Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

Podsumowanie

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Streszczenie

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

Wprowadzenie

Though the gastrointestinal (GI) tract is primarily dedicated to the processing and reabsorption of nutrients from food, the GI tract also maintains central roles in the integrity of the vascular, lymphatic, and nervous systems and of numerous other organs through its mucosal and submucosal immune system¹. The GI immune system has an influential role in both gastrointestinal and systemic health due to its constant exposure to foreign antigens from food, commensal bacteria, or invading pathogens^1,2. Thus, the GI immune system must maintain a delicate balance in which it tolerates non-pathogenic antigens while responding appropriately to pathogenic antigens^1,2. When the balance of tolerance and defense is disrupted, localized or systemic immune dysregulation and inflammation can occur resulting in a myriad of diseases^1,2,3.

The intestine harbors at least 70% of all lymphoid cells in the body⁴. Most primary immunologic interactions involve at least one of three immune stations in the intestine: 1) Peyer’s Patches, 2) Intraepithelial lymphocytes (IEL) and 3) lamina propria lymphocytes (LPL). Each of these is comprised of a complex interconnected network of immune cells that rapidly respond to normal immune challenges in the gut⁵. Restricted to the stroma above the muscularis mucosae, the loosely structured lamina propria is the connective tissue of the gut mucosa and includes scaffolding for the villus, the vasculature, lymphatic drainage, and mucosal nervous system, as well as many innate and adaptive immune subsets^6,7,8,9. LPL are comprised of CD4⁺ and CD8⁺ T cells in an approximate ratio of 2:1, plasma cells and myeloid lineage cells including, dendritic cells, mast cells, eosinophils and macrophages⁶.

There is a growing interest in understanding the immune dysregulation and inflammation of the gut as it pertains to various disease states. Such conditions as Crohn’s disease and ulcerative colitis all manifest varying levels of colonic inflammation^10,11,12. Additionally, patients with malignant or non-malignant disorders of the marrow or immune system who undergo an allogeneic bone marrow transplantation (allo-BMT) can develop various forms of colitis including 1) direct toxicity from conditioning regimens before BMT, 2) infections caused by immunosuppression after BMT and 3) graft-versus-host disease (GVHD) driven by donor-type T cells reacting to donor allo-antigens in the tissues after BMT^13,14,15. All these post-BMT complications result in significant alterations in the immune milieu of the intestines^16,17,18. The proposed method allows a dependable assessment of immune cell accumulation in the mouse colon and, when applied to murine recipients after BMT, facilitates an efficient assay of both donor and recipient immune cells involved in transplant tolerance^19,20. Additional causes of gut inflammation include malignancies, food allergies, or disruption of the gut microbiome. This protocol allows access of gut mononuclear cells from the colon and, with modifications, to leukocytes of the small intestine in any of these preclinical murine models.

A PubMed search using the search terms “intestine AND immune cell AND isolation” reveals over 200 publications describing methods for small intestine digestion to extract immune cells. However, a similar literature search for colon yields no well-delineated protocols specifying isolation of immune cells from the colon. This may be because the colon has more muscular and interstitial layers, rendering it more difficult to completely digest than the small intestine. Unlike existing protocols, this protocol specifically uses Collagenase E from Clostridium histolyticum without other bacterial collagenases (Collagenase D/ Collagenase I). We demonstrate that, using this protocol, digestion of the colonic tissue can be achieved while preserving the quality of isolated gut mononuclear immune cells (MNC) without the addition of anti-clumping reagents such as sodium versenate (EDTA), Dispase II, and deoxyribonuclease I (DNAse I)^21,22,23. This protocol is optimized to allow reproducible robust extraction of viable MNC from the murine colon for further directed studies and should lend itself to the study of immunology of the colon or (with modifications) the small intestine^24,25.

Protokół

All studies were conducted under rodent research protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Miami Miller School of Medicine, which meets the veterinary standards set by the American Association for Laboratory Animal Science (AALAS).

1. Preparation of Solutions

1. As described in Table 1, prepare the Colon Buffer, Silica-Based Density Separation Media 100%, Silica-Based Density Separation Media 66%, Silica-based Density Separation Media 44%, Collagenase E Digestion Buffer, and FACS Buffer.
   1. Prepare Colon Buffer the day prior to the procedure and store overnight at 4 °C.
   2. Prepare 100% Silica-Based Density Separation Media the day prior to the procedure and stored overnight at 4 °C, placing at room temperature the morning of the procedure to thaw.
   3. Prepare 66% and 44% Silica-Based Separation Media in the morning of the isolation, using room temperature 100% Silica-Based Density Separation Media and Colon Buffer.
   4. Measure the appropriate amount of Clostridium histolyticum-derived Collagenase E and store at -20 °C overnight prior to the procedure. The following morning, dissolve in the appropriate volume of Colon Buffer to derive Collagenase Digestion Buffer. For all incubations with Collagenase Digestion Buffer on day of the procedure, pre-warm the solutions to 37 °C.
2. For the entire of the protocol steps, keep one centrifuge at 20 °C and the rotation speed of 859 x g, with brakes inactivated (0 deceleration) for gradient centrifugation. Set another to 4 °C and rotation speed of 859 x g, with the standard deceleration, for wash steps.

