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

We have developed a 3D co-culture system that mimics the gut mucosal environment to study the serial crosstalk and to evaluate the drug responses which could be utilized for drug screening and for understanding mucosal cellular interactions. Different groups have developed several different culture system to model the gut mucosal environment by using very complex bioengineering techniques. But instead, we developed a co-culture system which is much easier to make using less expensive materials.

Our co-culture system revealed that a proteinogenic drug triggers coordinated responses among gut bacteria, epithelial and immune cells, activating host immunity likely with our bacterial metabolites and mediating cross-species communication. We aim to study both aerobic and anerobic bacterial strains and their metabolites to uncover immune modulating effects, paving the way for new immunotherapeutic strategies based on bacterial metabolites. To begin, open a Petri dish cover and set it aside.

Place a scalpel blade on the edge of the Petri dish bottom. Take a set of sterile tweezers and gently grab the insert bottom up to remove it from the packaging. With the other hand, take the sterile scalpel blade and bring it up to the insert.

With 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. Cut the PET membrane out and use the second set of tweezers to remove it. Then use the scalpel blade to scrape any white membrane shavings to clean the edge and rim of the insert.

Next, gently spread a 2-3 millimeter bead of silicone all the way around the insert bottom, being careful not to touch the membrane. Pick up the second set of sterile forceps and carefully lift the first insert without the membrane out of its protective pack. Bring the two inserts together bottom to bottom so that the bottom rims align.

When the Flipwell stack is glued, place it inside the original sterile pack or a deep Petri dish to dry for 72 hours. Use the lid from the specified Petri dishes to cover the stack assembly. To sterilize the stack assemblies, use sterile forceps to hang the Flipwell stack on the rim of the specified deep Petri dish rim.

Then, close the glass sash of the biosafety cabinet and turn on the ultraviolet light. To test for leakage prior to collagen coating, first add 500 microliters of sterile PBS or sterile deionized water. The next day, aspirate the liquid used for testing before drying the stacks for 1-2 hours inside the biosafety cabinet.

To coat the membrane with collagen, open the Petri dish lid and let the Flipwell hang off the Petri dish rim. Carefully add 200 microliters of collagen solution to one side of the insert stack. Aspirate the collagen solution after one hour, then pipette 200 microliters of sterile PBS After aspirating the PBS, cover the Petri dish with a lid and let the membrane dry for 60 minutes inside the cabinet.

When the membrane is dry, use sterile forceps or tweezers and flip the Flipwell to the opposite side and let it hang off the Petri dish rim. To make the bacterial insert, place two sets of sterile forceps and the 24-well inserts inside the biosafety cabinet. With sterile tweezers, lift the insert from its sterile pack.

With the second set of tweezers or forceps, break off the small plastic feet at the bottom of the insert. Test the 24-well insert by fitting it inside one of the sterile Flipwell co-culture insert stacks or the original 12-well inserts. To begin seeding the Flipwells, seed 500 microliters of the epithelial cells lines on each side of the Flipwell's apical side.

Cover the assemblies with a Petri dish lid, then incubate at 37 degrees Celsius with 5%carbon dioxide overnight until the cells attach. Open the Petri dish lid and aspirate the DMEM media from the apical side of the Flipwell stack. With sterile forceps, pick up the Flipwell carefully by the hook and rotate it by 180 degrees.

With the second set of sterile forceps, grab the Flipwell stack by the other hook now facing upward. Then lower the assembly back into the Petri dish and hang the hook over the Petri dish rim. Add 500 microliters of THP-1 cell suspension to the apical side of the co-culture insert stack.

Adjust the HT-29 media for the colon epithelial cells by adding media to the deep well Petri dish. To remove any air bubbles, hold the Flipwell stack by the arm with sterile forceps. Carefully lower a gavage needle in and under the insert assembly and very gently place the soft tip up to the air bubble.

Carefully and very slowly pull the syringe plunger up and watch the air bubble disappear slowly. Then carefully remove the needle and syringe from the Flipwell. After the air bubbles have been removed with a gavage needle, culture both cell types in the cell culture incubator.

With forceps, set the insert inside a sterile Petri dish and cover with the lid. Transfer the Petri dish with the Flipwells into the biosafety cabinet and set aside on the opposite side of the dish with bacterial inserts. Pipette out 300 microliters of the DMEM media from the apical side of the Flipwell to allow for the bacterial insert, then cover with the Petri dish lid.

Now, add 50 to 100 microliters of Bacillus subtilis culture to the 24-well bacterial inserts and insert it into the Flipwell top side. Rotate the arm and hang it from the rim of the Flipwell co-culture insert stack. Hold the stack with the second set of sterile forceps.

After a three-hour incubation, use sterile forceps to remove the bacterial insert from the Flipwell. Place the Flipwell inside a 50-milliliter conical tube without disassembly. To disassemble the Flipwell for electron microscopy, hold it with both hands and twist off each glued part of the stack.

Rotate the insert with the membrane and set it down With the membrane facing up. Hold the insert, carefully cut out the membrane with a scalpel blade, then place it inside a 1.7-milliliter tube containing paraform aldehyde solution. For confocal microscopy, add 300 microliters of sterile PBS to the apical side.

Carefully remove the stack from the Petri dish after pipetting out the PBS. Now, flip the Flipwell and add 300 microliters of PBS to the now facing upward RPMI side of the Flipwell co-culture insert stack, then aspirate the phosphate buffered saline. Using a 200-microliter pipette, create a large droplet of PBS.

Twist off the insert assembly to separate into the inserts. Then carefully position the Flipwell stack with THP-1 cells on top of the 200-microliter drop of PBS. Add 200 microliters of permeabilization buffer to the top side with colon epithelial cells and leave for 10 minutes.

For immunofluorescent staining, pipette 300 microliters of primary antibody to the top of the insert. Add 300 microliters of primary antibody to the well of a 12-well plate, then place the insert inside the well on top of the droplet. Cover the antibody well.

Staining showed dramatic increases of mucus secretion in the gut epithelial compartment after sepiapterin treatment, as indicated by the increases of the epithelial marker protein CK20 and mucosal protein MUC2. In THP-1 monocultures, sepiapterin treatment significantly increased the expression of the M1 macrophage marker CD80, while down modulating M2 macrophage marker CD163, indicating M1 polarization. Scanning electron microscopy revealed increased mucus secretion from epithelial cells in sep treated co-cultures.

Sepiapterin treatment induced macrophage morphology resembling M1 phenotype in both monocultures and co-cultures. Bacterial cells in sepiapterin-treated monocultures displayed wrinkled surfaces characteristic of biofilm formation.