Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells

The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (<35 years) as compared to older donors (>45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.     

Impact of individual platelet lysates on isolation and growth of human mesenchymal stromal cells

Background aimsCulture medium for mesenchymal stromal cells (MSC) is frequently supplemented with fetal calf serum (FCS). FCS can induce xenogeneic immune reactions, transmit bovine pathogens and has a high lot-to-lot variability that hampers reproducibility of results. Several studies have demonstrated that pooled human platelet lysate (HPL) provides an attractive alternative for FCS. However, little is known about the variation between different platelet lysates.MethodsWe compared activities of individual HPL on initial fibroblastoid colony-forming units (CFU-F), proliferation, in vitro differentiation and long-term culture. These data were correlated with chemokine profiles of HPL.ResultsIsolation of MSC with either HPL or FCS resulted in similar CFU-F frequency, colony morphology, immunophenotype and adipogenic differentiation potential. Osteogenic differentiation was even more pronounced in HPL than FCS. There were significant differences in MSC proliferation with different HPL, but it was always higher in comparison with FCS. Cell growth correlated with the concentration of platelet-derived growth factor (PDGF) and there was a moderate association with platelet counts. All HPL facilitated expansion for more than 20 population doublings.ConclusionsTaken together, reliable long-term expansion was possible with all HPL, although there was some variation in platelet lysates of individual units. Therefore the use of donor recipient-matched or autologous HPL is feasible for therapeutic MSC products.     

Preparation method and growth factor content of platelet concentrate influence the osteogenic differentiation of bone marrow stromal cells

Background aimsAn extensive debate about the clinical benefits of autologous platelet concentrates used as a treatment option for patients with orthopedic injuries is ongoing. The aim of this study was to determine whether different compositions of platelet concentrates may affect the osteogenic differentiation of bone marrow stromal cells (BMSC).MethodsPure platelet-rich plasma (P-PRP) and leukocyte-PRP (L-PRP) were characterized for platelet and leukocyte content. As an indicative marker of the delivery of growth factors (GFs), the release of basic fibroblast growth factor (bFGF) from platelet gel (PG) was measured at 1, 18, 48 and 72 h and at 7 d. The ability of different PGs to induce proliferation and differentiation of BMSC was evaluated by using bioactivity assays.ResultsThe platelet recovery was significantly higher in L-PRP, either fresh or frozen. PGs derived from L-PRP and P-PRP showed significant differences in terms of bFGF release and biological activity. bFGF release was faster both in fresh and frozen L-PRP preparations. Moreover, L-PRP samples were able to induce a significantly higher proliferation of BMSC compared with P-PRP or PPP samples. Even though all PG preparations allowed the deposition of mineral nodules in BMSC cultures, the mineralization activity correlated significantly with bFGF levels.ConclusionsThe biological activity of platelet concentrates differs according to preparation technique, which affects platelet and leukocyte content and GF availability. Because GF levels are not always optimal in subjects with defective bone healing, composition and bioactivity of PRP should be analyzed to test the reliability and potential effectiveness of the regenerative treatment.     

生长因子TGF-β1和bFGF促兔骨髓间质干细胞向韧带样细胞分化的实验研究

摘要目的：采用生长因子TGF-β1和bFGF诱导体外培养的兔骨髓间质干细胞（MSCs）,转化为韧带样细胞,并研究此种韧带样细胞的生物特性。方法：自幼兔四肢骨抽取骨髓分离纯化MSCs并培养、增殖;采用特定浓度TGF-β1（10ng／ml）和bFGF（25ng／mL）对MSCs进行诱导分化,观察生长因子对MSCs生长、形态的影响,使用MTT法绘制细胞生长曲线,使用天狼腥红染色法定量对比MSCs分泌胶原蛋白量。单纯培养和单一因子诱导组作为对照。结果：TGF-β1和bFGF联合使用组,细胞形态优于空白组及单一因子组,细胞增殖率、胶原分泌量也均高于对照组。结论：联合使用生长因子TGF-β1和bFGF刺激兔MSCs,能够促使兔MSCs定向转化为韧带样细胞,对组织工程前交叉韧带的构建具有积极意义。     

Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications

Mesenchymal stem cells (MSCs) are considered as emergent "universal" cells and various tissue repair programs using MSCs are in development. In vitro expansion of MSCs is conventionally achieved in medium containing fetal calf serum (FCS) and is increased by addition of growth factors. However, for widespread clinical applications, contact of MSCs with FCS must be minimized since it is a putative source of prion or virus transmission. Therefore, because platelets are a natural source of growth factors, we sought to investigate in vitro MSC expansion in response to platelet lysates (PL) obtained from platelet-rich plasma. Human MSCs were expanded in FCS (+/-bFGF)- or PL-supplemented medium through a process of subculture. We demonstrated that PL-containing medium is enriched by growth factors (platelet-derived growth factors (PDGFs), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), insulin-like growth factor-1 (IGF-1) ...) and showed that PL is able to promote MSC expansion, to decrease the time required to reach confluence, and to increase CFU-F size, as compared to the FCS medium. Furthermore, we demonstrated that MSCs cultured in the presence of PL maintain their osteogenic, chondrogenic, and adipogenic differentiation properties and retain their immunosuppressive activity. Therefore, we propose that PL may be a powerful and safe substitute for FCS in development of tissue- and cellular-engineered products in clinical settings using MSCs.     

Separation of mouse crushed muscle extract into distinct mitogenic activities by heparin affinity chromatography

Extracts from gently crushed adult mouse skeletal muscles (CMEs) contain potent myoblast mitogens, and may be used as a model system to investigate myotrophic factors released by adult muscles following injury. CME was separated into four peaks of mitogenic activity by heparin affinity chromatography. The fraction of CME that did not bind to heparin contained transferrin (Tf). Three peaks of mitogenic activity were eluted from the heparin-agarose columns at NaCl concentrations of 0.4 M, 0.9 M, and 2.0 M. A 46 kDa protein that shared antigenicity with the BB isoform of platelet-derived growth factor (PDGF-BB) was present in the 0.4 M NaCl eluant. Mitogenic activity in the 2.0 M NaCl peak eluted identically to purified basic fibroblast growth factor (bFGF), did not act additively to saturating amounts of purified bFGF, and was neutralized by anti-bFGF antibodies. The 0.9 M NaCl eluant acted additively to the combination of three known growth factors for myoblasts, bFGF, insulin-like growth factor I, and epidermal growth factor, to stimulate C2 myoblast proliferation, suggesting this fraction contains a mitogenic activity which does not utilize (and hence compete for) receptors for the known mitogens for myoblasts. Additionally, the 0.9 M NaCl eluant did not stimulate proliferation of fibroblast-like cells derived from muscle tissue. The unbound, 0.4 M NaCl, 0.9 M NaCl, and 2.0 M NaCl eluants from the heparin-agarose column acted additively to one another to stimulate myoblast proliferation. Our data suggest that Tf, PDGF-BB-like molecules, bFGF-like activity, and an uncharacterized heparin-binding myoblast mitogen could be released after muscle injury and act to stimulate satellite cell proliferation. © 1994 Wiley-Liss, Inc.     

Nanofiltration of growth media supplemented with human platelet lysates for pathogen-safe xeno-free expansion of mesenchymal stromal cells

Background aimsHuman platelet lysate can replace fetal bovine serum (FBS) for xeno-free ex vivo expansion of mesenchymal stromal cells (MSCs), but pooling of platelet concentrates (PCs) increases risks of pathogen transmission. We evaluated the feasibility of performing nanofiltration of platelet lysates and determined the impact on expansion of bone marrow–derived MSCs.MethodsPlatelet lysates were prepared by freeze-thawing of pathogen-reduced (Intercept) PCs suspended in 65% storage solution (SPP+) and 35% plasma, and by serum-conversion of PCs suspended in 100% plasma. Lysates were added to the MSC growth media at 10% (v/v), filtered and subjected to cascade nanofiltration on 35- and 19-nm Planova filters. Media supplemented with 10% starting platelet lysates or FBS were used as the controls. Impacts of nanofiltration on the growth media composition, removal of platelet extracellular vesicles (PEVs) and MSC expansion were evaluated.ResultsNanofiltration did not detrimentally affect contents of total protein and growth factors or the biochemical composition. The clearance factor of PEVs was >3 log values. Expansion, proliferation, membrane markers, differentiation potential and immunosuppressive properties of cells in nanofiltered media were consistently better than those expanded in FBS-supplemented media. Compared with FBS, chondrogenesis and osteogenesis genes were expressed more in nanofiltered media, and there were fewer senescent cells over six passages.ConclusionsNanofiltration of growth media supplemented with two types of platelet lysates, including one prepared from pathogen-reduced PCs, is technically feasible. These data support the possibility of developing pathogen-reduced xeno-free growth media for clinical-grade propagation of human cells.     

