Generation of vascularized human cardiac organoids for 3D in vitro modeling

Summary Here, we provide a protocol for next-generation human cardiac organoid modeling containing markers of vascularized tissues. We describe steps for cardiac differentiation, harvesting cardiac cells, and generating vascularized human cardiac organoids. We then detail downstream analysis of functional parameters and fluorescence labeling of human cardiac organoids. This protocol is useful for high throughput disease modeling, drug discovery, and providing mechanistic insight into cell-cell and cell-matrix interactions. For complete details on the use and execution of this protocol, please refer to Voges et al.1 and Mills et al.2

2. On ice, aliquot 500 mL of Matrigelä into sterile Eppendorf tubes.3. Store aliquots at À20 C until required.4. Prepare a working solution of Matrigelä: a. Thaw aliquot on ice for 1 h until liquid.b.Aliquot 49.5 mL PBS (minus magnesium and calcium) and store on ice until ready to mix.c.Check Matrigelä dilution factor as per manufacturer's instructions.E.g., Add 500 mL (or as instructed) of Matrigelä to ice-cold PBS for a 1:50 dilution (v/v).d.Rinse Eppendorf with PBS to transfer entire aliquot.
Note: working solution of Matrigelä can be stored at 4 C for 2 weeks.
5. Coat each T25 with 2.5 mL of Matrigelä as required.6. Leave coated flasks in biosafety cabinet for a minimum of 1 h before use.
Note: Alternatively, transfer coated flask to cell culture incubator at 37 C for minimum 30 min before use, or pre-coat the flask the night before and store in the fridge at 4 C. CRITICAL: It is critical to use pre-chilled PBS and ensure Matrigelä is always kept at 4 C otherwise it will quickly gel.
CRITICAL: When scaling to other culture vessel sizes it is critical that the surface area to volume ratio is maintained.CRITICAL: For some batches of Matrigelä with very different concentrations to standard, the dilution may require adjustment.See product data sheet for further details.

Maintenance of human pluripotent stem cells
Timing: 7 days 7. Culture human pluripotent stem cell lines on Matrigelä coated T25 flasks (see steps [1][2][3][4][5][6].This protocol can be used on both human embryonic stem cell and human induced pluripotent stem cell lines.8. On Day -4 (Friday) prepare T25 flask for differentiation: a. Aspirate mTeSRä PLUS medium from T25 culture flask.Flask should be $70% confluent.b.Wash cells with 3-5 mL of warmed (37 C) PBS (without Calcium or Magnesium).c. Aspirate PBS.d.Gently add 2 mL of room temperature (15 C-25 C) ReLeSRä to cover the cells.e. Aspirate the ReLeSRä within 1 min ($50s) so that the culture is left with a thin film of ReLeSRä.f.Incubate at 37 C for 5-8 min depending on the cell line.g.Tilt the flask and use 3 mL mTeSRä PLUS to wash the colonies to the bottom corner of the flask.
Note: No rock inhibitor is used for passaging hPSCs.
h.If colonies do not detach easily hold the plate firmly and tap the side of the flask firmly for 30-60 s. i. Slowly transfer the cell suspension to a sterile 15 mL canonical tube using a 10 mL serological pipette to maintain aggregates.j.Prepare aliquot for counting.k.Work carefully and do not over pipette the cell suspension to maintain aggregates.9. Work quickly to count cells within aggregates using a hemocytometer.
a. Add 10 mL of pre-warmed mTeSRä PLUS (37 C) to a new Matrigelä coated T25 flask.
b. Transfer required amount from the cell suspension canonical tube into the new flask, plating 400,000 cells per T25 flask.c.Gently move the flask from side to side to ensure cells spread evenly in the flask.d.Return cells to 37 C 5% CO2 for the weekend ($72 h).e.On Monday change the medium with 5mL of fresh, pre-warmed media: i Culture maintenance flasks of human pluripotent stem cells in mTeSRä PLUS.
ii Culture flasks going into differentiation in mTeSR TM -1, as the stabilized growth factors in mTeSRä PLUS can interfere with the first few days of differentiation.f.On Tuesday passage the maintenance flasks of the human pluripotent stem cells as per a-o, with daily 5 mL medium changes with mTesr1 until Friday.
Note: Each cell line will have variations in initial seeding density depending on growth rate (typically 300,000-500,000 per T25).Seed cells so that they are $70% confluent by Day 0 of differentiation.Rock inhibitor is not used at any point to passage stem cell lines.

