a pipette and a 200 µL tip, carefully rinse the bottom side of the insert 3x with 200 µL of PBS. For the top of the insert, wash by adding 200 µL of PBS to the top, aspirating, and repeating washing 3x.
5. Prepare all secondary antibodies (please see Table of Materials for the specific antibodies used) per manufacturer's recommendations or 1:1000 dilution in 1% BSA in PBS or other. Secondary antibody staining needs to be done in the dark; use a cardboard box or a drawer of a cabinet to create a light-free environment.
6. Using either a large droplet method or a 12-well plate method, prepare a new sterile plate. Incubate for 2 h at RT in a dark environment. Wash as described in 15.4.

16: Preparation for confocal imaging

1. If the membrane has not been labeled yet as described in step 14, label it to know cell type location. Place a small mark (dot, x, check, etc.) with a solvent-resistant marker at any location (edge preferred).
2. Remove the round cover slips carefully from the storage container and gently set them on the bench surface.
3. Dilute DAPI in PBS at 1:100 dilution. For dispensing DAPI with the droplet dispenser, add 1-3 droplets to the middle of the cover slip. Otherwise, pipette 1-3 droplets of DAPI into the middle of the cover slip.
4. Hold the insert with forceps. Carefully cut out the membrane with a scalpel blade in a circular motion all the way around the rim/bottom edge. Do not let the membrane fall onto the bench. Set the plastic insert down on the bench and hold the membrane with forceps while cutting the final fibers off with the scalpel blade.
5. Place the insert down on top of the DAPI. Dispense 1-3 droplets of DAPI (1:100 dilution) to the top of the membrane so both sides of the membrane have DAPI.
6. Lift up the second-round cover slip with fingers (wear PPEs). Do not use forceps as the cover slip is very fragile.
7. Align the second cover slip over the first cover slip and carefully lower it on top of the membrane.
8. Using a solvent-resistant marker, carefully label the cover slip side if needed to code the cell type/orientation (optional). If the membrane was labeled, skip this step.
9. Depending on the number of the coverslips to be imaged and the number of metal rings available, either mount the coverslips between the commercially made metal rings to hold the round coverslips together for imaging or store them in the dark until imaging.
10. To image, mount the cover slip with the membrane between the metal rings of the cell chamber and place it into the confocal microscope holder. Contact the confocal microscopy core prior to see what holder may be needed to image the round cover slips.
11. When ready to image, wrap each sandwich in an aluminum foil and take it to the facility.
12. Using the Z-stack feature, scan each side of the membrane individually using the proper wavelength depending on the fluorophores used. Flip and image the second side. To store the coverslips, keep them in the dark at 4 °C.

Results

To determine the biological relevance of Flipwell co-cultures of gut bacteria, colon epithelia, and macrophages, we treated the co-cultures with sepiapterin (SEP), a modulator of arginine metabolism, to promote the production of nitric oxide^9,10,11 in comparison to the control DMSO. We determined the effects of SEP treatment on each component of co-cultures using immunofluorescent (IF) microscopy and scanning electron microscopy...

Discussion

To recreate gut mucosal architecture in vitro, we have designed a simple co-culture system that comprises stratified layers of gut bacteria, mucus-producing colon epithelial cells, and macrophages. This multi-co-culture system has allowed us to study the effects of crosstalk between the bacterial secretome, gut epithelia, and macrophages on inducing immunogenic reprogramming and mucosal defense mechanisms.

