Cryopreservation of Sheep Red Blood Cells: 4. Titrations of The First Complement Component with Frozen Eac4 Cells

The cryoprotection of the sheep erythrocyte intermediate EAC4 cells, used as reagent in titration of the first complement component, Gl, was investigated. The cryoprotective agents tested were untreated polyvinylpyrrolidone (PVP), purified PVP, neutralized PVP and a hydroxyethylated potato starch of high viscosity, Avelex 1030, hydrolyzed for 40 min. Recovery of EAG4 cells after thawing was 80–90 %, with best results using untreated, purified or neutralized PVP. The EAG4 cells frozen in the presence of untreated PVP showed, however, increased susceptibility to the hemolytic action of Gl, whereas cells frozen with purified or neutralized PVP or with Avelex 1030 gave titers similar to that obtained with fresh cells. Gl titrations with frozen and thawed EAG4 cells gave more reproducible results than those obtained when titrations were performed with fresh separately prepared cells.     

Cryopreservation of Hen Red Blood Cells

The cryoprotection of hen erythrocytes, used as reagent in virus titration, was investigated. The cryoprotective agents tested were neutralized polyvinylpyrrolidone (PVP), dimethylsulfoxide (DMSO) and glycerol. Good results were obtained with PVP, especially with PVP K15 (average molecular weight 10 000), and with DMSO, especially when used in a final concentration of 10 %, whereas glycerol was unfit for use in the concentrations tested. The red cell concentration, the suspending buffer before freezing and the washing procedure after thawing were of importance. The cells could be frozen and stored for at least three months without any significant effect on the virus titer.     

Cryopreservation of sheep red blood cells. 1. Experiments with polyvinylpyrrolidone and other protective agents

The protection of sheep erythrocytes at freezing temperatures was investigated using glycerol, dimethylsulfoxide (DMSO), glucose and four different types of polyvinylpyrrolidone (PVP) as cryoprotective agents. Depending on type (molecular weight) and concentration good protection was obtained with PVP, whereas glycerol, DMSO and glucose were unsatisfactory. Recovery of cells after thawing was most successful when the cells had been frozen at a concentration of 1–2 × 10⁹ cells/ml. No cells tolerated freezing at −20 °G. Best results were obtained when the cells were frozen directly in liquid nitrogen (−196°G).