May 03, 2024
Patient PBMC collection and cryopreservation and cryorecovery
- 1University of Florida
Protocol Citation: Rebecca Wallings 2024. Patient PBMC collection and cryopreservation and cryorecovery. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld895ov5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 24, 2024
Last Modified: May 03, 2024
Protocol Integer ID: 98735
Abstract
Isolation of PBMC from human blood using CPT tubes, cryopreservation and cryorecovery of cells
PBMC collection and cryopreservation
PBMC collection and cryopreservation
Collect 8 mL of blood per BD Vacutainer CPT Cell Preparation Tube with Sodium Citrate (BD Biosciences, 362761).
Invert tubes 8-10 times and centrifuged at room temperature at 1500x g for 20 minutes with no brake.
Using a 10mL stripette , transfer the PBMC enriched layer to a new 50-mL conical tube, being sure not to touch the ficoll layer, and add MACS buffer (PBS, 0.5% bovine serum albumin, 20 mM EDTA, pH 7.2) to a final volume of 50 mL, followed by centrifugation at 1800x g for 10 minutes at room temperature.
Following removal of the supernatant, resuspend PBMCs in 10mL MACS buffer and count using a hemocytometer with Trypan blue at a 1:20 dilution (10uL of cells mixed with 10uL of 1:20 Trypan blue solution).
Centrifuge PBMCs for 10 min at 300x g at 4°C.
Aspirate supernatant and gently resuspend cell pellet in cryopreservation media (RPMI 1640, with 20% FBS and 10% DMSO) at a final concentration of 1 x 77 cells/mL in cryovials (Simport, T311-2).
Place cryovials in a room-temperature Mr. Frosty freezing container with isopropanol as per manufacturer’s instructions and store at -80°C overnight.
Remove cryovials from freezing containers and immediately placed into liquid nitrogen for long-term storage.
Cryorecovery
Cryorecovery
For cryorecovery, retrieve PBMCs from liquid nitrogen, and thaw at 37°C in water bath.
Once thawed (roughly 1 minute with agitation) slowly add contents of one cryovial to 25mL 37°C filter sterilized complete culture media (RPMI 1640 media, 10% heat-inactivated FBS, 1mM Penicillin-Streptomycin) in 50mL falcon and pellet via centrifugation at 300x g for 10 min at room temperature.
Remove supernatant and resuspend cells in 37°C complete culture media for cell counting using Trypan blue.