Manufacture of silicone inserts for 96 well plates
Timing: 2 days 10.SU-9 wafers are fabricated using photolithography.For details please see relevant publication. 3 11.Pre-heat oven to 65 C and clean SU-8 wafer with 80% ethanol, followed by water and dry with compressed air.Place clean SU-8 wafer in clean 15 mm petri dish (Figure 1A).12. Mix PDMS elastomer (Sylgard 184) with the curing agent at a 10 to 1 ratio (w/w): a. E.g., Weigh out 45 g of PDMS base agent into large weigh boat slowly due to viscous nature.b.Weigh 4.5 g of PDMS curing agent.c.Mix thoroughly with spatula for several min.13.Once mixed thoroughly, add platinum catalyst at 0.555 mL per gram of total combined PDMS elastomer and mix thoroughly with a spatula for several min.
CRITICAL: To ensure even distribution of the catalyst, add slowly while moving the pipette tip through the PDMS elastomer.Uneven catalyst incorporation will result in premature polymer crosslinking, visualized as yellow polymer (Figure 1B), if this occurs discard mixture and repeat steps 10 & 11.
CRITICAL: The catalyst is a hazardous chemical and the proper safety precautions should be taken.
14. To remove bubbles from the PDMS elastomer, place weigh boat in the vacuum desiccator on a flat surface for 30 min with regular re-pressurizing (Figure 1C).
Alternatives: elastomer can be combined in any vessel, but a large weigh boat maximizes the surface area and increases the speed of removing bubbles.
15. Once bubbles are removed, add 42 g (of 45 g made up in step 11) of PDMS elastomer to the SU-8 wafer on the precision balance and return to the vacuum desiccator for 40 min (Figure 1D).16.Visually inspect that no bubbles have remained in the pillar forming holes of the SU-8 wafer.Use syringe to expel air directly above stubborn bubbles to remove (Figure 1E).17.Place SU-8 wafer + PDMS in oven for 35 min to cure.Once cured, remove the SU-8 wafer and store at room temperature (15 C-25 C) overnight ($18 h).CRITICAL: For successful plate fabrication, ensure plate is level in the oven by using a spirit level.Temperature of curing and ratio of base/curator agent will impact elastic modulus of the resulting polymer and therefore the downstream force analysis.
18. On day 2, cut and remove excess PDMS from around the SU-8 wafer + PDMS edges with a scalpel.19.Transfer SU-8 wafer into 245 mm square tissue culture dish and cover with 80% ethanol.
Alternatives: any large flat dish can be used that has enough room to place pipette tips around the perimeter of the SU-8 wafer.20.Slide pipette tips between SU-8 wafer and cured PDMS elastomer to slowly separate.
a. Once PDMS is removed from the wafer, place on a cutting mat as in step 21. b.Meanwhile dry the SU-8 wafer with a non-particle producing tissue wipe and compressed air before storing (Figure 1F).21.Prepare 2 g of elastomer mixture at a ratio of 10 to 1 base to curing agent in a 50 mL canonical and mix thoroughly.a. Centrifuge at 300 3 g for 3 min to quickly remove bubbles.b.Using a P200 pipette, transfer enough PDMS to cover the surface of the well (approx.10 mL) in each of a new 96 well culture plate.This will be the PDMS glue.22. Clean the back and pillar side of the PDMS sheet with sticky tape to remove dust and debris, and place on cutting mat with the pole side facing up (Figure 1G).23.Using a 6 mm biopsy punch, cut individual well inserts out of the sheet and use the biopsy punch to also release and stick down one per well of 96 well plate with the PDMS glue as in step 20 (Figure 1G).24.Store plate at room temperature (15 C-25 C) overnight ($18 h) to ensure well inserts cure to the 96 well plate before use (Figure 1H).a. Day before use, cover plate in 80% ethanol inside biosafety hood for > 2 h and sterilize with 2 3 20 min UV exposures.b.Rotate plates 90 between the UV exposures to ensure complete sterilization. .Note: Aliquots are stable when stored at À20 C for 12 months.Freeze thaw is not recommended for product stability. .Continued (F) After PDMS is cured in oven, separation of PDMS from SU-8 is required.SU-8 is transferred to a large square petri dish and covered in 80% Ethanol.Pipette tips are used to slowly separate the cured PDMS layer.(G) Once the PDMS sheet is removed from SU-8, cleaned and sealed with sticky tape, a 0.5 mm biopsy punch is used to cut individual inserts.(H) PDMS inserts are transferred and glued into a 96 well culture plate using degassed PDMS glue and left to cure overnight ($18 h).

