Culture of mouse and human liver and pancreas organoids: Isolation of duct cells ???? TIMING 4h  

1| Choose from the following options depending on the tissue to process. Mouse liver, mouse pancreas and human pancreas are processed using option A; for human liver follow option B.

(A) Mouse liver, mouse pancreas and human pancreas dissociation

(i)        Tissue collection. For mouse tissue collection, euthanize the mice using an appropriate ethically approved method, and then remove the liver or pancreas as entire organs by standard surgical procedures. For human tissue collection, after surgical excision keep the tissue cold at 4°C in Basal media until ready for processing. ΔCRITICAL STEP The common methods of mouse euthanasia do not adversely affect organoid isolation or culture.

nPAUSE POINT Mouse and human tissue can be stored in Basal medium at 4°C for up to 48h.

(ii)      Transfer the tissue to the biological safety cabinet and place it into a 100mm Petri dish. Pre-warm Digestion solution to 37°C.

(iii)    Mince tissue into pieces of roughly 0.5mm³ using fine scissors. Transfer minced tissue to a 15ml centrifuge tube and add ~10ml of ice cold Wash medium. Pipette up and down with a 10ml pipette to remove red blood cells and fat (which will float to the top of the solution).  

(iv)    Allow tissue pieces to settle and discard ~7.5ml of the supernatant, including any floating pieces of fat. Repeat this wash step once.

(v)      Remove any remaining wash medium and add ~10ml of the Digestion solution prewarmed to 37°C. Incubate digestion mixture on a shaker at 37°C for 45min. ΔCRITICAL STEP Care should be taken to have the correct amounts of Collagenase and Dispase in the Digestion solution, as inaccuracies will have a large impact on the success of the duct isolation.

(vi)    Pipette Digestion solution up and down with a 10ml pipette and check an aliquot of the solution for the presence of clean duct structures using a brightfield microscope (for examples see Fig. 2A and B, day 0 Duct isolation panels). If none are present, return the solution to the shaker at 37°C. Perform this check every 20-30min. ΔCRITICAL STEP Generally a large proportion of ducts will appear once no more tissue remains to the naked eye, which will require ~2h total incubation. 

(vii)   When the ductal structures appear, transfer the supernatant to a fresh 15ml centrifuge tube, add ice cold Wash medium to increase the volume to 15ml and pellet the material at 100-300g for 5min at 8°C. Discard supernatant and add cold Wash medium to a volume of 15ml, repeat the pelleting procedure again to wash out any remaining Digestion solution. 

(viii) If you wish to cell sort to enrich for ductal cells, follow Box 1 instructions.  If sorting is not required, proceed to next step.

(ix)     Enrichment of duct stem cells by hand-picking. Under aseptic conditions, resuspend the material from step 1(A)(viii) in 5ml of cold Wash medium and transfer it to a sterile 100mm petri dish. Using a brightfield microscope, identify and collect ducts using a 200µl pipette, transfer material to a 15ml centrifuge tube. ΔCRITICAL STEP The goal is not to achieve a pure duct culture but rather to enrich the ducts from any contaminating structures/cells.

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(x)      Following the picking of all ducts, add 10ml of Basal Medium to the 15ml centrifuge tube and pellet the material by centrifuging at 100-200g for 5min at 8°C. Remove and discard the supernatant before washing the pellet again with 10ml of Basal medium, again pelleting the material by centrifuging at 100-200g for 5min at 8°C. The pellet now contains isolated duct fragments that can be directly cultured (see Fig. 2 for example).

(xi)     Proceed to step 2.

(B) Human liver dissociation ???? TIMING 2h

(i)        After surgical excision the tissue is kept cold at 4°C in Basal media until processing.  nPAUSE POINT Tissue can be stored in Basal medium at 4°C for up to 48h.

(ii)      Under aseptic conditions, place tissue into a 100mm Petri dish and mince into pieces of roughly 0.5-1mm³ using fine scissors.

(iii)    Transfer minced tissue into a 15ml centrifuge tube and add ~10ml of ice cold Wash medium. Gently pipette up and down with a 10ml pipette to wash the liver pieces. Allow tissue pieces to settle and discard ~7.5ml of the supernatant, including any blood cells and floating pieces of fat. Repeat this wash step once.

(iv)    Remove as much supernatant as possible from the pellet, add ~4-5ml per gram of tissue of pre-warmed human liver Digestion solution and incubate at 37°C. ΔCRITICAL STEP Care should be taken to have the correct amount of Collagenase in the Digestion solution, as inaccuracies will have a large impact on the success of the dissociation. 

(v)      After 30min of incubation, pipette the digestion solution up and down vigorously with a 10ml pipette and check an aliquot of the solution for the presence of single cells. If few single cells are present then return the solution to 37°C. Perform this check every 10min for up to a maximum of 90min. Stop the digestion when the suspension contains 800% single cells. ΔCRITICAL STEP Take great care not to over-digest the material. 

(vi)    When digestion is complete, add cold Wash medium to increase the volume to 15ml. Filter the digested material through a 70µm filter, and add ice cold Wash medium to increase the volume to 50ml.

(vii)   Pellet the material by centrifuging at 300g for 5min at 8°C. Discard the supernatant and add cold Wash medium to a volume of 15ml. Transfer the resuspended material into a 15ml centrifuge tube.

