Serum Replacement by hPL

It has been known for some time that human platelet lysate (hPL) is a true alternative to the controversial fetal calf serum (FBS), which is still used excessively in cell culture. In fact, it is now known that the factors that make FBS such an excellent media additive are derived from activated bovine platelets and enter the serum during the manufacturing process. The approach of directly accessing the platelet content is therefore obvious.

Human platelet lysate is obtained by breaking down the cell structure of human platelets (“lysing” the cells) and then separating everything which is dissolved inside the cell from the solid cell components by centrifugation. Platelet lysate is therefore a diverse mixture of molecules from the platelet interior.
In recent years, platelet lysates have already been successfully used for various human and animal cells and cell lines – and were able to achieve comparable or improved growth results.

We are convinced that the use of FBS will be increasingly questioned in the coming years and that, in addition to defined, synthetic media formulations, xeno-free serum alternatives such as hPL will become established in the long term.

The desire to demand and promote sustainability and animal welfare in all aspects of our lives should not be lost, especially in scientific and medical research. The time is right for a change in the cultivation of human cells; away from animal components and those obtained under questionable conditions and towards xeno-free alternatives.

The starting material for hPL are expired human platelet concentrates produced by blood donation services intended for medical applications. For transfusion purposes, platelet concentrates must be only a few days old. If a period of 5 days is exceeded, they have to be discarded. It is estimated that 5-20% of platelet concentrates are not used as intended. These platelet concentrates can be used for the preparation of hPL and thus serve a new purpose as a serum substitute in cell culture.

Expired platelet concentrates are “pooled” into larger volumes, lysed by freezing and thawing processes and cell debris and solid aggregates are removed by centrifugation.

Human platelet lysate is a complex mixture of proteins, low molecular weight substances, vitamins, minerals and electrolytes. In addition to the platelet content, it also contains components of blood plasma (plasma proteins, various protease inhibitors and carriers and fibrinogen), since platelets are commonly stored in plasma.

What is particularly important, however, is that platelets harbor a variety of potent mitogenic growth factors and cytokines such as EGF, bFGF, IGF-1, HGF, PDGF-AA, PDGF-BB, PDGF-AB, TGF-β1, BDNF and VEGF.

In general, it can be stated that hPL is suitable for the in vitro cultivation of almost all human and animal cells.

Platelet lysates have already been successfully used in the propagation and maintenance of a wide variety of adherent cells and cells in suspension. Particularly well studied is the outstanding performance associated with human mesenchymal stem cells (MSCs). Other human cells, such as endothelial cells, fibroblasts, keratinocytes and various cancer cell lines also grow very well with hPL. For animal cells, too, there are now many examples demonstrating the suitability of hPL as an FBS substitute.

If your cell line is not included in our list, it is (also) up to you to find out whether the cells you are working with are suitable for cultivation with hPL. And ideally, you let us know at which concentration hPL is most effective.

We at neoFroxx look forward to your feedback and will be glad to share your experiences with other users. The more feedback we get, the better. For you, for other cell culture users and especially for the general exit from FBS consumption.

If you prefer this information as a pdf, you can download the hPL cell line overview here.

Human platelet lysate is a poorly defined media additive, just like fetal calf serum. But hPL has some distinct advantages over FBS:

• No animal suffering
• Very high quality due to high-quality and extensively tested starting material
• Greater safety; no transmission of animal pathogens; negative testing for a wide range of viruses
• Highbatch stability with sufficient pool size (>50 donors) and thus elimination of time-consuming batch testing
• Better reproducibility
• Better performance; hPL enables faster growth with lower volumes: the final concentration is often only 2-5 %
• Xeno-free; first choice for in vitro cultivation of human cells
• Can be manufactured according to GMP; suitable for clinical trials and clinical applications in cell therapy
• Good availability and stable price
• Sustainable

As with FBS, hPL is strictly speaking a further processed waste product. However, with the big difference that the “raw material” for hPL is a high-quality medical product, not a by-product of mass slaughter – and that no unnecessary animal suffering is accepted for its extraction. Thus, there are both qualitative and ethical reasons for serum replacement with hPL.

Watch out when buying lysate! The partly different starting materials as well as the processing of the platelets, which varies from manufacturer to manufacturer, lead to significant differences in the quality of the final product. hPL is not (necessarily) equal to hPL. For important criteria such as pool size, fibrinogen content and subsequent testing for cell culture-relevant parameters such as mycoplasma and endotoxins, there is no manufacturer-independent quality standard.

• Fibrinogen content. Since human platelet concentrates are usually stored in blood plasma, the platelet lysate contains clotting factors (fibrinogen). Although these can be removed from the hPL in a further processing step, human platelet lysates are frequently offered in which the fibrinogen is contained. These products require the addition of anticoagulants such as heparin to prevent gelation of the medium. Of course, if the inexpensive porcine heparin is used for this purpose, the finished culture medium is anything but xeno-free.
  Synthetic heparins are available but expensive. In addition, heparin at higher concentrations is known to have a negative effect (e.g., reduced proliferation, impaired differentiation and cell migration, lower viability) on human cells.
  We recommend the usage of fibrinogen-depleted (FD) hPL products, which do not require the addition of heparin and are ready-to-use. Depending on the depletion method, FD-hPLs may contain traces of heparin. In products declared as xeno-free, these heparin traces should be of synthetic origin.
• Pool size. If the hPL is generated from a donor pool that is too small (<50), batch fluctuations must be expected.
• Immunoglobulins. In hPL, the content of immunoglobulins is higher than in FBS.

• Available in “fibrinogen-depleted”, ready-to-use (no heparin required)
  
• 100 % Xeno-free
• Batch stability by pools of 150 to 300 healthy donors
• Manufactured in FDA-licensed blood centers or EU blood centers: Tests negative for hepatitis B, HIV, hepatitis C, human T-lymphotropic virus 1 and 2, Trypanosoma cruzi, West Nile virus, syphilis and Zika virus
• Excellent performance due to optimized manufacturing process
• Available in GMP grade: Manufactured, tested and released in compliance with GMP guidelines; DMF available
• Tested for mycoplasma, endotoxins, sterility and MSC performance

At the beginning of the switch, we recommend trying out different hPL concentrations to find the optimal concentration for the respective cell type and requirement.

Typical concentrations are in the range of 1-10% and in this case, more does not equal better! Once the concentration has been determined, no further testing (batch testing) is necessary.

• Lindl & G. Gstraunthaler, Fetales oder fatales Kälberserum? Das Schmuddelkind der Zellkultur, Laborjournal 7-8/2019
• Rauch et al., Alternatives to the Use of Fetal Bovine Serum: Human Platelet Lysates as a Serum Substitute in Cell Culture Media, ALTEX 28, 4 /2011
• van der Valk et al., Fetal Bovine Serum (FBS): Past – Present – Future, ALTEX 35(1), 2018
• Gstraunthaler & T. Lindl, Auf der Suche nach brauchbaren Serumalternativen, BIOspektrum 06/2017
• Fernandez-Rebollo et al., Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells, Scientific Reports 11/2017
• Bieback et al., Gaps in the knowledge of human platelet lysate as a cell culture supplement for cell therapy: a joint publication from the AABB and the International Society for Cell & Gene Therapy, TRANSFUSION Vol. 59, 2019
• Burnouf et al., Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation? Biomaterials 76, 2016