PluriFreeze Protocol

Materials

*or equivalent

Equipment

• NucleoCounter® NC-200™ Automated Cell Counter (ChemoMetec) 

• CO₂ Incubator, 37°C, humidified, 5-7.5% CO₂ 

• Laminar Flow Biohazard Safety Cabinet certified for Level II handling of biological materials 

• Inverted Phase Contrast Microscope, 4x-10x objectives 

• Low-speed centrifuge with swinging bucket rotor capable of 150 x g 

• Pipet-Aid with appropriate serological pipettes 

• Pipettor with appropriate filter/aerosol barrier tips 

• Liquid nitrogen (LN₂) vapor-phase storage system 

• Refrigerator (2-8°C) 

• Freezer (-20°C) 

• Controlled rate freezer (or isopropanol freezing container or equivalent)

• CryoPod™ Carrier for Biological Material (Azenta Life Sciences) 

• ThawSTAR (BioLife Solutions) or 37°C water bath

Adherent hPSC culture is detached from the culture vessel using non-enzymatic cell dissociation with some single cells but mostly small multicellular aggregates (<100 μm in diameter). hPSCs are ready to passage when the majority of colonies are large with compact colony centers and well-defined colony edges. Leading up to the day  of harvest, cultures should be monitored closely for these changes using phase contrast microscopy, and the pH of the culture medium should be monitored to ensure it remains between 6.7 and 7.4, with an optimal target of pH 6.9.

1. Aspirate medium from culture vessel.

2. Add DPBS containing no calcium and no magnesium (DPBS-/-) to the culture vessel and rock gently to rinse residual medium from the growth surface. Fully aspirate DPBS-/-.

3. Add pre-warmed Versene supplemented with 10 μM Y-27632 to the culture vessel and ensure the growth surface is completely covered in liquid. See Table 1 for reagent volumes.

4. Incubate the culture vessel at ambient temperature for 30 seconds.

5. Aspirate Versene.

6. Add the second amount of fresh Versene with 10 μM Y-27632 (refer to Table 1) to the culture vessel  and ensure the entire growth surface is coated with liquid.

7. Incubate the culture vessel in a 37°C incubator for 7-9 minutes, tilting the culture vessel periodically to ensure uniform reagent exposure during delamination.

Note: Incubation time may vary depending on the cell line; dissociation should be monitored under a light microscope until the optimal time is determined. Avoid overexposure with a non-enzymatic dissociation reagent (Versene). Cells should not be exposed to the dissociation reagent for more than 10 minutes.

8. Remove the culture vessel from the incubator and mechanically dislodge the cells from the growth surface by vigorously tapping the sides of the cultureware and in multiwell plates by gentle pipetting. Cells should be nearly or completely sloughed.

9. Gently add Essential 8™ Medium supplemented with 10 μM Y-27632 (refer to Table-1) and carefully pipette 3x across the growth surface to rinse the cells and mix the cell suspension to homogeneity.

Note: minimize pipetting to prevent excessive breakage of cell aggregates. The goal is not to create a single cell suspension, but to create a uniform suspension of cell aggregates.

10. Measure and record the precise volume of cell suspension and transfer to a 50 mL conical tube (or appropriate collection tube for centrifugation).

11. Transfer 200 μL cell sample to a microcentrifuge tube for assessment of cell number and viability. Perform a cell count using the Viability and Cell Count — A100 and B Assay on the NC-200™ automated cell counter.

12. Calculate the volume of cryomedia needed for desired target freezing density per vial.

1. Centrifuge conical tube(s) at 150 x g for 5 minutes at room temperature (15-25°C).

2. Place pre-chilled cryovials on ice or in a thermal rack and transfer to a biosafety cabinet. Gently aspirate the supernatant, taking care not to disrupt the cell pellet. Leave a residual amount of growth medium on the cell pellet (approximately the equivalent volume to the volume of the cell pellet).