Human Bone Morphogenetic Protein 4 (BMP4):
a. Quick spin the vial to ensure no product is lost.b.Reconstitute vial to 500 mg/mL in 4 mM hydrochloric acid.i. E.g., add 100 mL 4 mM Hydrochloric acid to 50 mg BMP4.c. Dilute this solution in 0.1% (w/v) bovine serum albumin (BSA) in milli-Q water to 20 mg/mL.d.Prepare 50 mL aliquots in sterilized 0.2 mL PCR tubes.e. Transfer PCR tubes to 50 mL conical tube and store at À20 C.
Note: Aliquots are stable when stored at À20 C for 3 months.Freeze thaw is not recommended for product stability.We keep thawed reagents at 4 C for 1 week maximum.Note: Aliquots are stable when stored at À20 C for 3 months.Freeze thaw is not recommended for product stability.Thawed reagents are stable at 4 C for 1 month.

Human basic fibroblast growth factor (FGF-2):
a. Quick spin the vial to ensure no product is lost.b.Reconstitute vial to 20 mg/mL in 0.1% (w/v) BSA in PBS.c. E.g., Add 1.25 mL 0.1% BSA in PBS to 25 mg stock and mix well.d.Prepare 50 mL aliquots in sterilized 0.2 mL PCR tubes.e. Transfer PCR tubes to 50 mL conical tube and store À20 C.
Note: Aliquots are stable when stored at À20 C for 3 months.Freeze thaw is not recommended for product stability.We keep thawed reagents at 4 C for 1 week maximum.
30.CHIR99021.a. Quick spin the vial to ensure no product is lost.b.Reconstitute vial to 10 mM in dimethyl sulfoxide (DMSO).i. E.g., Add 2.15 mL DMSO to 10 mg stock and mix well.c.Prepare 20 mL aliquots in sterilized 0.2 mL PCR tubes.d.Transfer PCR tubes to 50 mL conical tube and store À20 C.
Note: Aliquots are stable when stored at À20 C for 3 months.Freeze thaw is not recommended for product stability.We keep thawed reagents at 4 C for 1 week maximum.

IWP-4:
a. Quick spin the vial to ensure no product is lost.b.Reconstitute vial to 5 mM in DMSO.i. E.g., Add 4.03 mL DMSO to 10 mg stock and mix well.c.May require a freeze-thaw and mixing at 37 C to properly get into solution.d.Prepare 50 mL aliquots in sterilized 0.2 mL PCR tubes.e. Transfer PCR tubes to 50 mL conical tube and store À20 C.
Note: Aliquots are stable when stored at À20 C for 3 months.Another freeze thaw is not recommended for product stability.We keep thawed reagents at 4 C for 1 week maximum.
i. E.g., Add 2.5 mL 0.1% BSA in PBS to 50 mg PDGF-BB.b.Filter sterilize solution into 50 mL tube.c.Prepare 20mL aliquots in sterilized 0.2 mL PCR tubes.d.Transfer PCR tubes to 50 mL conical tube and store À20 C.
Note: Aliquots are stable when stored at À20 C for 3 months.Freeze thaw is not recommended for product stability.We keep thawed reagents at 4 C for 1 week maximum.Note: Aliquots are stable when stored at À20 C for 12 months.Freeze thaw is not recommended for product stability.We keep thawed reagents at 4 C for 1 week maximum.