2. Harvesting the Colon

1. Euthanize the mouse via CO₂ asphyxiation followed by AALAC-approved confirmatory method.
2. Place the mouse in a supine position and spray the fur with 70% ethanol. Using large tissue scissors, make a vertical midline incision and expose the intact peritoneum.
3. Using fine dissection scissors, open the peritoneum. Use forceps to move the small bowel to one side and expose the descending colon. Slightly pull upward on the descending colon to maximally expose the rectal portion of the colon. Cut the distal rectum deep in the pelvis and dissect and remove the entire colon as one unit, from the distal rectum to cecal cap.
4. Transfer the colon in 20 mL chilled Colon Buffer in a 50 mL polypropylene tube.

3. Cleaning the Colon

1. Place the colon on a moistened paper towel and extract the solid stool by applying mild pressure to the bowel wall with the blunt end of scissors or forceps.
2. Place the colon in a Petri dish and flush the gut with 10 mL of chilled Colon Buffer using a 10 mL syringe with 18 G blunt fill needle.
3. Transfer the colon to a Colon Buffer moistened paper towel and remove the mesentery and fat with the sharp end of scissors.
4. Place the colon in a Petri dish filled with 5-10 mL chilled Colon Buffer agitating manually to wash remaining colonic contents. Repeat 2-3 times.
5. Cut the colon longitudinally from its more muscular rectal end to the proximal colon (generating a single rectangular open colon piece) in a Petri dish filled with fresh chilled Colon Buffer. Discard existing media and refill with clean chilled Colon Buffer.
6. Wash the intestine 3 times by vigorously swirling it in the Petri dish and replacing the 5-10 mL of chilled Colon Buffer after with each wash.
7. Place the rectangular colon tissue on a paper towel moistened with Colon Buffer and cut it by slicing it horizontally and then into small fragments (3 mm x 3 mm sections).
8. Collect the colon fragments carefully using fine forceps into 20 mL chilled Colon Buffer in a 50 mL polypropylene conical tube.
9. Wash the colon fragments 3 times, each wash in 20 mL Colon Buffer, by vigorously swirling the tube for 30 s. Between each agitation, allow the tissue fragments to settle to the bottom of the tube. Decant or vacuum aspirate the supernatant while preventing tissue fragment loss in the aspiration process between each wash.
   NOTE: There is no need to change the tube after each wash.

4. Collagenase Digestion 1

1. Add 20 mL of the Collagenase Digestion Buffer to the washed colon fragments in the 50 mL polypropylene conical tube.
2. Place the closed 50 mL tube at 37 °C in an incubated orbital shaker with the rotation rate set at 2 x g for 60 min. Ensure the tissue fragments are in constant motion during agitation; if necessary, increase the rotation rate incrementally to ensure that no tissue fragments settle to the tube bottom.

5. Prepare Silica-based Separation Media Gradients

1. Prepare 66% and 44% Silica-based Density Separation Media, using 100% Silica-based Density Separation Media at 20 °C (room temperature) and Colon Buffer.
2. Pour 5 mL of 66% Silica-based Density Separation Media into each of 3 separate 15 mL polypropylene tubes. Prepare 3 tubes per colon. This forms the higher density base of the gradient isolation procedure, onto which lower density separation media will be layered to create the separation gradient.
3. Store at 20 °C until use.

6. Collection of Supernatant from Digestion 1

1. Collect only the supernatant using a 25 mL serological pipette and filter the supernatant through a 40 μm pore filtration fabric cell strainer placed into a clean 50 mL polypropylene conical tube, after Collagenase Digestion 1 is completed. Be careful not to aspirate any existing tissue fragments.
   NOTE: Retain any remaining visible tissue fragments in the tube. These will undergo second collagenase digestion (step 8).

7. Quenching Collagenase Digestion Buffer

1. Fill the 50 mL polypropylene tube completely with chilled Colon Buffer.
   NOTE: Collagenase is active at 37 °C; hence chilled buffer inactivates this enzyme.
2. Centrifuge the tube at 4 °C at 800 x g for 5 min.
   1. Discard the supernatant via vacuum aspiration. Wash cells with 25 mL of fresh Colon Buffer and centrifuge at 800 x g for 5 min.
   2. Resuspend the pellet in less than 1 mL of fresh chilled Colon Buffer.
   3. Place the 50 mL polypropylene conical tube on ice.

8. Collagenase Digestion 2

1. Repeat step 4 (Digestion 1) with the remaining tissue fragments retained from step 6.1.

9. Tissue Disaggregation Following Digestion 2

1. Flush the tissue fragments vigorously back and forth between the tube and a 10 mL syringe through an 18 G blunt-end needle.
2. Repeat this flush for a minimum of 7-8 complete passages, continuing until no gross tissue fragments or debris are visible.