3. Gently tap the bottom of the tube to loosen the cell pellet and resuspend cells in the residual growth medium.

4. Using a glass pipette, gently add a small amount of cold cryomedia (~1 mL) to the sides of the centrifuge tube, allowing the cryomedia to gravity settle into the cell suspension.

5. Using the same glass pipette, gently mix the cell suspension with the cryomedia by gently pipetting up and down and swirling the tube until the cells are in a homogenous solution. Minimize the amount of pipetting if possible.

6. Using a glass pipette, add the remaining amount of cryomedia and mix gently.

7. Transfer 1 mL of cell suspension into each labeled pre-chilled cryovial on ice or in a thermal rack.

8. Tightly seal the cryovial cap and place the filled vial(s) into a controlled rate freezer rack or isopropanol freezing container.

Controlled Rate Freezer

1. If appropriate, fill one cryovial with 1 mL PluriFreeze PF10 cryomedia to act as an instrument probe.

2. Follow the manufacturer’s procedures for operation of a controlled rate freezer, using default Program #1 for standard 2 mL cryovials with a 1 mL fill volume.

3. Once the freezing cycle is complete, quickly transfer cryovials to a temperature-equilibrated storage box in the CryoPod™ Carrier for Biological Material.

4. Using the CryoPod™, transfer the frozen cryovials into vapor-phase LN₂ storage.

Isopropanol Freezing Container

1. Incubate the freezing container with cryovials at 2-8°C for 15 minutes.

2. Transfer the freezing container with cryovials to a -80°C freezer and incubate for 15 minutes.

3. After 15 minutes at -80°C, initiate ice nucleation in the samples by forcefully tapping three times at the bottom and each side quadrant of the freezing container. Return the freezing container with samples to -80°C. Ensure ice nucleation has occurred by visually inspecting ice formation within the cryovials. If ice nucleation did not occur, incubate for additional 5 minute intervals before repeating mechanical agitation.

4. After 6–24 hours, transfer frozen cells to vapor phase LN₂ storage.

1. Prior to cell thaw, coat an appropriately sized tissue culture-treated culture vessel with 1 μg/cm2 rhlaminin-521 in DPBS with calcium chloride and magnesium chloride (DPBS+/+). Use Table 1 to determine the volume of DPBS-Laminin for each cultureware. For example, if using a T25 flask, add 250 μL of 10 mM (100 μg/mL) rhlaminin-521 stock solution to 4.75 mL DPBS+/+.

Notes:

• Cells grown in Essential 8 medium should not be cultured on gel-based matrices such as Geltrex  and Matrigel.

• Follow manufacturer recommendations for thawing rhlaminin-521. Do not thaw in a 37°C water bath.

• The coated culture flask should be incubated at 15-25°C for 12-24 hours prior to seeding with cells. Use care to ensure the flask is leveled and the solution covers the whole growth surface at an even depth to ensure uniform coating. Areas that are not properly wetted and stored level will not support adherent growth and will appear as “bald spots."

• On the day of seeding cells, incubate the coated culture flask at 37°C for 2 hours prior to seeding. Ensure the flask is level and solution coverage is even over the growth surface-plane to optimize uniform coating. Rock the flask gently at least once during incubation to ensure even surface exposure to the laminin coating for uniform cell adhesion.

• Other recombinant Laminin formulations such as iMatrix 511 (Matrixome/Reprocell NP891-011), Biolaminin 521 MX (Biolamina MX521-0501) or Biolaminin 521 CTG (Biolamina, CT521-0501) can be used for coating of culturware for hPSC thaw and expansion. Optimal Laminin concentration should be determined for each formulation and cell line starting from manufacturer recommendations.

2. Supplement complete Essential 8 medium with 10 μM Y27632 and warm an aliquot in 37 °C water bath for 15 min. Do not warm the entire bottle, only warm an amount sufficient for thaw.

Note: For all Essential 8 Medium preparations, aliquot an amount and warm to room temperature (15-25 °C). The medium  is a very sparse medium containing labile recombinant growth factors that degrade quickly in warm temperatures (37°C).  Consequently, medium warm-up should only be performed for cell thaw and should not exceed 15 min.

