Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 May 4.
Published in final edited form as: Methods Mol Biol. 2020;2121:185–198. doi: 10.1007/978-1-0716-0338-3_16

Establishing Human Intestinal Enteroid/Organoid Lines from Preterm Infant and Adult Tissue

Christopher J Stewart 1, Mary K Estes 2,3, Sasirekha Ramani 2
PMCID: PMC8094111  NIHMSID: NIHMS1693429  PMID: 32147796

Abstract

Self-organizing mini-intestines cultured ex vivo from intestinal biopsy/resected samples, termed intestinal organoids or enteroids, present a unique opportunity for mechanistic investigation of health and disease of the intestinal epithelium. These patient-derived epithelial cultures are nontransformed, retain the genetic background of the patient, maintain regional specificity, differentiate into all major cell types of the intestinal epithelium, and are physiologically active. The biological relevance of human intestinal enteroids also circumvents the need for animal models for studies on the human gastrointestinal epithelium. Coculture with human endogenous microbes allows for exciting new studies on microbial–host interactions.

While the popularity of organoids/enteroids for human research has risen drastically over the past decade, existing work and published methods are primarily limited to adult tissue. Here, we describe a concise and effective method for the establishment neonatal enteroids (including preterm and term) from surgically resected tissue or biopsy material. While the protocol works on adult tissue/biopsies, it has been specifically adopted and optimised for neonatal tissue. We detail the procedure at each stage ranging from human tissue collection and extraction of stem cells from the tissue, to passaging and general maintenance of organoid/enteroid lines, and how to freeze and revive lines as needed.

Keywords: Stem cell, Crypt, Intestine, Enteroid, Organoid, Preterm, Infant, Adult, Cell culture, Media

1. Introduction

Innate lymphoid cells (ILCs) are tissue resident populations that occupy specific niches in peripheral tissues including the intestine. Here they form a first line of defense against pathogens and are responsible for expressing cytokines critical for maintaining the integrity of the intestinal mucosa. Intestinal ILC interact with the gut microbiome disruption, which has been implicated in a wide variety of diseases including inflammatory bowel disease and intestinal cancer. Therefore, it is important understand how ILCs modulate the gut microenvironment in humans and one methodology that might provide insight into ILC function within the gut microenvironment in human is through enteroid cultures.

Epithelial cells line all surfaces of the human body and are of major importance to health and disease. The epithelium provides a barrier from exogenous material coming into contact with the external (e.g., skin) or internal (e.g., intestinal lumen) surfaces. Thus, the epithelial barrier is positioned at the interface where host and non-host meets and, as such, is central for absorption, secretion, susceptibility to pathogens, and immune regulation. Of particular importance to human health and disease are epithelial cells of the intestine, which consists of four sequential sections: duodenum, jejunum, ileum, and the colon. Self-organizing mini-intestines cultured ex vivo from intestinal biopsy/resected samples, termed intestinal organoids or enteroids, present a unique opportunity for mechanistic investigation into health and disease at the epithelial level [1]. As per the 2012 guidelines on nomenclature for intestinal in vitro cultures, an enteroid is defined as a multilobulated structure with a lumen that develops from an enterosphere by formation of budding crypts; thus, we will use the term enteroid from herein [2].

Human intestinal enteroids are generated from isolated intestinal crypts that contain Lgr5+ stem cells and established cultures retain segment-specific properties (e.g., ileal enteroids will be distinct from colonic enteroids) [3, 4]. Enteroids are composed of all the major cell types of the intestinal epithelium (enterocyte, Paneth, goblet, neuroendocrine, and stem) and are physiologically active (e.g., secrete mucin; demonstrate fluid secretion and swelling in response to enterotoxins) [5, 6]. Furthermore, human intestinal enteroids retain the genetic and potentially the epigenetic susceptibility and immune programming of the host. Enteroids are therefore a robust and relevant model to systematically explore host responses to stimuli (e.g., microbial colonization) and drug responses. To this end, recent developments have been made in coculturing bacteria with human intestinal enteroids, allowing mechanistic studies of host–microbial interactions [7, 8]. The use of human intestinal enteroids further facilitates human specific studies of the biology of the gastrointestinal epithelium and thus circumvent the need for animal models while supporting the 3Rs framework (Replacement, Reduction and Refinement) [1]. Of particular note, mechanistic investigation of preterm infants has been challenging due to the inability to recapitulate the extent of human prematurity (e.g., equivalent to 23 week gestation) in animal models. Therefore, enteroids derived from preterm infants may facilitate important and potentially paradigm shifting research.

