pZerve^™

pZerve is a cryopreservation solution that does not contain dimethyl sulfoxide (DMSO), fetal bovine serum or other animal protein. Optimal recovery is achieved through elimination of these harmful components. pZerve is ready to use, does not require any dilution or further processing, and can be used for cells cultured in serum free medium or with serum containing medium.

pZerve has been successfully used with the following cell types including, but not limited to:

• Mast Cells
• Dendritic Cells
• Mesenchymal Cells
• LNCaP – Metastatic Prostate Adenocarcinoma, Human
• LS 180 – Colon, Adenocarcinoma, Human
• 293 – Transformed Primary Embryonal Kidney, Human

Recommended Freezing and Thawing Protocol

Freezing:
1. Examine the culture for healthy growth, confluency, etc. and the absence of contamination.
2. If freezing adherent cells, remove using 0.25% trypsin for 1 to 3 minutes at 37 °C.
NOTE: Some cell lines grown in serum-free medium may be sensitive to 0.25% trypsin and therefore, may require less trypsin or the addition of a trypsin inhibitor. Wash cells after incubating with trypsin.
3. Perform a cell count to determine the total number of viable cells. Cell viability should be greater than 80%, and cells should be in late log phase or pre-confluency growth phase.
4. Centrifuge cells at 600 to 800 RPM for 10 minutes. Remove supernatant and save 3 to 5 mL for sterility testing (e.g. thioglycollate, brain heart infusion, etc.) and mycoplasma testing.
5. Resuspend the cells gently in an appropriate volume of pZerve at a concentration of 1x10⁶ - 1x10⁷ cells/ml. Some cell types such as hybridomas and myelomas may require an increase in cell density.
6. Dispense the cell suspension in 1 to 2 mL aliquots in plastic or glass ampules.
7. Seal ampules and store at room temperature for 30 minutes with occasional, gentle agitation to expose cells completely to cryopreservative.
8. Place ampules in an insulated container and store in a -20°C freezer for one hour.  Remove insulation and transfer to -70 °C freezer for one hour. Do not store at -70 °C for more than two hours. Transfer vials to vapor phase of liquid nitrogen and store for 24 hours before transferring to liquid phase. The suggested optimum cooling rate is 1 °C per minute for most cell types.

Recovery:
1. Remove vials from freezer and rapidly thaw in a 37 °C water bath.
2. Wipe vials with 70% ethanol.
3. Transfer cells to a culture flask and slowly add the appropriate volume of growth medium (2 to 5 mL).
4. Accurate viability counts (i.e., trypan blue dye exclusion) should be performed after at least 2 hours recovery at 37 °C.
5. Medium should be changed within 24 hours.

If desired, pZerve may be removed by washing in the following manner:
1. Transfer cells to a 15ml centrifuge tube and slowly add 2 to 3 mL of complete growth medium (cells are more fragile after thawing).
2. Centrifuge at 400 to 600 RPM for approximately 5 minutes.
3. Decant and transfer the cells to a culture flask with the appropriate volume of growth medium.
4. Cell viability count should be performed at least two hours after recovery.

IMPORTANT: Before terminating a culture, it is recommended that you test entire freeze/thaw cycle to ensure sterility of the culture and cell viability before long term storage.

pZerve is a trademark of Protide Pharmaceuticals, Inc.