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Meniscus regeneration could be enhanced by targeting meniscus cells and mesenchymal stromal cells (MSCs) with the right growth factors. Combining these growth factors with the Collagen Meniscus Implant (CMI®) could accelerate cell ingrowth and tissue formation in the implant and thereby improve clinical outcomes. Using a transwell migration assay and a micro‐wound assay, the effect of insulin‐like growth factor‐1, platelet‐derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF‐β1), fibroblast growth factor, and platelet lysate (PL) on migration and proliferation of meniscus cells and MSCs was assessed. The formation of extracellular matrix under influence of the above‐mentioned growth factors was assessed after 28 days of culture of both MSCs and meniscus cells. As a proof of concept, the CMI® was functionalized with a VEGF binding peptide and coated with platelet‐rich plasma (PRP) for clinical application. Our results demonstrate that PDGF, TGF‐β1, and PL stimulate migration, proliferation, and/or extracellular matrix production of meniscus cells and MSCs. Additionally, the CMI® was successfully functionalized with a VEGF binding peptide and PRP which increased migration of meniscus cell and MSC into the implant. This study demonstrates proof of concept of functionalizing the CMI® with growth factor binding peptides. A CMI® functionalized with the right growth factors holds great potential for meniscus replacement after partial meniscectomy.

Platelet products (PP) and bone-marrow aspirate are popular sources of osteoinductive signalling molecules and osteogenic bone marrow mesenchymal stromal cells (BM-MSCs) used in the treatment of impaired bone healing. However, the combined use of PP and BM-MSCs in clinical studies has reported mixed results. Understanding the cellular and molecular interactions between PP and BM-MSCs plays the important role of guiding future research and clinical application. This systematic review investigates the effects of PP on the biophysiological functions of BM-MSCs in in vitro human studies, including (i) proliferation, (ii) migration, (iii) differentiation, (iv) growth factor/cytokine/protein expression, (v) immunomodulation, (vi) chemotactic effect on haematopoietic stem cells, (vii) response to apoptotic stress, and (viii) gene expression. In vitro studies in human have demonstrated the multi-faceted ‘priming effect’ of PP on the biophysiological functions of BM-MSCs. PP has been shown to improve proliferation, migration, osteogenic differentiation, reaction to apoptotic stress as well as immunomodulatory, pro-angiogenic and pro-inflammatory capacities of BM-MSCs. Several factors are highlighted that restrict the transferability of these findings into clinical practice. Therefore, more collaborative in vitro research in humans modelled to reflect clinical practice is required to better understand the effects of PP exposure on the biophysiological function(s) of BM-MSCs in human.

Purpose: Transfusion of red blood cells (RBCs) is a supportive and common treatment in surgical care, trauma, and anemia. However, in vivo production of RBC seems to be a suitable alternative for blood transfusions due to the limitation of blood resources, the possibility of disease transmission, immune reactions, and the presence of rare blood groups. Cell cultures require serum-free or culture media supplemented with highly expensive animal serum, which can transmit xenoviruses. Platelet lysate (PL) can be considered as a suitable alternative containing a high level of growth factors and a low production cost. Methods: Three-step culture media supplemented with PL or fetal bovine serum (FBS) were used for proliferation and differentiation of CD34+ umbilical cord blood stem cells to erythrocytes in co-culture with bone marrow mesenchymal stem cells (BM-MSCs). The cells were cultivated for 15 days and cell proliferation and expansion were assessed using cell counts at different days. Erythroid differentiation genes, CD71 and glycophorin A expression levels were evaluated. Results: Maximum hematopoietic stem cells (HSCs) proliferation was observed on day 15 in PL-containing medium (99±17×103 -fold). Gene expression and surface markers showed higher differentiation of cells in PL-containing medium. Conclusion: The results of this study indicate that PL can enhance erythroid proliferation and differentiation of CD34+ HSCs. PL can also be used as a proper alternative for FBS in the culture medium and HSCs differentiation.

BACKGROUND Allogeneic platelet (PLT) infusion is a strategy to raise Factor V (FV) levels in patients with congenital FV deficiency. However, since FV is labile in vitro, we hypothesized that FV activity could be low in PLT units. STUDY DESIGN AND METHODS FV activity was tested using a prothrombin time-based platform in the supernatant and platelet lysate (PL) of apheresis PLT units (16 units stored in PLT additive solution with acetate and phosphate [PAS-C] and 10 units stored in plasma only), on post-collection days 3-6. Statistical analysis was performed using Student's t test (P

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are being investigated for their potential in the treatment of musculoskeletal injuries, including tendon and ligament lesions, and cartilage lesions. Culture expansion of cells has traditionally been performed in medium supplemented with fetal bovine serum (FBS), however, concerns regarding the antigenicity and potential viral or prion contamination of FBS have prompted interest in alternative medium supplements. Platelet lysate (PL) contains elevated concentrations of growth factors, including transforming growth factor-β (TGF-β), platelet-derived growth factors, and fibroblast growth factor, released from the α-granules of platelets; therefore, PL could be an ideal medium supplement. The effect of PL on mesenchymal stromal cell (MSC) growth and differentiation has not been fully elucidated. We hypothesized that PL medium would contain significantly higher amounts of TGF-β1 than FBS medium and would be associated with enhanced osteogenic and chondrogenic differentiation. MSCs were isolated from bone marrow collected from five adult horses. Cells were cultured in traditional medium supplemented with FBS or in medium supplemented with fibrinogen depleted-PL (FD-PL). Immunophenotyping was performed using flow cytometry. Trilineage differentiation was assessed through histology and gene expression analysis using quantitative reverse transcription-polymerase chain reaction. TGF-β1 was quantified in both medium types. The immunophenotypes of BM-MSCs cultured in FBS and FD-PL medium were similar with both culture types containing cells positive for stromal cell markers [cluster of differentiation 29 (CD29), CD44, CD90, CD105, and major histocompatibility complex I (MHCI)] and negative for exclusion markers (CD45, CD79α, and MHCII). Despite significantly higher TGF-β1 concentration in FD-PL medium, chondrogenic and osteogenic differentiation were not significantly different between FBS and FD-PL supplemented cultures. PL is an appropriate alternative medium supplement for the culture of equine BM-MSCs up to passage 3. However, despite increased TGF-β1 concentration in FD-PL medium, significant changes in chondrogenic differentiation compared with FBS medium should not be expected.

The aim of this work is to investigate the capability of PRP as an adjuvant therapy to autologous chondrocyte implantation (ACI) in combination with multi-axial load with respect to cartilage regeneration. Articular cartilage shows poor repair capacity and therapies for cartilage defects are still lacking. Well-established operative treatments include ACI, and growing evidence shows the beneficial effects of PRP. Platelets contain numerous growth factors, among them transforming growth factor beta (TGF-β). Dynamic mechanical loading is known to be essential for tissue formation, improving extracellular matrix (ECM) production. For our ACI model monolayer expanded human chondrocytes were seeded into polyurethane scaffolds and embedded in fibrin (hChondro), in PRP-Gel (PRP), or in fibrin with platelet lysate (PL), which was added to the media once a week with a concentration of 50 vol%. The groups were either exposed to static conditions or multi-axial forces in a ball-joint bioreactor for 1 h per day over 2 weeks, mimicking ACI under physiological load. The culture medium was collected and analyzed for glycosaminoglycan (GAG), nitrite and transforming growth factor beta 1 (TGF-β1) content. The cell-scaffold constructs were collected for DNA and GAG quantification; the expression of chondrogenic genes, TGF-β and related receptors, as well as inflammatory genes, were analyzed using qPCR. Loading conditions showed superior chondrogenic differentiation (upregulation of COL2A1, ACAN, COMP and PRG4 expression) than static conditions. PRP and PL groups combined with mechanical loading showed upregulation of COL2A1, ACAN and COMP. The highest amount of total TGF-β1 was quantified in the PL group. Latent TGF-β1 was activated in all loaded groups, while the highest amount was found in the PL group. Load increased TGFBR1/TGFBR2 mRNA ratio, with further increases in response to supplements. In general, loading increased nitrite release into the media. However, over time, the media nitrite content was lower in the PL group compared to the control group. Based on these experiments, we conclude that chondrogenic differentiation is strongest when simulated ACI is performed in combination with dynamic mechanical loading and PRP-gel or PL supplementation. An inflammatory reaction was reduced by PRP and PL, which could be one of the major therapeutic effects. Loading presumably can enhance the action of TGF-β1, which was predominantly activated in loaded PL groups. The combination of load and PRP represents an effective and promising synergy concerning chondrocyte-based cartilage repair.

