Home / dmso bladder instillation protocol / Isolation of Human PBMC from Whole Blood

Isolation of Human PBMC from Whole Blood - dmso bladder instillation protocol

Cryopreservation of Human PBMC

1. Principle

Peripheral blood mononuclear cells (PBMC) can be frozen and stored in a cryoprotective media containing 10% dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS), then thawed rapidly.
Cells frozen and thawed in this manner should have an acceptable yield and viability post-storage.

2. Materials and Equipment

1. 2X freezing medium consisting of 20% DMSO in Fetal Bovine Serum (FBS)
2. Cell culture media consisting of RPMI-1640 plus 10% FBS, 10mM Penicillin Streptomycin, 10mM L- glutamine (Complete RPMI; cRPMI)
3. Cryovials
4. Mr. Frosty freezing container with isopropanol
5. -80(C Freezer
6. Liquid nitrogen freezer
7. Biosafety cabinet
8. Hemocytometer or ViCell counter
9. Centrifuge

3. Procedure

1. Label enough cryovials to freeze the entire sample. Label should include study number, sample identification, the date, and “PBMC”. (See note below.)
2. Count cells using the ViCell (or Hemocytometer with viable cell exclusion dye)
3. Resuspend the cells at about 2X107 cells/ml in cell culture media at room temperature. (See note below)
4. Add dropwise enough 2X freezing medium at room temperature to double the volume of the cell suspension. Gently swirl the tube when adding the freezing medium.
5. Slowly remove the cell suspension into a pipette and dispense 1ml per cryovial.
6. Place the cryovials in a room temperature Mr. Frosty freezing container and label the container.
7. Place the freezing container as soon as possible into the -80(C freezer.
8. Transfer the cryovials to liquid nitrogen tank after 1 to 14 days.

Note: No less than 3 vials should be frozen per sample. If there are less than 3X107 cells recovered from a patient sample, evenly divide between 3 cryovials. Below is an example of how to calculate in order to freeze:

Suppose the Vicell count is 37X106 cells/ml. Since the volume of cells I sampled from is 1ml, I have 37X106 cells in total. I round this to 4 X 107. This means that I will make 4 vials. Each vial is 1ml., so I need 4ml total volume to fill these vials. Half the volume (2ml) is cRPMI, the other half is 2X freezing media (2ml). The cells are already in 1ml complete media, so I add one more to make 2ml cRPMI volume. Then I double that with 2X freezing media to make 4ml total, and put 1ml into each of 4 vials.

In case you have 3X107 or less cells: The cells are suspended in 1ml cRPMI already. Add 2ml 2X freezing media and put 1ml into each of 3 vials.

What is the recommended solution for continuous bladder irrigation? Ensure that continuous bladder irrigation (CBI) and indwelling urethral catheter (IDC) are patent – only 0.9% Sodium Chloride 2000ml solution to be used as irrigant for CBI Ensure Bladder Irrigation Chart is maintained- balances to be recorded on FBC