The use of enteroids for scientific investigation continues to rise and the technology is widely applicable to a range of research. The methods described in this chapter detail the procedure at each stage of enteroid generation, starting from human tissue collection, extraction of crypts containing stem cells from the tissue, passaging and general maintenance of the lines, and methods to freeze and revive lines as needed (Fig. 1). This protocol has been optimized for working with neonatal tissue (including preterm and term) but is also effective on adult tissue/biopsies. These protocols can be performed on both resected tissue and biopsies, but for the purposes of this book chapter we discuss the generation of enteroids from surgically resected tissue. Additionally, this protocol should work with tissue from any region of the intestine.

Fig. 1.

Fig. 1

Open in a new tab

Schematic showing the main stages in the generation of enteroid lines from intestinal tissue

2. Materials

Ensure all solutions and reagents are tissue culture grade. Perform all steps inside of a tissue culture laboratory hood. Use filter tips where possible. Diligently follow all waste disposal regulations when disposing of waste materials. When making up the media, each stock can be made ahead of time and frozen at −80 °C. In our experience, it is best to avoid storing media for more than 4 months where possible, although media stored beyond this timeframe may still be used.

2.1. Enteroid Media

  1. Complete Media Growth Factor negative (CMGF−): 500 ml Advanced DMEM/F12, 5 ml GlutaMAX 100×, 5 ml HEPES 1 M. Aliquot ~50 ml per sterile 50 ml falcon tube.

  2. Complete Media Growth Factor positive (CMGF+) in a volume of 500 ml: 78 ml CMGF−, 250 ml Wnt3A-conditioned media produced from ATCC CRL-2647 cells (ATCC,), 100 ml R-spondin-conditioned media (Trevigen Inc.), 50 ml Noggin-conditioned media [9], 10 ml B27 (50×), 5 ml N2 (100×), 5 ml nicotinamide (10 mM), 1 ml N Acetylcysteine (1 mM), 500 μl Gastrin (10 nM), 500 μl A83 (500 nM), 166 μl SB202190 (10 μM), 50 μl EGF (50 ng/mL) [4]. Vacuum filter (0.2 μm) and aliquot into 15 ml and/or 50 ml falcon tubes of varying volumes depending upon ultimately needs and throughput.

  3. High Wnt media in a volume of 500 ml: 250 ml CMGF+, 250 ml Wnt3A-conditioned media. Vacuum-filter (0.2 μm) and aliquot into 15 ml and/or 50 ml falcon tubes of varying volumes depending upon ultimately needs and throughput.

  4. Differentiation media in a volume of 500 ml: 458 ml CMGF−, 25 ml Noggin conditioned media, 10 ml B27 (50×), 5 ml N2 (100×), 1 ml N Acetylcysteine (500 mM), 500 μl Gastrin (100 μM), 500 μl A83 (500 μM), 50 μl EGF (500 μg/mL).

  5. Enteroid freezing media: 10% DMSO in CMGF+ containing Y-27632 (10 μM). Alternatively can use Dulbecco’s modified Eagle medium/F12, 10% fetal bovine serum, and 10% dimethyl sulfoxide [10].

  6. Matrigel Growth Factor Reduced Basement Membrane Matrix.

2.2. Extraction of Stem Cells from Tissue

  1. Incomplete chelating solution (ICS) in a volume of 500 ml: Dissolve 2.49 g Na2HPO4·2H2O (sodium phosphate dibasic dihydrate), 2.7 g KH2PO4 (potassium dihydrogen phosphate), 14 g NaCl (sodium chloride), 0.3 g KCl (potassium chloride), 37.5 g sucrose, and 25 g D-sorbitol in 500 ml tissue culture grade water. Vacuum-filter (0.2 μm) and aliquot 50 ml per sterile 50 ml falcon tube.

