A. Cryopreservation of human or mouse primary tissue derived organoids
We recommend freezing 100 – 200 µL of ECM containing organoids per 1 mL of freezing media. For example, if culturing organoids in 50 µL of ECM per well of a 24-well plate, 2-4 wells should be pooled and frozen in a single vial.
Procedure
- Collect organoids and remove from ECM by mechanical dissociation.
- Wash once in DMEM:F12 (ATCC 30-2006) and centrifuge at 500 x g for 5 minutes to pellet.
- Aspirate supernatant.
- Resuspend pellet in cold Stem Cell Freezing Media.
- Immediately transfer 1 mL of the suspension to pre-labeled cryovials.
- Place the cryovials into an ATCC CoolCell (ATCC ACS-6000) freezing container.
- Place the freezing container at -80°C for 24 hours.
- Remove vials from freezing container and transfer to LN2 vapor phase for long-term storage.
B. Cryopreservation of hESC or hiPSCs
Refer to the ATCC Stem Cell Culture Guide for more information.
This protocol is designed for the cryopreservation of cells cultured in a 6 cm dish, using Stem Cell Dissociation Reagent (ATCC ACS-3010) to detach the cell colonies from the dish. Stem Cell Dissociation Reagent is stored as a 0.5 U/mL working solution in DMEM: F12 Medium (ATCC 30-2006).
Stem cell culture medium
Pluripotent Stem Cell SFM XF/FF (ATCC ACS-3002) is recommended for feederfree culture
For optimal results, cryopreserve stem cell colonies when the cell cultures are ≤80% confluent.
Recommended Dissociation Protocol
- Warm an aliquot of Stem Cell Dissociation Reagent working solution to room temperature.
- Aspirate and discard the stem cell culture medium.
- Rinse the cells once with 5 mL of DMEM:F12 (ATCC 30-2006) per 6-cm dish.
- Add 3 mL of Stem Cell Dissociation Reagent working solution to the dish.
- Incubate at 37°C for 10 to 15 minutes or until the edges of the individual colonies begin to loosen and fold back. View the dish under the microscope starting at 5 minutes as incubation time may vary depending on the cell line and colony size.
- Aspirate the Stem Cell Dissociation Reagent and gently rinse the colonies with 5 mL of DMEM: F12 Medium, taking care not to dislodge the cells during manipulation.
- Add 3 mL of stem cell culture medium to the dish, and detach the cells by pipetting up and down 3-4l times with a 1 mL tip. Take care not to overpipette the culture into a singlecell suspension as single cells will not establish colonies after seeding.
- Transfer the cell aggregates to a 15 mL conical tube.
- Add an additional 3 mL of stem cell culture medium to the dish to collect any remaining cells. Transfer this rinse to the 15 mL conical tube containing the cell aggregates.
- Centrifuge the cell aggregates at 200 x g for 5 minutes.
- Aspirate the supernatant and discard. Gently tap the bottom of the tube to loosen the cell pellet.1
Cryopreservation Protocol
- Detach stem cell colonies from the dish as described in the recommended dissociation protocol.
- Remove the Stem Cell Freezing Media from storage and swirl to mix. Keep cold. Decontaminate by dipping in or spraying with 70% alcohol.
- Add 2 mL of cold Stem Cell Freezing Media to the tube containing the cell pellet. Gently resuspend the pellet by pipetting up and down 5-6 times with a 1 mL tip, maintaining the cell aggregates.
- Immediately transfer 1 mL each of the cell suspension into two labeled cryovials.
- Freeze the cells gradually at a rate of -1°C/min until the temperature reaches -70°C to -80°C. A freezing container (eg, the CoolCell LX Alcohol-free cryopreservation container (ATCC ACS-6000)) may also be used.
- The cells should not be left at -80°C for more than 24 to 48 hours. Once at -80°C, frozen cryovials should be transferred to the vapor phase of liquid nitrogen for long-term storage.
Handling Procedure for Frozen Cells and Initiation of Cultures
- 30 Minutes Prior to Handling Cells - Pre-warm the appropriate stem cell culture medium at 37°C for at least 30 minutes before adding to cells.
- Remove cryovial of frozen cells from liquid nitrogen storage.
- Thaw the cells by gentle swirling in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 1 to 2 minutes). Remove the cryovial from the water bath when only a few ice crystals are remaining.
- Sterilize the cryovial by rinsing with 70% ethanol.
- Using a 1 mL or 5 mL pipette, gently transfer the cell suspension to a 15 mL conical tube.
- Slowly add 4 mL stem cell culture medium dropwise, to the conical tube. Use an additional 1 mL of media to rinse the cryovial and transfer the liquid to the 15 mL conical tube. Shake the conical tube gently to mix the cells while adding media.
- Gently pipette the cells up and down several times to mix thoroughly. Avoid breaking apart the aggregates into a single-cell suspension.
- Centrifuge the cells at 200 x g for 5 minutes.
- Aspirate the supernatant and discard. Gently tap on the bottom of the tube to loosen the cell pellet.
- Add 1 mL of stem cell culture medium that has been supplemented with ROCK Inhibitor Y27632 (ATCC ACS-3030) to a final concentration of 10 µM. Gently resuspend the pellet by pipetting up and down 5 to 6 times with a 1 mL tip, maintaining the cell aggregates.
- Plate the cells as desired under feeder-dependent or feeder-free culture conditions. The presence of 10 µM ROCK Inhibitor Y27632 in the stem cell culture medium is recommended.