This in vitro study was carried out to evaluate the potential antibacterial properties of canine non-transfusional hemo-components. Therapeutic formulations commonly used for regenerative medicine purposes (platelet-rich plasma, platelet gel, platelet lysate, fibrin glue), considering both leukocyte-rich and leukocyte-poor formulations, but also platelet-poor plasma and activating substances (thrombin, calcium gluconate), were tested to detect elements with potential antimicrobial properties. The antibacterial effect was tested on different bacterial strains (Staphylococcus aureus subspecies aureus, Staphylococcus cohnii subspecies cohnii, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae subspecies pneumoniae) isolated from canine wounds and classified as susceptible, multidrug-, extensively, and pandrug-resistant bacteria toward a known panel of human and veterinary antibiotics. The evaluation was carried out by agar gel diffusion method (Kirby–Bauer) and micro-inhibition in broth using microplates and spectrophotometer reading. The study findings confirmed the hypothesized antibacterial properties of canine non-transfusional hemo-components. A more effective bacteriostatic effect was found against Gram-negative bacteria, drug-resistant too. The presence of leukocytes or platelets does not appear to be essential for the antibacterial effect. Further studies should be conducted to evaluate the exact mechanism of action of the antimicrobial activity. However, non-transfusional hemo-components could be a useful natural aid in controlling bacterial infections in dogs.

International audience; Mesenchymal stem cells (MSCs) have received significant attention in recent years due to their large potential for cell therapy. Indeed, they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, several factors may restrict their use for clinical applications: the requirement of invasive procedures for their isolation, their limited numbers, and their heterogeneity according to the tissue of origin or donor. In addition, MSCs often present early signs of replicative senescence limiting their expansion in vitro, and their therapeutic capacity in vivo. Due to the clinical potential of MSCs, a considerable number of methods to differentiate induced pluripotent stem cells (iPSCs) into MSCs have emerged. iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations. Additionally, the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells. In this review, we analyze the main current protocols used to differentiate human iPSCs into MSCs, which we classify into five different categories: MSC Switch, Embryoid Body Formation, Specific Differentiation, Pathway Inhibitor, and Platelet Lysate. We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization. Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added. The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.

The development of antimicrobial resistant bacteria and the lack of novel antibiotic strategies to combat those bacteria is an ever-present problem in both veterinary and human medicine. The goal of this study is to evaluate platelet lysate (PL) as a biological alternative antimicrobial product. Platelet lysate is an acellular platelet-derived product rich in growth factors and cytokines that is manufactured via plateletpheresis and pooled from donor horses. In the current study, we sought to define the antimicrobial properties of PL on select gram-positive and gram-negative bacteria. Results from an end-point in vitro assay showed that PL did not support bacterial growth, and in fact significantly reduced bacterial content compared to normal growth media. An in vitro assay was then utilized to further determine the effects on bacterial growth dynamics and showed that all strains exhibited a slower growth rate and lower yield in the presence of PL. The specific effects of PL were unique for each bacterial strain: E. coli and P. aeruginosa growth was affected in a concentration-dependent manner, such that higher amounts of PL had a greater effect, while this was not true for S. aureus or E. faecalis. Furthermore, the onset of exponential growth was delayed for E. coli and P. aeruginosa in the presence of PL, which has significant clinical implications for developing a dosing schedule. In conclusion, our findings demonstrate the potential value of PL as a broad-spectrum antimicrobial that would offer an alternative to traditional antibiotics for the treatment of bacterial infection in equine species.

Se desarrolló un hidrogel con matriz de β-quitosano que retarde la liberación del factor de crecimiento derivado de plaquetas – BB (PDGF – BB), el cual forma parte del lisado plaquetario (LP), obtenido a partir del plasma rico en plaquetas (PRP). Para la elaboración del hidrogelse preparó la solución de β-quitosano 1,5% (p/v) en ácido acético 2 % (v/v), LP al 6 % o 12 % (v/v) y glutaraldehído a una concentración final de 0,03M. El β-quitosano usado como materia prima, se caracterizó por tener 12,47 % de humedad relativa promedio, grado de desacetilación promedio de 94,53 % y peso molecular promedio de 145,94 kDa; atributos importantes que permitieron formular un hidrogel de consistencia semi-sólida, incoloro y fácil de manipular. La liberación retardada se logró con el hidrogel con 12 % de LP, pues la liberación de PDGF – BB no difiere significativamente (p < 0,05) al variar el pH de 6,5 (5 491,42 pg de PDGF – BB/mL de LP) a 7,4 (5 387,39 pg de PDGF – BB/mL de LP) luego de 32 horas. Por el contrario, el hidrogel con 6 % de LP, sí mostró una diferencia significativa en la cantidad de PDGF – BB liberado al variar el pH de 6,5 (12 687,66 pg de PDGF – BB/mL de LP) a 7,4 (9 396,58 pg de PDGF – BB/mL de LP) luego de 32 horas. A hydrogel with β-chitosan matrix that retards the release of platelet-derived growth factor - BB (PDGF - BB), which is part of the platelet lysate (LP) obtained from platelet rich plasma (PRP), was developed. The hydrogel was composed of a solution of 1.5 % (w/v) β-chitosan in 2 % (v/v) acetic acid, 6% or 12 % LP (v/v) and glutaraldehyde 0.03M. The β-chitosan used as raw material had 12.47 % of average relative humidity, 94.53 of average deacetylation degree and an estimated average molecular weight of 145.94 kDa; important attributes to formulate a hydrogel of semi-solid consistency, colorless and easy to handle. Delayed release was achieved with the 12 % LP hydrogel since the release of PDGF – BB does not differ significantly (p

AimThis study aimed to test whether human platelet lysate (HPL) has neurotrophic ability for corneal nerve regeneration.MethodsWe measured the neurotrophic factors in human peripheral serum (HPS) and two commercially available HPLs, UltraGRO and PLTMax. In vitro, we compared the growth rates, neuronal differentiation and immunostaining of neuron markers in mouse neuroblastoma cell line (Neuro-2a) and primary culture of mouse trigeminal ganglion cells that were cultivated in different concentrations of fetal bovine serum, HPS and HPL. In vivo, we created corneal wounds on Sprague Dawley rats with a rotating burr and evaluated the effects of topical HPL on wound healing and corneal nerve regeneration by in vivo confocal microscopy and corneal aesthesiometry.ResultsHPLs had significantly higher concentrations of various neurotrophic factors compared with HPS (pConclusionHPL has significantly higher concentrations of neurotrophic factors compared with HPS. It showed not only in vitro but also in vivo corneal neurotrophic abilities. Our results suggest that HPL may have a potential role in the treatment of diseases related to corneal nerve damage or degeneration.

Traumatic brain injury (TBI) leads to major brain anatomopathological damages underlined by neuroinflammation, oxidative stress and progressive neurodegeneration, ultimately leading to motor and cognitive deterioration. The multiple pathological events resulting from TBI can be addressed not by a single therapeutic approach, but rather by a synergistic biotherapy capable of activating a complementary set of signalling pathways and providing synergistic neuroprotective, anti-inflammatory, antioxidative, and neurorestorative activities. Human platelet lysate might fulfil these requirements as it is composed of a plethora of biomolecules readily accessible as a TBI biotherapy. In the present study, we tested the therapeutic potential of human platelet lysate using in vitro and in vivo models of TBI. We first prepared and characterized platelet lysate from clinical-grade human platelet concentrates. Platelets were pelletized, lysed by three freeze-thaw cycles, and centrifuged. The supernatant was purified by 56°C 30 min heat treatment and spun to obtain the heat-treated platelet pellet lysate that was characterized by ELISA and proteomic analyses. Two mouse models were used to investigate platelet lysate neuroprotective potential. The injury was induced by an in-house manual controlled scratching of the animals’ cortex or by controlled cortical impact injury. The platelet lysate treatment was performed by topical application of 60 µl in the lesioned area, followed by daily 60 µl intranasal administration from Day 1 to 6 post-injury. Platelet lysate proteomics identified over 1000 proteins including growth factors, neurotrophins, and antioxidants. ELISA detected several neurotrophic and angiogenic factors at ∼1–50 ng/ml levels. We demonstrate, using two mouse models of TBI, that topical application and intranasal platelet lysate consistently improved mouse motor function in the beam and rotarod tests, mitigated cortical neuroinflammation, and oxidative stress in the injury area, as revealed by downregulation of pro-inflammatory genes and the reduction in reactive oxygen species levels. Moreover, platelet lysate treatment reduced the loss of cortical synaptic proteins. Unbiased proteomic analyses revealed that heat-treated platelet pellet lysate reversed several pathways promoted by both controlled cortical impact and cortical brain scratch and related to transport, postsynaptic density, mitochondria or lipid metabolism. The present data strongly support, for the first time, that human platelet lysate is a reliable and effective therapeutic source of neurorestorative factors. Therefore, brain administration of platelet lysate is a therapeutical strategy that deserves serious and urgent consideration for universal brain trauma treatment., Nebie et al. show the therapeutic potential of sequential topical and intranasal administration of human platelet lysate (HPL) in two mouse models of traumatic brain injury. This HPL biotherapy reduced cortical inflammation, oxidative stress and synaptic impairments at the injury site and improved motor and cognitive functions.

