Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Summary

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Abstract

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo^1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin^3-5 or lysed by freeze/thaw cycles^6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.^1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors^18,19 and a current lack of standardized protocols for platelet preparation.^15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Protocol

1. Starting material

Start with platelet rich plasma (PRP) units prepared by cytapheresis or derived from buffy coats.

2. Sterility check

For sterility check take a sample of 20 mL from each PRP unit by transferring the volume to a connected small bag (Baxter). Disconnect this bag by welding.

3. Freezing of PRP units

Immediately after preparation, freeze the PRP units down to at least -20°C in the original storage bag without further manipulation.

4. Thawing of HPL units

When ....

Discussion

In some regions platelet rich plasma (PRP) may be obtained from buffy-coats otherwise being a waste product of packed red blood cell production from tested blood donations (Figure 1). Optimally, PRP is used immediately for further preparation of pHPL, as in outdated platelet concentrates the availability of growth factors may be reduced due to platelet storage lesions and degradation²⁰. It is further recommended to produce PRP by matching platelets of blood group O with plasma of blood group AB to avoid possib.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Double bag (2 x 3.5L)	MacoPharma	VDL 8000XQ	originally for ascites puncture
50 mL Falcon tube	Falcon BD	2098
50 mL Stripettes	Costar	4501
Plasma bags (600mL)	Baxter Internationl Inc.	R4R2021
Sterile tubing welder	Terumo Medical Corp.	TSCD-II
Welding equipment	Fresenius Kabi	Compo Seal
Water bath
Sterile scissors
Clamps
Centrifuge