One of the important cautions for creating 3D Flipwells is that the assembly mu...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.4% PFA (see recipe in Reagents and Solutions)	described in text
10-ml serological pipette	Corning	4488
12 well TC Plate,  Costar	Corning	3513
15-ml conical tubes, SpectraTube	Alkali Scientific	C2715
200 µl tips	USA Scientific	1111-1700
24-well inserts - Culture Inserts for 24-well plates, PET 0.4 µm	BrandTech Scientific	782710
3D Flipwells pre-made, UV sterilized	Make in lab
4% PFA (see recipe in Reagents and Solutions)	described in text
50 mM Sepiapterin stock (SEP, See recipe in Reagents and Solution)	Career Henan Chemical Co	CAS#: 17094-01-8
50-ml conical tubes	Greiner Bio-One	227261
5-ml serological pipette	Corning	4487
70-95% Ethanol (Ethyl Alcohol)	Decon Labs	04-355-151
Aluminum foil	Fisher Scientific	01-213-100
Animal Feeding needles, 20 gauge 1.5” length, single use	Fisher Scientific	01-208-87
Antibody Dilution Buffer (see recipe in Reagents and Solutions)	described in text
Attofluor Cell Chamber	ThermoFisher Scientific	A7816
Autoclave tape	Fisher Scientific	15-901-111
Bacillus subtilis wt (3A1T) strains in glycerol stock ()	Bacillus Genetic Stock Center (https:// bgsc. org/
BioGrip Pink Nitrile powder free latex free gloves	Top Quality Manufacturing	P2810
Blocking Buffer (see recipe in Reagents and Solutions - 10% Normal Goat Serum in PBS)	described in text
Blocking Buffer (see recipe in Reagents and Solutions)	described in text
Brightfield microscope (Nikon or equivalent) Eclipse Ts2	Nikon	TS2
Caco-2 cells	ATCC	HTB-37
Cell culture incubator, 37°C, 5% CO2 (Eppendorf CellXpert)	Eppendorf	6734010015
Centrifuge (Eppendorf, Centrifuge 5804/5804 R or equivalent	Eppendorf	22622501
Centrifuge tubes 50 ml SpectraTube	Alkali Scientific	C2750
Culture inserts for 12-well plate, PET, 0.4 µm, sterile	BrandTech Scientific, BRAND Insert 2in1, BIO-CERT	782730
DAPI VectaShield mounting medium	Vector Laboratories	H-1000
Deep petri dish bottoms, 100 x 25 mm Deep Polystyrene Stackable Petri Dish, sterile	USA Scientific	8609-0625
Drummond Pipet Aid XP	Drummond	4-000-101
Eppendorf 5804 Centrifuge with a swing bucket rotor for 50 ml conical tubes	Eppendorf	22622501
Forceps, stainless steel, sterile autoclaved, 2x sets	VWR	82027-438
Forceps, stainless steel, sterile autoclaved, 2x sets, Specimen Forceps	VWR	82027-438
Glass flask 500 ml	Corning	4980-500
Hemacytometer (Neubauer with cover slips)	Thermo Fisher Scientific	50-312-90
HT-29-MTX-E12 cells	Millipore Sigma	12040401
ImmunoPen (optional)	Fisher Scientific	4021761EA
Kevlar-based or other cut-resistant gloves (optional)	Hardware store or Amazon
Lab tape or shipping tape	Fisher Scientific	159015R
Laminar Flow hood 1300 Series A2 BSC Class II	ThermoFisher Scientific	1375
Laminar flow hood with UV light SterilGARD	The Baker Company	SG403A-HE
Markers solvent proof	VWR	52877-310 or 95042-566
Media for Caco-2 cells (DMEM High Glucose-based, see recipe in Reagents and Solutions)	described in  text
Media for HT-29 cells (DMEM High Glucose, see recipe in Reagents and Solutions)	described in text
Media for THP-1 cells (RPMI, see recipe in Reagents and Solutions)	described in text
Microcentrifuge tube, 1.7 ml, sterile	Alkali Scientific	C3017-ST
Microcentrifuge tubes	Thermo Fisher	3451
Miller LB media (see recipe in Reagents and Solutions)	Sigma-Aldrich	L3522
Needle nose pliers, 2x sets	Hardware store or Amazon
Nitrile powder free latex free gloves Aurelia Amazing	MidSci	92885
Petri dish lids, sterile Falcon Petri Dish sterile 100x15 mm style	Corning Inc	351029