PDGFRβ triggered by bFGF promotes the proliferation and migration of endothelial progenitor cells via p-ERK signalling

We have examined the effects of bFGF (basic fibroblast growth factor) on p-ERK (phosphorylated extracellular signal-regulated kinase) through PDGFRβ (platelet-derived growth factor receptor β) in the proliferation and migration of EPCs (endothelial progenitor cells). EPC migration was detected using the Transwell system. The expression of PDGFRβ mRNA and protein, total ERK and p-ERK proteins was respectively assessed by real-time PCR and Western blottings. bFGF promote the proliferation and migration of EPCs, the effects of bFGF being implemented by activating ERK signalling through the expression of PDGFRβ, whereas an anti-bFGF antibody and inhibitor of PDGF (platelet-derived growth factor) receptor kinase (AG1296) could respectively decrease the expression of PDGFRβ mRNA and protein and p-ERK protein. Total ERK protein did not change under the same experimental conditions, and an inhibitor of p-ERK (PD98059) inhibited the proliferation and migration of EPCs. The findings strongly suggest that a PDGFRβ/p-ERK signalling pathway triggered by bFGF plays an important role in the proliferation and migration of EPCs.     

人主动脉硫酸乙酰肝素蛋白聚糖对培养的人血管壁细胞生长的作用

通过培养的人主动脉平滑肌细胞（ｈＡＳＭＣ）及脐静脉内皮细胞（ｈＵＶＥＣ）,应用３Ｈ－ＴｄＲ参入、Ｎｏｒｔｈｅｒｎｂｌｏｔ分析、逆转录多聚酶链反应（ＲＴ－ＰＣＲ）、放射免疫分析（ＲＩＡ）、和紫外比色法等技术观察了人主动脉中硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对ｈＡＳＭＣ和ｈＵＶＥＣＤＮＡ合成的作用及对血小板源生长因子（ＰＤＧＦ）、ＰＤＧＦ受体、转化生长因子β（ＴＧＦ－β）、内皮素－１（ＥＴ－１）或碱性成纤维细胞生长因子（ｂＦＧＦ）基因表达和肾素－血管紧张系统（ＲＡＳ）的影响,结果显示,ＨＳＰＧ明显抑制培养的ｈＡＳＭＣ基础的ＤＮＡ合成（ｃｐｍ值为：１０３８５±３２６３ｖｓ,２５５４１±６４２１,Ｐ＜０．０１）及外源性ＰＤＧＦ诱导的ＤＮＡ合成（ｃｐｍ值为：９８７８±１９４７ｖｓ．１３４８１±４４ｌ０,Ｐ＜０．０５）;抑制ＰＤＧＦＡ链、ＴＧＦ－Ｂｐ和ＥＴ－１ｍＲＮＡ表达,提高ＰＤＧＦａ和β受体ｍＲＮＡ的表达;显著降低ｈＡＳＭＣ培养液中血管紧张素Ⅱ（ＡｎｇⅡ）的浓度和血管紧张素转换酶（ＡＣＥ）的活性,推测ＨＳＰＧ抑制ＰＤＧＦＡ链、ＴＧＦ－β及ＥＴ－１ｍＲＮＡ表达,降低ＡＣＥ活性及ＡｎｇⅡ浓度是其抑制ｈＡＳＭＣ增殖的重要机     

Density-dependent inhibitory effect of transforming growth factor-β on human fibroblasts involves the down-regulation of platelet-derived growth factor α-receptors

We have previously found that transforming growth factor-β1 (TGF-β1) inhibits the mitogenic activity of platelet-derived growth factor (PDGF) in cultures of human neonatal fibroblasts in a density-dependent fashion. In the present investigation we determined the effect of TGF-β1 on the PDGF α-receptor, which binds all PDGF isoforms, as well as on the β-receptor, which binds only PDGF-BB with high affinity. We found that the inhibitory effect of TGF-β1 on PDGF-AA-induced mitogenesis was density-dependent; when dense cell cultures were preincubated with TGF-β1, there was an complete inhibition of ³H-thymidine incorporation, whereas the effect was less in sparse cultures. A similar density-dependent effect of TGF-β1 was seen in PDGF-BB treated cells, although less pronounced. The binding of ¹²⁵I-labeled PDGF-AA and PDGF-BB to the α-receptor was significantly reduced after treatment with TGF-β1 in dense cultures, whereas the sparse cultures were less affected. A decrease of α-receptor mRNA was also seen. The levels of β-receptor protein and mRNA were unaffected. We conclude that the growth inhibitory effect of TGF-β1 is cell density-dependent and involves down-regulation of PDGF α-receptors. © 1993 Wiley-Liss, Inc.     

