Cryopreservation and Recovery of Cells

I. Steps for Programmed Cryopreservation (Requires Gradual Cooling)

1. Prepare Freezing Medium: Recommended ratios for freezing medium: 55% (basic culture medium) + 40% (serum) + 5% (DMSO) or 90% fetal bovine serum + 10% DMSO. It is recommended to use Pricella® General Freezing Medium, Cat.No.: PB180436.

2. Count Prepared Cell Suspension: Calculate the total number of cells.

3. Centrifuge Cells: After centrifugation, aspirate the supernatant as thoroughly as possible. Centrifuge at 1200 rpm (250g) for 3 minutes.

4. Resuspend Cells in Freezing Medium: Adjust the cell concentration to 3×10^6-1×10^7 cells/mL.

5. Aliquot into Cryovials: Dispense 1-1.5 mL per cryovial.

6. Use Programmed Freezing Box: Place the filled cryovials into the programmed freezing box and transfer directly to a -80°C freezer overnight.

7. If No Programmed Freezing Box is Available: Follow this sequential cooling process: Room temperature → 4°C for 20 minutes → -20°C for 30 minutes → -80°C overnight → liquid nitrogen for long-term storage. Ensure cooling during transfer to avoid temperature fluctuations or thawing of the cells.

8. Transfer to Liquid Nitrogen: Quickly transfer the cryovials from the -80°C freezer to liquid nitrogen for long-term storage.

Precautions:

1. DMSO Handling: DMSO releases heat when mixed with the medium. Allow the freezing medium to cool before use to prevent cell damage.

2. Supernatant Removal: After centrifugation, thoroughly aspirate the supernatant to minimize residual culture medium, which could dilute the freezing medium.

3. Avoid Manual Gradient Cooling: Manual gradient cooling is not recommended due to unstable temperatures, which can reduce cell viability.

4. Isopropanol in Freezing Box: Ensure the isopropanol level in the freezing box is above the minimum mark. Replace isopropanol after every five uses to maintain effectiveness. Allow the freezing box to return to room temperature before reuse; do not use it directly from the freezer.

5. Minimize Room Temperature Exposure: After adding the freezing medium to the cell suspension, minimize the time spent at room temperature, as DMSO can damage cells at this temperature. Immediately transfer the aliquoted cryovials to the programmed freezing box and place in the -80°C freezer without an intermediate 4°C step.

6. Post-Freezing Transfer: After overnight storage at -80°C, transfer to liquid nitrogen for long-term storage. Keep the freezing box cold during the transfer to avoid prolonged exposure to room temperature, which can thaw the cells.

7. No Liquid Nitrogen Tank: If no liquid nitrogen tank is available, store the cryovials at the back of the -80°C freezer to minimize temperature fluctuations from frequent door opening.

8. Viability Testing: After cryopreservation, retrieve one vial for thawing to test cell viability. Although cells can theoretically be stored long-term in liquid nitrogen, for safety, revive and culture cells after six months to observe growth before continuing storage.

II. Steps for Non-Programmed Cryopreservation (Using Serum-Free Freezing Medium)

1. Warm Freezing Medium: Take the freezing medium out of the freezer and allow it to warm to room temperature. It is recommended to use Pricella® Freezing Medium (Serum-free & animal origin-free), Cat.No.: PB180438.

2. Count Prepared Cell Suspension: Calculate the total number of cells.

3. Centrifuge Cells: After centrifugation, aspirate the supernatant as thoroughly as possible. Centrifuge at 1200 rpm (250g) for 3 minutes.

4. Resuspend Cells in Serum-Free Freezing Medium: Adjust the cell concentration to 3×10^6-1×10^7 cells/mL using the Freezing Medium (Serum-free & animal origin-free) (PB180438).

5. Aliquot into Cryovials: Dispense 1-1.5 mL per cryovial.

6. Direct Transfer to -80°C: Place the filled cryovials directly into the -80°C freezer overnight without using a programmed freezing box.

7. Transfer to Liquid Nitrogen: During the transfer to liquid nitrogen, keep the cryovials cold to avoid direct exposure to room temperature. Quickly move the cryovials to the liquid nitrogen tank for long-term storage.

Precautions:

1. Cooling During Transfer: Since a freezing box is not used, ensure cryovials are kept cold during transfer to liquid nitrogen. Do not handle directly with hands; use dry ice or a small amount of liquid nitrogen for protection. Follow safety precautions during this process.

2. Quick Transfer: Minimize exposure to room temperature during transfer to prevent thawing.

3. Storage Position in -80°C Freezer: If no liquid nitrogen tank is available, store cryovials at the back of the -80°C freezer to maintain stable temperatures despite frequent door openings.

III. Steps for Cell Recovery

1. Prepare Water Bath and Medium: Preheat a water bath to 37°C. Prepare a sterile centrifuge tube with 9 mL of sterile culture medium and have clean disposable PE gloves ready.

2. Thaw Cells in Water Bath: Remove cells from the liquid nitrogen tank and place them inside PE gloves. Quickly immerse the cryovials in the water bath, shaking to speed up the thawing process. Complete thawing within 1 minute.

3. Centrifuge Thawed Cells: In a biosafety cabinet, transfer the thawed cell suspension to the centrifuge tube containing fresh culture medium. Centrifuge at 1200 rpm for 3 minutes, then remove the supernatant.

4. Resuspend and Culture Cells: Resuspend the cell pellet in an appropriate volume of complete culture medium and transfer to a sterile container (culture flask or dish). Supplement with additional culture medium as needed and place in an incubator for cultivation.

Precautions:

1. If the water bath is not in the same room as the liquid nitrogen tank, keep the cryovials cold during transfer to the water bath to prevent surface thawing.

2. Shake the cells rapidly during the thawing process to speed up dissolution.

3. Keep thawed cells at room temperature for the shortest possible time. Centrifuge immediately to remove DMSO.

4. Observe adherent cells 24 hours after recovery. If the density reaches 80%, proceed with normal passaging; if not, continue cultivation and change the medium after 48 hours.

5. Do not centrifuge and change the medium within the first 3 days after recovery.

6. For cells not sensitive to DMSO, centrifugation can be skipped to reduce handling steps and potential contamination. Transfer the thawed cell suspension directly into a T25 flask, add fresh culture medium, and incubate. However, replace the medium with fresh culture medium after 12-24 hours to remove dead cells.