Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

Summary

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

Abstract

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

Introduction

Inflammatory bowel disease (IBD) is a chronic, remitting, and relapsing disease characterized by episodes of inflammation and clinical quiescence. The etiology of IBD is multifactorial, but key characteristic features of the disease include defective barrier function and increased permeability of the intestinal epithelium, in addition to proinflammatory signaling cascades activated within the epithelial compartment^1,2. Several in vitro and in vivo models have been used to recapitulate the epithelial response during IBD, including cell culture and murine models of inflammation³. However, all these systems have important shortcomings that limit their ability to recapitulate the epithelial changes during IBD⁴. Most cell lines used to study IBD are transformed, have the ability to form a monolayer, and can differentiate³ but intrinsically propagate differently than non-transformed intestinal epithelial cells in the host. Several different murine models of inflammation are used to study IBD, some of which include knockout models, infectious models, chemical inflammatory models, and T-cell transfer models^5,6,7,8. While each can study certain etiological aspects of IBD, such as genetic predispositions, barrier dysfunction, immune deregulation, and the microbiome, they are limited in their ability to study the multifactorial nature of the disease.

Intestinal organoids, including enteroids and colonoids, have been established over the last decade as a useful in vitro model for studying not only the dynamics of intestinal stem cells but also their role the barrier integrity and function of the intestinal epithelium play in intestinal homeostasis and disease. These entities have significantly contributed to our understanding of the pathogenesis of IBD⁹ and have opened new opportunities for personalized medicine. Colonoids, or stem cell-derived, self-organizing tissue cultures from the colon, have been developed from both murine and human tissue in a process that allows stem cells located within intestinal crypts to propagate and be maintained indefinitely¹⁰. The stem cell niche in vivo relies on extracellular factors to support its growth, notably the canonical Wnt signaling and bone-morphogenic protein signaling pathways¹¹. The addition of these factors promotes the health and longevity of colonoids but also drives the culture toward a stem cell-like state that is not reflective of the in vivo epithelial cellular architecture, which consists of both self-renewing and terminally differentiated cells^12,13. While the functionality of the intestinal epithelium is dependent upon the continual crosstalk between the stem cell compartment and differentiated cells, the ability to have both in a colonoid culture system is fairly limited. Despite these limitations, the organoid culture system remains the gold standard to study the intrinsic properties of the epithelium ex vivo. Nonetheless, alternative culture strategies may need to be considered to answer the scientific question at hand.

It has been shown that mice on a continuous 7 day regimen of dextran sodium sulfate (DSS) develop both epithelial inflammation and barrier dysfunction¹⁴. Furthermore, mitochondrial biogenesis failure and metabolic reprogramming within the intestinal epithelium, which have been shown to be evident in human IBD, have also been captured in this DSS model of colitis¹⁵. However, our preliminary data demonstrate that the characteristics of mitochondrial biogenesis failure are not retained in colonoids derived from the crypts of DSS-treated animals (Supplementary Figure 1). Thus, alternative culture methods must be used when examining how inflammation drives epithelial changes during murine intestinal inflammation. Here, we outline a protocol we have developed that describes 1) how to isolate crypts from whole colonic tissue for the establishment of murine colonoids, 2) how to terminally differentiate this cell population to reflect the cell population as it stands in vivo, and 3) how to induce inflammation in this in vitro model. To study drug interactions within the inflamed epithelium, we have developed a protocol to establish 2-dimensonal (2D) monolayers from murine colonoids that can be basally treated with inflammatory mediators and apically treated with drug therapies.

Protocol

All the experimentation using murine tissues described herein was approved by the Institutional Review Board at the University of Pittsburgh and conducted in accordance with the guidelines set forth by the Animal Research and Care Committee at the University of Pittsburgh and UPMC.

1. Preparation for culture

NOTE: All the reagents are listed in the Table of Materials section and all solution compositions can be found in the solution composition table (Table 1).

