Rapidly resuscitate cells, slowly dilute and re-inoculate at high cell density
Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition
Operation method
Scheme 20.2 Resuscitation of Frozen Cell Experiments
Principle
Quickly resuscitate the cells, slowly dilute, and re-inoculate at a high cell density (Fig. 20.9). Move Materials Caveat 1. When removing cryotubes from liquid nitrogen, lab coats and gloves must be worn. If the tube is submerged in liquid nitrogen, a mask or goggles must be worn, as well as a lab coat and gloves. Cryopreservation tubes stored in liquid nitrogen, including small plastic tubes, may inhale liquid nitrogen and may explode violently when resuscitated. In this case, a plastic bucket with a lid must be used for resuscitation to control the explosion. 2. Plastic cryotubes should be carefully inspected before use to prevent cracking of the tube wall or mismatch of the screw threads resulting in a poor seal. 3. When using DMSO as the cryoprotectant, the cryoprotectant should be pre-cooled at 4℃ before use, and the protectant should be washed off immediately after thawing. Common Problems 1. Check cell viability. Resuspend the cells with 1 ml of culture medium, and check the cell viability with Taipan blue dye exclusion method. 2. DMSO is highly toxic and special care should be taken when using it. In addition, the later the cells are added before freezing, the better, and after thawing, it should be removed as soon as possible to avoid toxicity to the cells. For more product details, please visit Aladdin Scientific website.
Aseptic
Culture flasks (if centrifugation is required)
Growth medium
Pipette, 1 ml, 10 ml
Syringe and 19-gauge needle (if you use glass cryotubes)
Non-sterile
Protective gloves and face mask
Sterile 37°C water, 10 cm deep, in a clean, ethanol-wiped bucket with a lid
Tweezers
70% ethanol
Cotton swabs
1% naphthalene black (amide black) or 0.4% Taipan blue
Procedure
1. Check the cryopreservation catalog to determine the location of the tubes to be resuscitated.
2. Collect all materials, prepare the medium and label the culture bottles.
3. Remove the cryopreserved tubes from the cryopreservation jar, check the labels to determine if they are the desired cryopreserved tubes, and if the tubes are not submerged in liquid nitrogen, place the tubes in a beaker of plastic sterilized water in a bucket at 37°C. Avoid submerging the water over the official cap if possible, as it increases the chance of contamination.
Safety tips
When removing the cryotubes from the liquid nitrogen, you must wear a lab coat and gloves. If the cryostat tubes are submerged in liquid nitrogen, a mask or goggles must be stayed on and lab coat and gloves must also be worn. Cryopreservation tubes stored in liquid nitrogen, including small plastic tubes, may inhale liquid nitrogen and may explode violently when resuscitated. In this case, a plastic bucket with a lid must be used for resuscitation to contain the explosion.
4. When the tubes are resuscitated, the labels are checked again to make sure they are the desired cells; the tubes are then thoroughly scrubbed with 70% ethanol, and then opened on an ultra-clean bench.
5. Transfer the contents of the cryotubes to culture flasks using a 1 ml pipette.
6. Slowly add the medium to the cell suspension: add dropwise at a rate of 10 ml every 2 min. Add drop by drop at first, then you can speed up and gradually dilute the cells and cell protectants.
For cells that require removal of the cell protectant by centrifugation:
(a) Slowly dilute the cells in a centrifuge tube or conventional container according to step 6.
(b) Centrifuge at 100 g for 2 min.
(c) Discard the supernatant culture medium containing cell protectant.
(d) Resuspend the cells with fresh medium.
(e) Inoculate into culture flasks.
7. The residue in the cryopreservation tubes can be stained with naphthalene black or Tapan blue to determine cell viability (see protocol 22.3.1).
8. Check after 24h:
(a) For adherent monolayers, confirm that the cells are adherent and estimate cell viability based on a photograph of the cells at the predicted density (cells/cm2) (see Sections 13.6.1, 16.4.5; Color Figure 4).
(b) For suspension-grown cells, check the appearance (clear cytoplasm, lack of cellular granules) and dilute to routine inoculum concentrations. The inoculum concentration may be more accurate if the cytotechnology is performed and cell survival is estimated (see Section 22.3.1), in which case the cells can be diluted to the conventional inoculum concentration of surviving cells.
Source Animal Cell Culture: A Guide to Basic Techniques (5th Edition)