Phorbol myristate acetate (PMA) (100 ng/μl, see recipe in Reagents and Solutions)	InvivoGen	Tlrl-pma
Pipet tips (10 µl, 200 µl, 1000 µl) PurePoint or equivalent	Alkali Scientific	ST1010-CS, ST1200-CS, ST1000-CS
Plastic wrap	Grocery store
Purecol working collagenase solution (100 µg/mL)	Advanced BioMatrix	5005-100ML
Round cover slips #1 1 oz (Corning Float Glass or Fisherbrand)	Corning or Fisherbrand	50143822 or 12-546-2 25CIR-2
Scalpel blades, sterile, disposable	Mopec or Feather #10	AJ136 or 2975#10
Sepiapterin (SEP, 100 µM, see recipe in Reagents and Solution)	Career Henan Chemical Co	CAS#: 17094-01-8
Shaking incubator set for 220 rpm 37°C	Eppendorf New Brunswick Scientific	M12990080
Silicone Adhesive Loctite Clear Silicone IDH or 3M Marine Grade Silicone Sealant Clear with a nosetip	Loctite or 3M	1745658 or PN08019
Single channel pipet Eppendorf Research Plus, 200 µl	Eppendorf
Single channel pipettes (10 µl, 200 µl, 1000 µl)	Eppendorf	2231001122
Spectrophotometer NanoDropOneC	Thermo Fisher Scientific	13-400-519
Sterile deionized water
Sterile phosphate buffered serine (PBS) without calcium or magnesium	Fisher Scientific	BSS-PBS-1X6
Syringe 3 ml or 5 ml with Luer-Lok Tip	BD	309585 or 309603
THP-1 cells	ATCC	TIB-202
Transfer pipets, sterile	Fisherbrand	13-711-20
Triton X-100	Millipore Sigma	X100
Trypan blue solution	VWR	97063-702
Trypsin-EDTA 0.25%	Thermo Fisher Scientific	25200-056
Wipes Kimwipes Professional with LintGuard Anti-Stat Polyshield	Kimberly-Clark	34120
Primary Antibodies:
Anti-human CD163	Abcam	156769
Anti-human CD68	Novus Biologicals	NB100-683-0.1 mg
Anti-human CD80, E3Q9V	Cell Signaling Technologies	15416
Anti-human CK20	Life Technologies	PA5-82875
Anti-human MUC2 (996/1)	ThermoFisher	MA5-12345
Secondary Antibodies:
Alexa Fluor 488 Goat anti-Rabbit IgG (for CD80 and CK20)	ThermoFisher	A11008
Alexa Fluor 594 Goat anti-Mouse IgG (for MUC2 and CD68 or CD163)	ThermoFisher	A11005
Reagents and Solutions:
Caco-2 Medium:
DMEM with high glucose (4.5g/L) and 1% (v:v) GlutaMax	Thermo Fisher Scientific	35050061
FBS 10% (v:v) heat-inactivated	Sigma	F2242-500ML
Non-essential amino acids 1% for Caco-2 media	Thermo Fisher Scientific	11140076
Pen Strep 1% (v:v)	Sigma	F2242-500ML
HT-29-MTX Medium
DMEM with high glucose (4.5g/L) and 1% (v:v) GlutaMax	Thermo Fisher Scientific	35050061
FBS 10% (v:v) heat-inactivated	Sigma	F2242-500ML
HEPES (10 mM final concentration)	Thermo Fisher Scientific	15630080
Pen Strep 1% (v:v)	Sigma	F2242-500ML
THP-1 Medium:
FBS (10% v:v) heat-inactivated fetal bovine serum	Sigma	F2242-500ML
Glucose (4.5 g/L final concentration)	Thermo Fisher Scientific	A249001
GlutaMax 1% (v:v)  2 mM final concentration	Thermo Fisher Scientific	35050061
HEPES (10 mM final concentration)	Thermo Fisher Scientific	15630080
Pen Strep 1% (v:v)	Sigma	F2242-500ML
Roswell Park Memorial Institute (RPMI)-1640 medium	Thermo Fisher Scientific	11875119
Sodium Pyruvate (1 mM final concentration)	Thermo Fisher Scientific	11360070
Purecol working collagenase solution (dilution to 1:30 100 µg/ml):
Purecol 3 mg/ml 1:30 to 100 µg/ml	Advanced BioMatrix	5005-100ML
sterile deionized water
L-Sepiapterin stock solution (50 mM):
L-Sepiapterin (SEP)	Career Henan Chemical Co	CAS#: 17094-01-8
Phorbol myristate acetate stock solution (PMA, 5 mg/mL)	InvivoGen	Tlrl-pma
BSA 1% Bovine serum albumin	Sigma-Aldrich	5470
DMSO	Sigma	D2650
Miller LB	Sigma-Aldrich	L3522
Normal Serum from the same species as the secondary antibody	Sigma	NS02L-1ML
Paraformaldehyde	Sigma-Aldrich	441244