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Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells

      Abstract

      Culture media for therapeutic cell preparations—such as mesenchymal stromal cells (MSCs)—usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL—such as “xenogen-free,” “animal-free” and “serum free”—are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations.

      Key Words

      Introduction

      Overall, the composition of cell culture media still closely resembles formulas developed in the pioneering work of the 1950s: Harry Eagle described a basal medium (Eagle's minimal essential medium), which comprised of a mixture of 29 essential components including 13 amino acids, nine vitamins, D-glucose and six inorganic salts (
      • Eagle H.
      Nutrition needs of mammalian cells in tissue culture.
      ). In the early days of cell culture, this basal medium was supplemented with human or horse serum to support the in vitro growth of human carcinoma cells or murine fibroblasts. To date, fetal bovine serum (FBS; alternatively termed fetal calf serum [FCS]) is the most commonly used serum additive that is capable of supporting growth of a variety of cell types.
      Over the past decades, mesenchymal stromal cells (MSCs) have received much attention for their potential role in regenerative medicine and cellular therapies (
      • Dominici M.
      • Le Blanc K.
      • Mueller I.
      • Slaper-Cortenbach I.
      • Marini F.
      • Krause D.
      • et al.
      Minimal criteria for defining multipotent mesenchymal stromal cells: the International Society for Cellular Therapy position statement.
      ,
      • Horwitz E.M.
      • Le B.K.
      • Dominici M.
      • Mueller I.
      • Slaper-Cortenbach I.
      • Marini F.C.
      • et al.
      Clarification of the nomenclature for MSC: the International Society for Cellular Therapy position statement.
      ). They can be easily culture-expanded, they harbor differentiation capacity toward mesodermal lineages and they reveal a variety of immunomodulatory features. The first clinical trial with the use of culture-expanded MSCs was performed in 1995 (
      • Lazarus H.M.
      • Haynesworth S.E.
      • Gerson S.L.
      • Rosenthal N.S.
      • Caplan A.I.
      Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use.
      ). In this study, MSCs were obtained from 23 patients who were then reinfused intravenously to demonstrate that these cells can be expanded in vitro and were then transplanted without toxicity (
      • Lazarus H.M.
      • Haynesworth S.E.
      • Gerson S.L.
      • Rosenthal N.S.
      • Caplan A.I.
      Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use.
      ). By 2013, the public clinical trials database (http://clinicaltrials.gov) registered 338 clinical trials that used MSCs for a wide range of therapeutic applications. MSCs can hardly be purified directly from tissue and therefore they are usually culture-expanded in vitro to attain sufficient cell numbers for clinical applications. Most of the protocols that have been reported in the literature use FBS-supplemented media to raise and expand human MSCs. However, since concerns have been raised regarding the safety of FBS-based culture media, protocols intended to raise cells for the clinical application should—according to Good Manufacturing Practice—avoid usage of animal sera.
      Culture conditions exert major impact on cells cultured in vitro. Because of the wide range of therapeutic applications of MSCs and to their worldwide use, we aimed to discuss the relevance of serum substitutes for MSC cultures. Accordingly, this article summarises limitations and improvements of culture media with particular emphasis on human platelet lysate (hPL) (Table I).
      Table IDifferent types of culture media.
      Type of culture mediaDefinition
      Xeno-free mediaAll components are either synthetic or derived from the same species corresponding to the species of cellular origin and/or recipient of the transplant. For application with human cells, particularly in clinical therapy, “xeno-free” means that it comprises exclusively human components and chemically defined substances. Addition of recombinant proteins and/or recombinant growth factors is often considered as acceptable for this definition. Human serum or hPL are examples for xenogen-free supplements.
      Serum-free mediaDoes not comprise any serum, either from animals or humans. Per definition, it may include recombinant growth factors and even animal components that are not serum-derived. In this context, hPL comprises plasma but not serum. However, definition of hPL to be “serum-free” might be misleading because plasma has a composition very similar to serum.
      Animal-free mediaMedia are completely devoid of any animal substances. Animal-free media may contain recombinant proteins. Please note that coating of culture dishes is not necessarily animal-free. Often, coating is performed with serum or serum proteins; therefore culture conditions are not “animal-free.” For hPL, the label “animal-free” may be misunderstood under the perception that humans are also mammals. However, many groups conventionally use this definition to discern culture condition with supplements derived from animals as opposed to humans.
      Animal-free culture conditionsCulture conditions are completely devoid of animal substances, including potential surface coating. They may comprise recombinant proteins, such as growth factors, which are sometimes difficult to standardize and to define.
      Fully defined synthetic culture media without growth factorsThese media comprise only pure synthetic substances with known activities. They do not rely on recombinant growth factors, which may comprise traces of other proteins or vary in activity. Only such media can be completely standardized.

      Fully defined synthetic culture media: an unmet goal

      Efforts have been made to replace serum supplements and to design more standardized and better-defined serum-free formulations. They must comprise all nutrients, amino acids, lipids hormones, vitamins, buffer substances and growth factors that are essential to maintain all physiological functions and to facilitate cellular proliferation. Over the past decade, various serum-free and animal-free media have been described (
      • Barnes D.
      • Sato G.
      Methods for growth of cultured cells in serum-free medium.
      ,
      • Bjare U.
      Serum-free cell culture.
      ,
      • Froud S.J.
      The development, benefits and disadvantages of serum-free media.
      ,
      • Gstraunthaler G.
      Alternatives to the use of fetal bovine serum: serum-free cell culture.
      ,
      • Jayme D.W.
      • Epstein D.A.
      • Conrad D.R.
      Fetal bovine serum alternatives.
      ,
      • Zimmerman A.M.
      • Vierck J.L.
      • O'Reilly B.A.
      • Dodson M.V.
      Formulation of a defined medium to maintain cell health and viability in vitro.
      ,
      • Chase L.G.
      • Yang S.
      • Zachar V.
      • Yang Z.
      • Lakshmipathy U.
      • Bradford J.
      • et al.
      Development and characterization of a clinically compliant xeno-free culture medium in good manufacturing practice for human multipotent mesenchymal stem cells.
      ,
      • Patrikoski M.
      • Juntunen M.
      • Boucher S.
      • Campbell A.
      • Vemuri M.C.
      • Mannerstrom B.
      • et al.
      Development of fully defined xeno-free culture system for the preparation and propagation of cell therapy-compliant human adipose stem cells.
      ). In fact, it has been suggested that specific serum-free culture media can be used for expansion of human MSCs (
      • Chase L.G.
      • Lakshmipathy U.
      • Solchaga L.A.
      • Rao M.S.
      • Vemuri M.C.
      A novel serum-free medium for the expansion of human mesenchymal stem cells.
      ). However, these media often failed to support the initial isolation and expansion steps: particularly on untreated culture flasks, the cell adhesion and initial outgrowth of fibroblastoid colony-forming units were largely impaired. Therefore, peptides and serum proteins have been used to coat the culture dishes upfront—at the expense of fully standardized and serum-free culture conditions. In general, precisely defined synthetic media provide a better controlled cell culture environment, but optimization of the concentration of individual compounds is a tedious and expensive task that is ongoing (
      • Barnes D.
      • Sato G.
      Serum-free cell culture: a unifying approach.
      ).