Induction of Fibroblast Growth Factor Receptor-1 mRNA and Protein by Platelet-Derived Growth Factor BB

Expression levels of growth factor receptors are subject to complex regulation, which is of consequence for their signaling capacity in physiological and pathological processes. We examined the regulation of expression levels of fibroblast growth factor receptor 1 (FGFR-1) in human fibroblasts treated with a panel of growth regulatory factors. Only platelet-derived growth factor BB (PDGF-BB) treatment had a significant effect and induced FGFR-1 mRNA levels fourfold, with a peak around 8 h of stimulation. The increase in mRNA levels was followed by an increased synthesis of FGFR-1 protein, which responded to basic FGF (bFGF) stimulation with induction of kinase activity and biological signaling. Thus, murine brain endothelial cells displayed an augmented induction of plasminogen activator activity in response to bFGF, following treatment with PDGF-BB. These data suggest that PDGF-BB could support FGFR-1-mediated biological responses in processes such as angiogenesis.     

Electrochemical immunosensor based on electron transfer mediated by graphene oxide initiated silver enhancement

A new electrochemical immunosensor for the detection of protein biomarker platelet-derived growth factor BB (PDGF-BB) was developed based on graphene oxide (GO) initiated silver enhancement. The immunosensor was fabricated based on the traditional sandwich protocol using secondary anti-PDGF-BB antibody (Ab(2)) modified GO as label. Gold electrode was first modified with self-assembled monolayer (SAM) to block the electron transfer between the electrode and K(3)Fe(CN)(6) solution. After the immobilization of primary anti-PDGF-BB antibody (Ab(1)) onto electrode via aminidation to the carboxylic group of SAM and the formation of the sandwich immuno-structure onto electrode surface, the electrode was immersed into silver enhancement solution for silver deposition. The deposited metal silver onto GO then mediated electron transfer across the SAM, producing redox current. The resulting immunosensor displays a wide range of linear response, low detection limit, good reproducibility and stability. The immunosensor was used to the detection of PDGF-BB contents in serum samples with satisfactory results.     

Epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-bb can substitute for fetal bovine serum and compete with human platelet-rich plasma in the ex vivo expansion of mesenchymal stromal cells derived from adipose tissue

Background aimsHuman mesenchymal stromal cells (MSC) are multipotent cells possessing self-renewal capacity, long-term viability and multilineage potential. We analyzed the effect of four different medium supplements on the expansion and differentiation of adipose tissue-derived MSC (ADSC) in order to avoid the use of xenogeneic serum.MethodsWe compared fetal bovine serum (FBS) with 10% human platelet-rich plasma (hPRP), 3% human platelet-poor plasma (hPPP) and with a cytokine cocktail composed of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor-bb (PDGFbb) added to 3% hPPP. This mixture was developed testing EGF, bFGF, granulocyte–colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF-I), PDGFbb and transforming growth factor (TGF)-β1 added alone or in combination with hPPP.ResultsOur data demonstrate that the addition of EGF, bFGF and PDGFbb, in a medium supplemented with hPPP, obtainable from 150–200 mL whole autologous blood, supports ADSC expansion better than FBS, as confirmed by cumulative population doublings (cPD; 15.0 ± 0.5 versus 9.4 ± 2.8). The addition of human platelet-rich plasma (hPRP) further improved ADSC proliferation (cPD 20.0 ± 1.2), but the achievement of hPRP presented a major drawback, requiring 1000–1200 mL autologous or donor whole blood. The medium supplements did not influence ADSC phenotype: they expressed CD105, CD90 and CD44 lacking hematopoietic antigens. The exposure to the proposed cocktail or to hPRP increased adipogenic and osteogenic differentiation.ConclusionsThe addition of EGF, bFGF and PDGFbb to hPPP could ensure a sufficient number of ADSC for clinical applications, avoiding the use of animal serum and representing a novel approach in regenerative medicine.     

