Organoids as a Model to Study Infectious Disease

Abstract

The advent of the gastric organoid culture system has provided a new model to emulate native epithelial tissue in vitro. Gastric organoids grow from isolated epithelial stem cells and develop into three dimensional structures that can be used to study host physiology. Here we describe current laboratory protocols for growing gastric organoids and the microinjection of pathogens such as Helicobacter pylori into the lumen of gastric organoids in order to study the cellular response following infection.

1. Introduction

In 2009, Sato et al. established long-term epithelial primary culture methods under which isolated gastrointestinal (GI) stem cells grow to form three dimensional structures [1]. These three dimensional epithelial structures, referred to as “organoids,” have proved useful in studying biological processes that occur in native tissue. In the past, GI disease studies have chiefly been limited to in vivo animal models or in vitro immortalized cancer cell lines. Whereas in vivo models have the cellular diversity that is necessary to support tissue function, approaches for experimental manipulation are limited. Conversely, in vitro cancer cell lines are simple to work with, but have pathologic genetics and function in a more homogeneous cell population. GI organoids represent a significant advancement in our ability to replicate the gastrointestinal environment in terms of cellular diversity of normal epithelial cells, and can be an applicable model to bridge the gap between human and mouse research. Human and mouse gastric organoids have value in studying gastric physiology and disease, especially in examining Helicobacter pylori (H. pylori) infection [2–4]. Injection of bacteria into human and mouse intestinal organoids results in a similar pathogenic response as with in vivo tissue [5–7]. Thus the use of GI organoids expands our current understanding of bacterial–host interactions and allows for a deeper understanding of toxic factors or pathogens that mediate disease [3, 4].

Here we describe our current protocol to inject H. pylori into organoids derived from the mouse gastric corpus, as an example of microinjection to study organoid epithelial cell-pathogen interaction.

2. Materials

Prepare all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations.

2.1. Generation and Maintenance of Gastric Organoid Cultures Derived from Mouse Stomach Tissue

1. Microdissecting curved scissors.

2. #7 fine point curved forceps.

3. Silicon dish (Sylgard^® 184 Silicone Elastomer Kit prepared in a glass culture dish).

4. Dissection microscope.

5. Matrigel Matrix (Corning), basement membrane, growth factor reduced, phenol red free.

6. Sterile Dulbecco’s phosphate-buffered saline (DPBS) without calcium or magnesium, phenol red free, (Corning). Store at 4 °C.

7. 250 mM EDTA stock in ultrapure water.

8. Dissociation buffer: 43.3 mM sucrose (Fisher), 54.9 mM d-sorbitol (Sigma), in DPBS.

9. Sterile 24-well cell culture plate with lid.

10. Sterile 2-well chambered cover glass slides.

11. Sterile 5 mL round bottom polystyrene test tube, with snap cap.

12. Sterile 15 mL centrifuge tubes.

13. Sterile 1 mL Tuberculin syringe with 26G × 3/8 in.

14. Mouse gastric organoid culture growth medium: Advanced Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (DMEM/F12, Invitrogen), 2 mM l-glutamine (Invitrogen), 10 mM HEPES (Sigma), 100 U/mL penicillin/100 mg/mL streptomycin (Thermo Scientific), 1× N2 medium supplement (Invitrogen), 1× B27 medium supplement (Invitrogen), 10% R-spondin conditioned medium, 10% Noggin conditioned medium, 50% Wnt3a conditioned medium, 50 ng/mL epidermal growth factor (EGF) (PeproTech), 10 nM Human [Leu15]-Gastrin I (Sigma) (see Note 1).

15. 70% Ethanol.

2.2. Helicobacter pylori Culture

1. Columbia Agar Base (Thermo Scientific): 0.0425 g/mL in ultrapure water.

2. Defibrinated horse blood (Colorado Serum Company).

3. β-cyclodextrin (Sigma).

4. Cycloheximide (Sigma), stock solution 50 mg/mL in 95% ethanol.

5. Vancomycin (Sigma), stock solution 30 mg/mL in ultrapure water.

6. Trimethoprim (Sigma), stock solution 40 mg/mL in DMSO.

7. Sterile petri dishes 100 × 15 mm.

8. 2 L Erlenmeyer flask.

9. H. pylori Sydney Strain 1 (SS1).

10. Microaerophilic chamber (BD Diagnostic Systems).

11. BD GasPak EZ (BD Diagnostic Systems).

12. Brucella broth (BD Diagnostic Systems).

13. Fetal bovine serum (HyClone).

14. H. pylori plate preparation: Suspend 21.25 g Columbia Agar Base into 475 mL of ultrapure water, in a 2 L Erlenmeyer flask. Add 1 g β-cyclodextrin. Use a stir bar to mix. Autoclave agar solution at 121 °C for 20 min. Cool agar to 55 °C in a water bath for at least 1 h. In a biosafety hood, add to cooled agar 500 μL cycloheximide (Final: 50 μg/mL), 83 μL vancomycin (Final: 5 μg/mL), 125 μL trimethoprim (Final: 10 μg/mL) and 25 mL defibrinated horse blood (Final: 5%). Mix by swirling the flask by hand and with a serological pipet add 25 mL of blood agar medium per plate, avoiding bubbles (this protocol makes approximately 20 plates). Wait until agar is set before storing at 4°C, upside down in a plastic sleeve. Plates can be stored for 2 weeks.

