A true FBS Substitute – Human Platelet Lysate in Cell Culture

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.

Fetal bovine serum, as the name implies, comes from unborn calves. It is a profitable by-product of the meat industry. In most European countries, the extraction of FBS for commercial purposes is prohibited and, due to the mostly common indoor husbandry, pregnant animals are also rarely led to slaughter unnoticed. In contrast, in the main countries of origin for FBS (Brazil, New Zealand, USA), cattle are kept outdoors in large herds, so that a significant proportion of females (about 8%) go to slaughter pregnant. If during the slaughter process it is noticed that the cow is pregnant, the fetus is cut out of the womb and placed in a separate room. Under aseptic conditions – as far as possible – the heart of the fetus is exposed, punctured with a wide tip cannula, and the blood is collected while the heart is still beating. Since the amount of blood in the fetuses is still quite small, this cruel procedure is the method of choice to obtain as much blood as possible.

Surely, the fetuses would have died either way, but the question is whether we should accept the suffering and pain of these unfortunate animals.

By the way, the worldwide demand for FBS is estimated at about 800,000 L per year. And for one liter of FBS 3-4 cattle fetuses are required!

How is hPL produced?

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.

Whole blood is usually processed into three different types of blood products: Red blood cell concentrates, fresh plasma preparations and platelet concentrates.

An anticoagulant is added to the blood and it is separated into the individual fractions by centrifugation. After centrifugation, the erythrocytes (red blood cells, approx. 45 % of the blood volume) are located in the lowest layer, while the cell-free blood plasma (approx. 55 %), as the lightest component, floats at the very top. During centrifugation, the so-called buffy coat is formed between erythrocytes and plasma; a brownish boundary layer consisting of white blood cells (leukocytes) and platelets (thrombocytes). The buffy coat is freed of white blood cells for further use as (leukocyte-depleted) platelet concentrates.

What exactly is in there?

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.

Why not use synthetic media? Serum-free, xeno-free, chemically defined media unfortunately have a few key drawbacks that cause them to fail some cells and cell lines. First of all, practically every cell type needs its own special medium that is optimally adapted to the specific requirements of the cells. Finding or developing such a medium takes time.

In addition, the synthetic media available today do not perfectly support the initial isolation and expansion steps of adherent cells – unless one switches to specially coated cell culture vessels. And the coating, that makes the surface attractive for cell attachment, is most often made up of animal peptides and serum proteins again. Bye-bye defined and xeno-free conditions…

For which cells is hPL particularly suitable?

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.

  • Adipose-derived Stem Cells (ADSC)
  • Chondrocytes
  • Corneal Keratocytes
  • Dental Follicular Cells (DFSC)
  • Dental Pulp Stem Cells (DPSC)
  • Fibroblasts (Dermal Fibroblasts, Foreskin Fibroblasts)
  • Gamma-Delta-T-Cells
  • Head and Neck Squamous Cell Carcinoma (HNSCC)
  • Hematopoietic Stem Cells (HSC)
  • Lymphocytes from Blood
  • Macrophages/Monocytes
  • Mesenchymal Stem Cells from Adipose Tissue (MSC-AT), Bone Marrow (MSC-BM), Mononuclear Cells (MSC-MNC) and Umbilical Cord (MSC-UC)
  • Mesenchymal Stem Cells differentiated from iPSCs
  • Neural Crest Cells
  • Periodontal Ligaments Cells (PDL)
  • Stem Cells from Sweat Glands (SGSC)
  • Umbilical Vein Endothelial Cells (HUVEC)
  • Bone Osteosarcoma Cell Lines (HOS(TE85), U-2 OS)
  • Breast Cancer Cell Lines (MCF-7, BT-20, HBL100)
  • Cervical Cancer Cell Line (HeLa)
  • Chordoma cell lines
  • Colon Adenocarcinoma Cell Line (LS 180)
  • Colorectal Adenocarcinoma Cell Line (HROC24)
  • Dermal Keratinocytes (NCTC 2544, HaCaT)
  • Embryonal Lung Fibroblasts (MRC-5)
  • Embryonic Kidney Cell Line (HEK-293)
  • Epithelial Colorectal Adenocarcinoma Cell Line (Caco-2)
  • Epithelial Mammary Gland; Breast/Duct (ZR-75-1)
  • Fetal Foreskin Fibroblasts (HFFF2)
  • Gastric Carcinoma Cell Line (HGC-27)
  • Gingival Fibroblasts (HGF-1)
  • Glioblastoma Multiforme Cell Line (U-251 MG)
  • Hematopoietic Stem Cells (HSC)
  • Human Epithelial Cell Line Type 2 (HEp-2)
  • Leukemia Cell Lines (THP-1, HL-60, Jurkat, KG-1, Kasumi-1, K562)
  • Lung Adenocarcinoma Cell Line (A-549)
  • Lung Large Cell Carcinoma (LCC)
  • Lymphocytes (immortalized)
  • Melanoma Cell Line
  • MSCs containing catalytic subunit of telomerase (hMSC-TERT)
  • Pancreas Adenocarcinoma Cell Line (Panc-1)
  • Retinal Pigmented Epithelium (ARPE-19)
  • Renal Clear Cell Carcinoma Cell Line (RCC-ER)
  • Umbilical Vein Endothelial Cells (HUVEC)
  • Urinary Bladder Carcinoma Cell Line (5637)
  • Bovine Corneal Endothelial Cells (CEC)
  • Murine Astrocytes
  • Murine Mesenchymal Stem Cells
  • Murine Microglia
  • Rat Mesenchymal Stem Cells
  • Sprague-Dawley Rat Spiral Ganglions (SGC)
  • African Green Monkey Fibroblast (COS-7)
  • African Green Monkey Kidney (Vero)
  • Chinese Hamster Ovary Epithelial (CHO)
  • Mouse Adenocarcinoma (RAG)
  • Mouse Fibroblast (L929)
  • Mouse Mammary Tumor (MMT 060562)
  • Mouse Microglia (BV-2)
  • Mouse Myeloma (Sp2O-Ag14)
  • Mouse Neuroblastoma (Neuro-2a)
  • Rabbit Cornea, Statens Seruminstitut (SIRC)
  • Rat Adrenal gland (PC-12)
  • Rat Testis (R2C)

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.

Benefits over FBS

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.

Disadvantages?

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.
Fibrinogen-depleted human Platelet Lysate for cell culture applications

hPL from neoFroxx

  • 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
BezeichnungHeparin-Zugabe erforderlich?AnwendungsbereichUrsprungArtikelnummerDownloads
Humanes Plättchenlysat für die Zellbiologie, Fibrinogen-depleted & Gamma-irradiated, GMP gradeneinHergestellt nach GMP; für klinische Studien und Zelltherapie-AnwendungenUS2322TDA
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Humanes Plättchenlysat für die Zellbiologie, Fibrinogen-depleted, GMP gradeneinHergestellt nach GMP; für klinische Studien und Zelltherapie-AnwendungenUS2320TDA
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Humanes Plättchenlysat (US) Premiumqualität für die Zellbiologie, Fibrinogen-depletedneinForschungsanwendungen / prä-klinische StudienUS2319TDA
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Humanes Plättchenlysat (EU) Premiumqualität für die Zellbiologie, Fibrinogen-depletedneinForschungEU2515TDA
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Humanes Plättchenlysat Premiumqualität für die ZellbiologiejaForschungEU2304TDA
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What do I have to consider when switching from FBS to hPL?

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.

The topic is of particular interest to you and you would like to learn more? We recommend the following articles:

  • 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