The time following removal of the cell vial from LN₂ storage until the cells are placed in growth conditions is when cells are most vulnerable to degradation. Plan and work efficiently to minimize hold times, use gentle handling techniques, and minimize bubble exposure during liquid handling and transfers.

1. Remove hPSCs from liquid nitrogen storage and maintain cryogenic temperature (<-150°C) until final thaw.

2. Thaw hPSCs quickly in a 37°C water bath or mechanical thawing device (i.e., ThawSTAR).

Note:

• When using a water bath, immerse the vial into the warm water using care to NOT submerge the vial below the level of the cap-threads. Swirl the sample gently and remove it from the water bath when a small ice crystal is still visible.

• The approximate thaw time for a 1 mL sample in cryovial format is 2 min.

• The cryovial should be cool to the touch and should not be warmed further.

3. Upon removing the vial from the thawing device or the water bath, wipe the outside of the cell vial with 70% isopropanol or 70% ethanol and transfer to the biosafety cabinet.

4. Using a 2 mL serological pipette, transfer the cell/cryomedia mix to a sterile 50 mL conical tube.

Note: Using a 2 mL serological pipette instead of a P1000 micropipettor will minimize unwanted dissociation of cell aggregates. If you use a P1000 micropette tip, a large opening tip (special order) is recommended to reduce shear stress.

5. Slowly, over several minutes, add 5 mL of pre-warmed Essential 8 medium supplemented with Y-27632 dropwise to the cell contents in the 50 mL conical tube, gently swirling and mixing as the medium is added. For best results, submerge the pipette tip below the liquid surface in the receiving tube close to, but not touching, the bottom of the tube.

6. Pipette up and down once using a 5 mL serological pipette to mix and measure the precise volume of cell suspension.

7. Transfer a 200 μL sample to a microcentrifuge tube for determination of cell viability and number. Perform  a cell count using the Viability and Cell Count – A100 and B Assay on the NC-200 Automated Cell Counter.

8. Centrifuge cell suspension at 150 x g for 5 minutes at room temperature (15-25°C).

9. Aspirate the supernatant, being careful not to disturb the cell pellet.

10. Gently resuspend the cell pellet in 5 mL Essential 8 medium supplemented with Y-27632. Pipette gently to maintain the cells as aggregates until a homogeneous cell suspension is obtained.

11.  Transfer a 200 μL sample to a microcentrifuge tube for determination of cell viability and number. Perform a cell count using the Viability and Cell Count – A100 and B Assay on the NC-200 Automated Cell Counter.

12. Remove cultureware from the incubator and aspirate off the DPBS-Laminin solution from the culture vessel and add the combined cell suspension. Do not allow the coated surface inside the flask to dry.

Note: The coating matrix does not require washing before use.

13. Plate hPSCs into the appropriate tissue culture-treated culture vessel at a density of 15,000 to 20,000 cells/cm² and incubate at 37°C, 7.5% CO₂, humidified.

Note:

• Cells will adhere immediately on laminin so cell distribution movement patterns should be performed immediately after cell suspension is added to the culture vessel. Immediately orient the flask to the flat growth position and move in an appropriate pattern to cover the growth surface completely and uniformly. For a multi-well plate or T-flask move the plate in the X-Y plane in several short back and forth motions or use a “figure-eight” pattern, ensuring the wave front of the liquid covers the well or bounces off the corners of the flask.

• Cells may be inspected microscopically for cell distribution and general appearance.

14. At intervals of 24 ± 2 hours, perform daily medium exchanges using complete Essential 8 medium without Y-27632 equilibrated at room temperature (15-25°C).

Note: Feeding twice in one day (doubling feeding) and/or transfer to an incubator with a 5% CO₂ atmosphere on day 3 may be needed if cells are reaching confluence by or before day 4. The acidity of the medium, as observed by the yellowing of the phenol red indicator or via pH meter and the health of cell colonies should be monitored closely. Operators must determine the optimal seeding density, feeding schedule, and CO₂ concentrations for their cell line.