1. Prepare mouse wash medium as described in Table 1. Store at 4°C for up to 2 months.
2. Prepare crypt isolation buffer 1 (CIB1) as described in Table 1. Store at 4°C for up to 2 months.
   ​CAUTION: Dithiothreitol (DTT) is considered hazardous by the OSHA Hazard Communication Standard due to the potential acute toxicity if ingested and skin and eye damage if those areas are exposed to it. Protective gloves, eye protection, and face protection should be used when handling this chemical.
3. Prepare crypt isolation buffer 2 (CIB2) as described in Table 1. Store at 4°C for up to 2 months.
4. Prepare L-WRN-conditioned medium according to a previously published protocol¹⁶.
5. Prepare complete colonoid medium as described in Table 1. Complete colonoid growth medium can be stored for up to 5 days at 4°C without a loss of activity.
6. Prepare the differentiation medium as described in Table 1. This protocol was modified from a previously published paper¹⁷. The differentiation medium can be stored for up to 5 days at 4°C without a loss of activity.
7. Prepare the inflammation mediator medium as described in Table 1. This protocol was modified from a previously published paper¹⁸. The inflammation mediator medium can be stored for up to 5 days at 4°C without a loss of activity.
8. Prepare the murine monolayer medium. Omit Y-27632 when challenging the monolayers with inflammatory factors and/or drug(s). The murine monolayer medium can be stored for up to 5 days at 4°C without a loss of activity.
9. Prepare the inflammation mediator monolayer medium. The inflammation mediator monolayer medium can be stored for up to 5 days at 4°C without a loss of activity.

2. Crypt isolation from murine colonic tissue

NOTE: Transfer the tissue on ice. Take the appropriate amount of basement membrane matrix out of −20°C storage, and thaw on ice. Each 24-well is plated with 15 µL of basement membrane matrix. Prepare CIB1, CIB2, and complete colonoid growth medium as described in section 1.