Routine cell culture demands the use of animal-derived products, mainly fetal bovine serum and swine or bovine trypsin. According to the 3Rs principle and to the European Centre for the Validation of Alternative Methods, animal-free substitutes are strongly recommended for in vitro methods. In this study, the HEp-2 cell line was adapted to different totally animal-free culture systems, such as a serum-free complete medium (VP-SFM), human platelet lysate and a synthetic trypsin (TrypLE™ Express); afterward, cell growth was assessed with the xCELLigence instrument. Animal-free products provided promising results, with performances similar or preferable to the common reagents; therefore their use could be encouraged for both ethical and technical advantages.

Mesenchymal stem cells (MSCs) are of great interest for their use in cell-based therapies due to their multipotent differentiation and immunomodulatory capacities. In consequence of limited numbers following their isolation from the donor tissue, MSCs require extensive expansion performed in traditional 2D cell culture setups to reach adequate amounts for therapeutic use. However, prolonged culture of MSCs in vitro has been shown to decrease their differentiation potential and alter their immunomodulatory properties. For that reason, preservation of these physiological characteristics of MSCs throughout their in vitro culture is essential for improving the efficiency of therapeutic and in vitro modeling applications. With this objective in mind, many studies already investigated certain parameters for enhancing current standard MSC culture protocols with regard to the effects of specific culture media components or culture conditions. Although there is a lot of diversity in the final therapeutic uses of the cells, the primary stage of standard isolation and expansion is imperative. Therefore, we want to review on approaches for optimizing standard MSC culture protocols during this essential primary step of in vitro expansion. The reviewed studies investigate and suggest improvements focused on culture media components (amino acids, ascorbic acid, glucose level, growth factors, lipids, platelet lysate, trace elements, serum, and xenogeneic components) as well as culture conditions and processes (hypoxia, cell seeding, and dissociation during passaging), in order to preserve the MSC phenotype and functionality during the primary phase of in vitro culture.

Chronic wounds affect millions of patients worldwide, and it is estimated that this number will increase steadily in the future due to population ageing. The research of new therapeutic approaches to wound healing includes the development of nanofibrous meshes and the use of platelet lysate (PL) to stimulate skin regeneration. This study considers a combination of a degradable electrospun nanofibrous blend of poly(L-lactide-co-ε-caprolactone) and poly(ε-caprolactone) (PLCL/PCL) membranes (NF) and fibrin loaded with various concentrations of PL aimed at the development of bioactive skin wound healing dressings. The cytocompatibility of the NF membranes, as well as the effect of PL, was evaluated in both monocultures and co-cultures of human keratinocytes and human endothelial cells. We determined that the keratinocytes were able to adhere on all the membranes, and their increased proliferation and differentiation was observed on the membranes that contained fibrin with at least 50% of PL (Fbg + PL) after 14 days. With respect to the co-culture experiments, the membranes with fibrin with 20% of PL were observed to enhance the metabolic activity of endothelial cells and their migration, and the proliferation and differentiation of keratinocytes. The results suggest that the newly developed NF combined with fibrin and PL, described in the study, provides a promising dressing for chronic wound healing purposes.

Cell-based therapies have been emerged to find innovative solutions for corneal endothelial dysfunction. The aim of this study is to investigate the suitability of a blended scaffold containing human platelet lysate (HPL) and fibrin not only for cultivating human corneal endothelial cells (HCECs) but also for serving as a scaffold for the respected cells. We isolated HCECs from human donors and encapsulated the cells with three concentrations of HPL/Fibrin scaffold, namely HPL/Fibrin 1, HPL/Fibrin 2 and HPL/Fibrin 3, by adding 28.9, 57.8 and 86.7 mg/dl of fibrinogen to HPL to obtain a final percentage of 10, 20 and 30 % of fibrinogen, respectively. SEM imaging and swelling test were done to characterize the scaffolds. Cell viability assay and cell counting were performed on the cells. HCECs were characterized by morphology and immunocytochemistry. SEM imaging on freeze-dried scaffolds showed higher porosity of HPL/Fibrin 1 and HPL/Fibrin 2 than HPL/Fibrin 3, but larger pores were observed only in HPL/Fibrin 1. Cellular attachment and morphology on HPL/Fibrin 1 were appropriate by SEM imaging. A higher swelling rate was observed in HPL/Fibrin 1. After 3 and 5 days, higher numbers of cells were observed specifically in HPL/Fibrin 1. A higher expression of Na+/K+-ATPase, ZO-1 and vimentin proteins was detected in the HPL/Fibrin 1-cultured HCECs as compared with control (no scaffold). HPL/Fibrin can be used as a suitable scaffold for HCECs while preserving the cells viability. Further investigations are necessitated to approve the beneficial effects of the suggested scaffold for delivering and transplantation of cultivated HCECs into the anterior chamber of the eye.

Background Glioblastoma, the most common primary malignant brain tumor, is nearly universally fatal by 5 years. Dendritic cell vaccines are promising but often limited clinically by antigen choice, dendritic cell potency, and/or manufacturing yield. We optimized vaccine manufacture, generating potent mature autologous dendritic cells pulsed with allogeneic glioblastoma lysates. Methods Platelet lysate-based supplement was used to establish human glioblastoma cell lines. Phenotype and genotype were assessed. An improved culture technique to generate mature dendritic cells from glioblastoma patients’ monocytes was developed. The ability of T cells stimulated with autologous dendritic cells pulsed with allogeneic glioblastoma cell lysate to kill HLA-A2-matched glioblastoma cells was assessed. Results Glioblastoma cell lines established with platelet lysate supplement grew faster and expressed more stem-like markers than lines grown in neural stem cell media or in the presence of serum. They expressed a variety of glioma-associated antigens and had genomic abnormalities characteristic of glioblastoma stable up to 15 doublings. Unlike standard culture techniques, our optimized technique produced high levels of mature dendritic cells from glioblastoma patients’ monocytes. Autologous T cells stimulated with mature dendritic cells pulsed with allogeneic glioblastoma cell line lysate briskly killed HLA-A2-matched glioblastoma cells. Conclusions Our glioblastoma culture method provides a renewable source for a broad spectrum glioblastoma neoantigens while our dendritic cell culture technique results in more mature dendritic cells in glioblastoma patients than standard techniques. This broadly applicable strategy could be easily integrated into patient care.

Purpose To develop a human platelet lysate (hPL)-based bioink that is transparent and also encompasses the regenerative properties of hPL to facilitate wound healing. Methods The effect of different batches of hPLand fetal bovine serum (FBS) on corneal epithelial cell growth and scratch assay was first examined using Incucyte Zoom. Various combinations of human fibrinogen (concentration range from 0.2 to 5 mg/mL) and thrombin (concentration from 1 to 10 U/mL) were combined with hPL to generate nine types of potential bioink. Rheology, transparency, and cell compatibility of bioinks were assessed and compared. The final selected bioink was used in an ex vivo corneal model to examine its ability in re-epithelization. Results No significant difference was detected in cell proliferation and wound healing tests between different hPL batches at the same concentration. Scratch assay data showed that hPL had significantly higher effect on wound healing than FBS. Comparing across the nine bioinks, bioink 5 consisting of 10% hPL, 2 mg/mL fibrinogen, and 5 U/mL thrombin demonstrated all required mechanical and cellular properties and was able to regenerate the full-thickness epithelium ex vivo. Conclusions The results showed that a transparent and adhesive bioink can be generated by combining hPL, fibrinogen, and thrombin together. The bioink can be directly applied to a human cornea to promote corneal re-epithelization with huge potential applications in corneal injuries. Translational relevance The developed transparent hPL-based ink with its adhesive and healing ability showed that it could be used as a new treatment option for corneal injuries.

