A Transwell Tissue Model for Studying Interactions Between Human Oral Biofilms and Epithelium

Begin with a multi-species bacterial oral biofilm grown on a mineral disc submerged in a buffer.

Sonicate to disrupt the biofilm matrix and generate a uniform single-cell suspension.

Add this bacterial suspension onto a transwell insert containing a human oral tissue model with epithelial cells grown on the membrane.

Place the insert in a fresh multiwell plate with media.

During incubation, the bacteria adhere to the epithelial surface and colonize, forming biofilms that mimic an oral infection.

This triggers the expression of inflammatory signaling molecules in the epithelial cells.

After incubation, remove the insert and discard the residual suspension.

Using a needle, excise the epithelial tissue along with its supporting membrane and transfer it into a tube containing lysis buffer and glass beads.

Homogenize to mechanically lyse the epithelial cells and bacteria, releasing intracellular contents into the buffer.

The lysate is ready for analysis of epithelial responses to oral biofilms.

Meanwhile, to remove the biofilms from the hydroxyapatite discs, use a 19 gauge needle and tweezers to lift the discs from the bottom of the 24 well plate. Transfer the discs into a bijoux containing 1 milliliter of sterile DPBS.

Sonicate the discs at 35 kilohertz for 10 minutes in a sonication water bath. After acclimatization, use tweezers to remove the tissue inserts from the 12 well plate. Pipette 100 microliters of the biofilm sonicate suspension directly onto the tissue models.

Transfer the inserts to another 12 well plate containing 1 milliliter of fresh maintenance media. Incubate the tissue models for 24 hours at 37 degrees Celsius and 5% carbon dioxide. For tissue processing, prepare 350 microliters of RLT-lysis buffer, containing 1% β-mercaptoethanol in a 2 milliliter screw cap O-ring tube.

Add approximately 100 microliters of 0.5 millimeter acid washed glass beads to the tube. Using tweezers, remove the inserts containing the tissue from the media and discard any remaining microbial suspension from the insert. Hold the insert inverted at eye level, then use a 19 gauge needle to carefully slice the tissue and membrane from the bottom of the insert.

Transfer the tissue and membrane into the prepared RLT buffer. Homogenize the tissue for 30 seconds, using a benchtop bead beater homogenizer.