1. Euthanize a C57BL/6J mouse (6-8 weeks old) according to the Institutional Animal Care and Use Committee-approved protocol, and extract the distal end (approximately 5-7 cm) of the colon using clean tweezers and scissors.
   NOTE: In this study, the subjects were euthanized using carbon dioxide chamber incubation for 2-3 min followed by cervical dislocation.
   1. For this, place the mouse on its back, make a horizontal incision just below the ribcage and then a vertical incision from the middle of the horizontal incision toward the base of the tail to expose the abdominal cavity.
   2. With tweezers, move the small intestine out of the field, identify the colon, and follow it down to the base of the tail. Break the pelvis with scissors to fully expose the distal colon if needed. Use the scissors to cut the most distal 5-7 cm of the colon.
2. Place the colon tissue on a clean surface. Remove the excess serosal fat that surrounds the colon. Remove the fecal matter by flushing the luminal contents of the colon with Dulbecco's phosphate-buffered saline (DPBS) using a syringe attached to an 18 G 1 1/2 needle. Then, place the colon in a 50 mL conical tube containing 10 mL of ice-cold DPBS, and place it on ice.
   NOTE: If isolating the colon from more than one animal, place the tissue on ice before continuing to the next animal.
3. Transfer the colon tissue to a Petri dish with 10 mL of ice-cold DPBS, and irrigate the lumen twice with DPBS using a P1,000 pipette.
4. Open the colon longitudinally using scissors, and gently shake using tweezers for ~30 s in the DPBS to remove any remaining fecal contents.
5. Transfer the tissue to a new Petri dish with 10 mL of ice-cold DPBS, and again gently shake using tweezers prior to cutting the colon into 2 cm pieces using scissors and placing them in a 50 mL conical tube with 7 mL of ice-cold CIB1. Incubate the tissue in CIB1 on ice for 20 min.
   NOTE: The remaining steps are performed inside a biological safety cabinet.
6. Transfer the tissue using tweezers to a pre-warmed 50 mL conical tube containing 7 mL of CIB2, and incubate in a water bath at 37°C for 5 min.
7. Remove the CIB2 from the 50 mL conical tube by letting the tissue settle at the bottom of the conical tube and then pipetting off the CIB2. Then, add 10 mL of ice-cold mouse wash medium to the same conical tube.
8. Obtain a new 50 mL conical tube, and place a 70 µm filter on top of the open tube.
9. While holding the tube with the colon contents, rotate the hand approximately 45°. Shake the tube in a rocking and vigorous manner for 45 s to release the crypts. Then, pour the crypt suspension through the 70 µm cell strainer into a new 50 mL conical tube.
10. Using tweezers, place the colon tissue back in the empty 50 mL conical tube, and add 10 mL of ice-cold mouse wash medium. Obtain a new 70 µm cell strainer, and place it on top of the 50 mL conical tube containing the previous filtered solution.
11. Again, pick up the 50 mL tube containing the crypt fragments, and shake it in an identical manner as in step 2.9. Filter the medium through the 70 µm cell strainer into the 50 mL conical tube to combine with the previously filtered crypts.
    NOTE: If the crypt yield is low, shake the colon tissue an additional time by placing the tissue in an empty 50 mL conical tube with 10 mL of mouse wash medium. Shake the tissue for 45-50 s to release the crypts. Also, if the crypt yield is low, then increase the vigorousness of the shaking. Finally, if the overall crypt yield is low, both the pipets and tubes can be coated with fetal bovine serum (FBS) to prevent the adherence of the tissue to the plastic.
12. Centrifuge the 50 mL conical tube containing the filtered crypts at 300 x g for 5 min at 4°C.
    NOTE: A pellet should be observed at the bottom of the 50 mL conical tube.
13. Decant the medium by pipetting, resuspend the crypts by pipetting up and down with 10 mL of ice-cold mouse wash medium, and transfer to a 15 mL conical tube. Centrifuge at 300 x g for 5 min at 4°C.
14. To obtain a cleaner preparation, wash the crypts with an additional 10 mL of mouse wash medium. Decant the previous medium, and resuspend the crypts by pipetting up and down in 10 mL of mouse wash medium. Centrifuge at 300 x g for 5 min at 4°C using a 15 mL conical tube.
    NOTE: Caution should be taken when pipetting off the medium because, at times, the pellet is not centrifuged tightly. If the pellet is not tight, pipette off most of the medium, leaving 500 µL to 1 mL. Then, resuspend the pellet by pipetting in 10 mL of mouse wash medium, and transfer the solution to a 15 mL conical tube. Centrifuging in a smaller conical tube can improve the tightness of the pellet.
15. Decant the medium, and resuspend the crypts by pipetting up and down with 10 mL of ice-cold mouse wash medium. Once resuspended, count the crypts under a bright-field microscope by immediately pipetting 10 µL of the medium onto a glass slide.
    1. Place two 10 µL drops on separate ends of the slide, and count the number of crypts in each droplet. Average the numbers between the two drops to determine the number of crypts per 10 µL. Multiply this number by 100 to get the number of crypts in 1 mL.
       NOTE: For accurate counting, 10 µL of the crypt suspension must be immediately removed after resuspension. If not, the crypts will fall to the bottom of the tube, which will result in an inaccurate count.
16. With a goal of plating 300-500 crypts per well, transfer the appropriate volume of crypts resuspended in the mouse wash medium to a new 15 mL conical tube and bring to 10 mL with ice-cold mouse wash medium. Centrifuge the tube at 300 x g for 5 min at 4°C. A small pellet should be visible at the bottom of the tube.
17. Remove the supernatant with a power pipette. Use a P200 pipet to carefully remove any residual medium, leaving only the pellet.
18. Resuspend the pellet in the appropriate amount of ice-cold basement membrane matrix by slowly pipetting up and down. Be careful not to introduce bubbles when resuspending the crypt pellet. Plate 300-500 crypts/15 µL of basement membrane matrix in each well of a plate. After plating, invert the tissue culture plate, and place it in a 37°C incubator for 10-20 min.
19. Revert the plate, and add 500 µL of the complete mouse colonoid medium to each well. Change the medium every other day until they are ready for passaging. Passage the colonoids every 3-5 days.

3. Passaging the colonoids

NOTE: Each well can generally be passaged 1:4 to 1:6 according to the density of the original well. Take the appropriate amount of basement membrane matrix out of −20°C, and place it on ice to thaw. Colonoids can be used for experiments after two passages. When passaging colonoids, the steps are performed in a biological safety cabinet to prevent contamination.