      FBS: the gold standard for cell culture

      To date, FBS is the most widely used serum supplement for in vitro culture of eukaryotic cells. It has been estimated that the annual worldwide production is approximately 500,000 L of raw FBS, which equates harvesting of more than one million bovine fetuses per year (
      • Hodgson J.
      To treat or not to treat: that is the question for serum.
      ,
      • Jochems C.E.
      • van der Valk J.B.
      • Stafleu F.R.
      • Baumans V.
      The use of fetal bovine serum: ethical or scientific problem?.
      ). Generally, FBS is produced from the blood drawn from a bovine fetus that is obtained from pregnant cows sent to slaughter. The fetus—usually at approximately 6 months of fetal development (
      • Tekkatte C.
      • Gunasingh G.P.
      • Cherian K.M.
      • Sankaranarayanan K.
      “Humanized” stem cell culture techniques: the animal serum controversy.
      )—is separated at the abattoir, and the fetal blood is collected under aseptic conditions. This is usually performed by puncturing of the heart. The blood is chilled, allowed to clot and serum is then separated from the fibrin-clotted mass and red blood cells by centrifugation (Figure 1A). Thereby, FBS can be produced in relatively large quantities, and large batches of pretested serum can be generated and distributed on a commercial basis.
      Figure thumbnail gr1
      Figure 1Generation of FBS and human platelet lysate. (A) FBS is produced from the blood of bovine fetuses during slaughter of pregnant cows. The blood is chilled, allowed to clot and serum is then separated from the fibrin-clotted mass and red blood cells by centrifugation and filtration (the calf scheme has been modified with kind permission from Dr William Dee Whittier, Production Management Medicine and Veterinary Extension, VA Tech, Blacksburg, VA, USA). (B) hPL is generated from common platelet units by means of a simple freeze-thaw procedure. Platelet units are aliquoted, twice frozen at −80°C, re-thawed at 37°C and centrifuged to remove cell fragments. The supernatant is then filtered and finally supplemented with heparin to avoid gel formation.
      Because of its relatively easy production and rich content of growth factors, FBS became the “most universally applicable cell culture additive for the stimulation of cell proliferation and biological production” (
      • Jayme D.W.
      • Epstein D.A.
      • Conrad D.R.
      Fetal bovine serum alternatives.
      ). Other advantages in the use of FBS in cell culture include the following (Table II): i) it is effective on most types of human and animal cells; ii) FBS is implemented in many existing culture protocols for MSCs (
      • Sundin M.
      • Ringden O.
      • Sundberg B.
      • Nava S.
      • Gotherstrom C.
      • Le B.K.
      No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients.
      ,
      • Horwitz E.M.
      • Gordon P.L.
      • Koo W.K.
      • Marx J.C.
      • Neel M.D.
      • McNall R.Y.
      • et al.
      Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone.
      ); iii) FBS is rich in fetal growth factors and hormones that stimulate cellular proliferation and maintenance (
      • Gospodarowicz D.
      • Ferrara N.
      • Schweigerer L.
      • Neufeld G.
      Structural characterization and biological functions of fibroblast growth factor.
      ,
      • Heldin C.H.
      • Betsholtz C.
      • Johnsson A.
      • Nister M.
      • Ek B.
      • Ronnstrand L.
      • et al.
      Platelet-derived growth factor: mechanism of action and relation to oncogenes.
      ,
      • Carpenter G.
      • Cohen S.
      Epidermal growth factor.
      ,
      • Cohen S.
      Epidermal growth factor.
      ); and iv) it facilitates differentiation toward various lineages. For example, it is traditionally supplemented in differentiation media for osteogenic, adipogenic and chondrogenic lineage. The advantageous effect of FBS in comparison to adult bovine serum might rely on the enormous regenerative needs during fetal development.
      Table IIAdvantages and disadvantages of FBS and hPL.
      FBShPL
      Advantages:
       Broadly applicable for many different cell typesApplicable for a wide range of different cell types. Thus far, it is particularly used for human MSCs, endothelial cells and fibroblasts.
       Rich in growth factorsEnriched in growth factors of the platelet fraction (such as platelet-derived growth factor).
       –Proliferation of MSCs—particularly of MSCs derived from adipose tissue—is significantly faster than in FBS.
       FBS is abundantly available (as a by-product during slaughters of pregnant cows)hPL is easily generated by freeze-thaw procedures (waste product after expiration date of platelet units).
       Commercially available (high lot-to-lot variation necessitates pretesting, facilitating more profit-yielding commercial advertisement).
       Clinical trials have been performed with relatively few side effects. However, human alternatives are preferable.Has been used in clinical trials—no critical side effects were reported.
       –No risk of xenogeneic immune reactions or transmission of bovine pathogens.
       –Can be used in autologous settings to reduce risks of contamination or immune reactions.
      Disadvantages:
       The ingredients are not precisely defined.Not precisely defined; yet, platelet units might be more standardized than bovine fetal blood.
       High lot-to-lot variation (even in pooled batches).Variation exists between individual hPLs, which can be reduced by pooling.
       –Thus far, hPL is rarely distributed commercially.
       Can evoke severe immunological reactions against xenogenic serum antigens.Immunological reactions are possible in allogeneic settings.
       High endotoxin content
       Potential source of microbial contaminants, such as fungi, bacteria, viruses or prions.Danger of transmission of human diseases by known or unknown viruses such as human immunodeficiency virus and human T-lymphotropic virus (quarantine storage cannot completely exclude this risk). Contamination with mycoplasma should be excluded.
       Animal welfare concerns during the bleeding procedure of bovine fetuses.