  2. Complete chelation solution (CCS) in a volume of 50 ml: Prepare the CCS by combining 40 ml sterile water, 10 ml 5× ICS, and 60 μl 1 M DTT. The addition of DDT to ICS should be done immediately prior to use.

3. Methods

Perform all steps inside of a tissue culture laboratory hood. Use filter tips where possible. Diligently follow all waste disposal regulations when disposing of waste materials. All reagents should be on ice and centrifuges should be cooled to 4 °C, unless otherwise stated.

3.1. Collection and Freezing of Resected Tissue

  1. Resected tissue should be collected in ice-cold CMGF− or PBS (enough to cover the entire sample) and placed in ice. The resected tissue should be stored on ice for as short a time as possible and ideally stem cells will be isolated within a few hours (see Note 1).

  2. If same day extraction is not possible, resected tissue can be frozen, but this may reduce the crypt yield (see Note 1).

  3. To freeze the resected tissue for later enteroid generation, first place the tissue in a small sterile dish (e.g., 35 mm diameter) containing enough PBS to cover the base of the dish and cut the tissue into ~5 mm2 fractions.

  4. Place each ~5 mm2 tissue fraction into a cryovial with 500 μl freezing media.

  5. Put each vial into a CoolCell (or other freezing containers that allow standardized controlled rate of −1 °C/min) and place the CoolCell in −80 °C overnight.

  6. Move each cryovial to liquid nitrogen storage the next day.

3.2. Extraction of Stem Cells from Tissue

For the below protocol, use wide-bore 1 ml low-retention filter tips. All tubes and solutions should be kept on ice throughout the procedure, unless otherwise stated. Set the centrifuge to 4 °C before beginning the protocol.

  1. Before starting this protocol, it is important to ensure reagents have been given sufficient time to thaw and that a 24-well plate has been incubated at 37 °C (see Note 2).

  2. Place the resected tissue in a puddle of CMGF− (or PBS) in small sterile dish (e.g., 35 mm diameter). If the resected tissue is fresh and plentiful, use sterile scissors to take a ~5 mm2 specimen. If the resected tissue is frozen in freezing media, remove the cryovial from liquid nitrogen and place in a 37 °C incubator for 2–5 min to thaw, then proceed as below.

  3. Mince tissue into small pieces (see Note 3) using sterile scissors (i.e., cleaned with 70% ethanol). Sterile forceps can also be used if needed.

  4. Transfer minced tissue sample into a 15 ml falcon containing 10 ml CCS and wash tissue sample by inverting tube ~10 times (see Note 4).

  5. Allow tissue sample to settle down to the bottom of the falcon tube (typically ~3–5 min) and then remove supernatant carefully using a pipette (see Note 5).

  6. Add 10 ml of CSS containing 100 μl of Fungizone (1:100) + 100 μl Pen/strep (1:100) and invert the falcon ~10 times.

  7. Allow minced tissue to settle down to the bottom of the falcon tube (typically ~3–5 min) and then remove supernatant carefully using a pipette (see Note 5).

  8. Transfer minced tissue to a new 15 ml tube containing 10 ml CCS and wash tissue sample by inverting ~10 times.

  9. Allow minced tissue to settle down to the bottom of the falcon tube (typically ~3–5 min) and then remove supernatant carefully using a pipette (see Notes 5 and 6).

  10. Pour CCS to 3 ml mark on the 15 ml falcon tube.

  11. Transfer the entire contents (i.e., minced tissue sample in 3 ml CSS) to a single well of a 6-well plate and add 180 μl of 0.5 M EDTA.

