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Isolation and cryopreservation of functionally competent equine leucocytes
H Zerbe1, L F F Castilho, F Engelke
1Clinic for Bovine Obstetrics and Gynaecology, School of Veterinary Medicine Hannover, Bischofsholer Dumm, Hannover, Germany. holm.zerbe@tiho-hannover.de
Summary
Cryopreservation effectively preserves functional equine neutrophils (PMN) for treating mares with endometritis. This method ensures viable, potent immune cells are available for therapeutic applications.
Area of Science:
- Veterinary Immunology
- Reproductive Medicine in Equines
Background:
- Equine endometritis necessitates functional polymorphonuclear neutrophil granulocytes (PMN).
- A reliable method for cryopreserving these neutrophils is crucial for effective local therapy in mares.
Purpose of the Study:
- To develop and validate a cryopreservation technique for functionally competent equine neutrophils.
- To assess the viability and function of cryopreserved neutrophils for potential therapeutic use.
Main Methods:
- Leukocyte separation from equine whole blood using hypotonic lysis, outperforming dextrose sedimentation.
- Cryopreservation of neutrophils in equine plasma with 5% dimethyl sulfoxide (DMSO) using a controlled cooling rate (1°C/min).
Main Results:
- Hypotonic lysis yielded superior leukocyte separation compared to dextrose sedimentation.
- Controlled-rate freezing (-70°C at 1°C/min) maximized post-thaw cell viability.
- Cryopreserved neutrophils retained high phagocytic capacity and enhanced reactive oxygen species (ROS) generation for up to 6 hours post-thaw.
Conclusions:
- Cryopreservation is a viable method for preserving functional equine neutrophils (PMN).
- This technique can provide a ready supply of competent neutrophils for treating mares suffering from endometritis.