D-glucose at 1 M:
a. Reconstitute D-glucose to 1 M in milli-Q water.i. E.g., Add 50 mL milli-Q water to 9 g D-glucose.b.Filter sterilize solution into 50 mL conical tube.c.Store at 4 C for up to 3 months.37. Aprotinin: a. Reconstitute vial to 33 mg/mL in PBS.i. E.g., Add 15.1 mL PBS to 500 mg stock.b.Aliquot stock solution in 50 mL into sterilized 1.5 mL Eppendorf tubes.c.Transfer PCR tubes to 50 mL conical tube and store À20 C.
Note: A aliquots are stable when stored at À80 C for 6 months.Freeze thaw is not recommended for product stability.Use on day of thaw.
i. E.g., Add 50 mL 20% FBS in PBS to 100 mg of collagenase I. c. Filter sterilize solution into 50 mL conical tube.d.Aliquot stock solution in 5 mL into 15 mL conical tubes.

Protocol
e. Store at À20 C.
Note: Aliquots are stable when stored at À20 C for 12 months.Freeze thaw is not recommended for product stability.Use on the day of thaw.
Note: Occasionally the concentration of the collagenase I needs to be adjusted based on the enzymatic activity of a particular batch.This section achieves the differentiation of cardiac cell types over a 15-day protocol.
CRITICAL: Thaw growth factors before use.Add growth factors and small molecules immediately prior to use and add to warm medium (37 C).
d. Wash with RPMI base media on Day 0 before adding differentiation media.2. On Day 3, wash cell with RPMI base medium to remove cell debris: a. Prepare 10 mL RPMI/B27 minus insulin medium per T25.b.Add final concentration 5 mM IWP4.c. E.g., add 10 mL IWP4.
CRITICAL: Add IWP4 immediately prior to use and warm to 37 C.
Note: Cardiomyocyte beating is generally observed from day 10 onwards.
4. On Day 13, perform media change with RPMI/B27 (plus insulin) with no factors.5. On Day 15, harvest cells for organoid generation as outline below.

Harvest of differentiated cardiac cells for organoids
Timing: 2 h

Protocol
This section describes the generation of human cardiac organoids.
6. Prepare PDMS culture plates from step 23 for use: a. Inside biosafety hood, ensure no residual ethanol remains in the well inserts.b.Coat inserts with 3% BSA in PBS (approx.5-10 mL per well) for > 60 min at room temperature (15 C-25 C).
Begin cell dissociation 7. Aspirate medium from T25. 8. Add 2.5 mL collagenase I (step 36) per T25 and incubate for 1 h at 37 C 5% CO2.a. Gently tap the side of flask every 10 min to help dissociation.9. Add 7.5 mL PBS (no calcium, no magnesium) to T25 and titrate liquid and cells up and down $5 times to disrupt cells with a 10 mL serological pipette.10.Collect cell solution into 50 mL conical tube.11.Centrifuge at 300 3 g for 3 min.12. Aspirate supernatant.13.Re-suspend cell pellet in 5 mL 0.25% Trypsin with EDTA per T25.14. Gently agitate cells in water bath at 37 C for 10 min.15.Neutralize enzyme with 5 mL organoid harvest medium (see Table above) per T25.16.Filter cells through 100 mm Corning cell strainer and perform cell count.