      On the other hand, FBS bears also a number of disadvantages (Table II): i) many compounds of FBS have not yet been identified, and, for many identified substances, the function of cultured cells is still unclear (
      • van der Valk J.
      • Mellor D.
      • Brands R.
      • Fischer R.
      • Gruber F.
      • Gstraunthaler G.
      • et al.
      The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture.
      ). ii) FBS batches reveal significant lot-to-lot variability; this makes pre-testing of each batch necessary (
      • Jochems C.E.
      • van der Valk J.B.
      • Stafleu F.R.
      • Baumans V.
      The use of fetal bovine serum: ethical or scientific problem?.
      ). iii) The high endotoxin content of FBS also raises questions regarding the suitability and safety (
      • Even M.S.
      • Sandusky C.B.
      • Barnard N.D.
      Serum-free hybridoma culture: ethical, scientific and safety considerations.
      ). iv) FBS may provoke immunological response against xenogenic serum antigens (
      • Horwitz E.M.
      • Gordon P.L.
      • Koo W.K.
      • Marx J.C.
      • Neel M.D.
      • McNall R.Y.
      • et al.
      Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone.
      ). It has been estimated that approximately 7–30 mg of bovine protein are transferred per 108 MSCs (
      • Spees J.L.
      • Gregory C.A.
      • Singh H.
      • Tucker H.A.
      • Peister A.
      • Lynch P.J.
      • et al.
      Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy.
      ). Spees et al. (
      • Spees J.L.
      • Gregory C.A.
      • Singh H.
      • Tucker H.A.
      • Peister A.
      • Lynch P.J.
      • et al.
      Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy.
      ) showed that FBS-expanded MSCs may evoke immune responses in patients even on first administration and particularly if repeated administrations were required. Several studies reported anaphylaxis and immune reactions in the patients transplanted with cells exposed to animal-derived products (
      • Selvaggi T.A.
      • Walker R.E.
      • Fleisher T.A.
      Development of antibodies to fetal calf serum with arthus-like reactions in human immunodeficiency virus-infected patients given syngeneic lymphocyte infusions.
      ,
      • Mackensen A.
      • Drager R.
      • Schlesier M.
      • Mertelsmann R.
      • Lindemann A.
      Presence of IgE antibodies to bovine serum albumin in a patient developing anaphylaxis after vaccination with human peptide-pulsed dendritic cells.
      ,
      • Tonti G.A.
      • Mannello F.
      From bone marrow to therapeutic applications: different behaviour and genetic/epigenetic stability during mesenchymal stem cell expansion in autologous and foetal bovine sera?.
      ). v) FBS is also a potential source of microbial contaminants, such as fungi, bacteria, viruses or prions (
      • Erickson G.A.
      • Bolin S.R.
      • Landgraf J.G.
      Viral contamination of fetal bovine serum used for tissue culture: risks and concerns.
      ). It has been estimated that 20–50% of commercially available FBS is virally contaminated, particularly with bovine viral diarrhea virus (
      • Even M.S.
      • Sandusky C.B.
      • Barnard N.D.
      Serum-free hybridoma culture: ethical, scientific and safety considerations.
      ). Even though bovine spongiform encephalopathy is currently not a common threat, the danger of xenogeneic infections—which may cross the species barrier—remains. Thus, expansion of MSCs in a clinical setting should avoid usage of any animal sera. Accordingly, if desired to be translated for a clinical setting, FBS-based protocols that have been successfully applied to raise MSCs for pre-clinical applications must be modified and re-evaluated, which might be very time-consuming.
      Apart from safety and scientific concerns, the methods adopted for harvesting blood from the fetal calf have raised animal welfare concerns (
      • Jochems C.E.
      • van der Valk J.B.
      • Stafleu F.R.
      • Baumans V.
      The use of fetal bovine serum: ethical or scientific problem?.
      ,
      • Mellor D.J.
      • Gregory N.G.
      Responsiveness, behavioural arousal and awareness in fetal and newborn lambs: experimental, practical and therapeutic implications.
      ). As mentioned above, fetuses probably are exposed to pain and discomfort, which makes the current practice of fetal blood harvest questionable (
      • Mellor D.J.
      • Gregory N.G.
      Responsiveness, behavioural arousal and awareness in fetal and newborn lambs: experimental, practical and therapeutic implications.
      ). Several measures should be taken to minimize nociception and suffering of the animals, such as ensuring that the calf is unconscious and desensitized during the cardiac puncture and blood collection, but these safeguards are not always warranted (
      • Tekkatte C.
      • Gunasingh G.P.
      • Cherian K.M.
      • Sankaranarayanan K.
      “Humanized” stem cell culture techniques: the animal serum controversy.
      ).
      Despite these disadvantages, FBS usage is still tolerated—at least in phase I clinical trials (
      • Halme D.G.
      • Kessler D.A.
      FDA regulation of stem-cell-based therapies.
      )—but its use must be highly regulated in regenerative medicine (
      • van der Valk J.
      • Mellor D.
      • Brands R.
      • Fischer R.
      • Gruber F.
      • Gstraunthaler G.
      • et al.
      The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture.
      ). For therapeutic applications, FBS derived from countries without any incidence of bovine spongiform encephalopathy is preferred. Veterinary control of animal-derived products largely follows the regulations set by the European Union (DG SANCO; EU 142/2011) and the United States Department of Agriculture (USDA). Thus, it is possible to use FBS for culture expansion of therapeutic cell preparations; however, human alternatives are preferable (
      • Jayme D.W.
      • Epstein D.A.
      • Conrad D.R.
      Fetal bovine serum alternatives.
      ,
      • Bieback K.
      • Hecker A.
      • Kocaomer A.
      • Lannert H.
      • Schallmoser K.
      • Strunk D.
      • et al.
      Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow.
      ,
      • Kocaoemer A.
      • Kern S.
      • Kluter H.
      • Bieback K.
      Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue.
      ).