  12. Place the 6-well plate on a rotary (orbital) shaker in 4 °C room (~500 g) for 30 min (see Note 7).

  13. Transfer the entire contents to a 15 ml falcon tube and add 5 ml CCS and 2 ml FBS (FBS should be at RT) to the falcon tube. The 10 cm dish should be set aside for use in step 16.

  14. Pulse-vortex for ~5 s.

  15. Allow tissue to settle and transfer supernatant to a 15 ml tube. This supernatant should contain the crypt cells. Spin down supernatant at 1000 × g, for 5 min, at 4 °C.

  16. While waiting for the centrifuge, pour CCS to the 3 ml mark on the 15 ml falcon containing the minced tissue and transfer the entire contents of the falcon back to the same single well of a 6-well plate. Add 250 μl of 0.5 M EDTA and place back on orbital shaker in 4 °C room for 30 min (see Note 7).

  17. When centrifugation is complete, carefully remove supernatant and resuspend the pellet in 2 ml CMGF− (see Note 8). The pellet contains the crypt cells. Keep the resuspended crypts on ice while doing the additional EDTA step.

  18. Once the minced tissue from the second EDTA incubation (step 16) has been on the shaker for 30 min, transfer the entire contents to a 15 ml falcon tube.

  19. Vortex vigorously for ~10 s.

  20. Add 5 ml CCS and 2 ml FBS (FBS should be at RT) to the 15 ml falcon tube.

  21. Pulse-vortex for ~5 s.

  22. Allow minced tissue to settle and transfer supernatant to a new 15 ml falcon tube and spin down supernatant at 1000 × g, for 5 min, at 4 °C.

  23. Biopsy samples can now be discarded through the appropriate waste disposal regulations.

  24. When centrifugation is complete, carefully remove supernatant and resuspend the pellet (containing crypt cells) in 1 ml CMGF− (see Note 8). Then, using the same pipette tip, transfer the resuspended crypts to the 15 ml falcon tube on ice containing the first crypt factions (i.e., from step 17).

  25. Add 5 ml of CMGF− to the falcon tube and pipet up and down ~10 times to mix.

  26. Centrifuge the falcon tube at 1000 × g, for 5 min, at 4 °C.

  27. Carefully remove as much supernatant as possible, ensuring the pellet (containing the crypt cells) is not disturbed.

  28. Using cold tips from the fridge, prelube tip with ice-cold CMGF− (i.e., pipet and expel CMGF− into the tip) and then add ~100 μl thawed Matrigel to the pellet in the 15 ml falcon tube (see Note 9). Gently mix the Matrigel up and down ~10 times (avoiding air bubbles), then add ~30 μl per well to a new prewarmed 24-well plate (see Note 10 and Fig. 2).

  29. Incubate at 37 °C 5% CO2 for 20–30 min to polymerize the Matrigel.

  30. Carefully add 500 μl of prewarmed High Wnt containing 10 μM Y-27632 + Pen/Strep (1:100) + Fungizone (1:100) (see Note 11).

  31. Place plate back into 37 °C incubator and check for enteroid growth daily (Fig. 3). See Note 12 for a detailed description on managing the initial phases of enteroid growth.

Fig. 2.

Fig. 2

Open in a new tab

Images taken down a microscope lens showing enteroids at various stages of growth. (a) Enteroid growing from previously frozen tissue after 7 days under 10× magnification (blue arrow points to the enteroid). (b) Enteroid growing from previously frozen tissue after 11 days under 10× magnification (blue arrow points to the enteroid). (c) Healthy Enteroids prior to passaging after 7 days of growth. (d) Unhealthy Enteroids beginning to turn black, signifying they are dying. (e) Contaminated well with microbial growth in both the Matrigel and in the media (4× magnification)

Fig. 3.

Fig. 3

Open in a new tab

Schematic representation of a 24-well tissue culture plate showing four possibilities for plating the Matrigel containing enteroids. Each well will have a total volume of ~30 μl. It is advantageous to have multiple Matrigel dots to maximize the contact surface area between the Matrigel and the media. When plating multiple dots, it is important to leave sufficient space between each dot to ensure they do not join together

3.3. Maintenance/Passaging

  1. Remove all media from wells, taking care not to dislodge the Matrigel plug.

  2. Add 500 μl cold CMGF− to the first well and mechanically break up Matrigel by pipetting using a P1000 up and down ~10 times.