10. Filter Cells

1. Pass the tissue disaggregation suspension through a 40 μm-pore filtration fabric cell strainer into a clean 50 mL polypropylene tube.
2. Wash the filtration fabric cell strainer with 10 mL chilled Colon Buffer to recover any cells ensnared in the filter.

11. Quenching Collagenase Digestion

1. Fill the 50 mL polypropylene conical tube to the rim with chilled Colon Buffer.
   NOTE: The temperature of Colon Buffer is critical to ensure quenching of collagenase activity.
2. Spin at 4 °C and 800 x g for 5 min.
3. Discard the supernatant via vacuum aspiration.
4. Wash by resuspending in 25 mL of fresh chilled Colon Buffer, followed by centrifugation at 4 °C, 800 x g for 5 min.
5. Discard the supernatant via vacuum aspiration.
6. Pool the resuspended pellet from Collagenase Digestion 1 (step 7) to its corresponding tube from step 11.4.
7. Repeat Step 11.4 (wash and centrifugation).

12. Silica-based Density Separation Media Gradient Separation

NOTE: Perform steps 12-18 as quickly as possible, to ensure rapid quenching of collagenase activity.

1. Following step 11.7, resuspend each pellet in 24 mL total of 44% Silica-based Density Separation Media per colon.
2. Slowly layer 8 mL of the media from step 12.1 onto each of three tubes prepared at step 5.2 (containing 66% Silica-based Density Separation Media), using a 10 mL serological pipette. Maintain a steady and slow flow of the 44% Density Separation Media while layering the gradient in order to avoid disruption of the interface.
3. Carefully balance all tubes within the centrifuge buckets using a weigh scale or a balance.
4. Spin the tubes 20 min at 859 x g in a centrifuge without brake at 20 °C. Allow the rotors to come to complete rest before removing tubes, taking care not to disrupt the cells at the gradient interface.

13. Collect Mononuclear Cells from the Gradient Interface

1. Visualize the gradient interface (near the 5 mL mark), where typically a 1-2 mm thick white band (containing MNC) is present.
   NOTE: One may or may not see a white band. However, MNC will be at this interface and should cloud the clarity of the gradient interface.
2. Vacuum aspirate and discard the top 7 mL of the top gradient to allow easier pipette access to the interface.
3. Using continuous manual suction and steady rotating wrist motion, collect the interface layer of cells into a clean 50 mL polypropylene conical tube. Collect until the interface between the 2 gradients is clear and refractile (clear of cells).
4. Fill the collection tube with 50 mL of chilled FACS Buffer. Spin at 4 °C, 800 x g for 5 min.
5. Aspirate the supernatant via vacuum aspiration and resuspend the pellet in 1 mL of FACS Buffer.
6. Count the cells on a hemocytometer at a 1:2 dilution using appropriate dead cell exclusion methods.
7. Proceed to FACS staining or other assays with freshly isolated colonic MNC.

Wyniki

When working with murine colon disease models, it is helpful to be able to both quantify and qualitatively assess, among the MNC of the colon, multiple immune cell subsets involved in the inflammatory process. The single-cell suspension of MNC obtained through the application of this protocol facilitates such phenotypic characterization in a robust and reproducible manner. As a proof of principle for the application of this isolation method under diverse experimental settings, we retrieve...

Dyskusje

This visual protocol describes well-tolerated methods for the isolation of colonic mononuclear cells including lamina propria lymphocytes (LPL). Given that this protocol was optimized in evaluating severe post-transplant mouse colitis models where inflammatory cytokines and tissue injury lend themselves to poor viability of recovered MNC, we anticipate that these methods can be translated to other applications requiring phenotypic analysis of colonic MNC. These include but, are not limited to assessing colon inflammation...

Materiały

Name	Company	Catalog Number	Comments
60 mm Petri DIsh	Thermo Scientific	150288
1x PBS	Corning	21-040-CV
10x PBS	Lonza BioWhittaker	BW17-517Q
10 mL Disposable Serological Pipette	Corning	4100
10 mL Syringe	Becton Dickinson	302995
15 mL Non-Sterile Conical Tubes	TruLine	TR2002
18 G Blunt Needle	Becton Dickinson	305180
25 mL Disposable Serological Pipette	Corning	4250
40 μm pore size Cell Strainer	Corning	352340
50 mL Falcon Tube	Corning	21008-951
Bovine Serum Albumin (BSA)	Sigma	A4503-1KG
Fixation Buffer	Biolegend	420801
E. coli Collagenase E from Clostridium histolyticum	Sigma	C2139
EDTA, 0.5 M Sterile Solution	Amresco	E177-500ML
Fetal Bovine Serum	Thermo /Fisher Scientific -HyCLone	SV30014.03
HEPES	GE Healthcare-HyClone	SH30237.01
Percoll	GE Healthcare-Life Sciences	1708901
RPMI Medium	Corning	17-105-CV
Sodium Azide	VWR Life Science Amresco	97064-646
Trypan Blue	Lonza BioWhittaker	17-942E