Materials and Reagents

500 ml filter units with 0.22 μm filter (Fisher, catalog number: 974102)
24-well tissue culture plates (Thermo Fisher, catalog number: 142475)
T75 tissue culture flasks (Thermo Fisher, catalog number: 156499)
2 ml pipettes (Fisher, catalog number: 13-678-25C)
10 cm dishes (Thermo Fisher, catalog number: 150464)
15 ml Falcon tubes (Corning, catalog number: 35296)
1 ml pipettes (Fisher, catalog number: 13-678-25B)
Forceps (Fisher Scientific, catalog number: XX6200006P)
Razor blade (Fisher Scientific, catalog number: 12-640)
Glass slides (VWR, catalog number: 89500-466)
SIMS cells (available per request)
Sterile DMEM medium + 4.5g/L high glucose + L-glutamine without sodium pyruvate (Invitrogen, catalog number: 11965-092)
Sterile 1x PBS-Ca⁺²-Mg⁺² free (Thermo Fisher, catalog number: 110010031) or 1x HBSS-Ca⁺²-Mg⁺² free (Thermo Fisher, catalog number: 14175079)
Penicillin-Streptomycin (Thermo Fisher, catalog number: 15140122)
Sterile USDA-approved FBS (Sigma-Aldrich, catalog number: F2442-500ML)
0.25% Trypsin-EDTA (Sigma-Aldrich, catalog number: T4049-100ML)
Epidermal growth factor (EGF) (Sigma-Aldrich)
Glutamax (Gibco/ThermoFisher, catalog number: 35050061)
Fibroblast growth factor-2 (FGF2) (R&D, catalog number: 3139FB025)
N2 (Thermo Fisher, catalog number: 17502048)
Dexamethasone (VWR, catalog number: 80056-298)
Triiodothyronine (Sigma-Aldrich, catalog number: 709719)
Retinoic Acid (Thermo Fisher, catalog number: AA4454077)
Hydrocortisone (Sigma-Aldrich, catalog number: H0888)
Cholera toxin (Sigma-Aldrich, catalog number: C8052)
Calcium, 99%, Granular (Fisher, catalog number: AC201180050)
Growth factor reduced Matrigel (BD Biosciences, catalog number: 354230)
Collagen Type I, Rat Tail (Millipore, catalog number: 2790888)
4% PFA (VWR, catalog number: AAJ61899-AK)
Trypan blue 0.4% (Thermo Fisher, catalog number: 15250061)
O.C.T. Compound (Fisher scientific, catalog number: 23-730-571)
SIMS subculture media (see Recipes)
Differentiation media (see Recipes)

Equipment

Water bath (Thermo Scientific, catalog number: TSGP02)
Vacuum pump aspirator (standard)
CO₂ incubator (standard)
Cryotome (standard)
Brightfield microscope (standard)
Confocal laser scanning microscope (standard)

Software

ImageJ software (http://imagej.net, available online for download)

Procedure

SIMS subculture
1. Pre-warm cell medium, 1x PBS-Ca⁺²-Mg⁺² free or 1x HBSS- Ca⁺²-Mg⁺² free and 0.25% trypsin-EDTA in a water bath at 37 °C.
2. Aspirate off old medium with vacuum aspirator or use 10 ml pipettes to manually aspirate from the T75 cell culture flask.
3. Wash twice with 10 ml of sterile 1x PBS/HBSS-Ca⁺²-Mg⁺, aspirate off completely-either manually with a 2 ml pipette or with an aspirator and sterile fresh 2 ml glass pipette.
4. Add 3 ml of pre-warmed 0.25% Trypsin-EDTA per T75 flask.
5. Incubate plate with cells at 37 °C for 5-7 min. Tap and gently shake the plate to dislodge cells.
6. Neutralize trypsin by adding fresh ~3-5 ml culture medium using a 10 ml pipette. Pipette gently up and down several times to re-suspend cells and transfer to a 15 ml Falcon tube.
7. Centrifuge at 100 x g for 3 min, wash 2 times with 1x PBS-Ca⁺²-Mg⁺² free, and take up the cells in desired amount of culture medium.
8. Plate up to 1 million cells to get 95% confluency in 4-5 days in a T75 tissue culture flask.
  Note: Cell viability (~90-95%) is determined by trypan blue cell count.
Organoid differentiation culture
1. Resuspend SIMS cells in SIMS medium at 0.4 x 10⁶ cells/ml.
2. Thaw out growth factor reduced matrigel matrix on ice for 10 min before culturing cells. Both collagen and matrigel need to remain on ice until they are resuspended with cells for plating.
3. Add 50 μl of cell solution (10,000 cells) to 100 µl of 60:40 ratio of Type I rat tail collagen to growth factor reduced Matrigel on ice. Mix gently while slowly pipetting up and down using a 200 μl pipette.
  Notes:
  1. Aliquot collagen first then Matrigel. Do not mix with pipette until you add cells to avoid bubbles.
  2. Avoid fast aspiration or suspension of aliquoted matrix mix to avoid bubble formation.
4. Deposit cell/collagen/matrigel mix in the center of 24-well tissue culture plate on ice and incubate for 20 min at 37 °C.
5. Add up to 1 ml differentiation medium.
6. Change medium every 2-3 days.
7. Cultures are maintained up to 14 days as the cells reach maximum confluency within the matrix, and the matrix starts breaking down.
8. Organoid formation can be seen starting Day 5 of culture (Figure 1).
  
