ガットオンチップマイクロ流体デバイスにおけるマイクロ加工ヒト腸絨毛と生活Microbiomeの共培養

This overall goal of this protocol is to demonstrate the villus growth of human intestinal cells under peristalsis-like motions and flow in a gut-on-a-chip microfluidic device, as well as to show how to co-culture these cells with living gut microbiome. So this method can help the key questions in the field of the gastroenterology, clinical microbiology, and the pharmaceutical sciences to identify the disease mechanism and to validate the efficacy and toxicity of the new drug compounds. The main advantage of this technique is that the researchers can establish the viable and functional post-microbiome ecosystem of the human living intestine in vitro using a microphysiological system.

The implications of this technique can extend towards diseases such as ulcerative colitis or Crohn's disease, since the stable co-culture of the host cells with the microbiome can emulate the complexities of these diseases. Though this method can provide an insight into the complex interactions between the gut microbiome and the immune system in the intestine, it can also be applied to other organ systems where there are host microbe interactions, such as the skin, your genital tract, or the oral cavity. To begin, prepare the gut-on-a-chip microfluidic device as described in the accompanying text protocol.

The device consists of a transparent gas-permeable silicon polymer containing two parallel microchannels, separated by a flexible, porous PDMS membrane. On each side of the channels are vacuum chambers, which can be used to apply cyclic mechanical strain. Sterilize the device by using a one-milliliter syringe to flow 70%ethanol through the channels.

Then, dry the device in a 60 degree Celsius dry oven overnight. The next day, expose the gut-on-a-chip microsystem, along with its connected tubing, to UV light and ozone simultaneously for 40 minutes. Cool down the device while remaining under the UV light of a biosafety cabinet for 15 minutes.

Next, load 100 microliters of an extracellular matrix coding solution into a one milliliter syringe, and dispense it into the upper microchannel of the device. Repeat this process to coat the lower microchannel as well. Place the whole setup in a 37 degree Celsius humidified 5%CO2 incubator for one hour.

During the incubation, de-gas pre-warmed complete culture medium by passing it through a 0.45 micron filter in a 50-milliliter filtration system for one minute. Gently tap on the filtered medium for one minute to remove any bubbles or dissolved gas in the medium. Then, transfer the de-gassed, complete medium into two three-milliliter disposable syringes, and incubate the syringes for one hour.

Next, connect the syringes to the upper and lower microchannels of the gut-on-a-chip, and wash out the coding solution by flowing complete medium into the upper and lower microchannels with a gentle pulse. Then, use a syringe pump to flow the de-gassed, complete-cell culture medium into the upper microchannel of the device, while incubating the chip overnight at 37 degrees Celsius and 5%CO2. Harvest a fully confluent T75 flask containing Caco-2 intestinal epithelial cells by first rinsing the cells with 10 milliliters of calcium and magnesium-free PBS.

Then, aspirate the PBS and repeat the rinse two more times. Next, add one milliliter of 0.05%Trypsin-EDTA solution and incubate the flask for five minutes at 37 degrees Celsius. Once detached, add 10 milliliters of pre-warmed complete-cell culture medium, and pipette the cell suspension up and down three to five times before transferring it to a centrifuge tube.

Spin down the cell suspension. Then, remove the supernatant, and re-suspend the cells at 150, 000 cells per centimeters squared in pre-warmed complete medium. Draw up 500 microliters of the cell suspension into a one-milliliter disposable syringe, equipped with a 25-gauge, 5/8-inch needle.

Then, dispense 100 microliters of the cell suspension into the upper microchannel through the outlet tubing. Next, clamp all of the inlets and outlets that are connected to the upper and lower microchannels using binder clips. Incubate the whole setup for one hour to allow the intestinal epithelial cells to adhere to the surface of the porous membrane.

Then, remove the clamps and connect the upper channel tubing to a culture medium filled syringe that has been placed on a syringe pump. Resume the flow of culture medium to the upper microchannel at 30 microliters per hour until the cells from an intact monolayer. Once the cells form a monolayer, connect the lower channel to the second syringe and perfuse the culture medium into both the upper lumen and lower capillary microchannels at the same flow rate of 30 microliters per hour.

To create peristalsis-like motion in the chip, first turn on the vacuum pump. Connect the vacuum chambers of the device to the vacuum controller via tubing with a stainless steel connector. Then, set the controller so that it provides a cyclic sign stretching motion that is 10%of the mean cell strain at a frequenzy of 0.15 Hertz, then click start.

Keep flowing culture medium to both the upper and lower microchannels in the presence of mechanical deformations for at least 80 hours to induce villus growth. Prepare a 50/50 mixture of Autoclaved Lactobacilli MRS Broth and Reinforced Clostridial Medium. Then, re-suspend a freeze-dried probiotic bacterial mixture in 10 milliliters of the mixture.

Measure the optical density of the mixture at 600 nanometers and adjust the final cell density to approximately 0.2 optical density units. Aliquot three milliliters of the adjusted microbial cell suspension into disposable 10-milliliter sterile tubes. Place the aliquots into an anaerobic container and add two packs of anaerobic gas-generating sachets into the container.

Close the container lid tightly and incubate the container setup without shaking in an incubator overnight. Next, prepare de-gassed antibiotic-free cell culture medium and load it into three-milliliter disposable syringes. Transfer the gut-on-a-chip device containing micro-engineered villi into the biosafety cabinet.

Remove the syringes that are connected to the upper and lower microchannels of the device and replace them with syringes containing the de-gassed antibiotic-free cell culture medium. Once finished, place the device back into the CO2 incubator. Then, flow the antibiotic-free culture medium into the device for 12 hours prior to seating the microbiome.

Next, spin down the pre-cultured probiotic bacterial cell mixture. Aspirate the supernatant, then re-suspend the bacteria in the antibiotic-free DMEM. Load approximately 100 microliters of this cell suspension into a one-milliliter syringe attached to a 25-gauge, 5/8-inch needle.

Infuse the bacteria into the lumen side of the microchannel containing the previously germ-free intestinal villi. Allow the microbial cells to adhere to the apical surface of the intestinal villi for one and a half hours without flow by clamping the tubing. Then, perfuse the pre-warmed antibiotic-free culture medium into both the upper and lower microchannels at 40 microliters per hour with cyclic rhythmical deformations.

This co-culture protocol allows for the stable colonization of multiple species of probiotic bacteria in the intervillous space. Viable bacterial micro-colonies can be maintained for up to two weeks in co-culture. Shown here is a different co-culture consisting of both GFP labeled E.coli and intestinal villi.

The high-powered magnification clearly shows the location of bacterial micro-colonies at the 72-hour time point. While attempting this procedure, it is important to remember to gently and slowly inject the culture medium into the microfluidic channels to avoid damaging the epithelial cell monolayer in the early stages of the culture. Based on this method, a co-culture method with the human immune cells can also give some clues in terms of how the microbiome can crosstalk to the immune system and influences on the homeostasis optimum intestinal health.

After watching this video, you should have a good understanding of how to reconstitute the micro-environment of the human intestine and to demonstrate that co-culture between the microbiome and the human intestinal villi in vitro. Working with pathogenic bacteria and human cells are extremely dangerous, so don't forget to work inside biosafety cabinet.