  3. Move from well-to-well collecting the contents as you go.

  4. Transfer the CMGF− containing enteroids to a 1.5 ml or 2 ml Eppendorf on ice (see Note 13).

  5. With a new tip, add another 500 μl of cold CMGF− to the last well and work in reverse order from well-to-well, collecting the contents from all wells.

  6. Transfer the CMGF− containing enteroids to the same Eppendorf as before.

  7. Collect any residual CMGF−/enteroids from each well into to the Eppendorf.

  8. Using a 1 ml syringe containing a 25 G × 5/8 needle, draw up 1 ml from the Eppendorf containing enteroids in CMGF−. Then, with moderate force, expel the contents of the syringe back into the Eppendorf.

  9. Repeat the previous steps 1–3 times as necessary (see Note 14).

  10. Spin down the passaged enteroids at 1000 × g, for 5 min, at 4 °C.

  11. Being careful not to disrupt the enteroid pellet, remove all CMGF− (see Note 15).

  12. Using a cold pipette tip previously rinsed with CMGF−, add Matrigel to the Eppendorf containing the enteroids (see Note 16 for guidance on how to calculate the volume of Matrigel and how best to add this).

  13. Set a p200 pipette to 180 μl (i.e., the volume required to fill a row of a 24-well tissue culture plate, assuming ~30 μl per well) and add ~30 μl per well to a new prewarmed 24-well plate (see Note 10 and Fig. 2).

  14. Incubate at 37 °C 5% CO2 for 20–30 min to polymerize the Matrigel.

  15. Carefully add 500 μl of prewarmed High Wnt (see Note 11).

  16. Place plate back into 37 °C incubator and check for enteroid growth daily (Fig. 3). After 5–7 days the enteroids are usually ready to be passaged.

  17. As a general rule, enteroid media should be replenished 500 μl on Monday, 500 μl on Wednesday, and 600 μl on Friday (extra media on Friday overcomes the need to replenish media over the weekend).

3.4. Freezing

  1. As detailed in Note 12, it is important to make regular enteroid stocks, particularly during the early passages.

  2. Remove all media from wells, taking care not to dislodge the Matrigel plug.

  3. Add 500 μl cold CMGF− to the first well and mechanically break up Matrigel by pipetting P1000 up and down ~10 times.

  4. Move from well-to-well collecting the contents as you go.

  5. Transfer the CMGF− containing enteroids to a 1.5 ml or 2 ml Eppendorf on ice (see Note 13).

  6. With a new tip, add another 500 μl of cold CMGF− to the last well and work in reverse order from well-to-well, collecting the contents from all wells.

  7. Transfer the CMGF− containing enteroids to the same Eppendorf as before.

  8. Collect any residual CMGF−/enteroids from each well into to the Eppendorf.

  9. Centrifuge the passaged enteroids at 1000 × g, for 5 min, at 4 °C.

  10. Being careful not to disrupt the enteroid pellet, remove all CMGF− (see Note 15).

  11. Resuspend enteroids into freezing medium (each cryovial using 500 μl) at original ratio of 1–2 wells into 1 cryovial (see Note 17).

  12. Put each vial into a CoolCell (or other freezing containers that allow standardized controlled rate of −1 °C/min) and place the CoolCell in −80 °C overnight.

  13. Transfer cryovials into liquid nitrogen for long-term storage.

3.5. Thawing and Regrowing Cultures

  1. Remove cryovials from liquid nitrogen.

  2. Place in 37 °C 5% CO2 incubator until partially thawed (see Note 18).

  3. Add 500–1000 μl of prewarmed (37 °C) High Wnt to the cryovial of enteroids and pipet up and down to fully defrost the cells and dilute the DMSO.

