I would like to know if it is possible to get red blood cells and/or platelets from blood previously directly frozen at -80°C. If not, what previous steps could be done before to freeze? Some information about procedures and/or references are welcome. Thank you very much in advance!
Dear, it will be better you separate them immediately and store separately till you want to use them because thawing will affect the vitality of the cells.
Was that product frozen using any Cryoprotectant (Like DMSO) ? If yes then washing it with decreasing concentrations of the DMSO and centrifugation might help us in seperation. Frozen platelets have 33% viability.
After freezing, leukocytes can be recovered. Regarding the platelets, it is difficult (i.e., impossible) to separate them from RBC's debris. As Emmanuel's suggestion, it is preferable to recover each cellular fraction before freezing.
Thank you very much for your help. Really, I was trying to get cells from frozen blood stored by others some time ago. I understand that it is not good idea to use this material. In any case, could you provide me with a protocol or reference to separate cells from fresh blood?
The methods of separating leukocytes and /or platelets from whole blood are different depending on the expected outcome (i.e., all leukocytes, single population [monocytes, lymphocytes], degree of purification and others).
The most straightforward non-selective method with a lover level of purification is the separation by sedimentation for a short time. As soon as a partial separation between RBCs and plasma has been reached, we can recover with a pipette the plasma which contains leukocytes, platelets as well as a poor amount of RBCs.
Selective methods are based on the use of a gradient of centrifugation that is reached by using hypertonic solution.
Vissers MC, Jester SA, Fantone JC. Rapid purification of human peripheral blood monocytes by centrifugation through Ficoll-Hypaque and Sepracell MN. J Immunol Methods 1988; 110: 203-7.
https://www.stemcell.com/lymphoprep.html
http://www.axis-shield-density-gradient-media.com/Leaflet%20Lymphoprep.pdf
Thank you very much Antonio for your information. I am also interested into obtain purified RBC. Is this method useful for collect both cells types with the same protocol?
Yes. After centrifugation, RBCs which are less dense do end up in the bottom of the tube.
Obviously, each cellular population must be carefully washed before use
Antonio, could you provide me with a protocol and/or reference for washing steps? Thanks again!
I think that in your case we must eliminate any trace of hypertonic medium (percoll, Hypaque etc.). So, after separation, add to the separated cells 6-8 mL of saline mix, centrifuge 10min at low/medium speed, discard the washing liquid [all these three times or more]. Some differences can be present regarding the cellular types, the washing solution, the speed of centrifugation. All these information can be easily found in the specific literature on internet as well as in the datasheet of the hypertonic solution
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