Human cardiac organoid fabrication
Timing: 3 h 17.Prepare PDMS culture plates for use in culture: a. Inside biosafety hood, aspirate 3% BSA (from step 6b) and ensure no residual liquid remains in the well inserts.
CRITICAL: Ensure insert has no remaining liquid or the organoid mixture will be subsequently diluted and compromised.
18. Perform cell counts and prepare cell aliquots per plate: a.For 1 3 96 well plate, calculate 110 cardiac organoids and 6.6 3 106 cells.b.Centrifuge cell suspension for 300 3 g for 3 min.19.On ice, prepare organoid matrix mixture as outlined in 'Organoid Matrix Mixture' table using a P200 pipette tip: Note: Avoid introducing bubbles into the matrix mixture by slow pipetting and keeping the pipette tip under liquid surface.
a. Prepare a 1.5 mL Eppendorf tube per 96 well plate mixture and place on ice.b.Aliquot collagen 1 slowly into 1.5 mL Eppendorf tube (volumes in 'Organoid Matrix Mixture' Table above).See Figure 2A for example.c.Add cold 10x DMEM and mix thoroughly with collagen using pipette tip (volumes in 'Organoid Matrix Mixture' CRITICAL: Reagents should be added in the order listed above and thoroughly mixed with a P200 pipette tip at each step.All reagents and Eppendorf tubes should be kept on ice constantly. 20. Aspirate supernatant from cardiomyocyte and stromal cell pellet and re-suspend in cold serum free medium (volumes in 'Organoid Matrix Mixture' Table above).21.Add cardiomyocyte and stromal cell suspension immediately to matrix mixture and mix thoroughly.
CRITICAL: Once cells and matrix are combined, proceed immediately to seeding mixture between the pillars in each well of the PDMS culture plate.
22. Place PDMS culture plate on ice.See Figure 2D for example.23.Seed mixture into each well of the PDMS culture insert throughout the entire seeding reservoir (i.e., around and between the pillars covering the entire oval) (volumes in 'Organoid Matrix Mixture' Table above).Note: Tilt plate towards user and pipette mixture from bottom rows working up to ensure mixture in the plate stays cold prior to centrifugation.
24. Centrifuge plate 100 3 g for 10 s with maximum acceleration and deceleration settings.See Figure 2E for example seeded plate.
Note: Transport plate to the centrifuge on ice to reduce premature setting of collagen.
25. Remove plate from centrifuge and check even cell spread under the microscope (Zeiss Primovert, 10X objective).
Note: Observe cell density and distribution around the pillars in several wells of the PDMS culture plate.
26.Return plate to incubator and store for $ 30 min at 37 C 5% CO2 until mixture turns opaque indicating that it has set.See Figure 2F

Force analysis of human cardiac organoids
Timing: 1 h

Protocol
This section describes the analysis of functional parameters of beating human cardiac organoids.
35.Switch on microscope with 4x objective and CO2 and temperature control.
Note: Use automated stage controller e.g., Leica Thunder microscope and LAS X Life Science Microscope Software.Use a plate chamber for environmental control.
CRITICAL: Wait for temperature and CO2 to stabilize before commencing imaging.
36.Set acquisition points for each organoid.37. Using software control, focus Z axis on the top of the organoid pole (Figure 3A).
Note: In software, use square ROI in field of view to set consistent acquisition points within the frame (Figure 3B).Use low magnification (4x) to capture the whole organoid within the frame.
CRITICAL: Inaccurate focus on the top of the pole will influence downstream analysis of contractile force.Overexposure will result in loss of fine detail and color saturation which will negatively affect pixel detection and downstream contractile force analysis.
38. Set acquisition to record 500 frames over 10 s video (50 fps) with bright-field acquisition 39.Export multipage TIF files using naming system 001, 002 etc.
Note: Files will be $250 MB per TIF.Note: Files will be on order of $10 MB per AVI.  4.
Note: Align replicate organoids with the same stain in a row on the slide for efficient imaging.Pause point: Fixed samples can be stored protected from light at 4 C for 2 days.

EXPECTED OUTCOMES
A differentiated culture is expected to generate multiple cardiac cell types and visually form a network of beating cardiomyocytes.The cellular composition of differentiated cardiac cells should be $70% cardiomyocytes and $30% stromal cells.A T25 is expected to generate 10-15 3 10 6 cells at harvest.>95% Cardiac organoid formation efficiency is expected by 7 days post-harvest.
Cardiac organoids are expected to generate 200-500 mN of force.Cardiac organoids are expected to comprise of multiple cell types by day 7 that arise from the stromal fraction and self-sort into in vivo like morphologies, including cardiomyocytes (cTnT), stromal cells (CD90), endothelial cells (CD31), pericytes (NG2), and epicardial cells (WT1) (refer to Figure 4).

QUANTIFICATION AND STATISTICAL ANALYSIS
Cardiac organoids were excluded from force analysis if the tissue was broken; a pole was broken or deformed; the tissue did not condense or the tissue had balled up or come off the pole.

