Dental plat biofilms are comprised of hundreds of microorganisms that can drive inflammation in their oral epithelium, leading to diseases such as gingivitis. We want to better understand these interactions, using organotypic tissue models. These systems provide useful platforms for assessing alternative treatment modalities in a controlled manner.
So the advancement of sequencing technologies in recent years is transforming the field of infection biology. These technologies allows us to truly investigate host pathogen interactions, using multiple OMICs techniques through the assessment of host and microbial signatures at a proteomic, transcriptomic, and metabolomic level. Our recent research has highlighted the importance of inter-kingdom and polymicrobial interactions in biofilm related infections.
Through the use of in-vitro model systems, we have been able to investigate host biofilm interactions, test novel anti-biofilm therapeutics, and profile microbial communities, using innovative technologies such as RAM and spectroscopy. Future studies, using these organotypic tissue models, will aim to achieve a multi-OMIC signature and profiling of the microorganism and host tissue and culture, together. We hope to uncover and untangle complex interactions between the host and specific bacteria and fungal species in our biofilm models.
To begin, remove the frozen stalks of porous beads containing streptococcus species from 80 degrees Celsius. Revive streptococcus species on Columbia blood auger base plates, containing 5%sterile defibrinated horse blood. Incubate the plates at 37 degrees Celsius and 5%carbon dioxide for 24 hours.
The next day, transfer three to four colonies from the plate to 10 milliliters of tryptone soy broth medium. Culture the broth at 37 degrees Celsius and 5%carbon dioxide for 16 to 18 hours. After incubation, centrifuge the cell suspensions at 3, 000 G for five minutes at 20 degrees Celsius.
Wash the cell pellets with 10 milliliters of sterile PBS. Re-suspend the pellets in 10 milliliters of PBS and standardize each streptococcus species suspension, individually, using a spectrophotometer set to 550 nanometers. Following standardization, dilute each streptococcus suspension, one to 10, to reach a concentration of one times 10 to the power of seven cells per milliliter in a one-to-one mix of Todd Hewitt broth and Roswell Park Memorial Institute medium.
Pipette 500 microliters of diluted cell suspensions to a 24 well microtiter plate, containing a 13 millimeter hydroxyapatite disc. Incubate the culture for 24 hours at 37 degrees Celsius and 5%carbon dioxide to allow biofilm formation. In a similar manner, culture for anaerobic microorganisms on a fastidious anaerobic auger base, containing 5%sterile defibrinated horse blood.
Standardize each anaerobic microorganism to a specific absorbance, and dilute them in a one-to-one mix of Todd Hewitt broth and Roswell Park Memorial Institute medium. After streptococcus biofilm maturation, carefully remove non-adhered cells and spent media from the wells. Add 500 microliters of standardized one times 10 to the power of seven cells per milliliter suspensions of four anaerobic microorganisms into each well.
Incubate the biofilms for 24 hours under anaerobic conditions at 37 degrees Celsius. The next day, remove non-adhered cells and spent media from the wells. Add 500 microliters of sterile one to one mix of Todd Hewitt broth and Roswell Park Memorial Institute medium.
Culture the biofilms under anaerobic conditions for 24 hours. On day seven, wash the biofilms twice with 500 microliters of sterile PBS for co-culture. To begin, unbox and transfer the human oral epithelium or HOE tissue and media to a class two safety cabinet.
Add one milliliter of the maintenance media supplied with the tissue to each well of a 12 well plate. Using sterile tweezers, remove the polycarbonate inserts containing the HOE tissue from the nutrient auger, and 24 well shipping plate. Transfer the tissue to the prepared 12 well plate.
Incubate the tissue models for 24 hours at 37 degrees Celsius, and 5%carbon dioxide to acclimatize them to laboratory conditions. 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 bijou, containing one 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 one 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%beam mercaptoethanol in a two 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 beader homogenizer. Extract RNA from the resulting lysate, following the manufacturer's instructions's RNA extraction kit.
Collect the remaining spent tissue media for proteomic analyses, using low and high throughput methodologies. The gene expression of inflammatory biomarkers in the HOE tissue was significantly upregulated after exposure to biofilm sonicate, compared to the unstimulated control. The largest fold changes were observed for CCL2, CXCL1, and CSF3.
IL8 mRNA expression in HOE tissue was increased 8.67 fold, following stimulation with biofilm sonicate, compared to controlled tissue. IL8 protein levels in the spent media were increased from 1.005 nanograms per milliliter in control tissue to 4.245 nanograms per milliliter in biofilm sonicate stimulated tissue.