15. 50% Glycerol.

2.3. Microinjection

1. Parafilm.

2. Nanoject II microinjector apparatus (Drummond Scientific).

3. P-2000 Micropipette Puller (Sutter Instruments).

4. Replacement Capillaries for Nanojet II Injectors (Drummond Scientific).

5. Micro scissors, straight, 7 cm long, 0.1 mm tips, 3 mm blades.

6. Mineral Oil.

7. Stereoscope.

8. Lucifer Yellow: a fluorescent dye of M.W. 457.24.

9. Long needle syringe.

3. Methods

3.1. Generation and Maintenance of Gastric Organoid Cultures Derived from Mouse Stomach Tissue

3.1.1. Culturing of Mouse Gastric Glands

1. Euthanize mice using a method approved by the institution where the research is to be conducted. We have euthanized mice with isoflurane inhalation to deep anesthesia or CO₂ inhalation, immediately followed by cervical dislocation.

2. Disinfect the exterior abdomen of the mouse with 70% ethanol. Make an incision into the abdominal cavity. Extend the incision to the rib cage by cutting the abdominal musculature on both sides. Grasp the forestomach and cut the esophagus and the proximal duodenum. Remove whole stomach from the abdominal cavity, rinse whole stomach in cold DPBS, then open stomach along the greater curvature. Rinse the opened stomach in cold DPBS to remove food debris.

3. Pin the opened stomach (luminal side down) on a silicon dish, add ice cold DPBS to cover the tissue to prevent the tissue from drying. Quickly but carefully, remove the serosal muscle layer and fat using surgical scissors and forceps under a dissection microscope.

4. Cut out the corpus from the opened stomach and mince into<5 mm pieces.

5. Place minced tissue into a 15 mL centrifuge tube with 5 mL cold DPBS.

6. Add EDTA (5–10 mM final concentration) to the tissue in cold DPBS and place on a rocker at 4 °C for 2 h.

7. At this point:

   Thaw the required amount of 500 μL Matrigel aliquots in 4°C or on ice.

   Prepare mouse gastric organoid culture growth medium.

8. After rocking, remove EDTA solution and add 5 mL solution of dissociation buffer.

9. Vigorously shake by hand for 1–2 min to dissociate whole glands from tissue. From this point work in a biosafety hood.

10. Under a microscope, pipette approximately 50 μL of tissue solution to visualize the isolated glands. Isolated glands should be visible and approximately 50% solution area should be glands. Otherwise repeat step 9.

11. Transfer solution containing the glands to 5 mL round-bottom tube, avoiding nondissociated tissue.

12. Centrifuge for 5 min in 4 °C at a speed of 150 × g. Remove all of the solution, avoiding the gland pellet at the bottom of the tube.

13. Add Matrigel to tube and gently mix, avoid the introduction of bubbles.

14. Pipette 30 μL of Matrigel containing glands per 24-well plate.

15. Transfer 24-well plate into a CO₂ incubator (5% CO₂, 37 °C) for 15–20 min to allow Matrigel to polymerize.

16. Overlay 500 μL of organoid culture medium per well.

17. Incubate the organoids in a CO₂ incubator (5% CO₂, 37 °C).

18. Refresh medium every 3–4 days. If cultures are healthy, approximately 30–40% or greater of glands should grow out into organoids. Evaluate growth daily.

19. Organoids should be passaged every 7–10 days, when organoids attain a size of >500 μm.

3.1.2. Passaging of Gastric Organoids for Microinjection

1. Remove medium and add approximately 1 mL cold DPBS per well.

2. Use a sterile pipette tip to help break up Matrigel, then transfer suspension to 5 mL round-bottom tube.

3. Centrifuge at 150× g for 5 min at 4 °C.

4. Aspirate supernatant.

5. Add 1 mL cold DPBS. Using 26 G syringe, gently draw up the organoids and DPBS. Eject quickly from syringe once to break up the organoids.

6. Refill the 5 mL round-bottom tube with cold DPBS.

7. Centrifuge at 150 × g for 5 min at 4 °C.

8. Remove supernatant. Add fresh Matrigel and gently mix, avoid introduction of bubbles.

9. Pipette 50 μL of Matrigel containing fractions of organoid per chambered cover glass well for experiments. Remainder of the Matrigel/organoid fraction mixture can be returned to a 24-well plate for regrowth (see Subheading 3.1.1, steps 14–19).

10. Transfer chambered cover glass into a CO₂ incubator (5% CO₂, 37 °C) for 15–20 min to allow Matrigel to polymerize.

11. Overlay 1 mL of organoid culture medium per well.

12. Incubate the organoids in a CO₂ incubator (5% CO₂, 37 °C).

13. Change medium every 3–4 days.

14. Grow organoids 3–5 days before experiment.

3.2. Helicobacter pylori Culture

1. In a biosafety cabinet, warm plates to room temperature.

2. Remove H. pylori frozen stock from a storage tube with a sterilized loop and dilute into Brucella broth at 2 × 10⁶ H. pylori per 100 μL. Pipette solution onto a blood agar plate and spread with loop around the plate.

3. Incubate H. pylori inoculated plates for 3–4 days upside down at 37 °C in a humidified microaerophilic chamber with a CO₂ GasPak. Check growth and incubate as needed.

4. Use a sterilized loop to harvest H. pylori from plates and suspend in Brucella broth supplemented with 10% fetal bovine serum and CO₂ GasPak in a humidified microaerophilic chamber in an incubator at 37 °C for 16–18 h (see Notes 2 and 3).

5. Collect bacteria by centrifugation at 2000 × g for 5 min and resuspend in Brucella broth without serum.

6. Dilute H. pylori in 50% glycerol (1:100 dilution) to calculate density in hemocytometer.

3.3. Microinjection

To make the following procedures clear to the naked eye, a high concentration of Lucifer yellow was used in this section, in addition to H. pylori. Lower LY concentrations can be used once procedures are established, or other dyes of interest substituted (see Note 4).

1. At least 1 day before H. pylori injection, replace organoid culture medium with 1 mL of the culture medium without penicillin/streptomycin.

2. Use a micropipette puller to pull replacement glass capillaries. Setting for pulling replacement glass capillaries is as follows:

   Heat	Filament	Velocity	Delay	Pull
   400	4	50	250	200

3. Trim pulled glass capillaries with micro scissors (glass capillary needle) to approximately 2–3 mm for LY [Fig. 1A (b)] or 5–6 mm for H. pylori [(Fig. 1A (c)] from tip. Inner capillary size on tip can be 5–8 μm [(Fig. 1B (b)] or 15–20 μm [(Fig. 1B (c)], respectively (see Notes 5 and 6).

4. Before attaching glass capillary needle to the Nanoject II, capillary is filled with mineral oil by using a long needle syringe, avoid air bubble contamination (see Note 7).

5. Gently slide capillary into place in Nanoject II.

6. Click “EMPTY” to squeeze mineral oil from the capillary to make sure no air is in the tip (see Note 7).

7. Set injection volume in NanojectII microinjector apparatus (instrument volume range 2.3–69.0 nL), i.e., 2.3 nL/slow for LY or 23 nL/slow for H. pylori.

8. Onto a sheet of Parafilm, pipette approximately 2 μL of LY (Fig. 2) or Brucella broth containing H. pylori and orient Nanoject II with attached capillary to the droplet under the stereoscope.

9. Press “FILL” to take up solution on Parafilm (Fig. 2).

10. Remove the tip from the solution, press “INJECT” to make sure solution comes out from tip (Fig. 2).

11. Place organoids, in 2-well chambered cover glass, on the stage of the stereoscope, and insert the glass capillary needle into an organoid (Figs. 3 and 4 “Before”).

12. Use Nanoject II microinjector apparatus, click “INJECT” once to inject 2.3 nL LY (20 mM) or 23 nL of Brucella broth containing 5 × 10⁹/mL H. pylori into an organoid with a diameter of approximately 500 μm (Figs. 3 and 4 “Injection”). This yields approximately 1 mM fluorescence or 1 × 10⁵ bacteria injected per organoid, respectively (see Notes 5, 6 and 8).

13. Slowly and gently remove Nanoject II microinjector from the organoid.

14. Incubate the organoids in a CO₂ incubator (5% CO₂, 37 C).

15. Perform proposed experiments (see Note 9).

Fig. 1.

Glass capillary needle. A. Stereoscopic image of original glass capillary (left), untrimmed pulled capillary (a), 2–3 mm trimmed capillary (b) and 5–6 trimmed capillary (c) in the left panel. Right panel shows low magnification image of 2–3 mm trimmed capillary on the stage micrometer (Microscope World, 25 mm KR812). B. High magnification of stereoscopic images of a pulled capillary (a), 2–3 mm trimmed capillary (b), and 5–6 trimmed capillary (c) in the left panel. The approximate diameters of the original capillary are inner: 500 μm and outer: 1100 μm

Fig. 2.

Filling solution of interest into a glass capillary needle. Upper panel shows 2 μL of Lucifer yellow on Parafilm under bright-field light microscopy before, during and after filling glass capillary needle using Nanoject II, while lower panel shows same events under fluorescence (GFP filter)

Fig. 3.

Microinjection of Lucifer yellow into a gastric organoid. Stereoscopic images during Lucifer yellow (LY) injection in bright-field (upper panel) or fluorescence (lower panel). Images taken before, during, and immediate, 1 or 3 days following microinjection of 2.3 nL LY using 2–3 mm trimmed capillary needle in Nanoject II. Retention of microinjected LY over 3 days confirmed maintenance of barrier integrity of gastric organoid

Fig. 4.

Microinjection of H. pylori into a gastric organoid. Stereoscopic images of a gastric organoid before, during, and after microinjection of H. pylori into the lumen. The bacteria are visible as a cloud (red arrows) inside the organoid and then spread out (diffuse) in the lumen