THAWING AND SAMPLE RETRIEVAL
- Frozen Stemell closed-system vial containing cells
- Water bath warmed to 37°C
- 70% ethanol
- Medical waste container
- Sterile gloves
- Cryogenic vial holder
- Sterile scissors
Option 1 (Recommended):
- Leave the vial in a vial holder in an “upward” position. Place the vial holder into the 37°C water bath. You must leave the vial in a sealed original vial to reduce the chance of contamination. Make sure the water doesn’t go over the top; furthermore, the waterline must be up to where the cells are in the vial. Perform this step immediately after removing the vial from the dry ice in the shipment or after removing the vial from your liquid nitrogen storage or -80°C freezer. Visually and precisely inspect the vial, gently swirling the vial in the water bath until only a small bit of ice remains; then the vial with the vial holder must be removed from the water bath. (Duration: ≈4–8 minutes.)
Note: Thaw the vial vertically with vent tubes pointing upward.
- Place the vial in a vial holder in an “upward” position and leave the vial at room temperature until it’s completely defrosted and thawed. Perform this step immediately after removing the vial from the dry ice in the shipment or after removing the vial from your liquid nitrogen storage or -80°C freezer. (Duration: ≈10–15 minutes.)
- DO NOT vortex the cells at any point while thawing or after thawing. Work quickly to maximize cell viability.
- DO NOT shake or invert the vial.
- Cut open the vent tube (the one with a filter) with clean scissors. (It’s usually the shortest tube.)
- Note regarding photos: The red fluid used for the “sample retrieval instructions” is solely for training purposes and is NOT an actual sample.
- Note: Please make sure to tap down the vial lightly after defrosting to ensure retrieval of the full sample from the vial. It is recommended to use an automatic thaw system for this purpose.
5. Remove the foil on the bottom of the retrieval port.
6. Thoroughly swab the retrieval port septum with a sterile alcohol wipe. Allow to air-dry at least 1 minute before accessing.
7. Use a non‐coring needle and/or proper anti‐coring technique (see over) to puncture the retrieval port septum and extract the sample. It is recommended to use an 18G needle for this port in order to retrieve the sample.
Proper Anti‐Coring Technique:
8a. Hold the needle with the opening of the needle tip away from the retrieval port septum.
8b. Insert the needle into the septum at a 45°–60°angle.
8c. Increase the angle of the needle gradually as the needle enters the vial.
9. It is recommended to use a syringe larger than the fill volume to be withdrawn.
10. Dispose of the vial and syringe according to institutional guidelines for medical waste.
Instructions if the cryopreservative solution (DMSO) needs to be removed prior to usage :
1. Using aseptic technique and under a certified and functional laminar flow hood, have a qualified technician retrieve the cells from the vial according to the instructions discussed above.
2. Transfer the cell suspension into a sterile 15 ml conical tube (centrifuge tube) using the same needle and syringe. Centrifuge the conical tube for 8 minutes at 1400 RPM. Secure the lid. Once the centrifugation is completed, move the conical tube to the laminar flow hood, carefully open the cap, remove the supernatant (DMSO) and leave the cell pellet at the bottom. (Because it’s a very small pellet, DO NOT get close to the cell pellet while you are aliquoting out the supernatant.) It’s important to leave a small volume so as to not disturb the pellet.
3. You may now add your solution (saline, PRP, etc.) to the pellet. Gently resuspend the cells. Add a necessary solution according to the clinic’s medical treatment protocol. Make sure to disrupt the pellet gently in the solution.
Note: Be aware that cell loss is expected and may be up to 12% during thaw and centrifugation. Recovery rates vary depending on technique.