Platelet lysate (PL) as a cost-effective cocktail of growth factors is an emerging ingredient in regenerative medicine, especially in cartilage tissue engineering. However, most studies fail to pay attention to PL's intrinsic characteristics and incorporate it directly with scaffolds or hydrogels by simple mixture. Currently, the particle size distribution of PL was determined to be scattered. Directly introducing PL into a thermosensitive poly(d,l-lactide)-poly(ethylene glycol)-poly(d,l-lactide) (PLEL) hydrogel disturbed its sol-gel transition. Electrostatic self-assembly heparin (Hep) and e-poly-l-lysine (EPL) nanoparticles (NPs) were fabricated to improve the dispersity of PL. Such PL-NPs-incorporated PLEL gels retained the initial gelling capacity and showed a long-term PL-releasing ability. Moreover, the PL-loaded composite hydrogels inhibited the inflammatory response and dedifferentiation of IL-1β-induced chondrocytes. For in vivo applications, the PLEL@PL-NPs system ameliorated the early cartilage degeneration and promoted cartilage repair in the late stage of osteoarthritis. RNA sequencing analysis indicated that PL's protective effects might be associated with modulating hyaluronan synthase 1 (HAS-1) expression. Taken together, these results suggest that well-dispersed PL by Hep/EPL NPs is a preferable approach for its incorporation into hydrogels and the constructed PLEL@PL-NPs system is a promising cell-free and stepwise treatment option for cartilage tissue engineering.

Platelets harbor the primary reservoir of circulating plasminogen activator inhibitor 1 (PAI-1), but the reportedly low functional activity of this pool of inhibitor has led to debate over its contribution to thrombus stability. Here we analyze the fate of PAI-1 secreted from activated platelets and examine its role in maintaining thrombus integrity. Activation of platelets results in translocation of PAI-1 to the outer leaflet of the membrane, with maximal exposure in response to strong dual agonist stimulation. PAI-1 is found to co-localize in the 'cap' of phosphatidylserine-exposing platelets with its co-factor, vitronectin, and fibrinogen. Inclusion of tirofiban or Gly-Pro-Arg-Pro significantly attenuated exposure of PAI-1, indicating a crucial role for integrin αIIbβ3 and fibrin in delivery of PAI-1 to the activated membrane. Separation of platelets post stimulation into soluble and cellular components revealed the presence of PAI-1 antigen and activity in both fractions, with approximately 40% of total platelet-derived PAI-1 remaining associated with the cellular fraction. Using a variety of fibrinolytic models, we found that platelets produce a strong stabilizing effect against tissue plasminogen activator (tPA)-mediated clot lysis. Platelet lysate, as well as soluble and cellular fractions, stabilize thrombi against premature degradation in a PAI-1-dependent manner. Our data show for the first time that a functional pool of PAI-1 is anchored to the membrane of stimulated platelets and regulates local fibrinolysis. We reveal a key role for integrin αIIbβ3 and fibrin in delivery of PAI-1 from platelet α-granules to the activated membrane. These data suggest that targeting platelet-associated PAI-1 may represent a viable target for novel profibrinolytic agents.

Platelets are a recognised potent source of transforming growth factor‐β1 (TGFβ1), a cytokine known to promote wound healing and regeneration by stimulating dermal fibroblast proliferation and extracellular matrix deposition. Platelet lysate has been advocated as a novel personalised therapeutic to treat persistent wounds, although the precise platelet‐derived growth factors responsible for these beneficial effects have not been fully elucidated. The aim of this study was to investigate the specific role of platelet‐derived TGFβ1 in cutaneous wound healing. Using a transgenic mouse with a targeted deletion of TGFβ1 in megakaryocytes and platelets (TGFβ1fl/fl.PF4‐Cre), we show for the first time that platelet‐derived TGFβ1 contributes to epidermal and dermal thickening and cellular turnover after excisional skin wounding. In vitro studies demonstrate that human dermal fibroblasts stimulated with platelet lysate containing high levels of platelet‐derived TGFβ1 did not exhibit enhanced collagen deposition or proliferation, suggesting that platelet‐derived TGFβ1 is not a key promoter of these wound healing processes. Interestingly, human keratinocytes displayed enhanced TGFβ1‐driven proliferation in response to platelet lysate, reminiscent of our in vivo findings. In summary, our novel findings define and emphasise an important role of platelet‐derived TGFβ1 in epidermal remodelling and regeneration processes during cutaneous wound healing.

Due to their capacity to proliferate, migrate, and differentiate, mesenchymal stem cells (MSCs) are considered to be good candidates for regenerative medicine applications. The mechanisms underlying proliferation and differentiation of MSCs have been studied. However, much less is known about the mechanisms regulating the migration of MSCs. Platelet lysate (PL), a supplement used to promote cell expansion, has been shown to promote MSCs migration; however, the underlying mechanism are unknown. Here, by using adipose-derived rat MSCs (rMSCs) and the scratch assay in the absence and presence of various BK channels modulators, we evaluated the role of BK channels in mediating the PL-stimulated migration of rMSCs. We found that 5% PL increased rMSCs migration, and this effect was blocked by the addition of the BK channel selective antagonist Iberiotoxin (IBTX). In the absence of PL, the BK channel agonist NS1619, stimulated rMSCs migration to similar level as 5% PL. Addition of both NS1619 and 5% PL resulted in an increase in rMSCs migration, that was higher than when either one was added individually. From whole-cell recordings, it was found that the addition of 5% PL increased the magnitude of BK current density. By using Western blot and flow cytometry, it was found that PL did not affect the expression of BK channels. Together, our results indicate that as shown in other cell types, activation of BK channels by themselves also promote rMSC migration, and show that activation of BK channels contribute to the observed PL-induced increase in migration of rMSC.

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%-20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Wound repair is a dynamic process during which crucial signaling pathways are regulated by growth factors and cytokines released by several kinds of cells directly involved in the healing process. However, the limited applications and heterogeneous clinical results of single growth factors in wound healing encouraged the use of a mixture of bioactive molecules such as platelet derivatives for best results in wound repair. An interesting platelet derivative, obtained from blood samples, is platelet lysate (PL), which has shown potential clinical application. PL is obtained from freezing and thawing of platelet-enriched blood samples. Intracellular calcium (Ca2+) signals play a central role in the control of endothelial cell survival, proliferation, motility, and differentiation. We investigated the role of Ca2+ signaling in the PL-driven endothelial healing process. In our experiments, the functional significance of Ca2+ signaling machinery was highlighted performing the scratch wound assay in presence of different inhibitors or specific RNAi. We also pointed out that the PL-induced generation of intracellular ROS (reactive oxygen species) via NOX4 (NADPH oxidase 4) is necessary for the activation of TRPM2 and the resulting Ca2+ entry from the extracellular space. This is the first report of the mechanism of wound repair in an endothelial cell model boosted by the PL-induced regulation of [Ca2+]i.

Background Within the past years, umbilical cord (UC) and amniotic membrane (AM) expanded in human platelet lysate (PL) have been found to become increasingly candidate of mesenchymal stromal cells (MSCs) in preclinical and clinical studies. Different sources of MSCs have different properties, and lead to different therapeutic applications. However, the similarity and differences between the AMMSCs and UCMSCs in PL remain unclear. Results In this study, we conduct a direct head-to-head comparison with regard to biological characteristics (morphology, immunophenotype, self-renewal capacity, and trilineage differentiation potential) and immunosuppression effects of AMMSCs and UCMSCs expanded in PL. Our results indicated that AMMSCs showed similar morphology, immunophenotype, proliferative capacity and colony efficiency with UCMSCs. Moreover, no significantly differences in osteogenic, chondrogenic and adipogenic differentiation potential were observed between the two types of cells. However, AMMSCs exhibited higher PGE2 expression and IDO activity compared with UCMSCs when primed by IFN-γ and (or) TNF-α induction, and AMMSCs showed a higher inhibitory effect on PBMCs proliferation than UCMSCs. Conclusion The results suggest that AMMSCs expanded in PL showed similar morphology, immunophenotype, self-renewal capacity, and trilineage differentiation potential with UCMSCs. However, AMMSCs possessed superior immunosuppression effects in comparison with UCMSCs. These results suggest that AMMSCs in PL might be more suitable than UCMSCs for treatment of immune diseases. This work provides a novel insight into choosing the appropriate source of MSCs for treatment of immune diseases. Electronic supplementary material The online version of this article (10.1186/s12860-018-0178-8) contains supplementary material, which is available to authorized users.