  4. Centrifuge the enteroids at 1000 × g, for 5 min, at 4 °C.

  5. Being careful not to disrupt the enteroid pellet, remove all CMGF− (see Note 15).

  6. Using a cold pipette tip prelubed with CMGF−, add 120 μl Matrigel to the Eppendorf containing the enteroids and add ~30 μl per well to a new prewarmed 24-well plate (see Note 10 and Fig. 2).

  7. Incubate at 37 °C 5% CO2 for 20–30 min to polymerize the Matrigel.

  8. Carefully add 500 μl of prewarmed High Wnt + Y-27632 (see Note 11).

  9. Place plate back into 37 °C incubator and check for enteroid growth daily (Fig. 3).

  10. As a general rule, enteroid media should be replenished 500 μl on Monday, 500 μl on Wednesday, and 600 μl on Friday (extra media on Friday overcomes the need to replenish media over the weekend).

4. Notes

  1. It is important that the tissue is completely submerged in the collection solution (either CMGF− or PBS) and remains on ice/cold packs or in the fridge until transferred to the lab. Notably, even in cold conditions the crypt yield will reduce over time and so it is important to process the tissue as soon as possible after surgery. Tissue processed within 3 h and without being previously frozen will yield the greatest enteroid number. If sufficient tissue allows, it is suggested to perform immediate extraction on a fresh 5 mm2 section, as well as freezing down additional 5 mm2 sections in enteroid freezing media in case the fresh extraction fails. Excess tissue can also be used for other analyses, including histology, metagenomic sequencing, transcriptomics, and others.

  2. Matrigel should be thawed at least the night before use where possible. If tissue arrives unexpectedly and no thawed Matrigel is available, it is possible to use Matrigel that has been in the fridge for ~4 h. A 24-well tissue culture plate should be incubated overnight in the 37 °C 5% CO2 incubator, but there is no harm in having multiple sealed 24-well plates in the 37 °C 5% CO2 incubator at any given time (if space permits) to ensure there is always a warm plate on hand. A 200 μl tip box should also be left in the fridge as will be needed for plating the Matrigel. Before beginning the protocol, setup a centrifuge to contain the necessary rotor at set at 4 °C, remove a premade aliquot of 4 ml FBS from the freezer to thaw, set the water bath to 37 °C, and thoroughly clean the tissue culture hood with 70% ethanol and other detergents as needed. The media can be placed in the 37 °C water bath at any point so long as sufficient time is given for the entire volume of media to reach at least 30 °C.

  3. It is important that the minced tissue is small enough to easily fit into a wide-bore pipette tip.

  4. Use a dedicated 15 ml falcon tube of FBS for lubing pipette tips (i.e., suck up and expel FBS before handling solutions containing tissue). This will avoid minced tissue sticking to the inside of the pipette tip.

  5. Although effort should be made to avoid bubbles where possible, inevitably bubbles will form. These can be problematic because some minced tissue will stick to bubbles and therefore not settle to the base of the falcon tube, resulting in these bits of tissue being lost when removing the supernatant after each wash step (i.e., ultimately reducing enteroid yield). In order to dislodge pieces of minced tissue from the bubbles and to encourage them to sink, repeatedly tap the base of the falcon tube onto the hood. Additionally, it is not necessary to remove all of the supernatant; it is better to leave a small volume of liquid in the falcon tube rather than risk losing minced tissue.

  6. If after the 3× wash steps the supernatant is still dirty (i.e., discolored), repeat additional wash steps with CSS until the supernatant is clear.

  7. If using a rotary (orbital) shaker located within a 4 °C room/fridge is not possible, an alternative is to place the 6-well plate on top of an ice bucked filled with ice and use a rotary (orbital) shaker on the bench.

  8. The supernatant at this point contains the stem cell containing crypts. After centrifugation, a faint pellet is usually visible; this is the crypt cells, and it is important not to disturb this pellet when removing supernatant. Therefore, it is better to leave a small amount of the supernatant (less than 1 ml) rather than risk losing crypt cells.

