Induction of Necrotizing Enterocolitis in Human Epithelial Enteroid Model

プロトコル

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Reagent Preparation

1. Prepare Culture Media stock solution for whole tissue collection: 1 L of Dulbecco's Modified Eagle Medium (DMEM), 110 mL of Fetal Bovine Serum (FBS), 11 mL of Penicillin-Streptomycin (final concentration 1%), and 1.1 mL of filter-sterilized insulin (final concentration 0.1%.) Store stock solution at 4 °C.
2. Prepare Chelating Buffer #1: 30 mL of Culture Media (as described in 1.1), 600 µL of 0.5 M Ethylenediaminetetraacetic acid (EDTA) (final concentration 10 mM), 300 µL of Gentamicin (final concentration 1%) and 60 µL of Amphotericin B (final concentration 0.2%). Store stock solution at 4 °C.
3. Prepare Chelating Buffer #2: 30 mL of Culture Media (as described in 1.1), 300 µL of 0.5 M EDTA (final concentration 5mM), 300 µL of Gentamicin (final concentration 1%) and 60 µL of Amphotericin B (final concentration 0.2%). Store stock solution at 4 °C.
4. Prepare Human Minigut Media: 41.4 mL of DMEM/F-12, 5 mL of FBS (final 10%), 500 µL of Penicillin-Streptomycin (final concentration 1%), 500 µL of L-glutamine (final concentration 1%), 500 µL of Gentamicin (final concentration 1%), 100 µL of Amphotericin B (final concentration 0.2%), 500 µL of 1 M N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (final concentration 10 mM), 500 µL of 100x N-2 supplement, and 1 mL of 50x B-27 supplement minus Vitamin A for a total volume of 50 mL. Store stock solution at -20 °C.
5. Prepare Human Minigut Media Complete: 10 mL of Human Minigut Media (prepared in 1.4), 10 µL of 100 µg/mL Wnt3a (final concentration 100 ng/mL), 10 µL of 100 µg/mL Noggin (final concentration 100 ng/mL), 10 µL of 1 mg/mL R-Spondin (final concentration 1 µg/mL), 10 µL of 500 µg/mL Epidermal Growth Factor (EGF) (final concentration 50 ng/mL), 10 µL of 1 M N-Acetylcysteine (final concentration 1 mM), 10 µL of 10 mM Y-27632 (final concentration 10 µM), 10 µL of 500 µM A-83 (final concentration 500 nM), 10 µL of 10 mM SB202190 (final concentration 10 µM), 100 µL of 1 M Nicotinamide (final concentration 10 mM) and 1 µL of 100 µM [leu] 15-gastrin 1 (final concentration 10 nM). Total volume 10 mL. Store stock solution at 4 °C.
   NOTE: Solutions must be used within 48 hours.

2. Crypt Isolation and Plating from Whole Tissue

1. At the time of collection in the operative suite, place the human small intestinal tissue sample in cold Dulbecco's Phosphate Buffered Saline (DPBS). Wash the specimen in cold DPBS until clear of stool and blood. Store specimen at 4 °C in RPMI 1640 Medium until ready for crypt isolation.
   NOTE: Tissue cannot be stored for more than 24 hours.
   1. Make sure that the specimen is clear of stool and blood. Using delicate dissecting scissors, remove any excess fat or surgical clips/staples, etc. Weigh the specimen.
      ​NOTE: Aim for a piece approximately 0.75-2.5 g.
2. Cut the specimen into 0.5 cm pieces and place in 30 mL of Chelating Buffer #1 (as prepared in step 1.2).
   1. Shake at low speed for 15 min at 4 °C.
   2. Filter tissue through 100 µm cell strainer and discard flow through.
3. Add the tissue to 30 mL of Chelating Buffer #2 (as prepared in step 1.3).
   1. Shake at low speed for 15 min at 4 °C.
   2. Filter tissue through a 100 µm cell strainer and discard flow through.
4. Thaw 500 mL of basement membrane matrix on ice for use in step 2.8.
5. Add tissue to 10 mL of cold DMEM in a 50 mL conical tube and shake vigorously by hand for 10 s.
   1. Filter through a 100 µm cell strainer and collect flow through (Label #1). Keep tube #1 on ice.
   2. Add tissue to another 10 mL of cold DMEM in a separate 50 mL conical tube and shake vigorously by hand for 10 seconds.
   3. Filter through a 100 µm cell strainer and collect flow through (label #2).
   4. Repeat two additional times until there are four conical tubes with flow through (labeled tubes #1-4).
6. Filter tube #1 solution through a 100 µm cell strainer and transfer flow through into a 15 mL conical tube (Label #1). Repeat for #2-4.
   1. Centrifuge 15 mL tubes #1-4 at 200 x g for 15 min at 4 °C.
7. In the laminar flow hood, remove the supernatant from tubes #1-4 and discard. Avoid disrupting the cloud of tissue immediately above the pellet, even if that means leaving some supernatant behind.
   1. By pipetting slowly, mix together the pellet with the leftover supernatant in tubes #1-4.
   2. Transfer the mixture from tubes #1-4 into one single 2 mL conical tube.
   3. Centrifuge the conical tube at 200 x g for 20 min at 4 °C.
8. Remove the supernatant and re-suspend the pellet in 500 µL of the basement membrane matrix.
   NOTE: Keep the basement membrane matrix on ice at all times and work quickly for the next steps. This product polymerizes very quickly at room temperature.
   1. Apply 50 µL of specimen/basement membrane matrix suspension to the center of a well in a 24-well plate. This should appear dome-shaped.
      NOTE: Use of chilled pipette tips aids in smoother transfer of the suspension, minimizing polymerization.
   2. Repeat 9 times to fill 10 total wells.
9. Place the 24-well plate in 37 °C, 5% CO2 incubator for 30 min to allow polymerization.
10. Add 500 µL of Human Minigut Media Complete (as prepared in step 1.5) to each well. Replace every 2 days.
11. Collect enteroids after 5-10 days, when budding is visualized. See Steps 4 and 5 for collection instructions.

3. Induction of Experimental NEC

1. Add 10 µL of 5 mg/mL of lipopolysaccharide (LPS) to 500 µL of Human Minigut Media Complete (as prepared in step 1.5) in each well on Day 0. Replace every 2 days until collection.

資料

Name	Company	Catalog Number	Comments
4% Paraformaldehyde	ThermoFisher	AAJ19943K2
Basement Membrane Matrix (Matrigel)	Corning	CB-40230C
DMEM/F-12	ThermoFisher	MT-16-405-CV
Dulbecco's Modified Eagle Medium (DMEM)	ThermoFisher	11-965-118
Dulbecco's Phosphate-Buffered Saline (DPBS)	ThermoFisher	14190-144
Epidermal Growth Factor (EGF)	Sigma	E9644-.2MG
Ethylenediaminetetraacetic acid (EDTA)	Sigma	EDS-500G
Fetal Bovine Serum (FBS)	Gemini Bio-Pro	100-125
Phosphate Buffered Saline (PBS)	Sigma	P5368-5X10PAK
RPMI 1640 Medium	Invitrogen	11875093
Tissue Processing Gel (Histogel)	ThermoFisher	22-110-678
Lipopolysaccharide (LPS)	Sigma	L2630-25MG
Gentamicin	Sigma	G5013-1G