This protocol presents the process of preparing a large pool of human platelet lysate, starting from platelet rich plasma. By performing one freeze thaw cycle, the platelets are lysed. Then at least 10 units of human platelet lysate are pooled into one batch of pooled human platelet lysate or PHPL.
This volume of about three liters is further portioned into aliquots and again frozen to deplete platelet fragments. These aliquots are thawed transferred into vials and centrifuged. The supernatant is the platelet fragment depleted PHPL, containing platelet derived growth factors used to supplement cell cultures.
The remaining platelet fragment pellet is discarded. Hi, I'm Kadina Sharmo from the laboratory of Dr.TRO in the stem cell research unit at the Medical University of Grads. Today we will show you a procedure for the preparation of a pool of human platelet isolates.
We will start from platelet rich plasma, performed several freeze power cycles, and then deplete the platelet fragments. We use this procedure in our laboratory to obtain a sufficient supplement for cell cultures as an alternative to fatal boen serum. So let's get started.
This protocol begins with platelet rich plasma, which is called PRP. This plasma might have been prepared by Cy Apheresis or derived from Buffy coats. People outside the transfusion medicine field should purchase PRP from a local blood bank or another authorized provider.
Check the sterility of the PRP unit by transferring 20 milliliters from each unit to a small connected bag And disconnecting the bag using the welder. This aliquot can be sent to a microbiology lab for sterility testing immediately after preparation. It's important to freeze the PRP units to at least minus 20 degrees in the original storage bag.
Once bacterial tests are negative, the next step can be performed. Taking care not to let the human platelet lysate, now called HPL become excessively warm fall the HPL units in a 37 degree water bath until the ice clots disappear. Platelets should be lysed after this freeze thaw step to prepare a standardized product for each platelet lysate pool.
Collect 10 to 15 thawed PRP units. Connect each consecutive HPL bag to the pooling double bag, and then transfer the HPL into these two bags. Disconnect the empty HPL bags using the welder.
Mix the contents of the two bags to get a final volume of three to four liters of pooled human platelet lysate. Now called PHPL. Connect a small bag and obtain a 20 milliliter sample of PHPL to check the sterility of the pooled product.
Aliquot samples of PHPL for further processing by connecting small bags to the pooling double bag. Transfer volumes of about 250 milliliters to the small storage bags, and then disconnect the filled bags Using the welder. Now perform an additional freeze thaw cycle to increase the rate of platelet fragmentation and the amount of released growth factors.
Do this by freezing the small bags of portioned PHBL at minus 20 degrees. Then thawing the bags in a 37 degree water bath. In the laminar flow hood.
Use sterile scissors to cut and open the tube of each bag, and then pour the PHPL into the 50 milliliter vials. Often platelet fragments tend to aggregate platelets may also induce alloimmunization. To avoid these conditions, remove the fragments from the PHPL through centrifugation at 4, 000 GS for 15 minutes in a refrigerated centrifuge.
Next, pipette the plasma supernatant into the final storage files and discard the platelet pellets to avoid having fragments in your cell Cultures freeze. 50 milliliter aliquots of PHPL once more at minus 20 degrees until further use. Now that platelet fragment depleted PHPL has been generated, the lysate characteristics can be analyzed.
Function and quality of the pooled platelet lysate are tested in cell cultures and proven by the efficient stimulation of cell proliferation, propagation of mesenchymal stromal cells or MSCs and of endothelial colony forming cells, E CFCs is successful using platelet fragment depleted PHPL. Please see our written protocol for the medium recipes here. Two applications for pooled human platelet lysate.
In mesenchymal stromal cell culture are shown MSCs from donor one were cultured with either PHPL or platelet fragment depleted PHPL. As indicated, more fragment contamination is shown here when compared with the fragment depleted PHPL alone. Platelet fragments do not sufficiently support MSC growth.
All three images were taken after 13 days of culture. The MSCs from donor two were cultured for nine days in platelet fragment depleted PHPL rather than platelet-rich plasma. Notice that PRP without platelet lysis does not sufficiently support MSC outgrowth.
Please also see the JoVE protocol by Andreas Reish, which describes the use of fragment depleted PHPL for isolating and culturing human umbilical cord blood derived MSCs. We've just shown you how to make PHPL to fully replace animal serum in your stem cell cultures. When doing this procedure, it's important to remember to remove platelet fragments before adding heparin and PHPL to your culture medium.
So that's it. Thanks for watching and good luck with your experiments.