  Figure 1. Collagen-matrigel embedded SIMS cells expand and form organoids by Day 5 (A) and 7 (B). Scale bars, 200 μm (4x magnification).
Organoid fixation
1. Wash matrix with ice cold 1x PBS-Ca⁺²-Mg⁺² free gently up to 3 times.
2. Cells can be fixed at any timepoint using 2% PFA in 1x PBS-Ca⁺²-Mg⁺² free for 20 min on ice or at 4 °C.
3. Rinse the matrix with ice cold 1x PBS-Ca⁺²-Mg⁺² free 3 times.
4. Incubate matrix in 1x PBS-Ca⁺²-Mg⁺² free for 10 min. Repeat this 3 times for a total of 30 minutes.
5. Using forceps, gently remove the matrix from the tissue culture plate.
  Note: You can cut the matrix into 2-3 pieces using a fine blade if you want multiple cryo blocks.
6. Embed with OCT (a full description of embedding can be found in Campbell et al., 2011).
  Note: Matrix will likely curl up in OCT if it is poured too quickly. Gently pour, and fix any curling using your forceps.
7. Optimal cutting thickness is between 12 and 16 μm.
8. Sections can be post fixed with 4% PFA, ice cold acetone, or acetone/methanol for staining optimization.
3D matrix staining
To conserve the 3D morphology, staining is performed on 1 mm³ pieces of the organoids embedded in matrix.
1. Post fixation, small pieces of matrix including organoids are cut using a sharp scalpel blade and pointed tweezers. Treat each chunk separately in a plastic dish or 24-well plate.
2. Matrix can be treated again with 2-4% PFA, acetone/methanol, or methanol treatments if necessary.
3. Matrix is treated like tissue slides for staining. An example is described in Athwal et al., 2019.
4. Mount each sample on glass slides with 1-2 spacers depending on the size of the matrix.
5. Capture images using a confocal microscope (Figure 2).
6. This staining can also be performed on cryosections of OCT embedded organoid matrix (Athwal et al., 2019). A complete description of the primary and secondary antibodies used in Figure 2 can be found in above-mentioned manuscript.
  
  Figure 2. 1 mm³ collagen/matrigel matrix with 3D grown SIMS organoids were stained with E-cadherin post-fixation with 4% PFA. Scale bar, 10 μm.

Data analysis

Brightfield images are taken starting Day 3 until the end of the experiment. Organoid diameter can be quantified using ImageJ. Each experiment is repeated 3 times with n = 5 images per day for quantification. Details on statistical analysis can be found in our previous manuscript (Athwal et al., 2019).

Notes

Cell death is noticed by Day 14 as cells become confluent within the matrix.
Serial passage of cells dissociated from organoids has not been performed with SIMS cell organoids.
Increase the volume of collagen/matrigel if cell number is increased.
Transduced or transfected SIMS cells are selected prior to the 3D culture assay.

Recipes

SIMS subculture medium (Table 1)

Table 1. SIMS subculture medium
Differentiation medium

Table 2. SIMS differentiation medium

Notes:
1. Make 100 μl aliquots of growth factor reduced matrigel and store at -20 °C.
2. Make 100 μl aliquots of rat tail collagen and store at 4 °C.

Acknowledgments

This work was supported by the National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) grants DE027034 and DE022557. This protocol was adapted from Maimets et al., 2016. Timeline of organoid formation using SIMS cells and changes in media recipe are described in our protocol.

Competing interests

There are no conflicts of interest.

References

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