Mesenchymal stem cells (MSCs) have immense potential for cell-based therapy of acute and chronic pathological conditions. MSC transplantation for cell-based therapy requires a substantial number of cells in the range of 0.5–2.5 × 106 cells/kg body weight of an individual. A prolific source of MSCs followed by in vitro propagation is therefore an absolute prerequisite for clinical applications. Umbilical cord tissue (UCT) is an abundantly available prolific source of MSC that are fetal in nature and have higher potential for ex-vivo expansion. However, the ex-vivo expansion of MSCs using a xenogeneic supplement such as fetal bovine serum (FBS) carries the risk of transmission of zoonotic infections and immunological reactions. We used platelet lysate (PL) as a xeno-free, allogeneic replacement for FBS and compared the biological and functional characteristics of MSC processed and expanded with PL and FBS by explant and enzymatic method. UCT-MSCs expanded using PL displayed typical immunophenotype, plasticity, immunomodulatory property and chromosomal stability. PL supplementation also showed 2-fold increase in MSC yield from explant culture with improved immunomodulatory activity as compared to enzymatically dissociated cultures. In conclusion, PL from expired platelets is a viable alternative to FBS for generating clinically relevant numbers of MSC from explant cultures over enzymatic method.

Human multipotent mesenchymal stromal cells (MSC) are isolated from a plethora of tissue sources for cell therapy purposes. In 2006, the International Society for Cellular Therapy (ISCT) published minimal guidelines to define MSC identity. Nevertheless, many independent studies demonstrated that cells meeting the ISCT criteria possessed heterogeneous phenotypes and functionalities, heavily influenced by culture conditions. In this study, human MSC derived from many adult (bone marrow and adipose tissue) or fetal (cord blood, Wharton’s jelly, umbilical cord perivascular compartment and amniotic fluid) tissues were investigated. Their immunophenotype was analyzed to define consistent source-specific markers by extensive flow cytometry analysis and real-time qRT-PCR. CD271+ subpopulations were detected in adult MSC, whereas NG2 was significantly more expressed in fetal MSC but failed validation on independent samples coming from an external laboratory. The highest number of CD271+ adult MSC were detected soon after isolation in serum-based culture conditions. Furthermore, heterogeneous percentages of CD271 expression were found in platelet lysate-based or serum-free culture conditions. Finally, CD271+ adult MSC showed high clonogenic and osteogenic properties as compared to CD271− cells. To conclude, in this phenotype-function correlation study CD271+ subpopulation confers heterogeneity on adult MSC, confirming the need of more specific markers to address MSC properties.

Background: Thrombocytosis in breast cancer (BC) patient was thought to play a role in the invasiveness of breast cancer stem cells (BCSCs). Modification of tumor microenvironment was proposed to increase the efficacy of anticancer therapy. This study was aimed to analyze the effect of platelet lysate (PL) as well as its PDGF-AB content as a tumor microenvironment on (CD24-/CD44+) BCSC proliferation.Methods: This was an experimental study that treated culture of BCSCs with PL from breast cancer (BC) patients or healthy donors. Venous blood from all subjects were subjected to prior hematology test and then processed to obtain platelet rich plasma (PRP). Platelet counts in PRP were determined. PRP was processed to obtain PL. PDGF-AB contents in PL were measured. PL at concentrations of 0.01% (v/v) was supplemented into DMEM-F12 medium and used for culturing BCSCs (CD24-/CD44+ cells). After 48 hours, total cell count, population doubling time (PDT), and cell viability were calculated and their correlation with platelet count and PDGF-AB levels were analyzed.Results: BC patients (n=5) had higher platelet counts and PDGF-AB levels in PL compared to healthy donors (n=15), (p=0.02). PL from BC patients could stimulate the proliferation of BCSCs higher than healthy donors (p

Purpose Articular cartilage has a poor capacity for self-repair, and thus still presents a major challenge in orthopedics. Mesenchymal stem cells (MSCs) are multipotent stem cells with the potential to differentiate into chondrocytes in the presence of transforming growth factor beta (TGF-β). Platelet lysate (PL) contains a relatively large number of growth factors, including TGF-β, and has been shown to ameliorate cartilage repair. Here, we investigated the ability of PL to direct chondrogenic differentiation of MSCs along with other standard differentiation components in a pellet culture system. Methods We isolated and expanded MSCs from human umbilical cords using a PL-supplemented medium and characterized the cells based on immunophenotype and potential for differentiation to adipocytes and osteocytes. We further cultured MSCs as pellets in a chondrogenic-differentiation medium supplemented with PL. After 21 days, the pellets were processed for histological analysis and stained with alician blue and acridine orange. The expression of SOX9 was investigated using RT-PCR. Results MSCs maintained their stemness characteristics in the PL-supplemented medium. However, the distribution of cells in the pellets cultured in the PL-supplemented chondrogenic differentiation medium had a greater similarity to cartilage tissue-derived chondrocytes than to the negative control. The intense alician blue staining indicated an increased production of mucopolysaccharides in the differentiated pellets, which also showed elevated expression of SOX9. Conclusions Our data suggest that MSCs could be differentiated to chondrocytes in the presence of PL and absence of exogenous TGF-β. Further research needs to be conducted to understand the exact role and potential of PL in chondrogenic differentiation and chondrocyte regeneration.

Mesenchymal stem cells (MSCs) may be easily isolated from the bone marrow, and possess multi-lineage differentiation potential and various therapeutic applications. The differentiation of MSCs into osteoblasts is a complex process that is regulated by multiple internal and external factors. In the present study, the differentiation of MSCs isolated from rabbit bone marrow into osteoblasts using different osteoblast inductive media in the presence of dexamethasone, bone morphogenetic protein 2 (BMP-2), 1,25-dihydroxyvitamin D3, transforming growth factor β (TGFβ), platelet lysate and cyclooxygenase 2 (COX2), respectively. Alkaline phosphatase (ALP) activity, mineralization, collagen type (Ct) I and osteocalcin activities, and the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), BMP-2 and Ct II were measured during the differentiation process in MSCs treated with different inducers. Rabbit MSCs were successfully isolated and were observed to be predominantly circular in shape after culture for 24 h. Following subculture for 5 days, the cells demonstrated a spindle shape. ALP, Ct I and osteocalcin activities were higher in cells cultured in dexamethasone, BMP-2 and TGFβ compared with the activities in control cells. Following differentiation, the dexamethasone, BMP-2 and TGFβ groups demonstrated significantly enhanced mineralization of MSCs detected by Alizarin Red S staining. The mRNA and protein expression levels of VEGF, BMP-2 and Ct II were significantly increased in the same groups compared with the levels in the control group. In conclusion, rabbit MSCs were successfully isolated from bone marrow and differentiated into osteoblasts indicated by raised ALP, Ct I and osteocalcin activities, mineralization and expression of osteogenesis-inducing genes and proteins. The present study revealed that dexamethasone, BMP-2 and TGFβ have a positive effect on cell differentiation.

Background Human platelet lysates (HPLs) are emerging as the new gold standard supplement of growth media for ex vivo expansion of cells for transplant. However, variations do exist in the way how HPLs are prepared. In particular, uncertainties still exist regarding the type of HPL most suitable for corneal endothelium cells (CEC) expansion, especially as these cells have limited proliferative capacity. Material and methods Three distinct HPL preparations were produced, with or without calcium chloride/glass beads activation, and with or without heat treatment at 56 °C for 30 min. These HPLs were used to supplement basal D-MEM growth medium, each at a protein concentration equivalent to that of 10% fetal bovine serum (FBS; control). Impact on CEC (BCE C/D-1b cells) in vitro morphology, viability and capacity to express Zonula occludens-1 (ZO-1) tight junction marker was assessed by Western blotting. Results BCE C/D-1b cells grown in all HPL supplements exhibited four of essential characteristic properties: adhesion capacity, microscopic morphology and viability similar to that observed when using 10% FBS. In addition, Western blots analysis revealed an expression of the ZO-1 marker by BCE C/D-1b cells in all conditions of culture. Conclusion CECs can expand ex vivo in a basal medium supplemented with the three HPLs without noticeable difference compared to FBS supplement. These data support further studies to evaluate the potential to use HPLs as a clinical-grade xeno-free supplement of CEC for corneal transplant.

