The overall goal of this experiment is to reconstruct a 3D inflammatory gingiva tissue model by using three types of human cells in vitro. This method can help study wound healing, tissue regeneration, inflammation, and screen potential medicines for periodontal diseases. The main advantage of this technique is that this is the first method to build an in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture.
In a 15-milliliter sterile tube, add collagen type I-A, 10%concentrated MEM-alpha, and 10%reconstruction buffer. Then, leave the collagen mixture on ice till the next use. Next, position the 24-well culture inserts with three micrometer pore size in a 24-well culture plate.
Then, use 25%trypsin-EDTA solution to detach the HGFs from the culture flask. Next, suspend the detached cells in 10 milliliters of culture medium. Then, count the number of cells using a Burker-Turk cell counter.
After isolating the desired cell number, centrifuge the cells at 190 times g for four minutes. Then, suspend the cell pellet with the collagen solution, and leave the tube on ice. Next, add 5 milliliters of cell-collagen mixture into the culture insert without producing any air bubbles.
Then, incubate the culture insert with the cell-collagen mixture at 37 degrees Celsius and 5%carbon dioxide for 10 to 30 minutes. After the incubation, add one milliliter of the culture medium into the well and 5 milliliter of the medium into the insert. Then, incubate the culture plate at 37 degrees Celsius with 5%carbon dioxide for a day.
Next, use 25%of trypsin-EDTA solution to detach the HaCaT cells from the culture plate. Once the cells are detached, add 10 milliliters of medium B to the cells. Then, count the cell number.
After isolating the desired cell number, centrifuge the cells at 190 times g for four minutes. After the centrifugation, dissolve the cell pellet in medium B.Next, aspirate the medium from the culture insert. Then, add 5 milliliters of HaCaT cell suspension over the HGF-collagen mixture.
Leave the culture insert in the incubator for a day. The next day, replace the culture medium. Add one milliliter of co-culture KSR medium supplemented with 5%fetal bovine serum into the culture well and 5 milliliter in the culture insert.
Then, incubate the culture plate at 37 degrees Celsius for 24 hours. The next day, replace the culture medium with co-culture KSR medium supplemented with 1%fetal bovine serum. Remove the entire culture medium from both the culture insert and the culture well after 24 hours.
Then, add fresh 7 to one milliliter of KSR medium in the culture well only, and continue culturing for two weeks. Remember to change the medium in the culture well twice every week. Prepare a one to three millimeter diameter tissue puncher, and sterilize the puncher in the autoclave at 121 degrees Celsius for 20 minutes.
After sterilizing, push the tissue puncher perpendicularly into GTE for about 30 micrometers deep to make a wound in the tissue. Next, mix 10 nanograms per milliliter of PMA, 10%reconstruction buffer, and 10%concentrated RPMI medium with collagen type I-A gel. Then, leave the collagen mixture on ice till next use.
Next, centrifuge the THP-1 cells at 190 times g for four minutes. After centrifugation, suspend the cell pellet in one milliliter of collagen solution and leave on ice. Then, add 10 to 30 microliters of the THP-1 cell-collagen mixture to the wounded area of the GTE.
Next, replace the culture medium with 7 to one milliliter KSR medium containing 10 nanograms per milliliter of PMA. Then, incubate the mixture at 37 degrees Celsius at 5%carbon dioxide for 72 hours. To characterize the reconstructed GTE, H and E staining and scanning electron microscopy are done.
The images show GTE exhibiting a gingiva-like tissue structure with multiple layers of epithelium and lamina propria. The images also show that the HGF cells in the reconstructed GTEs present distinct morphological characteristics of fibroblasts surrounded by collagen fibers. Then, immunofluorescence staining is done to study the expression of dermal and epithelial markers.
This is to validate if GTE and iGTE have the molecular features of the real inflammatory human gingiva. The images show vimentin and TE-7 are strongly present in the lamina propria of GTE. K19, a specific marker to junctional epithelium in periodontitis, though weakly expresses in the GTE epithelium, is strong enough in iGTE epithelium.
Finally, in order to generate the inflammatory foci in iGTE, PMA-treated THP-1 monocytes are injected in the wounded area of the tissue. At 72 hours after PMA treatment, THP-1 cells are differentiated into macrophage-like cells. Whole-mount immunostaining confirmed that these cells express two important macrophage markers, CD14 and vimentin, and formed the inflammatory foci.
Once mastered, this technique can be done in three to four weeks if it is performed properly. While attempting this procedure, it's important to remember to avoid contamination. Following this procedure, other methods, like using LPS to stimulate inflammatory responses, can be performed in order to answer additional questions, like how to induce inflammation through the host-biofilm interactions.
After its development, this technique paved the way for researchers in the field of inflammation, tissue regeneration, and wound healing to explore the cellular and molecular mechanism and screen potential medicines for periodontal diseases. After watching this video, you should have a good understanding of how to build a 3D inflammatory gingiva tissue model in vitro.