1. Remove the medium from the wells to be passaged.
   1. If there is debris within the basement membrane matrix, which can happen during the initial isolation, the following protocol can be used to clean up the debris:
      1. Add 1 mL of ice-cold 0.1% bovine serum albumin (BSA) in DPBS without Ca²⁺ or Mg²⁺ to each well. Let it sit for 5 min in the biological safety cabinet.
      2. Pipette up and down three to seven times using a P1000 pipette to disrupt the basement membrane matrix, and collect the contents of the wells in a 15 mL conical tube. Centrifuge the colonoids in a total of 5-10 mL of 0.1% BSA in DPBS without Ca²⁺ or Mg²⁺. Make up to the appropriate volume with 0.1% BSA.
      3. Centrifuge at 150 x g for 5 min at 4°C to pellet the colonoids but not the debris.
      4. Decant the DPBS by pipetting, leaving the pellet. Add 1 mL of room temperature (RT) enzymatic dissociation reagent, and pipette three to five times.
      5. Place the 15 mL conical tube with the enzymatic dissociation reagent and disrupted colonoids in a 37°C water bath for 3-4 min.
      6. Remove the tube from the water bath, pipette 3-5 times, and then bring to 10 mL with mouse wash medium. Proceed to step 3.6.
2. Place 500 µL of RT enzymatic dissociation reagent in each well, and let it sit for approximately 1 min before pipetting up and down three to seven times to disrupt the basement membrane matrix.
3. Place the plate in a 37°C incubator for 3-4 min.
4. Remove the plate from the incubator, and pipette up and down three to seven times using a P1000 pipette to dissociate the colonoids.
5. Transfer the contents into a 15 mL conical tube and make up to 10 mL with ice-cold mouse wash medium.
6. Centrifuge the sample at 300 x g for 5 min at 4°C.
7. Remove the medium using a power pipette and a P200 pipette as needed so that only a pellet is left in the conical tube.
8. Resuspend in an appropriate amount of ice-cold basement membrane matrix by gently pipetting up and down according to how many wells are to be plated. Do not introduce bubbles when pipetting. Place the colonoid fragments in 15 µL of basement membrane matrix drops per well.
9. Plate the crypts, invert the tissue culture plate, and then place the plate in a 37°C incubator for 10-20 min.
10. Finally, revert the plate, and add 500 µL of complete mouse colonoid medium to each well. Change the medium every other day until they have grown large enough to be passaged again in approximately 3-5 days.

4. Terminally differentiating the colonoid cells

1. Passage the colonoids as above, and add 500 µL of the complete mouse colonoid medium to each well.
2. At least 48 h after passaging, remove the complete mouse colonoid medium, and add 500 µL of the differentiating medium to each well (see section 1).
3. Incubate the colonoids with differentiation media for 48 h, after which they can be used in experiments, including the collection of RNA and protein.
   ​NOTE: Colonoids can be assessed for differentiation by collecting RNA and assessing the expression of Lgr5 and Ki67, a stem cell marker and cell proliferation marker, respectively, via quantitative reverse transcriptase-polymerase chain reaction (qPCR). Terminally differentiated cells should have a relatively low expression of Lgr5 and Ki67 compared to colonoids grown in traditional colonoid medium, where the cells are more stem-like. The duration of time that the colonoids need to incubate in the differentiation medium may need to be optimized for each individual laboratory. Refer to Supplementary Table 1 for the list of primers.

5. Inducing inflammation in differentiated colonoids with inflammatory mediators

1. After the colonoids have been incubated for 2-3 days with the differentiation medium, remove the existing medium, and add 500 µL of the inflammation mediator medium to each well.
2. Allow the wells to incubate for 24-72 h before collecting the RNA and protein (or assessing other downstream parameters). Change the medium every day.

6. Intestinal epithelial monolayers derived from established murine colonoids

NOTE: Murine intestinal epithelial monolayers are derived from murine colonoids that have been passaged a minimum of two times. To allow for successful monolayer formation in 3-5 days, it is imperative not to separate the colonoids into single cells. Fragmented organoids that have been enzymatically dissociated into cellular clusters are ideal for growth.