      Human alternatives for FBS

      Over the past 15 years, various human alternatives have been tested for their ability to sustain proliferation and differentiation of cells in culture. The use of human serum (HS) may be the most straightforward “humanized” approach. Several studies demonstrated that the use of autologous HS for culture expansion of MSCs is feasible without compromising differentiation capacity or the MSC cell surface immunophenotype (
      • Stute N.
      • Holtz K.
      • Bubenheim M.
      • Lange C.
      • Blake F.
      • Zander A.R.
      Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use.
      ,
      • Koller M.R.
      • Maher R.J.
      • Manchel I.
      • Oxender M.
      • Smith A.K.
      Alternatives to animal sera for human bone marrow cell expansion: human serum and serum-free media.
      ,
      • Yamaguchi M.
      • Hirayama F.
      • Wakamoto S.
      • Fujihara M.
      • Murahashi H.
      • Sato N.
      • et al.
      Bone marrow stromal cells prepared using AB serum and bFGF for hematopoietic stem cells expansion.
      ). However, it has also been reported that this method is not always reliable, particularly in studies with allogeneic HS: proliferation of MSCs was often rather low, and the cells hardly reached confluence (
      • Spees J.L.
      • Gregory C.A.
      • Singh H.
      • Tucker H.A.
      • Peister A.
      • Lynch P.J.
      • et al.
      Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy.
      ,
      • Bieback K.
      • Hecker A.
      • Kocaomer A.
      • Lannert H.
      • Schallmoser K.
      • Strunk D.
      • et al.
      Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow.
      ). Platelet-rich plasma (PRP) has been shown to enhance proliferation of MSCs in culture (
      • Vogel J.P.
      • Szalay K.
      • Geiger F.
      • Kramer M.
      • Richter W.
      • Kasten P.
      Platelet-rich plasma improves expansion of human mesenchymal stem cells and retains differentiation capacity and in vivo bone formation in calcium phosphate ceramics.
      ). However, the debris in PRP may disturb cell culture, and not all growth factors are released without thrombocyte activation. Bieback et al. (
      • Kocaoemer A.
      • Kern S.
      • Kluter H.
      • Bieback K.
      Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue.
      ,
      • Nesselmann C.
      • Ma N.
      • Bieback K.
      • Wagner W.
      • Ho A.
      • Konttinen Y.T.
      • et al.
      Mesenchymal stem cells and cardiac repair.
      ) demonstrated the use of thrombin-activated platelet-rich plasma (tPRP) for isolating and expanding human MSCs. They demonstrated that pooled human HS and tPRP provide a significantly higher proliferative effect on adipose tissue–derived MSCs than FBS: in early passages, HS and tPRP MSCs showed a 66-fold and 68-fold expansion, respectively, whereas expansion in FCS was only 24-fold. For generation of tPRP, platelets were activated by human thrombin (
      • Kocaoemer A.
      • Kern S.
      • Kluter H.
      • Bieback K.
      Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue.
      ). The fibrin clot can be removed subsequently by means of centrifugation (
      • Tekkatte C.
      • Gunasingh G.P.
      • Cherian K.M.
      • Sankaranarayanan K.
      “Humanized” stem cell culture techniques: the animal serum controversy.
      ). tPRP contains several growth factors derived from platelets that have been shown to enhance proliferation of MSCs (
      • Kocaoemer A.
      • Kern S.
      • Kluter H.
      • Bieback K.
      Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue.
      ,
      • Marx R.E.
      Platelet-rich plasma: evidence to support its use.
      ,
      • Müller I.
      • Kordowich S.
      • Holzwarth C.
      • Spano C.
      • Isensee G.
      • Staiber A.
      • et al.
      Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM.
      ,
      • Ng F.
      • Boucher S.
      • Koh S.
      • Sastry K.S.
      • Chase L.
      • Lakshmipathy U.
      • et al.
      PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages.
      ). Furthermore, thrombin-cleaved osteopontin promotes cell attachment and spreading (
      • Senger D.R.
      • Perruzzi C.A.
      • Papadopoulos-Sergiou A.
      • Van de W.L.
      Adhesive properties of osteopontin: regulation by a naturally occurring thrombin-cleavage in close proximity to the GRGDS cell-binding domain.
      ). It should be noted that several groups freeze their PRP before addition to culture medium, which then closely resembles human platelet lysate (hPL), and thus the nomenclature should be used accordingly.

