A simple method to optimize peripheral blood mononuclear cell preparation from cynomolgus monkeys and improve mixed lymphocyte reactions.
BACKGROUND
The standard Ficoll-Hypaque method used to isolate peripheral blood mononuclear cells (PBMC) gives very variable results when used with cynomolgus monkey blood. We have improved the method by using special cell processing tubes (CP-tubes), originally developed for clinical use.


METHODS
Blood samples were collected from cynomolgus monkeys and processed for PBMC preparation using either the classical Ficoll-Hypaque method or CP-tubes following various centrifugation protocols. The preparations were compared according to their cellular content as well as their response in the mixed lymphocyte reaction (MLR).


RESULTS
Good PBMC separation was achieved in >90% of samples by centrifugation of blood samples in CP-tubes for 40-45 min at 1650 x g and 20 degrees C. For the remaining samples, poor PBMC separation was probably due to low corpuscular hemoglobin concentrations (< 28 g/dl), but this could be rectified by using one to two additional centrifugations. The PBMC preparations thus obtained showed lower red blood cell (RBC) and polymorphonuclear (PMN) cell contamination, reacted well to mitogen and showed improved MLR stimulation indices vs. standard Ficoll-Hypaque-PBMC-derived preparations. The inhibitory effect of Cyclosporine-A (CsA) was within the low nanomolar range with both methods.


CONCLUSIONS
These results demonstrate that the use of CP-tubes is a practical way of improving PBMC separation and MLR responses with cynomolgus monkey blood.
Bigaud
Marc
M
Novartis Pharma AG, Transplantation Research, WSJ-386 1.55A, CH-4002 Basel, Switzerland. marc.bigaud@pharma.novartis.com
Maurer
Claudine
C
Vedrine
Corrine
C
Puissant
Bénédicte
B
Blancher
Antoine
A
D000818:Animals; D001772:Blood Cell Count; D001773:Blood Cells / Q000166:cytology; D002469:Cell Separation / Q000379:methods*; D016572:Cyclosporine / Q000494:pharmacology; D005362:Ficoll; D005782:Gels / Q000737:chemistry; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D007959:Lymphocyte Culture Test, Mixed; D008252:Macaca fascicularis / Q000097:blood*; D011091:Polyesters / Q000737:chemistry
D005782:Gels; D011091:Polyesters; D005362:Ficoll; D016572:Cyclosporine