Homo sapiens (Erythrocytes (red blood cells))

(Cryopreservation of Human Red Blood Cells (Erythrocytes))


This protocol utilizes an ice-binding protein from the Arctic yeast Leucosporidium sp. (LeIBP) that is created in a Pichia expression system. The use of LeIBP significantly reduced freeze-thaw-induced hemolysis and better preserved cell size distribution.


• Human blood
• LeIBP (see references)
• Heparinized blood collection tubes
• Centrifuge
• Phosphate-buffered saline (PBS)
• Glycerol
• Sodium chloride (NaCl)
• 2 ml cryovials


1. Centrifuge the blood at 2000 x g for 10 min.
2. Remove the plasma supernatant and the buffy coat.
3. Wash the RBC pellet three times with equal volumes of PBS. In the third wash, adjust the hematocrit to 70%.
4. Drop-by-drop, add equal volumes of freezing solution containing 80 % (w/v) glycerol and 0.9 % NaCl.
5. Centrifuge the RBCs at 2000 x g for 10 min and remove the supernatant to yield a final pellet containing 70 % hematocrit and 40 % (w/v) glycerol.
6. Mix 75 µl of glycerolized RBCs with 25 µl of LeIBP solution (0.8 mg / ml LeIBP and 40% glycerol in PBS) in a 2 ml cryovial.
7. Immerse the tubes in liquid nitrogen for 10 min, then store the frozen tubes in liquid nitrogen vapor.
8. To thaw, place in a 45°C water bath for 1 min.


Lee SG, Koh HY, Lee JH, Kang SH, Kim HJ. Cryopreservative Effects of the Recombinant Ice-Binding Protein from the Arctic Yeast Leucosporidium sp. on Red Blood Cells. Appl Biochem Biotechnol. 2012 Jun;167(4):824-34.

Park, K. S., Lee, J. H., Park, S. I., Do, H., Kim, E. J., Kang, S. H. & Kim, H. J. (2012) Characterization of a homodimeric ice-binding protein from Leucosporidium sp. Cryobiology, 64, 286–296.

Added by: Carlton Hoyt on July 25, 2012

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