LIMITATIONS
Cardiac organoid generation relies on successful differentiation of human pluripotent stem cells.Some stem cell lines have reduced differentiation efficiency along some lineages and therefore the protocol may be unsuccessful.In our experience the consistency in growth, passaging, confluence prior to differentiation and high quality cultures with no partially differentiated cells overcomes limitations with particular cell lines.This is potentially the most variable and important step in making this protocol work successfully and generate the required multi-cellular populations.

Potential solution
Make sure to optimize initial seeding density for every hPSC line so that starting density on Day 0 is $70% confluent.hPSCs should also be adapted to feeder-free culture on Matrigelä in mTeSRä medium.Ensure growth factors are fresh.

Potential solution
Ensure matrix is made up according to instructions and kept on ice during preparation.Look for the distinct color changes described above to indicate all reagents are working.Collagen1 will oxidize with continual opening of tubes, so aliquot from stock bottles into a working tube ($5mL).10x DMEM may precipitate out of solution, at which point it should be made fresh.Accurate cell counts are also required to generate the optimal cell to matrix ratio for the organoid to form.No condensation of organoids can also result with too much cell death following dissociation of the 2D differentiation, or because of the differentiation yielding very high cardiomyocyte proportions (see potential solution 1).Also ensure there is no residual BSA in the insert as this will also negatively impact and dilute the organoid matrix.

Problem 3
Cardiac organoids lose structural integrity over culture time and break (protocol steps 29-34).

Potential solution
This is usually due to insufficient matrix to support the cells in the organoid.Typically reagents in the 'Organoid Matrix Mixture' need replacing, the collagen I solution has formed a concentration gradient over long periods of time and the concentration is not accurate or the cardiac organoids do not contain the correct fibroblastic populations.Flow cytometry profiling is used to determine the ratio of fibroblasts to non-fibroblast cell populations.

Potential solution
This typically results from low cardiomyocyte fractions in the differentiation cultures, cardiomyocyte death during the dissociation or seeding phases and/or old/incorrectly formulated media.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, James Hudson, James.Hudson@qimrberghofer.edu.au.

Materials availability
The specifications to generate the SU-8 wafer are attached (Data S2).

Figure 1 .
Figure 1.Preparation of PDMS culture inserts (A) Preparation of custom SU-8 wafer for PDMS casting.Wafer thickness 1 mm, 0.7 mm pillar height, 0.2 mm pillar width and 0.5 mm pillar length.Cleaned and transferred to 15mm petri dish.(B) Mixing of crosslinker and PDMS base agent with example of incorrect catalyst addition leaving yellow crosslinked debris.(C) Degassing of PDMS in a weigh boat.(D) Degassing of PDMS coating the SU-8 wafer.(E) Final inspection of PDMS and removal of stubborn bubbles with air extrusion.

Figure 1
Figure1.Continued (F) After PDMS is cured in oven, separation of PDMS from SU-8 is required.SU-8 is transferred to a large square petri dish and covered in 80% Ethanol.Pipette tips are used to slowly separate the cured PDMS layer.(G) Once the PDMS sheet is removed from SU-8, cleaned and sealed with sticky tape, a 0.5 mm biopsy punch is used to cut individual inserts.(H) PDMS inserts are transferred and glued into a 96 well culture plate using degassed PDMS glue and left to cure overnight ($18 h).
33. Insulin, human recombinant: a.Stock vial comes at 4 mg/mL in zinc solution.b.Prepare 2-5 mL aliquots in sterilized 0.2 mL PCR tubes.c.Transfer PCR tubes to 50 mL conical tube and store À20 C. Note: Aliquots are stable when stored at À20 C for 12 months.Freeze thaw is not recommended for product stability.For use, dilute insulin 1:500 in DMEM and use this fresh in weaning medium.34.B-27 supplement (both with and without insulin): a. Thaw stock 10 mL supplement bottles at room temperature (15 C-25 C). b.Aliquot supplements into 1 mL aliquots in 1.5 mL Eppendorf tubes.c.Store at À20 C. Freeze thaw is not recommended for product stability.Thaw aliquot immediately before use.35.Palmitic acid: a. Reconstitute vial to 0.1 M in DMSO.i. E.g., Add 19.4 mL DMSO to 500 mg Palmitic acid.b.Prepare 100 mL aliquots in sterilized 0.2 mL PCR tubes.c.Transfer PCR tubes to 50 mL conical tube and store À20 C.