1. Prepare all the reagents prior to beginning the experiment. Thaw the basement membrane matrix on ice. Prepare the murine monolayer medium as described in section 1 . Dilute the thawed basement membrane 1:20 with the cold murine monolayer medium. Keep on ice.
2. Use sterile forceps to place a 0.4 µm transparent cell culture insert into a single well of a 24-well tissue culture plate (Table of Materials). Grab a corner prong of the insert, and transfer it into the well. Repeat until the appropriate number of cell culture inserts are available for culture.
   NOTE: Make sure to coat an additional control well with the basement membrane matrix only. This cell culture insert will be used to measure the background resistance of the cell culture insert without cells present, as described in the subsequent step (step 6.3).
3. Using a P200 pipet, cover the apical (top) surface of each cell culture insert with 150 µL of diluted basement membrane matrix. Place the pipet tip in the center of the insert without touching the membrane of the insert, and gently expel the solution. Place the plate in a sterile 37°C incubator with 5% CO₂ for at least 1 h.
4. Gently remove the basement membrane matrix prior to use, and allow the cell culture inserts to dry in a biological safety cabinet for a minimum of 10 min.
   1. To remove the basement membrane matrix, rotate the 24-well plate at a 45° angle. Place a single finger on the corner of the prong to stabilize the insert, and using a P200 pipet, gently place the pipet tip along the bottom side of the insert and remove the matrix.
   2. Remove any excess residue by washing each cell culture insert with 150 µL of sterile DPBS without Ca²⁺ or Mg²⁺.
5. After 10 min of drying, add 400 µL of the murine monolayer medium to the bottom of the cell culture insert and 75 µL on top. Repeat until all the cell culture inserts are immersed. Leave the plate in the biological safety cabinet, or place it back in the incubator until needed.
6. Remove the 24-well plate of mature (organoids on Day 3-5 of growth) intestinal colonoids from the 37°C, 5% CO₂ incubator, and place it under the biological safety cabinet. Remove the medium using sterile tips, and wash each well with 1 mL of RT sterile DPBS (without Ca²⁺ or Mg²⁺).
   NOTE: An individual well of 75-125 mature colonoids is split at a ratio of 1:4.
7. Remove the DPBS without Ca²⁺ or Mg²⁺ using sterile tips, and digest each plug of the basement membrane matrix by placing 500 µL of RT enzymatic dissociation reagent in each well to be passaged.
8. Pipet each well up and down approximately 6-10 times to break up the plug. Do not scratch the bottom of the plate to remove the plug. Instead, rotate the 24-well plate at a 45° angle, place the tip of the pipet at the edge of the plug, and gently dislodge it.
9. Place the 24-well plate back in a sterile 37°C, 5% CO₂ incubator for 3-4 min.
10. Remove the plate, and pipet the enzymatic dissociation reagent up and down five to seven times for each well under a biological safety cabinet. Transfer the disassociated colonoids from each well into a sterile, 15 mL conical tube. Bring up to a volume of 10 mL with ice-cold mouse wash medium.
11. Centrifuge the partially digested colonoids at 300 x g at 4°C for 5 min in a swinging bucket centrifuge.
12. Under the biological safety cabinet, remove the supernatant from the pellet using a 10 mL pipet. Remove any remaining medium using a P200 pipet. Resuspend the colonoid pellet in murine monolayer medium. Add 75 µL of medium per each cell culture insert of fragmented colonoids to be plated.
13. Add 75 µL of colonoid suspension to the center of each cell culture insert. Before adding the colonoid suspension to each well, gently pipet up and down once or twice before removing the 75 µL, which allows for more uniform plating.
14. Place the 24-well plate with cell culture inserts on a rotating platform for 10 min to further allow for uniform dispersion across the cell culture insert.
15. Place the plate in a sterile 37°C, 5% CO₂ incubator.
16. The next day (Day 1), dislodge the unattached colonoid fragments by pipetting the medium up and down three to five times with a P200 pipet. Gently place the tip alongside the inside of the cell culture insert as to not disrupt the attached intestinal cells. Do not introduce bubbles into the medium, as this prevents the removal of all the unattached fragments.
17. Immediately remove the medium and colonoid mixture. Repeat until all the cell culture inserts have been cleaned.
18. Gently add 150 µL of pre-warmed murine monolayer medium to the apical side of each cell culture insert. Add 400 µL of warmed murine monolayer medium to each well of a new 24-well plate. Transfer the cell culture insert over to the new plate by grabbing its prong using sterile forceps. Change the medium every other day until confluency.
    ​NOTE: The colonoid fragments should form a confluent monolayer 3-5 days post-plating.

7. Measuring the net resistance of the epithelial monolayers using a voltohmmeter on days 3 - 5 of monolayer culture

NOTE: The chopstick electrodes resemble forceps and are asymmetrical in length. The longer arm of the probe is the basolateral electrode, and the shorter arm is the apical electrode. The probe can be difficult to insert between the interior and exterior of the cell culture insert. Placing the probe at a slight angle upon insertion, followed by vertical straightening of the probe, will prevent the probe from becoming stuck. Make sure to read the net resistance of each cell culture insert at a similar angle, as this can impact the values.