Objectives Adipose-derived mesenchymal stem cells (ADMSCs) are a promising strategy for orthopaedic applications, particularly in bone repair. Ex vivo expansion of ADMSCs is required to obtain sufficient cell numbers. Xenogenic supplements should be avoided in order to minimise the risk of infections and immunological reactions. Human platelet lysate and human plasma may be an excellent material source for ADMSC expansion. In the present study, use of blood products after their recommended transfusion date to prepare human platelet lysate (HPL) and human plasma (Hplasma) was evaluated for in vitro culture expansion and osteogenesis of ADMSCs. Methods Human ADMSCs were cultured in medium supplemented with HPL, Hplasma and a combination of HPL and Hplasma (HPL+Hplasma). Characteristics of these ADMSCs, including osteogenesis, were evaluated in comparison with those cultured in fetal bovine serum (FBS). Results HPL and HPL+Hplasma had a significantly greater growth-promoting effect than FBS, while Hplasma exhibited a similar growth-promoting effect to that of FBS. ADMSCs cultured in HPL and/or Hplasma generated more colony-forming unit fibroblasts (CFU-F) than those cultured in FBS. After long-term culture, ADMSCs cultured in HPL and/or Hplasma showed reduced cellular senescence, retained typical cell phenotypes, and retained differentiation capacities into osteogenic and adipogenic lineages. Conclusion HPL and Hplasma prepared from blood products after their recommended transfusion date can be used as an alternative and effective source for large-scale ex vivo expansion of ADMSCs. Cite this article: J. Phetfong, T. Tawonsawatruk, K. Seenprachawong, A. Srisarin, C. Isarankura-Na-Ayudhya, A. Supokawej. Re-using blood products as an alternative supplement in the optimisation of clinical-grade adipose-derived mesenchymal stem cell culture. Bone Joint Res 2017;6:414–422. DOI: 10.1302/2046-3758.67.BJR-2016-0342.R1.

Objective:To determine the optimal environmental conditions for chondrogenic differentiation of human adipose tissue–derived mesenchymal stromal/stem cells (AMSCs). In this investigation we specifically investigate the role of oxygen tension and 3-dimensional (3D) culture systems.Design:Both AMSCs and primary human chondrocytes were cultured for 21 days in chondrogenic media under normoxic (21% oxygen) or hypoxic (2% oxygen) conditions using 2 distinct 3D culture methods (high-density pellets and poly-ε-caprolactone [PCL] scaffolds). Histologic analysis of chondro-pellets and the expression of chondrocyte-related genes as measured by reverse transcriptase quantitative polymerase chain reaction were used to evaluate the efficiency of differentiation.Results:AMSCs are capable of expressing established cartilage markers including COL2A1, ACAN, and DCN when grown in chondrogenic differentiation media as determined by gene expression and histologic analysis of cartilage markers. Expression of several cartilage-related genes was enhanced by low oxygen tension, including ACAN and HAPLN1. The pellet culture environment also promoted the expression of hypoxia-inducible cartilage markers compared with cells grown on 3D scaffolds.Conclusions:Cell type–specific effects of low oxygen and 3D environments indicate that mesenchymal cell fate and differentiation potential is remarkably sensitive to oxygen. Genetic programming of AMSCs to a chondrocytic phenotype is effective under hypoxic conditions as evidenced by increased expression of cartilage-related biomarkers and biosynthesis of a glycosaminoglycan-positive matrix. Lower local oxygen levels within cartilage pellets may be a significant driver of chondrogenic differentiation.

Human mesenchymal stem cells (MSCs) are promising candidates for therapeutic applications such as tissue engineering. However, one of the main challenges is to improve oxygen supply to hypoxic areas to reduce oxygen gradient formation while preserving MSC differentiation potential and viability. For this purpose, a marine hemoglobin, HEMOXCell, was evaluated as an oxygen carrier for culturing human bone marrow MSCs in vitro for future three-dimensional culture applications. Impact of HEMOXCell on cell growth and viability was assessed in human platelet lysate (hPL)-supplemented media. Maintenance of MSC features, such as multipotency and expression of MSC specific markers, was further investigated by biochemical assays and flow cytometry analysis. Our experimental results highlight its oxygenator potential and indicate that an optimal concentration of 0.025 g/L HEMOXCell induces a 25%-increase of the cell growth rate, preserves MSC phenotype, and maintains MSC differentiation properties; a two-fold higher concentration induces cell detachment without altering cell viability. Our data suggest the potential interest of HEMOXCell as a natural oxygen carrier for tissue engineering applications to oxygenate hypoxic areas and to maintain cell viability, functions and "stemness." These features will be further tested within three-dimensional scaffolds.

BACKGROUND Over the past decades, the focus on the regenerative properties of platelets (PLTs) has intensified and many PLT-derived growth factors are readily used in medical settings. A general lack of standardization in the preparation of these growth factors remains, however, and this study therefore examines the dynamics of growth factors throughout the freeze-thaw procedure. STUDY DESIGN AND METHODS Plateletpheresis (PA) and PLT-poor plasma (PPP) samples were collected from 10 healthy donors. PA was lysed to produce PLT lysate (PL) for 1, 3, 5, 10, and 30 freeze-thaw cycles. The resulting growth factor and cytokine concentrations from PPP, PA, and PL of different cycles were analyzed and compared using enzyme-linked immunosorbent assay and multiplex bead assays. RESULTS PL produced by the freeze-thaw procedure resulted in approximately four- to 10-fold enrichment of transforming growth factor-β1, epidermal growth factor, PLT-derived growth factor (PDGF)-AB/BB, PLT factor-4, and fibroblast growth factor-2. The increase in concentrations plateaued at Cycles 3 and 5 and in some cases declined with further cycles. The concentrations of insulin-like growth factor-1, hepatocyte growth factor, vascular endothelial growth factor, and bone morphogenetic protein-2 in PL were essentially comparable to those in PPP. CONCLUSION Using the freeze-thaw method, optimal preparation of PL with regard to the concentration of growth factors was achieved at Cycles 3 to 5. Based on our findings, the clinical significance of using a greater number of cycles is likely limited.

Background Platelet lysates (PL) represent a promising replacement for xenogenic growth supplement for adipose-derived stem cell (ASC) expansions. However, fresh platelets from human blood donors are not clinically feasible for large-scale cell expansion based on their limited supply. Therefore, we tested PLs prepared via three methods from outdated buffy coat–derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. Methods PLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat–derived PCs stored in the platelet additive solution, InterSol. Three types of PLs were prepared from outdated PCs with platelets suspended in either (1) InterSol (not manipulated), (2) InterSol + supplemented with plasma or (3) plasma alone (InterSol removed). Using these PLs, we compared ASC population doubling time, cell yield, differentiation potential and cell surface markers. Gene expression profiles were analyzed using microarray assays, and growth factor concentrations in the cell culture medium were measured using enzyme-linked immunosorbent assay (ELISA). Results Of the three PL compositions produced from outdated PCs, removal of Intersol and resuspension in plasma prior to the first freezing process was overall the best. This specific outdated PL induced ASC growth kinetics, surface markers, plastic adherence and differentiation potentials comparable with PL from fresh platelets. ASCs expanded in PL from fresh versus outdated PCs exhibited different expressions of 17 overlapping genes, of which 10 were involved in cellular proliferation, although not significantly reflected by cell growth. Only minor differences in growth factor turnover were observed. Conclusion PLs from outdated platelets may be an efficient and reliable source of human growth supplement allowing for large-scale ASC expansion for clinical use.

The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition.

