The gut organ culture system combines the advantages of in vitro and in vivo assays and thus serves as an intermediate experimental step between simple in vitro assays to complex in vivo experiments. The main advantage of this technique is the combination of tight experimental control with the preservation of intestinal physiology. This method provides insight for analyzing intestinal responses to specific stimulus with high level of control over the host, microbial, and the environmental components.
Demonstrating the procedure will be Alon Shemesh, a Masters student from my laboratory, and Dr.Sivan Amidror, our lab manager. To begin, insert the blunt-end needles to the appropriate position within the device mold and cast approximately 20 grams of polydimethylsiloxane or PDMS mix, for one set of the device and lid. Place the molds in a vacuum chamber for 30 minutes to remove air bubbles from the PDMS mix, then incubate the molds at 55 degrees Celsius overnight to complete PDMS polymerization.
When the PDMS is set, pull out the needles from the mold and carefully release the culture device and lid from the plastic molds. Remove PDMS residues from the well outline using a surgical blade. Attach the PDMS device and the device cover onto a cover glass of 75 by 50 millimeter microslides using non-toxic silicon adhesive and leave the parts to set overnight.
Apply the glued to the smooth side of the device. Insert 12, 22 gauge needles for the lumen and 12, 18 gauge needles for the well. Fix all the needles in place using silicone and let it set overnight.
Purge the input syringes and make sure that the well medium flows out from all tubes into a waste glass, then purge the input syringes and makes sure that the stimulations flow out of all the tubes into a waste glass, taking care to not contaminate the different stimulations. After sacrificing the mouse, use sharp scissors and forceps to dissect it and take out the digestive tract from the stomach to the anus, by cutting all the fat and connective tissues. Cut the colon and place it on a new plate.
Minimize contact while holding the tissue gently and only at the edges. Perform the colon flush under a dissection microscope. Gently flush the colon content with sterile IMDM with the prepared 10 milliliter syringe as described in the text.
After removing the feces from the intestinal tissue, place the colon in a new six well plate filled with 0.5 milliliters of sterile IMDM. Next, take the colon tissue and carefully connect it to the 22 gauge needle, making a tight tie with the two threads. Maintain the correct orientation of the colon to the lumen flow, such that the proximal and distal is equal to input and output, respectively.
Repeat colon flushing and needle tying for all the tissues. Connect the input and output tubes to the device, then begin the experiment by starting the pumps at the desired rates. Mucus-filled goblet cells in the colonic epithelium and mucus secretion within the lumen were detected as well as proliferating IEC in the colonic crypts, as indicated by KI67 staining.
These results showed that the gut culture system maintains intestinal function and structure ex-vivo. After two hours of segmented filamentous bacteria, or SFB, introduction, typical SFB filaments were detected in close association with small intestine villi using fluorescence in situ hybridization. Additionally, a transmission electron microscopy presented SFB within a few microns of the small intestine epithelium brush border.
Gene expression profiles of whole tissue samples were produced in triplicate two hours after infusion with SFB. Control cultures were infused with fecal suspensions of germ-free or Bacteroides fragilis monocolonized mice. These changes persuaded by SFB were mainly of small amplitude compared to the germ-free control.
The orientation of the colon is highly important. Take extra care when tying the colon on the needle. Proper tie will prevent the contamination of the well with the lumen content.
A wide range of readout techniques can follow this protocol, such as next generation sequencing, imaging, cell sorting, and many more. This readouts provide novel insight into host microbiome interactions in health and disease.