  9. As a general rule, a fresh (i.e., where the tissue sample was collected within 3 h postsurgery) ~0.5 mm2 sized piece of tissue will yield ~4 wells in a 24-well plate. The yield will be reduced in tissue that has been left on ice overnight and/or frozen; therefore, less Matrigel (~70 μl) should be added, aiming for ~2–3 wells in a 24 well plate. It is important that the Matrigel is not diluted (at least a Matrigel ratio of 3:1), so consider using a 200 μl pipette tip to carefully remove as much CMGF− as possible. If there is some residual CMGF− in the falcon tube, then use less Matrigel to account for this. For example, if 120 μl is to be plated into 4 well, but ~30 μl of CMGF− remains in the falcon tube, then add ~90 μl of Matrigel.

  10. It is important to plate around 30 μl per well in a 24-well tissue culture plate. For optimal enteroid growth it is suggested to plate 2× ~15 μl or 3× ~10 μl dots per well. It is important to avoid getting bubbles in the Matrigel, so take extra care when resuspending the enteroid pellet into the Matrigel solution.

  11. When adding enteroid media, slowly pipet down side of well taking care not to disturb the Matrigel plug. Forcefully expelling media onto the Matrigel may cause the plug to dislodge. If the lines are precious (i.e., no spare tissue in frozen storage) or within the initial weeks of growth, add 10 μM Y-27632 + Pen/Strep (1:100) + Fungizone (1:100) as required.

  12. Wells that are contaminated will usually appear yellow and cloudy. It might be possible to directly observe fungal growth on the surface of the media. To limit the risk of contamination, High Wnt media containing 10 μM Y-27632 + Pen/Strep (1:100) + Fungizone (1:100) can be used until passage 2. Continue adding Y-27632 if enteroids are not growing well or appear to be dying (Fig. 1). If an isolated well is contaminated, remove all media from the well and wash the well in question with 700 μl 10% bleach and/or 700 μl 70% ethanol, then wash twice with PBS. Leave well empty and mark on the plate the well that was contaminated so it is not used again. If enteroids show widespread contamination (Fig. 1) and the no stocks have been generated previously, it might be possible to save the line by passing the enteroids by adding Normocure. Specifically, passage the enteroids and incubate with 2 ml High Wnt media containing 10 μM Y-27632 and 4 μl Normocure for 30 min on ice. Centrifuge the Eppendorf tube at 1000 × g, for 5 min, at 4 °C, remove supernatant, and suspend the enteroid pellet into ~120 μl Matrigel (assuming 4 wells to be plated). Spike ~2 μl of Normocure into the Matrigel and add 500 μl per well of High Wnt media containing 10 μM Y-27632 + Pen/Strep (1:100) + Fungizone (1:100) + 100 μg/ml Normocure. Treat contaminated enteroids for three passages. The first passage should be performed when many enteroids can be observed and are showing good growth, which is typically 5–20 days following enteroid extraction. It is important to regularly freeze enteroids, particularly during the early passages; freeze down around 50% of the wells in the early passages. This will ensure a plentiful supply of enteroid stocks at early passages and will help to future-proof the line.

  13. If there are more than 12 wells of enteroids, use two Eppendorf tubes. This will avoid issues with having too much Matrigel in the CMGF− and ensure (1) a robust passage and (2) that a large Matrigel pellet does not occur after centrifugation.

  14. The optimal number of times to pass enteroids through the syringe and the force to apply will take practice and can vary from line to line. It is important to break up enteroids, but over-passaging (i.e., to many passages through the syringe or using too much force) will damage enteroids and cause them to die. As a general rule, it is better to err on the side of caution, passing through the syringe two times and with moderate pressure.

  15. If a layer of Matrigel can be seen directly above the enteroid pellet, remove as much CMGF− as possible then add another 1 ml of ice-cold CMGF−. Repeat the centrifugation and remove the CMGF− supernatant. This will avoid loss of enteroids that are stuck in the Matrigel layer and would otherwise be lost when removing the supernatant.