1. Place the voltohmmeter and all the reagents inside a biological safety cabinet.
   1. Plug the voltohmmeter into the nearest AC outlet, and plug the plastic modular connector of the electrode probe into the INPUT jack on the front display.
   2. To place the voltohmmeter at a 45° angle, swing the metal arm on the backside of the equipment out until it locks into place.
   3. Now, turn on the voltohmmeter by flipping the power switch UP on the front display. Lastly, flip the switch up toward OHMS to display the reading in this metric.
2. Remove the 24-well plate from the 37°C, 5% CO₂ incubator, and lay the plate flat in the biological safety cabinet. The transepithelial electrical resistance (TEER) is temperature sensitive. Only remove the plate immediately before the TEER is read.
3. Sterilize the electrode probe in pre-warmed 70% ethanol for 30 s to 1 min. Remove the probe, invert, and flick it one to two times to remove the excess ethanol. Allow the probe to air-dry for approximately 30 s.
4. Wash any remaining ethanol left on the probe with pre-warmed mouse wash medium.
   CAUTION: Do not let any of the remaining ethanol droplets higher on the probe fall into the cell culture insert. When washing the ethanol off with medium, do not let the medium go above the sterile ethanol field. This could cause contaminated medium to enter the cell culture insert.
5. Hold the probe perpendicular to the cell culture insert. Insert the basolateral electrode (longer end of the probe) on the outside of the cell culture insert until it touches the base of the 24-well plate. Slide the apical electrode (shorter end of the probe) into the cell culture insert. Do not vary the distance of the two electrodes from well to well. This will impact the TEER measurement.
6. Verify that the apical electrode is not touching the base of the cell culture insert before reading the TEER. Start with the background control cell culture insert, and record the value. The value will automatically be digitally displayed on the front of the voltohmmeter.
7. Repeat steps 7.5-7.6 for each cell culture insert.
   NOTE: If different experimental conditions exist between the cell culture inserts, sterilize the probe between each new condition. Start at step 7.1, and proceed numerically through the steps.
8. Place the 24-well plate back into the incubator once all the TEER measurements have been recorded.
9. The net values at 350 Ω or greater are indicative of monolayer formation. Repeat again on the subsequent days until monolayer formation has been achieved.
   ​NOTE: Generally, values of 1,000 Ω or greater can be captured on Day 3, but over time, they will all converge on a lower number around 350 Ω.

8. Calculating the TEER using net resistance measurements from the voltohmmeter

NOTE: Successful monolayer formation of the colonoids will give TEER measurements greater than 115 Ω·cm².

1. Take individual net resistance values, and subtract the net resistance from the background well. The cell culture inserts coated with the basement matrix membrane will give a net reading of around 100 Ω.
2. Take the background-subtracted net resistance measurements, and multiply them by the surface area of the cell culture insert. A 24-well cell culture insert has a surface area of 0.33 cm².
3. If the values are 115 Ω·cm² or greater, proceed to the downstream experimental assays.

9. Inducing inflammation in the epithelial monolayers with inflammatory mediators

1. Once successful monolayers have formed, add inflammatory mediators to the culture on the basal side of the culture. Prepare the inflammation mediator monolayer medium, as described in step 1.9, and add 400 μL to each well in a new 24-well plate.
2. Remove the medium from the apical side of the cell culture insert, and add 150 μL of the murine monolayer medium without Y-27632. Do not supplement this side with inflammatory mediators. However, if the cell death seems excessive, leave the Y-27632 in the media. This must be determined by the end user.
3. Transfer the cell culture inserts to a new 24-well plate, and place them into the wells supplemented with the inflammation mediator monolayer medium.
4. Stimulate the monolayers with inflammatory mediators for 24-72 h, depending on the experiment.
5. To test drug responses using this inflammatory model, supplement the monolayer medium on the apical side of the cell culture insert with the drug of choice. The drug can be added at any point in the experiment, depending on the mechanism of action of the drug and the downstream parameters to be assessed.

Results

The 3D intestinal colonoid culture system is an invaluable tool to study the intrinsic contribution of the epithelium to intestinal mucosal homeostasis. The described protocol provides detailed instructions on how to isolate crypts from C57BL/6J (WT) mice at 8 weeks of age and establish a long-term colonoid culture system that can be manipulated for multiple downstream applications. Upon the isolation and plating of crypts in the basement membrane matrix, the crypts appear dense and multicellular in structure when visual...

Discussion

Organoid development has revolutionized the way the scientific community studies organ systems in vitro with the ability to partially recapitulate cellular structure and function from an animal or human in a dish. Further, organoid systems derived from humans with diseases offer a promising tool for personalized medicine that could guide therapeutic decision-making. Here, we describes a crypt isolation protocol that works well and introduces key steps that allow for cleaning up excess debris in the isolatio...