Background: Multiple harvest explant method (MHEM) derived umbilical cord mesenchymal stem cells (UC-MSCs) was shown to undergo senescent beginning at passage-10 (P-10). However, for the same cells, there are no senescent data after cryopreservation and passage. Therefore, this study aimed to analyze the senescent profile of cryopreserved MHEM derived UC-MSCs after serial passages. Methods: MHEM derived UC-MSCs were isolated and cultured in platelet lysate (PL) containing medium as described previously. The cells were cryopreserved at passage-1 (P-1) in 10% dimethyl sulfoxide (DMSO) and 10% PL containing alpha minimal essential medium (αMEM). Cell density at cryopreservation was 500 000 cells/mL. After one month, the cells were thawed and recultured until P-8 in six 12-well plate. At 80-90% confluent, two wells were harvested and the cells were recultured into six wells, and the four remaining wells were subjected to senescent (β-galactosidase) staining, which was done for all passages. Random photographs were taken from all stained wells, and senescent percentage was recorded. Results: No senescent cells were observed at P-2. Senescent cells began to appear at P-3. Percentage (mean ± SD) of senescent cells from P-3 through P-8 were 0.04 ± 0.02, 1.18 ± 1.82, 0.14 ± 0.18, 0.49 ± 0.00, 0.58 ±0.91, and 0.07 ± 0.07, respectively. Results: No senescent cells were observed at P-2. Senescent cells began to appear at P-3. Percentage (mean ± SD) of senescent cells from P-3 through P-8 were 0.04 ± 0.02, 1.18 ± 1.82, 0.14 ± 0.18, 0.49 ± 0.00, 0.58 ±0.91, and 0.07 ± 0.07, respectively. Conclusion: No senescent cells were observed at P-2 (cumulative population doubling [CPD] > 9.38). Senescent cells began to appear at P-3 (CPD > 14.06), but in all passages until P-8 (CPD >34.34) the senescent percentage was below 5%.

Background & Aim Background The increasing request for culture expanded mesenchymal stromal cells (MSC) in the last years discloses the need to reduce costs and man power in the manufacturing process. This can be achieved by the use of closed, (semi-)automated bioreactor systems. We have decided to test a functionally closed hollow-fibre bioreactor system (Quantum®). Surveillance of metabolic parameters and adaptation of feeding in dependence of these is possible. However, the nature of the hollow-fibre surface does not support attachment of MSC without coating. Several biological substances for coating are available, either as isolated proteins (e.g. fibronectin, vitronectin), or as complex protein mixtures derived from human blood (e.g. cryoprecipitate, platelet lysate). The use of such closed systems additionally reduces the risk of contaminations. Methods, Results & Conclusion Methods MSC were cultured as conventional cell culture or in a platelet lysate (PL), cryoprecipitate (CP), vitronectin (VN), or fibronectin (FN) coated Quantum® bioreactor (TerumoBCT) for 5 - 15 days in alphaMEM (Lonza), supplemented with 5% - 10% platelet lysate (PL from IKT Ulm) and 1 i.u. heparin (B.Braun). The surface of the Quantum® bioreactor is 21,000 cm2. For Quantum®, the feed rate was adjusted based on daily measured lactate or glucose levels. For conventional cell culture, mainly 2 chamber CellSTACK® (Corning) with a surface of 1272 cm2 were used and complete medium exchange was performed twice a week. TrypZEAN® (Lonza) was used for detachment of cells. Results For results of the cell expansion experiments (N ≥ 3) using the different coating reagents refer to Table 1. Conclusion We showed that the Quantum® bioreactor is suitable for expansion of MSC using PL, CP, FN or VN as coating reagent. Despite higher doubling times for cells grown in the Quantum®, this system may help to save costs since less man power is needed to manage the system and several steps do not necessarily have to be performed in a class A cabinet. In addition, clinically relevant cell numbers could be achieved by the use of a single bioreactor (data not shown). Perspective Protocols for cell expansion in the Quantum® still may have to be optimized in order to increase its potential in saving man power and consumables.

Background and Purpose: Sporotrichosis is a subcutaneous and chronic fungal infection that is caused by a dimorphic fungus, namely Sporothrix schenckii sensu lato. Lymphocutaneous sporotrichosis is the most clinical form, which accounts for nearly 80% of the cases of cutaneous sporotrichosis. Platelets contain several substances with antimicrobial properties. Regarding this, the present study was performed to investigate the effect of blood-based biomaterials, especially platelets in the treatment of lymphocutaneous sporotrichosis. Materials and Methods: This study was performed on 12 golden hamsters, divided into three groups of control, platelet-rich plasma, and platelet lysate. For the purpose of the study, Sporothrix conidia suspension was injected subcutaneously on the back of the animals. After the induction of subcutaneous lesions, the Gomori methenamine silver method was applied to verify lymphocutaneous sporotrichosis. Subsequently, plasma-rich platelet and platelet lysate were injected into the created lesions in the animals in 3-day intervals (due to the short lifetime of platelets). In the final sage, skin tissue samples were examined to check for the presence of yeast cells and their quantification. Results: The data were indicative of the presence of yeast cells with/without bud in the tissue of lymphocutaneous sporotrichosis lesions in the infected animals. Histological investigation revealed that each of the two biomaterials under study (i.e., plasma-rich platelet and platelet lysate) played a positive role in the removal of the yeast cells of sporotrichosis. Conclusion: The results of this study showed that both plasma-rich platelet and platelet lysate were able to effectively prevent from the progression of cutaneous sporotrichosis. Accordingly, much attention has been given to new therapies, including treatment with blood-derived biomaterials.

The mechanobiological behavior of mesenchymal stem cells (MSCs) in two- (2D) or three-dimensional (3D) cultures relies on the formation of actin filaments which occur as stress fibers and depends on mitochondrial dynamics involving vimentin intermediate filaments. Here we investigate whether human platelet lysate (HPL), that can potentially replace fetal bovine serum for clinical-scale expansion of functional cells, can modulate the stress fiber formation, alter mitochondrial morphology, change membrane elasticity and modulate immune regulatory molecules IDO and GARP in amnion derived MSCs. We can provide evidence that culture supplementation with HPL led to a reduction of stress fiber formation in 2D cultured MSCs compared to a conventional growth medium (MSCGM). 3D MSC cultures, in contrast, showed decreased actin concentrations independent of HPL supplementation. When stress fibers were further segregated by their binding to focal adhesions, a reduction in ventral stress fibers was observed in response to HPL in 2D cultured MSCs, while the length of the individual ventral stress fibers increased. Dorsal stress fibers or transverse arcs were not affected. Interestingly, ventral stress fiber formation did not correlate with membrane elasticity. 2D cultured MSCs did not show differences in the Young’s modulus when propagated in the presence of HPL and further cultivation to passage 3 also had no effect on membrane elasticity. In addition, HPL reduced the mitochondrial mass of 2D cultured MSCs while the mitochondrial mass in 3D cultured MSCs was low initially. When mitochondria were segregated into punctuate, rods and networks, a cultivation-induced increase in punctuate and network mitochondria was observed in 2D cultured MSCs of passage 3. Finally, mRNA and protein expression of the immunomodulatory molecule IDO relied on stimulation of 2D culture MSCs with pro-inflammatory cytokines IFN-γ and TNF-α with no effect upon HPL supplementation. GARP mRNA and surface expression was constitutively expressed and did not respond to HPL supplementation or stimulation with IFN-γ and TNF-α. In conclusion, we can say that MSCs cultivated in 2D and 3D are sensitive to medium supplementation with HPL with changes in actin filament formation, mitochondrial dynamics and membrane elasticity that can have an impact on the immunomodulatory function of MSCs.

Fetal bovine serum (FBS) is widely used to culture mesenchymal stem cells (MSCs) in the laboratory; however, FBS has been linked to adverse immune-mediated reactions prompting the search for alternative cell culture medium. Platelet lysate (PL) as an FBS substitute has been shown to promote MSCs growth without compromising their functionality. Fibrinogen contained in PL has been shown to negatively impact the immune modulating properties of MSCs; therefore, we sought to deplete fibrinogen from PL and compare proliferation, viability, and immunomodulatory capacities of MSCs in FBS or PL without fibrinogen. We depleted fibrinogen from equine platelet lysate (ePL) and measured platelet-derived growth factor-beta (PDGF-β), transforming growth factor-beta (TGF-β) and tumor necrosis factor-alpha (TNF-α) through ELISA. First, we determined the ability of 10% ePL or fibrinogen-depleted lysate (fdePL) compared with 10% FBS to suppress monocyte activation by measuring TNF-α from culture supernatants. We then evaluated proliferation, viability, and immunomodulatory characteristics of bone marrow-derived MSCs (BM-MSCs) cultured in FBS or ePL with or without fibrinogen. Growth factor concentrations decreased in ePL after fibrinogen depletion. Lipopolysaccharide (LPS)-stimulated monocytes exposed to ePL and fdePL produced less TNF-α than LPS-stimulated monocytes in 10% FBS. BM-MSCs cultured in fdePL exhibited lower proliferation rates, but similar viability compared with BM-MSCs in ePL. BM-MSCs in fdePL did not effectively suppress TNF-α expression from LPS-stimulated monocytes compared with BM-MSCs in FBS. Depleting fibrinogen results in a lysate that suppresses TNF-α expression from LPS-stimulated monocytes, but that does not support proliferation and immune-modulatory capacity of BM-MSCs as effectively as nondepleted lysate.

