An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

Podsumowanie

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

Streszczenie

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

Wprowadzenie

The intestine is a highly complex organ containing a wide range of cell types (epithelial cells, immune system cells, neurons, and more) organized in a particular structure that allows cells to interact and communicate with one another and with the luminal content (microbiota, food, etc.)¹. Currently, the research toolbox available for analyzing host-microbiota interactions includes in vitro cell cultures and in vivo animal models². In vivo animal models provide a physiological tissue construction³ but with poor experimental control and limited ability to manipulate the experiment conditions. In vitro culture systems, on the other hand, use primary cells or cell lines that can be supplemented with microbes⁴, offering tight control over the experiment parameters but lack the cellular complexity and the tissue architecture. Modern in vitro assays allow the advanced use of healthy and pathological human tissue samples, like epithelial organoids derived from mouse or human sources^5,6, and samples that mimic the mucosal microenvironment⁷. Another example is the 'gut on a chip' assay, which includes the human colonic epithelial cell line (Caco2), extracellular matrix and microfluidic channels to mimic the physiological condition of the gut invariant⁸. However, as advanced and innovative as in vitro samples may be, they do not maintain a normal tissue architecture or naïve cellular composition.

To address that, Yissachar et al. recently developed an ex vivo organ culture system⁹ (Figure 1) that maintains intact gut fragments ex vivo, benefiting from the advantages of both in vivo and in vitro models. This ex vivo gut organ culture system is based on a custom-made culture device that supports a multiplexed culture of six colon tissues, allowing examining experimental inputs under comparable conditions while controlling the system's inputs and outputs. Recent works have demonstrated that this system is valuable for analyzing intestinal responses to individual gut bacteria⁹, whole human microbiota samples¹⁰ and microbial metabolites¹¹. This system allows, for the first time, the study of these early host-microbiota interactions with a high level of control over the host, microbial and environmental components. Furthermore, it allows monitoring and manipulating the system throughout the experiment, in real-time.

Figure 1: Schematics of the gut culture device. A whole intestinal tissue fragment is attached to the output and input ports of the chamber (top), with pumps regulating the medium flow inside the lumen and in the external medium chamber. The entire device (bottom) contains 6 such chambers. This figure has been modified from Yissachar et al. 2017. Please click here to view a larger version of this figure.

Protokół

This protocol follows the animal care guidelines approved by the ethics committee for animal welfare.

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) (3D files attached).
      NOTE: These plastic molds may be used for 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 air flow 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 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 dilutions¹², and culture under aerobic and/or anaerobic conditions.
   2. After calculating the bacterial load, dilute the bacterial cultures in 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 complete culture medium. The final volume depends on the experiment's duration and the flow rate; usually 1 mL/h plus 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 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 flow 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 12-14 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 the 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 and carefully connect it onto 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 min (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.

Wyniki

The gut organ culture system maintains tissue viability ex vivo. The evaluation of the tissue viability was done throughout the culture period. Colon tissue fragments were incubated in the gut organ culture system and fixed following 2/12/24 h culture. The intestinal epithelial cell (IEC) layer integrity was validated by immunofluorescence staining using E-cadherin and cytokeratin-18 antibodies. Likewise, mucus-filled goblet cells in the colonic epithelium and mucus secretion within the lumen were detected as we...

Dyskusje

This article describe an optimized protocol for ex vivo gut organ cultures that Yissachar et al. have recently developed (published⁹ and unpublished data). The gut organ culture system supports multiplexed culture of intact intestinal fragments while maintaining luminal flow. It provides full control over the intra- and extra-luminal environment (including stimulation dose, exposure time and flow rate) and preserves the naïve intestinal tissue structure and its cellular complexity

Materiały

Name	Company	Catalog Number	Comments
Device
18 Gauge Blunt Needle	Mcmaster	75165a754
22 Gauge Blunt Needle	Mcmaster	75165a758
All Purpose Adhesive Selant 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	Thermo Fisher Scientific	17504044
HEPES Buffer (1M)	Thermo Fisher Scientific	15630056
Iscove's Modified Dulbecco's Medium with Phenol Red (1x)	Thermo Fisher Scientific	12440061
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