Materials

Name	Company	Catalog Number	Comments
0.4-μM transparent transwell, 24-well	Greiner Bio-one	662-641
15-mL conical tubes	Thermo Fisher	12-565-269
50-mL conical tubes	Thermo Fisher	12-565-271
70-μM cell strainer	VWR	76327-100
Advanced DMEM/F12	Invitrogen	12634-010	Stock Concentration (1x); Final Concentration (1x)
B-27 supplement	Invitrogen	12587-010	Stock Concentration (50x); Final Concentration (1x)
Chopsticks Electrode Set for EVO	World Precision Instruments	STX2
Corning Matrigel GFR Membrane Mix	Corning	354-230	Stock Concentration (100%); Final Concentration (100%)
Dithiothreitol (DTT)	Sigma-Aldrich	D0632-5G	Stock Concentration (1 M); Final Concentration (1.5 mM); Solvent (ultrapure water)
DMEM high glucose	Thermo Fisher	11960-069	Stock Concentration (1x); Final Concentration (1x)
Dulbecco's phosphate-buffered saline without Calcium and Magnesium	Gibco	14190-144	Stock Concentration (1x); Final Concentration (1x)
Ethylenediaminetetraacetic acid (ETDA)	Sigma-Aldrich	E7889	Stock Concentration (0.5 M); Final Concentration (30 mM)
Fetal Bovine Serum	Bio-Techne	S11150H	Stock Concentration (100%); Final Concentration (1%)
Fisherbrand Superfrost Plus Microscope Slides, White, 25 x 75 mm	Thermo Fisher	12-550-15
G418	InvivoGen	ant-ga-1	Final Concentration (400 µg/µL)
Gentamicin Reagent	Gibco/Fisher	15750-060	Stock Concentration (50 mg/mL); Final Concentration (250 μg/mL)
GlutaMAX-1	Fisher Scientific	35050-061	Stock Concentration (100x); Final Concentration (1x)
HEPES 1 M	Gibco	15630-080	Stock Concentration (1 M); Final Concentration (10 mM)
hIFNγ	R&D Systems	285-IF	Stock Concentration (1000 ng/µL); Final Concentration (10 ng/mL); Solvent (ultrapure water)
hIL-1β	R&D Systems	201-LB	Stock Concentration (10 ng/µL); Final Concentration (20 ng/mL); Solvent (ultrapure water)
hTNFα	R&D Systems	210-TA	Stock Concentration (10 ng/µL); Final Concentration (40 ng/mL); Solvent (ultrapure water)
Hydrogen Peroxide	Sigma	H1009	Stock Concentration (30%); Final Concentration (0.003%); Solvent (Mouse wash media)
Hygromycin B Gold	InvivoGen	ant-hg-1	Final Concentration (400 µg/µL)
L-WRN Cell Line	ATCC	CRL-3276
mEGF	Novus	NBP2-35176	Stock Concentration (0.5 µg/µL); Final Concentration (50 ng/mL); Solvent (D-PBS + 1% BSA)
N-2 supplement	Invitrogen	17502-048	Stock Concentration (100x); Final Concentration (1x)
N-Acetyl-L-cysteine	Sigma	A9165-5G	Stock Concentration (500 mM); Final Concentration (1 mM); Solvent (ultrapure water)
Noggin	Peprotech	250-38	Stock Concentration (0.1 ng/µL); Final Concentration (100 ng/mL); Solvent (UltraPure water + 0.1% BSA)
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher	15140-122	Stock Concentration (100x); Final Concentration (1x)
Petri dishes (sterilized; 100 mm x 15 mm) Polystrene disposable	VWR	25384-342
Polystyrene Microplates, 24 well tissue culture treated, sterile	Greiner Bio-one	5666-2160
R-Spondin	R&D Systems	3474-RS-050	Stock Concentration (0.25 µg/µL); Final Concentration (500 ng/mL); Solvent (D-PBS + 1% BSA)
Tryp LE Express	Thermo Fisher	12604-013	Stock Concentration (10x); Final Concentration (1x); Solvent (1 mM EDTA)
UltraPure Water	Invitrogen	10977-023	Stock Concentration (1x); Final Concentration (1x)
Y-27632 dihyddrochloride	Abcam	ab120129	Stock Concentration (10 mM); Final Concentration (10 µM); Solvent (UltraPure Water)