Heparanase and platelet factor-4 induce smooth muscle cell proliferation and migration via bFGF release from the ECM

Basic fibroblast growth factor (bFGF) has been shown to play an instrumental role in the cascade of events leading to restenosis; however, the mechanisms of bFGF activation following vascular injury have remained elusive. We have demonstrated that heparanase and platelet factor-4 (PF4), released from activated platelets at the site of injury, liberate bFGF from the extracellular matrix (ECM) of vascular smooth muscle cells (SMC), resulting in the induction of SMC proliferation and migration. Increases in proliferation and migration were inhibited by treatment with a bFGF-neutralizing antibody, suggesting that proliferation and migration in response to heparanase or PF4 are mediated by bFGF activation. When platelets were seeded on top of SMCs, degranulation products were found to release bFGF from the ECM, increasing cell proliferation and cell migration. Again, these increases in SMC proliferation and migration were inhibited by treatment with an anti-bFGF antibody. Furthermore, these increases in proliferation were completely inhibited by treatment with an anti-heparanase antibody. Platelet degranulation products, such as heparanase and PF4, may liberate bFGF from extracellular sequestration, activating the growth factor and inducing the SMC proliferation and migration that contribute to the wound healing response following vascular injury.     

Platelet-derived growth factor-BB and basic fibroblast growth factor directly interact in vitro with high affinity.

Platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) are potent growth factors active on many cell types. The present study indicates that they directly interact in vitro. The interaction was investigated with overlay experiments, surface plasmon resonance experiments, and solid-phase immunoassays by immobilizing one factor or the other and by steady-state fluorescence analysis. The interaction observed was specific, dose-dependent, and saturable, and the bFGF/PDGF-BB binding stoichiometry was found to be 2:1. K(D)(1) for the first step equilibrium and the overall K(D) values were found to be in the nanomolar and in the picomolar range, respectively. Basic FGF/PDGF-BB interaction was strongly reduced as a function of time of PDGF-BB proteolysis. Furthermore, docking analysis suggested that the PDGF-BB region interacting with bFGF may overlap, at least in part, with the PDGF-BB receptor-binding site. This hypothesis was supported by surface plasmon resonance experiments showing that an anti-PDGF-BB antibody, known to inhibit PDGF-BB binding with its receptor, strongly reduced bFGF/PDGF-BB interaction, whereas a control antibody was ineffective. According to these data, the observed bFGF.PDGF-BB complex formation might explain, at least in part, previous observations showing that PDGF-BB chemotactic and mitogenic activity on smooth muscle cells are strongly inhibited in the presence of bFGF.     

Modulation of growth factor responsiveness of murine mammary carcinoma cells by cell matrix interactions: correlation of cell proliferation and spreading.

We have examined the role of growth factors and extracellular matrix in the proliferation and cell adhesion of a murine mammary carcinoma, SP1, and a stable highly metastatic variant, SP1-3M. On fibronectin, both cell types proliferated strongly in response to basic fibroblast growth factor (bFGF) and platelet-derived growth factor BB (PDGF-BB) after culture for 24 h and 72 h. In contrast, on collagen type I, SP1 cells proliferated only weakly to PDGF-BB at either time, and SP1-3M cells showed a response to PDGF-BB only at 72 h. The proliferative response to bFGF was also consistently lower when the cells were cultured on collagen than on fibronectin. No significant proliferative responses were detected to epithelial growth factor (EGF), transforming growth factor-beta (TGF-beta), or estrogen on any substratum. The lack of responsiveness to PDGF-BB of cells cultured on collagen type I was not due to differences in numbers or affinity of PDGF receptors. We therefore examined the adhesion and spreading properties of SP1 and SP1-3M cells. Without exogenous growth factors, both cell lines adhered to fibronectin and laminin. SP1-3M cells did not bind to collagen type I, whereas SP1 cells did. Attachment to all three substrata was inhibited by anti-beta 1 integrin IgG, suggesting that the primary adhesion to these substrata is mediated by beta 1 integrins. SP1 and SP1-3M cells showed similar integrin patterns following immunoprecipitation by anti-beta 1 integrin IgG. bFGF stimulated increased adhesion and spreading of both SP1 and SP1-3M cells to collagen type I within 24 h, whereas PDGF-BB was less capable of this effect. Our results suggest that the proliferative response of SP1 and SP1-3M cells to PDGF-BB and bFGF is dependent on the extracellular matrix environment, and imply that modification of extracellular matrix and/or surface integrin receptors may regulate responsiveness to these growth factors in the SP1 tumor model.     

Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow–derived mesenchymal stromal cells

Background aimsA medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed.MethodsPlatelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells.ResultsAfter 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations.ConclusionsThe proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS.