An Ex Vivo Gut Organ Culture System to Study Host-Microbiota Interactions

Overview

In this video, we describe the methodology for setting up an ex vivo gut organ culture system to study the interactions between mouse intestinal tissue fragments and specific gut bacteria. The device consists of two pairs of input-output ports, the first connecting the intestinal tissue lumen and supplying the specific bacterial suspension into it, and the second continuously supplying media to the device's wells to maintain ex vivo tissue viability.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Experiment preparation

1. Fabrication of the gut organ culture device (3 days)
   1. Using a 3D printer, print the reusable plastic molds for the organ culture device (the device has 6 wells, with 24 small and large holes, and for the device cover lid).
      NOTE: These plastic molds may be used for the fabrication of numerous devices.
   2. Insert the blunt-end needles (22 G & 18 G) to the appropriate position within the device mold and cast approximately 20 g of polydimethylsiloxane (PDMS) mix (1:10 weight ratio, base to catalyst) for one set of the device and lid.
   3. Place the molds in a vacuum chamber for 30 min, to remove air bubbles from the PDMS mix.
   4. Incubate the molds at 55 °C overnight, to complete PDMS polymerization.
   5. When the PDMS is set, pull out the needles from the mold and carefully release the culture device and lid from the plastic molds.
   6. Remove PDMS residues from the well outline using a surgical blade. Attach the PDMS device and the device cover onto a cover glass (75 mm x 50 mm micro slides) using non-toxic silicon adhesive and leave the parts to set overnight (apply the glue to the smooth side of the device).
   7. Insert twelve 22 G needles for the lumen and twelve 18 G needles for the well. Fix all the needles in place using silicone and let it set overnight. Insert two 18 G needles into the cover lid for proper airflow in and out of the gut organ culture device.
   8. Check all the needles for leaks using a water-filled syringe. Check that there is no leaking from the wells by filling the wells with water.
   9. Place two surgical knots on each 22 G needle that will be connected to the colon. Place the device and the lid in an autoclave paper bag and sterilize in an autoclave.
2. Culture medium (0.5 h)
   1. In a biological hood, mix the following (in a 50 mL tube): 37 mL of Iscove's Modified Dulbecco's Medium (IMDM), 10 mL of KSR serum replacement, 1 mL of B27 supplement, 0.5 mL of N2 supplement, 0.5 mL of 1 M HEPES buffer, and 0.5 mL of non-essential amino acids.
   2. Filter, and store the complete medium at 4 °C.
3. Tubing and surgical tool preparation
   1. Cut the appropriate length of tubes for the input lumen, input well, output lumen, and output well (12 short tubes and 12 long tubes). Connect an appropriate adaptor to each side of the tube.
   2. Prepare the surgical tools: straight scissors, 4x thin forceps, and 2x sharp forceps.
   3. Place the tubes and the surgical tools in an autoclave paper bag and sterilize them using an autoclave.
4. Prepare the luminal input (desired stimulation - bacteria, stool, drugs, etc.).
   1. Before the experiment, determine the bacterial load of the bacterial culture, by serial dilutions, and culture under aerobic and/or anaerobic conditions.
   2. After calculating the bacterial load, dilute the bacterial cultures in a sterile tissue culture medium to obtain the required bacterial concentration. For fecal samples, filter using a 100 μm strainer.
      NOTE: For nonbacterial stimulation (drugs, metabolites, etc.), dilute the substance to the required concentration using the gut culture medium.

2. Experiment setup preparation

1. Inside the organ culture incubator, turn on the heater unit, and set it to 37 °C.
2. Set up the pumps as well as the input and output syringes.
3. Tissue culture medium input
   1. In a biological or laminar flow hood, fill the input well syringes with a complete culture medium. The final volume depends on the experiment's duration and the flow rate; usually 1 mL/h plus an additional medium for purge.
   2. Connect the tubes to the syringes using the Luer-lock adaptor. Place the filled syringes in the syringe pumps.
   3. Purge the input syringes. Make sure that the well medium flows out from all tubes into a waste glass.
4. Luminal input
   1. Fill the luminal input syringes with stimulation treatment (bacteria, drugs, etc.). The volume depends on the duration of the experiment and the flow rate (usually 30 µL/h plus an additional medium for purge).
   2. Connect the tubes to the syringes using the Luer-lock adaptor. Place the filled syringes in the syringe pumps.
   3. Purge input syringes. Make sure that the stimulation flows out of all the tubes into a waste glass. Be careful not to contaminate the different stimulations.
5. Outputs
   1. Place the empty syringes in the output syringe pumps (set pump mode to 'withdrawal').
6. Device setup
   1. Set the regulator that controls the gas mixture (95% O₂ + 5% CO₂) flow rate for a gentle, minimal flow.
   2. Examine the device in a laminar hood.
   3. Flush the needles of the device with sterile IMDM to wash the needles.
   4. Add 500 µL of sterile culture medium into each well of the device.
7. Preparation for tissue dissection
   1. Put the sterile surgical tools inside the laminar hood.
   2. Fill a 10 mL syringe with sterile IMDM and connect a sterile (autoclaved) 22 G blunt-end needle for flushing of the colon.

3. Organ cultures

1. Mice sacrifice and tissue dissection
   1. In a laminar hood, sacrifice 12–to 14-day-old mice by decapitation. Spray the mice with 70% ethanol and place the mice on a plastic plate.
   2. Using sharp scissors and forceps, dissect the mouse 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.
      NOTE: Minimize contact with the colon tissue. Do not touch the middle part of the colon tissue. Hold the tissue gently and only at the edges of the tissue.
2. Colon flush and wash
   1. Under a dissection microscope, gently flush the colon content with sterile IMDM (into the proximal side) with the prepared 10 mL syringe (step 2.7.2). After removing the feces from the intestinal tissue, place the colon in a new 6-well plate filled with 0.5 mL of sterile IMDM.
3. Connecting the colon to the device
   1. Take the tissue, carefully connect it to the 22 G needle, and make a tight tie with the two threads. At this point, it is imperative to maintain the correct orientation of the colon to the lumen flow (proximal = input, distal = output). Repeat steps 3.2-3.3 for all 6 tissues.
   2. Verify that the input needle Luer locks are empty from the medium. If not, empty them. Add stimulations to the input needle Luer lock (to avoid entry of air bubbles into the lumen). Repeat this step for each colon with the appropriate stimulation.
   3. Check that all the tissues are connected and place the cover lid on top of the device.
4. Connecting the organ culture device to the pumps
   1. Place the device into the pre-heated temperature-controlled chamber (37 °C).
5. Connect gas flow
   1. Connect the gas adaptor to the cover lid using the appropriate input needle.
   2. Connect the input and output tubes to the device.
      NOTE: Connect the proximal colon side to the input tubs.
6. Purge the lumen with the stimulation inputs.
   1. Gently flow luminal stimulation through the gut and verify medium flow in the output tubes.
   2. Wash external medium.
   3. Wash the external medium 3 times (set the pumps at a rate of 600 µL/min for both input and output well). Each wash takes 1 minute (starting by emptying the well).
7. Start the experiment.
   1. Start the pumps at the following rates:
      Flow rate: lumen: input- 30 µL/h, output- 35 µL/h
      External medium: input- 1000 µL/h, output- 950 µL/h
      NOTE: Experiment time can vary between 30 min to 24 h.
8. End of experiment (up to 24 h for colon organ cultures)
   1. Disconnect all the tubes from the device.
   2. Disconnect the tissues from the needles and continue to the desired readouts.

Materials

Name	Company	Catalog Number	Comments
Device
18-Gauge Blunt Needle	Mcmaster	75165a754
22-Gauge Blunt Needle	Mcmaster	75165a758
All Purpose Adhesive Sealant 100% Silicone	DAP	688
Cubic Vacuum Desiccator VDC-21+ 2 Shelves		AAAD4021
Glass Slide 1 mm Thick	Corning	2947-75X50
Mini Incubator im-10		AAH24315K
MPC 301E Vacuum PUMP		VI-412711
Plastic Quick Turn Tube Coupling Plugs	Mcmaster	51525k121
Plastic Quick Turn Tube Coupling Sockets	Mcmaster	52525k211
Sylgard 184 Silicone Elastomer	Dow		Polydimethylsiloxane, PDMS
Tubing	Mcmaster	6516t11
Zortrax M200	Zortrax		Zortrax Z-SUITE, Autodesk Fusion 360
Zortrax M200 Materials: z-ultrat	Zortrax
Medium
B27 Supplement (50x), Serum-Free T	Thermo Fisher Scientific	17504044
Iscove's Modified Dulbecco's Medium with Phenol Red (1x)	Thermo Fisher Scientific	12440061
HEPES Buffer (1M)	Thermo Fisher Scientific	15630056
Knock-Out Serum	Thermo Fisher Scientific	10828028
N2 Supplement (100x)	Thermo Fisher Scientific	A1370701
Non-Essential Amino Acid (100x)	Thermo Fisher Scientific	11140035
Surgical Tools
Large Scissors	Aseltech	11-00-10
Sharp Forceps	F.S.T	11297-10
Silk-Braided Surgical Thread	SMI	8010G
Straight Scissors	F.S.T	14091-09
Thin Forceps	F.S.T	11051-10
Organ System
0.1 µm Filter	Life Gene
0.22 µm Filter	Life Gene
5 mL Luer-Lock Syringe	B-D	309649
Greenough Stereo Microscope	ZEISS	Stemi 305
Recirculating Precision Air Heater "CUBE"		CUBE-2-LIS
Syringe Pump	New Era Pump Systems Inc	nep-ne-1600-em