It is essential to thaw cryopreserved cells correctly to maintain the cells' viability and functionality. Our "how-to" video provides step-by-step instructions for thawing cryopreserved cells intended for use in downstream culturing applications and when testing a representative sample for quantification.
This "how to" video closely follows the technical protocol and shows step-by-step instructions for properly thawing cryopreserved cells, which is crucial to ensuring the viability and functionality of the cells. By following the protocol, using good techniques, and working quickly you can ensure that a high proportion of cells survive the procedure.
0:09 Thawing cryopreserved cells properly is crucial to ensure the viability and functionality of the cells.
0:15 Using good technique and working quickly ensures that a high proportion of the cells survive the procedure.
0:22 Before starting, please make sure that you have all the necessary materials and equipment for successful thawing.
0:50 Warm thawing media in 37°C water bath.
0:55 Obtain the cryovial containing the cryopreserved cells from liquid nitrogen storage.
1:01 Place the cryovial on a bed of dry ice to retain a consistent temperature when transporting the cryovial to the water bath.
1:09 Quickly loosen cap slightly to relieve pressure within the tube, retighten, and place into a 37°C water bath immediately.
1:17 Hold the cryovial in the water without submerging the cap area.
1:21 Do not move the vial while thawing.
1:23 Avoid any agitation (no figure 8 movement, shaking, or flicking).
1:28 To prevent contamination, do not allow water to come in contact with the cryovial cap.
1:33 When a sliver of ice remains in the cryovial, it can be removed from the water bath.
1:38 Remove the vial from water bath.
1:41 Clean the outside of the vial with 70% ethanol and transfer the cryovial into a BSC.
1:48 Aspirate 5mL of thawing media using a serological pipette.
1:55 With the same pipette, gently aspirate the cryopreserved cell solution from the cryovial into the media within the pipette.
2:03 Let the cells diffuse with the thawing media.
2:07 Gently transfer the cryopreserved cells and thawing media in the pipette into a labeled conical tube.
2:18 Rinse the cryovial with an appropriate volume of thawing media from the conical tube.
2:31 Transfer the rinse to the conical tube containing the cells.
2:40 Slowly add an appropriate amount of thawing media to the cryovial.
2:47 Aseptically, take a sample for cell count and viability analysis.
2:53 Centrifuge the cell suspension at 300 RCF for 10 minutes at room temperature (the centrifugation speed and duration can vary depending on the cell and conical tube types).
3:04 Check the clarity of the supernatant and visibility of a complete cell pellet, and transfer into a BSC.
3:12 Aseptically aspirate the supernatant without disturbing the cell pellet.
3:25 Flick or tap the tube to loosen the cell pellet.
3:31 Gently re-suspend the cells in washing buffer or media.
3:49 Take sample for cell count and viability analysis.
How to Thaw Cryopreserved Cells (Vials) Technical Protocol
