The overall goal of the following experiment is to mimic the in vivo situation for stimulation of human intestinal mucosa. This is achieved by gluing a cave cylinder on the apical side of the tissue to prevent the stimulus from making basolateral contact. As a second step, the stimulus is applied, which will allow analysis of the elicited immune response.
Next, the stimulus is taken off the apical side of the tissue in order to keep it viable for 24 to 36 hours. Results obtained can show differential effects of various probiotic strains based on immunohistochemistry data, cytokine secretion, profile analysis, and so forth. The main advantage of this technique over existing methods like the ASIC chamber, is that it allows analysis of up to 20 conditions at the same time.
It allows extension of the culture time up to 36 hours, and also it provides enough material for various readout analysis such as D-N-A-R-N-A analysis, immunohistochemistry, cytokine secretion, et cetera. Though this metal can provide insight in the probiotic field, it can also be used for other technologies and to study the effect of other immunomodulators like biologics, antiinflammatory drugs, and so on. Intestinal mucosa tissue from healthy or inflammatory bowel disease individuals must be obtained during surgery.
Once the specimen is excised, quickly transferred to the lab, keeping it chilled in HBSS with pen strep. Then begin by washing the tissue and buffer and remove any mesenteric material. Next, separate the mucosa layer from the submucosa using sterile scissors and forceps while keeping the tissue in the HBSS buffer.
In a Petri dish, it is not necessary to keep the tissue fully emerged, but touch the mucosal surface as little as possible with the forceps. Next, cut the mucosa into strips at least one centimeter wide and as long as possible, use two sterile scalpels in a manner like cutting food. Once cut, gently wash, clean the mucosa in calcium and magnesium free HBSS.
Finally, extend the mucosa strips on a Petri dish with the apical sides facing upwards. Begin by preparing fresh TIS DMEM. Next, fill a 10 centimeter Petri dish with about 30 milliliters of the F-B-S-N-A and completely submerge an iron grid into the solution.
Keep the plate open and position plastic cylinders on the plate.Cover. Now siphon three microliters of surgical glue into a pipette and carefully apply the glue to one of the borders of a plastic cylinder while holding it firm with the forceps. Next, gently set the cylinder on the apical side of the mucosa as close to one of the strips borders as possible while the glue dries.
Dispense 850 to 1000 microliters of medium in the center well of the tissue plate, and support the iron grid on the edges of the plate central. Well using two scalpels, cut away the excess tissue from the borders of the cylinder. Now gently lift the cylinder with the attached tissue and place the basolateral side of the tissue on the iron grid in the center of the plate.
Next, proceed with tissue stimulation. Apply the stimuli of interest at the center of the plastic cylinder at the desired concentration. Do not disturb the cylinder or the mucosal surface with the pipette tip and uniformly cover the surface inside the cylinder.
Now incubate the tissue for up to three hours. Incubation for greater than three hours is also feasible using a smaller volume of stimuli solution. After the incubation, remove the stimuli from the apical surface of the tissue, but do not replace it with fresh, medium.
Adding medium would asphyxiate the tissue. Now fill the oxygen jar with one atmosphere of pressure of 100%oxygen for 24 hours of culture time. Always be careful when handling the oxygen tank and make sure to close it properly after use.
After the 24 hour stimulation culture, use a scalpel to carefully remove the cylinder from the tissue by gently scraping off the glue. Then for IHC or immunofluorescence assays, fix the tissue. Or for gene expression or protein analysis, snap freeze the tissue in liquid nitrogen.
Next, harvest the basal lateral medium for cytokine secretion profiling. Following this, transfer the medium to a centrifuge tube and pellet the debris using the outlined protocol. Healthy and IBD human intestinal mucosa were cultured for at least 24 hours preserving the wellbeing of the tissues.
This was only possible if at least 85%of the culture time took place in an oxygen-rich environment. Conventionally cultured mucosa does not fare as well. The healthy tissue was then stimulated with pro-inflammatory invasive salmonella.
Two hours after inoculation, destruction of the upper layer of the epithelium was already visible compared to controls. After 24 hours, the degeneration became quite extensive. A deeper discussion of experimental applications with published results is in the written manuscript After its development.
This technique paved the way for researchers working in the gastroenterology field to study host microbe interaction in the gastrointestinal tract. After watching this video, you should have a good understanding of how to keep a human intestinal mucosa in culture and stimulated topically. Don't forget that working with human samples can be extremely hazardous and the necessary precautions should always be taken when performing this procedure.