Figure 2 .
Figure 2. Preparation of human cardiac organoids (A) Acid solubilized Collagen 1 in Eppendorf tube.(B) Addition of 10x DMEM to Collagen, thoroughly mixed.(C) Addition of 0.1 M NaOH to matrix, thoroughly mixed.(D) PDMS organoid plate on ice in culture hood.(E) Example of organoid mixture in PDMS insert before gelation.Mixture is transparent and pink in color.(F) Example of organoid mixture in PDMS insert after gelation at 37 C. Mixture is opaque and white in color.(G) Example of organoid in culture after 2 days that has condensed, (H) had not condensed and (I) after 5 days in culture.
40.Use MATLAB script 'TIF2AVI' to batch convert multipage TIF files into AVI video files (Matlab m-files are provided in Data S1).See Methods video S1: Example of contractile cardiac organoid.
culture reagents: Growth factors, small molecules, and supplements Prepare on ice immediately before adding the cell suspension.May require adjustment if stock collagen I is supplied at a different concentration.Mix collagen I prior to use or aliquoting as it may slightly settle into a more viscous and less viscous phase over long time frames.
a. Heat 2 3 1 mL aliquots of B27-and 1 3 100 mL aliquot of palmitate in water bath at 37 C. b.Once warm, add 25 mL of warmed palmitate to each 1 mL B27-aliquot.Note: Aliquots should be completely thawed and warmed before addition or the palmitate will not go into solution.c.Shake at 37 C for 2 h at 1250x RPM covered with aluminum foil.Note: Solution should be clear with no precipitate to indicate proper conjugation.(Continued on next page) STAR Protocols 4, 102371, September 15, 2023 mTeSRä stem cell medium: add 0.5% Penicillin-Streptomycin to supplement and base media.[Store at 4 C until manufacturer's expiration date].Immunohistochemistry blocking buffer: add 125 mL Triton-X and 2.5 mL FBS to PBS.[Store at 4 C and use within 1 month].Make fresh with every use.STAR Protocols 4, 102371, September 15, 2023 Store at 4 C and use within 1 week once B27 and palmitate are added.*Note pre-conjugation step of palmitic acid to B27 supplement outlined in step 41.
Note: Color should change from clear to yellow to indicate solution is well mixed.d.Add 0.1 M NaOH and mix thoroughly (volumes in 'Organoid Matrix Mixture'Table above).See Figure 2C for example.Note: Color should change from yellow to pink to indicate solution is well mixed.e. Add Matrigelä and mix thoroughly (volumes in 'Organoid Matrix Mixture' Table above).
Cells will begin to condense the matrix around silicone pillars by 2 days after harvest.If no condensation has occurred after 7 days the organoids are unlikely to form.See Figures2G-2Ifor examples of condensed and minimally condensed organoid after 2 days in culture and typical organoid at 5 days in culture.29.On day 2 of organoid culture, aspirate medium and add 150 mL Maturation Medium per well (see tableabove).Medium is supplemented with 10 ng/mL PDGF-BB, 10 ng/mL FGF-2, and 33 mg/mL aprotinin.
for example.CRITICAL: Matrix should set before adding medium to ensure mixture does not flush out of insert.27.Add 150 mL per well of Serum Free medium (see table above).28.Return plate to incubator (day 0 of cardiac organoid culture).Note: Can adjust minpeakheight (cut off for detecting beat) and minpeakdistance (cut off for the speed of beat) parameters depending on your particular experiment or platform.These are to help eliminate incorrectly assigned peaks.
Note:44.Save changes.45.Run 'batchanalysis' Matlab script (Matlab m-files are provided in Data S1).This will generate a JPG file per video and an excel sheet with measurements of force contraction (Table1).d.Carefully place coverslip over sample and ensure no bubbles form.e. Set overnight ($18 h) at room temperature (15 C-25 C) before imaging.62. Proceed to confocal microscope for fluorescence image acquisition.Example in Figure

Table 1 .
Example of raw force analysis analyzed with Matlab