      Generation of hPL

      The use of hPL for MSC expansion was first described by Doucet et al. (
      • Doucet C.
      • Ernou I.
      • Zhang Y.
      • Llense J.R.
      • Begot L.
      • Holy X.
      • et al.
      Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications.
      ) in 2005. Since then, hPL has been proven as an extremely effective cell culture additive that is concurrently used in many laboratories and clinical trials (
      • von Bonin M.
      • Stolzel F.
      • Goedecke A.
      • Richter K.
      • Wuschek N.
      • Holig K.
      • et al.
      Treatment of refractory acute GVHD with third-party MSC expanded in platelet lysate-containing medium.
      ). hPL is prepared from PRP, either derived from pooled buffy coat–derived platelet concentrates of whole blood or from apheresis (
      • Rauch C.
      • Feifel E.
      • Amann E.M.
      • Spotl H.P.
      • Schennach H.
      • Pfaller W.
      • et al.
      Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media.
      ,
      • Schallmoser K.
      • Bartmann C.
      • Rohde E.
      • Reinisch A.
      • Kashofer K.
      • Stadelmeyer E.
      • et al.
      Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.
      ,
      • Horn P.
      • Bokermann G.
      • Cholewa D.
      • Bork S.
      • Walenda T.
      • Koch C.
      • et al.
      Impact of individual platelet lysates on isolation and growth of human mesenchymal stromal cells.
      ). In contrast to tPRP, which requires a more complicated manufacturing process, hPL can be generated from common platelet units by a simple freeze-thawing procedure (Figure 1B). This procedure is very simple, fast and effective. Variation between individual platelets can be reduced by pooling of platelet units. Within one single bag, usually a pool of five harvests from fresh blood are combined unless apheresis from single donors has been performed. hPL is often used at a concentration of 10%. A single platelet unit of approximately 250 mL would consequently facilitate generation of 2.5 L of culture medium. Protocols have been developed to generate large pools of hPL to balance the lot-to-lot variation (
      • Schallmoser K.
      • Bartmann C.
      • Rohde E.
      • Reinisch A.
      • Kashofer K.
      • Stadelmeyer E.
      • et al.
      Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.
      ,
      • Schallmoser K.
      • Strunk D.
      Preparation of pooled human platelet lysate (pHPL) as an efficient supplement for animal serum-free human stem cell cultures.
      ,
      • Schallmoser K.
      • Strunk D.
      Generation of a pool of human platelet lysate and efficient use in cell culture.
      ). For example, pooling of more than 50 units counteracts variation between individual hPLs. Furthermore, it provides larger volumes for generation of homogeneous culture medium. Alternatively, it is possible to use autologous hPL to minimize the risk of immunological reactions or infections (
      • Horn P.
      • Bokermann G.
      • Cholewa D.
      • Bork S.
      • Walenda T.
      • Koch C.
      • et al.
      Impact of individual platelet lysates on isolation and growth of human mesenchymal stromal cells.
      ). hPL can be produced according to Good Manufacturing Practice procedures and permits scale-up production of MSCs for clinical applications (
      • Müller I.
      • Kordowich S.
      • Holzwarth C.
      • Spano C.
      • Isensee G.
      • Staiber A.
      • et al.
      Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM.
      ,
      • Doucet C.
      • Ernou I.
      • Zhang Y.
      • Llense J.R.
      • Begot L.
      • Holy X.
      • et al.
      Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications.
      ,
      • Schallmoser K.
      • Bartmann C.
      • Rohde E.
      • Reinisch A.
      • Kashofer K.
      • Stadelmeyer E.
      • et al.
      Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.
      ,
      • Capelli C.
      • Domenghini M.
      • Borleri G.
      • Bellavita P.
      • Poma R.
      • Carobbio A.
      • et al.
      Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts.
      ,
      • Holzwarth C.
      • Vaegler M.
      • Gieseke F.
      • Pfister S.M.
      • Handgretinger R.
      • Kerst G.
      • et al.
      Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells.
      ,
      • Lange C.
      • Cakiroglu F.
      • Spiess A.N.
      • Cappallo-Obermann H.
      • Dierlamm J.
      • Zander A.R.
      Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine.
      ). Furthermore, hPL can be used for isolation of a broad range of different cell types such as human endothelial cells (
      • Denecke B.
      • Horsch L.D.
      • Radtke S.
      • Fischer J.C.
      • Horn P.A.
      • Giebel B.
      Human endothelial colony-forming cells expanded with an improved protocol are a useful endothelial cell source for scaffold-based tissue engineering.
      ,
      • Kilian O.
      • Flesch I.
      • Wenisch S.
      • Taborski B.
      • Jork A.
      • Schnettler R.
      • et al.
      Effects of platelet growth factors on human mesenchymal stem cells and human endothelial cells in vitro.
      ), human fibroblasts (
      • Mirabet V.
      • Solves P.
      • Minana M.D.
      • Encabo A.
      • Carbonell-Uberos F.
      • Blanquer A.
      • et al.
      Human platelet lysate enhances the proliferative activity of cultured human fibroblast-like cells from different tissues.
      ) and MSCs from various tissues (
      • Doucet C.
      • Ernou I.
      • Zhang Y.
      • Llense J.R.
      • Begot L.
      • Holy X.
      • et al.
      Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications.
      ,
      • Schallmoser K.
      • Bartmann C.
      • Rohde E.
      • Reinisch A.
      • Kashofer K.
      • Stadelmeyer E.
      • et al.
      Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.
      ,
      • Lange C.
      • Cakiroglu F.
      • Spiess A.N.
      • Cappallo-Obermann H.
      • Dierlamm J.
      • Zander A.R.
      Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine.
      ,
      • Bernardo M.E.
      • Avanzini M.A.
      • Perotti C.
      • Cometa A.M.
      • Moretta A.
      • Lenta E.
      • et al.
      Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.
      ).

      Pros and cons of hPL

      Human platelet lysate has several advantages and disadvantages in comparison to FBS (Table II): i) many studies indicated that hPL supports culture expansion of MSCs even better than FBS, HS, PRP or tPRP (
      • Bieback K.
      • Hecker A.
      • Kocaomer A.
      • Lannert H.
      • Schallmoser K.
      • Strunk D.
      • et al.
      Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow.
      ,
      • Müller I.
      • Kordowich S.
      • Holzwarth C.
      • Spano C.
      • Isensee G.
      • Staiber A.
      • et al.
      Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM.
      ,
      • Horn P.
      • Bokermann G.
      • Cholewa D.
      • Bork S.
      • Walenda T.
      • Koch C.
      • et al.
      Impact of individual platelet lysates on isolation and growth of human mesenchymal stromal cells.
      ,
      • Lange C.
      • Cakiroglu F.
      • Spiess A.N.
      • Cappallo-Obermann H.
      • Dierlamm J.
      • Zander A.R.
      Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine.
      ). Particularly, proliferation of adipose tissue–derived MSCs is largely increased with hPL (
      • Lohmann M.
      • Walenda G.
      • Hemeda H.
      • Joussen S.
      • Drescher W.
      • Jockenhoevel S.
      • et al.
      Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.
      ,
      • Cholewa D.
      • Stiehl T.
      • Schellenberg A.
      • Bokermann G.
      • Joussen S.
      • Koch C.
      • et al.
      Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density.
      ,
      • Walenda G.
      • Hemeda H.
      • Schneider R.K.
      • Merkel R.
      • Hoffmann B.
      • Wagner W.
      Human platelet lysate gel provides a novel 3D-matrix for enhanced culture expansion of mesenchymal stromal cells.
      ). This growth advantage was less pronounced with the use of bone marrow–derived MSCs, which indicates that these cell preparations may differ in their nutritional requirements. ii) Platelet units can be used after the maximal shelf life time of 5 days. Thereafter, because of the increased risk of platelet aggregation and particularly because of the increased risk of bacterial contamination, they should not be considered for platelet transfusion; however, the waste product is still suitable for generation of hPL (
      • Chan R.K.
      • Liu P.
      • Lew D.H.
      • Ibrahim S.I.
      • Srey R.
      • Valeri C.R.
      • et al.
      Expired liquid preserved platelet releasates retain proliferative activity.
      ). iii) hPL has been used for therapeutic applications, and no severe side effects were observed (
      • Sanchez A.R.
      • Sheridan P.J.
      • Kupp L.I.
      Is platelet-rich plasma the perfect enhancement factor? A current review.
      ,
      • Dugrillon A.
      • Eichler H.
      • Kern S.
      • Kluter H.
      Autologous concentrated platelet-rich plasma (cPRP) for local application in bone regeneration.
      ). iv) MSCs expanded in the presence of platelet-released growth factors retain their immunosuppressive properties (
      • Doucet C.
      • Ernou I.
      • Zhang Y.
      • Llense J.R.
      • Begot L.
      • Holy X.
      • et al.
      Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications.
      ). v) Because hPL is derived from humans, neither bovine viruses nor immune reactions against bovine proteins are a concern (
      • Doucet C.
      • Ernou I.
      • Zhang Y.
      • Llense J.R.
      • Begot L.
      • Holy X.
      • et al.
      Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications.
      ,
      • Johansson L.
      • Klinth J.
      • Holmqvist O.
      • Ohlson S.
      Platelet lysate: a replacement for fetal bovine serum in animal cell culture?.
      ). vi) hPL can be derived from the patient's blood without immunological problems or risk of infection with human diseases.
      Despite the above-mentioned advantages, hPL also has some limitations (Table II): i) hPL—just as FBS—is not precisely defined. Several factors contribute to variation in hPLs derived from individual platelet units (Figure 2) (
      • Horn P.
      • Bokermann G.
      • Cholewa D.
      • Bork S.
      • Walenda T.
      • Koch C.
      • et al.
      Impact of individual platelet lysates on isolation and growth of human mesenchymal stromal cells.
      ,
      • Lohmann M.
      • Walenda G.
      • Hemeda H.
      • Joussen S.
      • Drescher W.
      • Jockenhoevel S.
      • et al.
      Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.
      ). ii) To date, hPL is rarely distributed commercially. The ease of hPL generation and the high profit production of FBS make it less attractive for companies to commercialize platelet lysate. Furthermore, the use of human-derived products raises ethical constrains that must be considered. iii) Immunological reactions are less likely with the use of hPL than with FBS, but they may still occur, particularly in an allogeneic setting. iv) hPL may transmit human diseases such human immunodeficiency virus, hepatitis B and C, syphilis, human T-lymphotropic virus and cytomegalovirus. All blood products are routinely tested for these diseases, but this does not exclude the risk of infections. To further minimize the risk, it has been recommended to quarantine stored hPL and reanalyze the donor after 3 months for potential serum conversions (
      • Rauch C.
      • Feifel E.
      • Amann E.M.
      • Spotl H.P.
      • Schennach H.
      • Pfaller W.
      • et al.
      Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media.
      ,
      • Fekete N.
      • Gadelorge M.
      • Furst D.
      • Maurer C.
      • Dausend J.
      • Fleury-Cappellesso S.
      • et al.
      Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components.
      ). However, such testing and storage is hardly feasible in daily research routine. Furthermore, hPL might harbor mycoplasma contamination, which is not routinely tested but must be excluded for cell culture (authors' observation).
      Figure thumbnail gr2
      Figure 2Relevant parameters for the activity of human platelet lysates in MSC cultures.

      What are the relevant ingredients of hPL?

      Activated platelets are known to deliver a broad spectrum of cytokines involved in tissue repair (
      • Kaplan D.R.
      • Chao F.C.
      • Stiles C.D.
      • Antoniades H.N.
      • Scher C.D.
      Platelet alpha granules contain a growth factor for fibroblasts.
      ,
      • Ledent E.
      • Wasteson A.
      • Berlin G.
      Growth factor release during preparation and storage of platelet concentrates.
      ) such as epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor 1, platelet-derived growth factor, platelet factor 4, transforming growth factor-β and vascular endothelial growth factor (
      • Doucet C.
      • Ernou I.
      • Zhang Y.
      • Llense J.R.
      • Begot L.
      • Holy X.
      • et al.
      Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications.
      ,
      • Horn P.
      • Bokermann G.
      • Cholewa D.
      • Bork S.
      • Walenda T.
      • Koch C.
      • et al.
      Impact of individual platelet lysates on isolation and growth of human mesenchymal stromal cells.
      ,
      • Lohmann M.
      • Walenda G.
      • Hemeda H.
      • Joussen S.
      • Drescher W.
      • Jockenhoevel S.
      • et al.
      Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.
      ,
      • King S.M.
      • Reed G.L.
      Development of platelet secretory granules.
      ,
      • Reed G.L.
      • Fitzgerald M.L.
      • Polgar J.
      Molecular mechanisms of platelet exocytosis: insights into the “secrete” life of thrombocytes.
      ). Such growth factors have been shown to enhance the MSC proliferation rate and to maintain their multilineage differentiation potential under in vitro conditions in the absence of FBS and exogenous growth factors (
      • Rauch C.
      • Feifel E.
      • Amann E.M.
      • Spotl H.P.
      • Schennach H.
      • Pfaller W.
      • et al.
      Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media.
      ,
      • Marx R.E.
      • Carlson E.R.
      • Eichstaedt R.M.
      • Schimmele S.R.
      • Strauss J.E.
      • Georgeff K.R.
      Platelet-rich plasma: growth factor enhancement for bone grafts.
      ,
      • Anitua E.
      • Andia I.
      • Ardanza B.
      • Nurden P.
      • Nurden A.T.
      Autologous platelets as a source of proteins for healing and tissue regeneration.
      ,
      • Coppinger J.A.
      • Cagney G.
      • Toomey S.
      • Kislinger T.
      • Belton O.
      • McRedmond J.P.
      • et al.
      Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions.
      ,
      • Walenda G.
      • Abnaof K.
      • Joussen S.
      • Meurer S.
      • Smeets H.
      • Rath B.
      • et al.
      TGF-beta1 does not induce senescence of multipotent mesenchymal stromal cells and has similar effects in early and late passages.
      ). In addition, the impact of hPL on MSC osteoblastic differentiation is supported by numerous growth factors, which include bone morphogenic proteins 2, 4 and 6, interleukin 1, osteonectin, platelet-derived endothelial growth factor, platelet factor 4, transforming growth factor-β, insulin-like growth factor 1, basic fibroblast growth factor and platelet-derived growth factor, some of which are known to mediate osteoinductive effects (
      • Sipe J.B.
      • Zhang J.
      • Waits C.
      • Skikne B.
      • Garimella R.
      • Anderson H.C.
      Localization of bone morphogenetic proteins (BMPs)-2, -4, and -6 within megakaryocytes and platelets.
      ,
      • van den D.J.
      • Mooren R.
      • Vloon A.P.
      • Stoelinga P.J.
      • Jansen J.A.
      Platelet-rich plasma: quantification of growth factor levels and the effect on growth and differentiation of rat bone marrow cells.
      ,
      • Koch H.
      • Jadlowiec J.A.
      • Campbell P.G.
      Insulin-like growth factor-I induces early osteoblast gene expression in human mesenchymal stem cells.
      ). Without question, growth factors are essential, but they may not be the only relevant compounds in hPL which attribute to growth stimulation.

      New research perspectives: microRNAs and microvesicles in hPL

      MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate protein expression by degrading messenger RNA or repressing messenger RNA translation (
      • Bissels U.
      • Bosio A.
      • Wagner W.
      MicroRNAs are shaping the hematopoietic landscape.
      ). They have been shown to modulate MSC differentiation (
      • Bork S.
      • Horn P.
      • Castoldi M.
      • Hellwig I.
      • Ho A.D.
      • Wagner W.
      Adipogenic differentiation of human mesenchymal stromal cells is down-regulated by microRNA-369-5p and up-regulated by microRNA-371.
      ) and may contribute to regulation of the bone marrow niche (
      • Laine S.K.
      • Hentunen T.
      • Laitala-Leinonen T.
      Do microRNAs regulate bone marrow stem cell niche physiology?.
      ). Thus far, miRNA content has rarely been addressed in hPL, but it may be relevant for MSC cultures. Apparently, miRNAs are trafficed at least by two types of extracellular vesicles (EVs), exosomes and microvesicles (MVs), which are formed during platelet activation or secreted into the plasma by other cell types (
      • Heijnen H.F.
      • Schiel A.E.
      • Fijnheer R.
      • Geuze H.J.
      • Sixma J.J.
      Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules.
      ). Exosomes are rather small vesicles (70–120 nm) of endosomal origin that correspond to the intraluminal vesicles of multivesicular bodies (MVBs), which, on fusion of MVBs with the plasma membrane, are released as exosomes into the environment (
      • Johnstone R.M.
      • Adam M.
      • Hammond J.R.
      • Orr L.
      • Turbide C.
      Vesicle formation during reticulocyte maturation: association of plasma membrane activities with released vesicles (exosomes).
      ). Carrying a variety of different molecules, including miRNAs, exosomes appear as extracellular organelles involved in intercellular communication processes (
      • Ludwig A.K.
      • Giebel B.
      Exosomes: small vesicles participating in intercellular communication.
      ,
      • Schorey J.S.
      • Bhatnagar S.
      Exosome function: from tumor immunology to pathogen biology.
      ). In contrast, MVs are larger EVs—ranging from 100–1000 nm—which are shed from the plasma membrane. Even though exosomes and MVs can hardly be discriminated experimentally, MVs are believed to contain a different molecular composition than that of exosomes. However, without consideration of whether they are predominantly exosomes or MVs, platelet-derived EVs have been shown to participate in agglutination. EV fractions are enriched in coagulation factors and have been reported to contain a 50–100-fold higher pro-coagulant activity than do activated platelets (
      • Sinauridze E.I.
      • Kireev D.A.
      • Popenko N.Y.
      • Pichugin A.V.
      • Panteleev M.A.
      • Krymskaya O.V.
      • et al.
      Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets.
      ). Depending on the platelet unit and processing, different amounts of EVs appear to be released in stored platelet concentrates (
      • Pienimaeki-Roemer A.
      • Ruebsaamen K.
      • Boettcher A.
      • Orso E.
      • Scherer M.
      • Liebisch G.
      • et al.
      Stored platelets alter glycerophospholipid and sphingolipid species, which are differentially transferred to newly released extracellular vesicles.
      ). Further research will be required to better understand the role of miRNAs, exosomes and MVs for MSC growth, which may explain the above-mentioned limitations of synthetic culture conditions.

      Cellular aging of MSCs and hPL

      MSCs undergo replicative senescence during culture expansion (
      • Wagner W.
      • Bork S.
      • Lepperdinger G.
      • Joussen S.
      • Ma N.
      • Strunk D.
      • et al.
      How to track cellular aging of mesenchymal stromal cells?.
      ,
      • Wagner W.
      • Ho A.D.
      • Zenke M.
      Different facets of aging in human mesenchymal stem cells.
      ), and their long-term growth curves are affected by serum supplements: in particular, adipose tissue–derived MSCs reach a higher number of cumulative population doublings with hPL in comparison to FBS. However, because of the faster proliferation rate, they enter the senescent state after fewer days in the presence of hPL than in the presence of FBS (
      • Cholewa D.
      • Stiehl T.
      • Schellenberg A.
      • Bokermann G.
      • Joussen S.
      • Koch C.
      • et al.
      Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density.
      ). It has been speculated that the enormously enhanced growth rates in hPL might favor chromosomal aberrations, but this effect has not been proven thus far (
      • Schellenberg A.
      • Lin Q.
      • Schuler H.
      • Koch C.M.
      • Joussen S.
      • Denecke B.
      • et al.
      Replicative senescence of mesenchymal stem cells causes DNA-methylation changes which correlate with repressive histone marks.
      ). Long-term culture of MSCs in media supplemented either with FBS or pooled hPL induce similar gene expression (
      • Schallmoser K.
      • Bartmann C.
      • Rohde E.
      • Bork S.
      • Guelly C.
      • Obenauf A.C.
      • et al.
      Replicative senescence-associated gene expression changes in mesenchymal stromal cells are similar under different culture conditions.
      ) and DNA methylation changes (
      • Koch C.M.
      • Joussen S.
      • Schellenberg A.
      • Lin Q.
      • Zenke M.
      • Wagner W.
      Monitoring of cellular senescence by DNA-methylation at specific CpG sites.
      ) in expanded cells. Interestingly, the stimulatory effect of hPL was found to be dependent on the donor age of the platelet unit: we have demonstrated that hPLs generated from platelet units derived from younger donors stimulate proliferation and enhance osteogenic differentiation of MSCs more than do hPLs derived from elderly donors (
      • Lohmann M.
      • Walenda G.
      • Hemeda H.
      • Joussen S.
      • Drescher W.
      • Jockenhoevel S.
      • et al.
      Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.
      ). The nature of these age-associated factors is unclear. Yet, the same effect may also contribute to the above-mentioned predominance of FBS in comparison to serum from adult cows (
      • Kruman I.I.
      • Miakisheva S.N.
      • Gavriliuk B.K.
      Age-related changes in the growth-stimulating effect of serum on BHK-21 cells.
      ).

      Heparin concentration is critical

      In contrast to FBS, hPL comprises plasma with fibrinogen and all other clotting factors. The anticoagulant effect of citrate is reduced on addition to calcium-containing culture medium. Therefore, further additives, such as heparin, must be added to prevent gelatinization. Heparins are highly sulfated glycosaminoglycans that activate anti-thrombin III, an inhibitor of several enzymes of the coagulation cascade. They are subdivided into unfractionated heparins (3000–30,000 Da) and low-molecular-weight heparins (2000–12,000 Da) featuring different clinical effects, pharmacokinetics and pharmacodynamics (
      • Andersen J.C.
      Advances in anticoagulation therapy: the role of selective inhibitors of factor Xa and thrombin in thromboprophylaxis after major orthopedic surgery.
      ). We have recently demonstrated that unfractionated heparins and low-molecular-weight heparins can be used as additive in hPL media. However, their concentration was found to be critical (
      • Hemeda H.
      • Kalz J.
      • Walenda G.
      • Lohmann M.
      • Wagner W.
      Heparin concentration is critical for cell culture with human platelet lysate.
      ): expansion and differentiation of MSCs was significantly impaired with the use of heparins at high concentrations (
      • Hemeda H.
      • Kalz J.
      • Walenda G.
      • Lohmann M.
      • Wagner W.
      Heparin concentration is critical for cell culture with human platelet lysate.
      ). This is of particular relevance for cellular therapies that require optimized and standardized cell culture procedures.

      hPL can provide culture medium and biomaterial

      Traditionally, cell culture of adherent cells is performed on tissue culture plastic. On these culture dishes—which usually resemble biofunctionalized polystyrene surfaces—growth of MSCs is restricted to two dimensions and occurs preferentially at the rim of colonies as the result of contact inhibition (
      • Cholewa D.
      • Stiehl T.
      • Schellenberg A.
      • Bokermann G.
      • Joussen S.
      • Koch C.
      • et al.
      Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density.
      ). There is a growing perception that cell cultures can be improved by suitable three-dimensional scaffolds. Hydrogels and fibrous scaffolds facilitate cell growth without direct contract with rigid biomaterials (
      • Toh W.S.
      • Lim T.C.
      • Kurisawa M.
      • Spector M.
      Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment.
      ,
      • Leisten I.
      • Kramann R.
      • Ventura Ferreira M.S.
      • Bovi M.
      • Neuss S.
      • Ziegler P.
      • et al.
      3D co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds as a model of the hematopoietic niche.
      ). In particular, fibrin gels raise high hopes for therapeutic application because they are naturally occurring, biocompatible and biodegradable. Furthermore, they have been shown to support proliferation of various cell types (
      • Jockenhoevel S.
      • Zund G.
      • Hoerstrup S.P.
      • Chalabi K.
      • Sachweh J.S.
      • Demircan L.
      • et al.
      Fibrin gel: advantages of a new scaffold in cardiovascular tissue engineering.
      ,
      • Bensaid W.
      • Triffitt J.T.
      • Blanchat C.
      • Oudina K.
      • Sedel L.
      • Petite H.
      A biodegradable fibrin scaffold for mesenchymal stem cell transplantation.
      ). Without adding anticoagulants, hPL-supplemented media form soft and translucent gels within 1 hour. These hPL gels provide a suitable matrix to largely increase culture expansion of MSCs (
      • Walenda G.
      • Hemeda H.
      • Schneider R.K.
      • Merkel R.
      • Hoffmann B.
      • Wagner W.
      Human platelet lysate gel provides a novel 3D-matrix for enhanced culture expansion of mesenchymal stromal cells.
      ). In addition, the very soft and viscous mechanical consistency of hPL gel allows a simple passaging procedure without the need of separating cells from their matrix or additional washing steps (Figure 3). Expansion rates on passaging with the use of this method were similar as compared with the conventional method on tissue culture plastic with trypsin. In our previous work, cell recovery rates after passaging with the use of the pipetting method were very high, and we did not observe obvious impact on cellular activity and viability; however, this aspect should be further analyzed and optimized in future studies. Cell culture with hPL gel provides various new perspectives: it supports formation of MSC colonies and increases their proliferation rate. Furthermore, higher cell densities can be obtained and passaging procedures for culture expansion of MSCs can be performed non-enzymatically (
      • Walenda G.
      • Hemeda H.
      • Schneider R.K.
      • Merkel R.
      • Hoffmann B.
      • Wagner W.
      Human platelet lysate gel provides a novel 3D-matrix for enhanced culture expansion of mesenchymal stromal cells.
      ). It is even possible to directly apply cells embedded in hPL gel to further optimize cellular integration in a three-dimensional context; yet, proof of concept must be demonstrated in a clinical setting.
      Figure thumbnail gr3
      Figure 3Culture expansion and passaging of MSCs in hPL gel. Scheme of culture expansion on tissue culture plastic (TCP) or gelatinized medium-hPL mixture (hPL-gel): MSCs on TCP are conventionally passaged after reaching semi-confluence with trypsin-ethylenediaminetetraacetic acid. On hPL gel, the cells reveal less contact inhibition and can be harvested together with the gel by pipetting without separation of MSCs from their matrix
      (
      • Walenda G.
      • Hemeda H.
      • Schneider R.K.
      • Merkel R.
      • Hoffmann B.
      • Wagner W.
      Human platelet lysate gel provides a novel 3D-matrix for enhanced culture expansion of mesenchymal stromal cells.
      )
      .

      Conclusions

      The right choice of serum or serum replacement is crucial for cell culture. Each type of culture supplement has advantages and disadvantages, and they convey severe effects on function and composition of cell preparations. hPL-cultured cells have been used in the clinic; for example, they have been used for treatment of graft-versus-host disease (NCT01764100) and for lumbar intervertebral degenerative disc disease (NCT01513694). Thus far, no critical side effects have been reported on treatment with such cell preparations. However, the relevant molecular factors are not fully unraveled. Batch-to-batch variation can be minimized by generation of large pools of approximately 40–50 donations per batch (
      • Bieback K.
      • Hecker A.
      • Kocaomer A.
      • Lannert H.
      • Schallmoser K.
      • Strunk D.
      • et al.
      Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow.
      ,
      • Schallmoser K.
      • Bartmann C.
      • Rohde E.
      • Reinisch A.
      • Kashofer K.
      • Stadelmeyer E.
      • et al.
      Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.
      ,
      • Schallmoser K.
      • Rohde E.
      • Reinisch A.
      • Bartmann C.
      • Thaler D.
      • Drexler C.
      • et al.
      Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum.
      ); yet, this also increases the risk of infection with human viruses. Ongoing research is required to understand the interplay of growth factors, metabolites and possibly other constituents such as miRNAs or EVs. This will open new perspectives that seem to be required for further development of fully defined synthetic culture media.

      Acknowledgments

      This work was supported by the German Research Foundation (WA 1706/2-1 and WA 1706/3-2) and was co-founded by the European Union, the State North Rhine Westphalia within the BioNRW2 program (StemCellFactory) and the Stem Cell Network NRW.
      Disclosure of interests: RWTH Aachen has applied for a patent for the method of hPL gel (2011; EP 11171595.9–2401). The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.

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