  16. Typically we want to at least double the number of wells after each passage, not accounting for wells that are frozen. For example, if you have 8 wells of healthy/dense enteroids and wish to freeze down 3 wells, then the remaining 5 wells will be passaged. Thus, these 5 wells will be split into ~10–15 wells. This estimate will need to be adjusted according to the enteroid density (i.e., dense enteroid wells should be more than doubled and vice versa). To calculate the amount of Matrigel needed, decide how many wells you intend to seed and assuming 30 μl per well, simply multiply 30 μl by the number of wells to be seeded. There will be some residual CMGF− in the Eppendorf so consider subtracting ~20–50 μl from the amount of Matrigel to add. If the volume of Matrigel to be added is <200 μl, use the same 200 μl pipette tip to immediately mix the enteroids into the Matrigel and then plate ~30 μl per well in a prewarmed tissue culture plate (Fig. 2). If the volume of Matrigel to be added is >200 μl, use a 1000 μl pipette tip to add the Matrigel to the Eppendorf containing enteroids and then use a 200 μl pipette tip set at 180 μl to mix the enteroids into the Matrigel, before plating ~30 μl per well in a prewarmed tissue culture plate (Fig. 2).

  17. To calculate how much enteroid freezing media to add to the enteroids, decide how many cryovials will be generated and then resuspend the enteroids in 500 μl multiplied by the number of cryovials. As a general rule, if the enteroids are healthy and very dense, then aim for 1 well into 1 cryovial, otherwise a ratio of 1.5 or 2 wells per cryovial should be used.

  18. Instead of the 37 °C 5% CO2 incubator, it is also possible to quickly thaw the enteroids by holding the cryovial under running distilled or warm water. As a rule of thumb, “freeze slow and thaw fast.”

Acknowledgments

This work was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. 794771 to C.J.S. and Public Health Service Grant U19AI116497 from the National Institutes of Health to M.K.E. We thank Xi-lei Zeng for her expertise and help with all enteroid cultures.

References

  • 1.Blutt SE, Crawford SE, Ramani S et al. (2017) Engineered human gastrointestinal cultures to study the microbiome and infectious diseases. Cell Mol Gastroenterol Hepatol 5:241–251 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Stelzner M, Helmrath M, Dunn JCY et al. (2012) A nomenclature for intestinal in vitro cultures. Am J Physiol Gastrointest Liver Physiol 302:G1359–G1363 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Sato T, Vries RG, Snippert HJ et al. (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265 [DOI] [PubMed] [Google Scholar]
  • 4.Sato T, Stange DE, Ferrante M et al. (2011) Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141:1762–1772 [DOI] [PubMed] [Google Scholar]
  • 5.Foulke-Abel J, In J, Kovbasnjuk O et al. (2014) Human enteroids as an ex-vivo model of host-pathogen interactions in the gastrointestinal tract. Exp Biol Med (Maywood) 239(9):1124–1134. 10.1177/1535370214529398 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Kovbasnjuk O, Zachos NC, In J et al. (2013) Human enteroids: preclinical models of non-inflammatory diarrhea. Stem Cell Res Ther 4(Suppl 1):S3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Jalili-Firoozinezhad S, Gazzaniga FS, Calamari EL et al. (2019) Complex human gut microbiome cultured in anaerobic human intestine chips. Nat Biomed Eng 3(7):520–531 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Fofanova TY, Stewart C, Auchtung JM, et al. (2019) A novel human enteroid-anaerobe co-culture system to study microbial-host interaction under physiological hypoxia. bioRxiv: 555755 [Google Scholar]
  • 9.Heijmans J, van Lidth de Jeude JF, Koo B-K et al. (2013) ER stress causes rapid loss of intestinal epithelial stemness through activation of the unfolded protein response. Cell Rep 3:1128–1139 [DOI] [PubMed] [Google Scholar]
  • 10.Tsai Y-H, Czerwinski M, Wu A et al. (2018) A method for cryogenic preservation of human biopsy specimens and subsequent organoid culture. Cell Mol Gastroenterol Hepatol 6:218–222.e7 [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES