A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Summary

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

Abstract

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.

Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.

To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.

We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

Protocol

1. Obtaining and Preparing the Tissue

1. Tissue (healthy or IBD mucosa) is obtained during surgery. Once the specimen is excised transfer to the lab as soon as possible, keeping it in Ca⁺⁺/Mg⁺⁺-free HBSS buffer supplemented with Pen/Strep at 4 °C or on ice.

*The size of the specimen depends on the availability of the tissue: the part of the tissue obtained, is strictly what is not needed for diagnosis and can vary greatly between healthy and IBD subjects. Healthy tissue is excised from patients undergoing surgery for colon cancer.

2. Wash the tissue gently and clean it of all mesenteric material. Separate the mucosa layer from the submucosa using sterile scissors and forceps while keeping the tissue in HBSS buffer in a Petri dish (not necessarily immerged). Touch the mucosal surface with the forceps as little as possible.
3. Cut mucosa in stripes at least 1 cm wide and as long as possible. Use two sterile scalpels, much as though you were cutting your food.
4. Wash clean mucosa gently in HBSS and extend it on a Petri dish with the apical side facing upwards.

2. Mounting the Tissue

1. Prepare fresh medium (tisDMEM). Use DMEM supplemented with Glutamine (2 mM) and NaPyr (1 mM), and add 15% FBS-Na (Fetal Bovine Serum - North American), 1% ITS-X and 200 ng/ml EGF at the time of use.
2. Fill a 10 cm Petri dish with the rest of the FBS-Na (use about 30 ml so that metal grids will be completely submerged) and submerge the metal grids. Keep the plate open and support the plastic cylinders on its tap.

*The metal grids are made of iron. The mesh is square-shaped with a side of 0.5 mm.

3. Take 3 μl of surgical glue with a 10 or 20 μl pipette. Apply it carefully to one of the borders of a plastic cylinder while holding it firm with the forceps.
4. Gently place the cylinder on the apical side of the mucosa, as close to one of the borders of the strip as possible.
5. To give the glue time to firmly attach the cylinder on the tissue, prepare the centre-well organ culture plate: Dispense 850 μl-1 ml of medium in the centre well and support the metal grid on the edges of the plate's central well.
6. Cut away excess tissue from the borders of the cylinder using two sterile scalpels as before.
7. Gently lift the cylinder with the attached tissue and place the basolateral side on the metal grid in the centre of the plate.

3. Stimulation

1. Use the stimuli of your choice in your preferred concentration.
2. Apply appropriate volume in the centre of the plastic cylinder without disturbing the cylinder or the mucosal surface with the pipette tip. Make sure your chosen volume uniformly covers the surface inside the cylinder. Indicated volumes:
   Large plastic cylinder: 200 μl
   Medium plastic cylinder: 100 μl
   Small plastic cylinder: 50 μl
3. Incubate for up to 3 hr in a conventional incubator.

*If you wish to incubate for longer time points, make sure you use a much smaller volume of stimuli (10-20 μl) and directly place the tissue in an atmosphere of 100% O₂ in the pressure of 1 Atm (see 3.5).

4. Take the medium with the stimuli off the apical surface of the tissue and DO NOT REPLACE with fresh medium, because the tissue does not get enough O₂ if a thick layer of medium on the apical face prevents gas exchange. Cover the plate.
5. Place the tissue in an atmosphere of 100% O₂ in the pressure of 1 Atm.

4. Harvesting

1. After 24 hr of total culture time (± 2.5 hr) carefully remove the cylinder from the tissue with the help of a scalpel gently scraping glue off and store it depending on the desired analysis (fix for immunohistochemistry and/or immunofluorescence assays, or snap freeze in liquid N2 for RNA purification and gene expression profiling or protein purification and Western blot assays).
2. Harvest the basolateral medium for cytokine secretion profiling. Centrifuge at 8,000 rpm for 7 min to pellet debris, transfer the supernatant in an Eppendorf tube (alternatively aliquot) and immediately store at -20 °C.

*if you want to keep supernatants for longer, store them at -80 °C.

Results

We succeeded in maintaining healthy and IBD human intestinal mucosa in culture for at least 24 hr preserving the wellbeing of the tissue. In accordance with previous observations ¹, this was only possible if the majority of the culture time (at least 85% of total) took place in O₂, as the tissue did not survive for 24 hr in conventional incubators (Figure 2). We have also shown that different responses of the explants can be observed on this model depending on the polarity of the st...

Discussion

The need for physiologically relevant models on which treatments for human intestinal mucosa can be validly tested has long been emphasized. Many researchers have identified potential artifacts and defects in both cell ⁵ and mouse models ⁶ used so far for that purpose. Indeed, even though promising preclinical data is often obtained, these rarely translate to significant clinical benefit.

The method described in this work, presents a relatively simple, yet effective and n...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
10X HBSS	Eurocalne	ECM4006XL
Peni/Strepto	Lonza	DE17602E
Gentamycin	Gibco	15750-037
DMEM	Lonza	BE12614
FBS-Na	Gibco	16000-044
100X ITS-X	Gibco	51500-056
EGF	Tebu-bio	100-15
L-Glutamine	Lonza	BE17605E
Non essential aminoacids 100X	Gibco	11140-035
NaPyr	Gibco	11360-039
Materials
Cloning cylinders 6x8 mm	BellCo	2090-00608
Cloning cylinders 8x8 mm	BellCo	2090-0080
Cloning cylinders 10x10 mm	BellCo	2090-01010
Metal grids	Home made
Centre well organ culture plate	BD Falcon	353037
Surgical glue (Vetbond)	3M	1469SB
Oxygen Jars	Home made
Oxygen tanks	Air Liquid Sanità