PBMC-isolation-and-cryopreservation.pdf - PBMC isolation...

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PBMC isolation and cryopreservation (version 2015 03 05) 1 Immune monitoring platform Centre Physiopathologie de Toulouse- Purpan (CPTP) PBMC isolation from buffy coats or whole blood PBMC cryopreservation Notes - Some critical steps are: - Perform the isolation and freezing of PBMCs less than 8 hours after the blood collection. Best results are obtained if the procedure is performed less than 2 hours after blood collection. - Minimize the time that the cells remain in a pellet or in contact with the Ficoll. - Wash the cells carefully after the Ficoll. - Do not stop during the cell freezing but add slowly the DMSO-containing solution to the cells (in most cases cells are first resuspended in FBS and DMSO-containing solution is then added dropwise). Most labs freeze cells either at 4°C or at room temperature. - The expected PBMC yields from whole blood for healthy populations are (source: HIV AIDS Network coordination PBMC processing SOP): Population Mononuclear Cell Yield Range (cells/mL blood) Adult (0.8 to 3.2) x 10 6 Pediatric less than 6 months (3 to 10) x 10 6 Pediatric 6 mo. to 2 years (2 to 9) x 10 6 Pediatric 2 to 5 years (1 to 6) x 10 6 Pediatric more than 5 years (0.8 to 4) x 10 6 Pediatric Unknown age (1 to 10) x 10 6 - Buffy coats are isolated from whole blood at the blood bank by an initial centrifugation without a density gradient. This initial spin concentrates red blood cells at the bottom and plasma on top, the buffy coat forms the interface and contains most of the leukocytes and platelets from the whole blood (although it still looks like blood). Each unit (~550 mL) of whole blood yields a ~60-45 mL buffy coat, which contains ~200- 800*10 6 mononuclear cells. The Purpan EFS blood bank performs this spin on the day of the blood collection, stores the buffy coat at 20°C and makes it available to the CPTP on the next day. - PBMCs are usually frozen right away after the Ficoll isolation and used subsequently in functional assays. However, for functional tests on fresh or frozen cells, a resting period (typically overnight) is often recommended after Ficoll isolation or cell thawing. Equipment, reagents and reagent preparation PBS without calcium and magnesium. Keep and use at room temperature (15 to 30 C) or non-complemented RPMI-1640 (may increase PBMC viability). Blood cell density gradient media: e.g. Ficoll-PAQUE Plus (GE Healthcare Pharmacia, endotoxin tested ref 17- 1440-02) or Pancoll human density 1,077 g/l (PanBiotech ref P04-60500). DMSO Grade culture (e.g. Sigma ref D2650). After opening, undiluted DMSO is stable at room temperature (15 to 30 C) when protected from light and moisture, for 6 months. Reagent may be aliquoted in small amounts to help preserve sterility. Label aliquots with “DMSO,” the date opened/aliquoted, the expiration
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