(viii) Pellet the material by centrifuging at 300g for 5min at 8°C. Remove and discard the supernatant and wash pellet again twice with Wash medium and once with 10ml of Basal medium for the final wash, each time pelleting the material by centrifuging at 300g for 5min at 8°C.

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(ix) If you wish to enrich for ductal cells prior to growth of organoids, sort cells as described in Box 1 prior to proceeding to step 2. If cell sorting is not required, proceed to step 2.  

Seeding of duct cells ???? TIMING 30 min  

ΔCRITICAL Pre-warm tissue culture plates at 37°C for 1h-overnight. For mouse cells, adherent plates can be used. For human cells, suspension plates are required to avoid cell attachment to the plate. 

2| Remove the supernatant and resuspend the desired number of cells or duct structures  (1000 cells or 50 duct structures per well of a 24-well plate) in an appropriate basement matrix for seeding (i.e. Matrigel for mouse material; BME2 for human material). Use a volume of 50µl/24-well and 25µl/48-well. ΔCRITICAL STEP Basement Matrix (BME2 and Matrigel) require thawing on ice at 4°C overnight.  Basement matrix will solidify at room temperature (25°C), so it is important to work quickly and keep the basement matrix cold throughout the process.

3| Seed the material by adding a droplet of basement matrix to the centre of each well, preventing the drop from touching the edges as this will cause the cells to attach to the plate. Incubate 5-10min at 37°C or until the basement matrix is solidified.

4| Overlay the droplet with Isolation medium (500µl/well for a 24-well plate and 250µl/well for a 48-well plate) according to the corresponding tissue as specified in the reagent set up and supplementary Table 1 [Query to AU: Please check, editor has changed text].

5| Incubate material under standard tissue culture conditions (37°C, 5% CO₂). 

6| In the case of mouse and human liver, after 3-4 days replace the Isolation medium with normal liver Expansion medium (without Wnt and Noggin). If seeding single cells, then continue to supplement the medium with 10µM of ROCK inhibitor [Y-27632] for the first 6 days in culture. Change medium every 3-4 days. Organoids should be visible within 7 days and ready for passaging before 14 days of culture.

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Cell culture: Passaging organoids for maintenance ???? TIMING 30-40min 

ΔCRITICAL Pre-warm multi-well plates for seeding at 37°C 1h-overnight.

7| Grow the organoids for 10-15 days after isolation or 5-7 days between passages. ΔCRITICAL STEP The interval between passages may differ slightly depending on the initial density of organoids seeded after isolation. Prevent organoids from overgrowing (i.e. the medium turning yellow). Similarly, do not passage the organoids too early. The general split ratio for a confluent well is 1:4 -1:6.

8| Disrupt the basement matrix containing the organoids (drops in centre of the well) by scraping and pipetting up and down using 500-1000ul of basal medium and a 1000µl pipette. Transfer the organoid suspension to a 15ml centrifuge tube, add cold Basal medium to the top (~1314ml) and pipette up and down 3-5 times to remove the basement matrix [Query to AU: Please add the volume of Basal medium used]. ΔCRITICAL STEP Do not pool more than 3 wells (24-well plates) or 6 wells (48-well plates) together so the basement matrix can be properly washed from the organoids.

9| Centrifuge the tube at 100-200g for 5min at 8°C. Aspirate the supernatant, leaving ~2ml of it in the tube. Resuspend the organoids in this remaining medium.

10| Flame a glass Pasteur pipette to narrow the aperture (by ~1/2). Using this narrowed Pasteur pipette, pipette up/down 5-10 times the 2ml of supernatant remaining in order to break the organoids. ΔCRITICAL STEP Do not dissociate the organoids into single cells (Fig. 2). If a Pasteur pipette cannot be narrowed, use 200µl filter tips and pipette up and down until organoids are completely disrupted into fragments. 

11| Add cold Basal medium to fill the tube and centrifuge the tube at 200-250g for 5min at 8°C.

12| Aspirate the supernatant completely. At this step it is possible to freeze the organoids for storage. If freezing organoids follow the procedure described in Box 3. ? TROUBLESHOOTING

13| Suspend the cell aggregates in the appropriate basement matrix (50 μl per well [24-well plates] or 25 μl per well [48-well plates]). Gently pipette up/down until the cell aggregates are completely resuspended. Then seed 50 μl or 25μl basement matrix suspension as a droplet into the centre of each required well on a pre-warmed 24/48-well plate. ΔCRITICAL STEP Work quickly and keep the suspension cold to prevent the matrix from solidifying in the tube. Avoid generating air bubbles.

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14| Incubate the plate for 5-10min at 37°C or until the basement matrix polymerises.

15| Overlay with the appropriate Expansion medium in each well (500 μl per well [24-well plates] or 250 μl per well [48-well plates]).

16| Change the medium every 3-4 days and passage organoids by repeating steps 8-15 every 5-7 days as desired. If you wish to differentiate the cells within liver organoids to hepatocytes, follow the procedure described in Box 4. If you wish to perform genetic manipulation on the organoids, proceed to step 17. If you wish to analyse organoids without performing genetic manipulation skip ahead to step 18. 

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