AimPlatelets provide substantial information about the proteolytic system profile in neurodegenerative diseases. Assessment of autophagy and proteasome target proteins in platelets may reflect tissue proteolytic machinery profile in central nervous system in Alzheimer’s diseases (AD). We aimed to demonstrate the optimum assay conditions and identify target proteins in platelet proteolytic machinery.MethodsPlatelet samples were obtained from clinically verified AD patients and age-matched non-demented control subjects that were recruited by University of Kansas Alzheimer’s disease Center.Autophagosome participating proteins in platelets were identified by Western blotting analysis. Standard gel electrophoresis and electro transfer apparatus were used for protein transfer onto the membrane. Several antibodies were tested to identify the best working antibodies, and their concentrations were optimized. An ELISA kit was used for platelet proteasome protein determination. Infrared imaging technology was used for visualizing the proteins on the membrane.ResultsAutophagosome participating proteins showed elevated levels in AD patient platelet cytosol. Only LC3-I autophagosome protein levels were significantly elevated. The concentrations of platelet lysate proteasome were assessed. AD patient’s proteasome levels were elevated but they were statistically not important as compared to controls.ConclusionsPlatelets can be used for assessing whether proteolytic system is functional. Blood-based sampling from human donors is less-invasive and analyzing platelet proteolytic system profile may help to develop pharmaceutical intervention approaches for neurodegenerative diseases in general.

Platelet lysate (PL), a blood product that contains high concentrations of growth factors (GFs), can be considered as a cost-effective source of multiple GFs. In this study, hyaluronic acid (HA) based microgels were developed for delivery of PL proteins. Spherical microgel were prepared using a water in oil emulsion method. First, hyaluronic acid was grafted with tyramine groups, after which prepared microdroplets were crosslinked via an enzymatic reaction in the presence of hydrogen peroxide and horseradish peroxidase. Because of electrostatic interactions, these microgels are promising carriers for positively charged proteins entrapment like most of the GFs. When microgels are incubated in PL solution, protein loading takes place which is mainly governed by nonspecific adsorption of plasma proteins. Although this hampered loading efficiency, loading could be increased by repeated washing and incubation steps. The loaded microgels presented a sustained release of PL growth factors for a period of two weeks. When PL enriched microgels were embedded in a HA bulk hydrogel, cell proliferation was higher compared to constructs without microgels. These findings suggest that the developed microgels are a potential candidate for sustained delivery of PL growth factors and present a solution to the issue of their short half-lives in vivo.

Some recent reports suggest that cryopreserved and thawed mesenchymal stromal cells (MSCs) may have impaired functional properties as compared to freshly harvested MSCs from continuous cultures. A cryopreservation step in the manufacturing process brings important benefits, since it enables immediate off-the-shelf access to the products and a completion of all quality testing before batch release and administration to the patient. Cryopreservation is also inevitable in MSC banking strategies. In this study, we present the results from the MSC stability testing program of our in-house manufactured clinical-grade allogeneic bone marrow-derived MSC product that is expanded in platelet lysate and frozen in passage 2. The current manufacturing protocol contains only one freezing step and the frozen MSC product is thawed bed-side at the clinic. We can conclude superior viability and cell recovery of the frozen and thawed MSC product utilizing the validated freezing and thawing protocols we have developed. The MSC phenotype and differentiation potential was generally found to be unaltered after thawing, but the thawed cells exhibited a 50% reduced, but not completely abolished, performance in an in vitro immunosuppression assay. The in vitro immunosuppression assay results should, however, be interpreted with caution, since the chosen assay mainly measures one specific immunosuppressive mechanism of MSCs to suppress T-cell proliferation. Since at least two freezing steps are usually necessary in MSC banking strategies, we went on to investigate the impact of repeated freezing on MSC quality attributes. We can conclude that two freezing steps with a preceding cell culture phase of at least one passage before freezing is feasible and does not substantially affect basic cell manufacturing parameters or quality attributes of the final frozen and thawed product. Our results suggest, however, that an exhaustive number of freezing steps (≥4) may induce earlier senescence. In conclusion, our results support the utilization of frozen MSC products and MSC banking strategies, but emphasize the need of always performing detailed studies on also the cryopreserved MSC counterpart and to carefully report the cryopreservation and thawing protocols.

Propagation ex vivo of mesenchymal stem cells (MSCs) requires culture medium supplementation. Fetal bovine serum (FBS) has long been the gold standard supplement, but its use is being questioned mainly due to ethical and safety issues. The use of platelet lysate (PL) as substitute of FBS has been proposed but little is known about its effects on equine MSCs characteristics including their immune profile. The aim of this work was to investigate for the first time the effect of allogenic PL on the immunogenic and immunomodulatory gene expression profile of equine bone marrow derived MSCs (eBM-MSCs) as well as on their proliferation ability, phenotype markers, and viability post-cryopreservation. The eBM-MSCs (n = 3) cultures were supplemented with 20% of allogeneic pooled concentrated PL (CPL; 591 × 103 platelets/μL) or basal PL (BPL; 177 × 103 platelets/μL) from three donors, using 10% FBS supplementation as control. The proliferative ability of eBM-MSCs under the three conditions was evaluated by calculating the cell doubling times (DT) up to passage 3 (P3) and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at P3. Viability of eBM-MSCs post-cryopreserved with CPL or FBS was assessed at 15, 30 and 60 days. The gene expression profile of eBM-MSCs was evaluated in P3 by RT-qPCR for characterization, immunogenic and immunomodulatory markers. The cells cultured in CPL had significantly higher ability to proliferate than with FBS or BPL (P

Previously, we have shown platelet lysate (PL) can be used as a non-xenogeneic serum supplement for generation of monocyte-derived dendritic cells (DCs). Since DC-based activation protocols are extremely sensitive to microenvironmental changes such as replacement of culture medium, we wanted to examine the behavior of DCs cultured in the presence of PL under various type-1 activation conditions and assess their type 1 polarization capacity. We compared the quality of DCs cultured in 10% PL-supplemented RPMI medium (plDCs) with clinical-grade DCs obtained using commercially available serum-free medium (sfDCs), frequently used in established DC vaccine protocols. The DC maturation protocols consisted of either monophosphoryl lipid A/IFN-γ, poly I:C/TNF-α/IFN-α or poly I:C/R848. In general, plDCs were inferior to sfDCs in most aspects of their functional type 1 polarization characteristics. After maturation, the expression of co-stimulatory, HLA class II and lymph node-homing molecules was strongly up-regulated, with some noticeable differences. The expression of CD80 and CD86 was more extensive on plDCs, which was particularly evident in case of CCR7. However, after observing their functional capacity, plDCs had significantly lower allo-stimulatory capacity both in terms of CD4+ and CD8+ T cell stimulation. The high expression of CCR7 corresponded to higher CCL-19 directed DC migration of plDCs compared to sfDCs. Finally, their capacity to induce granzyme B and IFN-γ production in CD8+ T cells was significantly reduced in comparison to sfDCs. Based on these findings, the use of PL as an alternative serum supplement for generation of monocyte-derived DC anti-tumor vaccines is questionable.Abbreviations: Ag: antigen; CCL: chemokine ligand; CCR: chemokine receptor; DC: dendritic cells; DC-SIGN: dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; FBS: fetal bovine serum; GMP: good manufacturing practice; IFN: interferon; IL: interleukin; MPLA: monophosphoryl lipid A; PGE: prostaglandin E; pI:C: polyinosinic:polycytidylic acid; pl: platelet lysate; sf: serum free; TLR: toll-like receptor; TNF: tumor necrosis factor.

Altres ajuts: BST is a member of the Spanish Cell Therapy Network (Red de Terapia Celular, TerCel, expedient No. RD16/0011/0028), awarded by the Generalitat de Catalunya as Consolidated Research Group and developed in the context of AdvanceCat with the support of ACCIÓ (Catalonia Trade & Investment; Generalitat de Catalunya) under the Catalonian ERDF operational program (European Regional Development Fund) 2014-2020. Background: Contrary to the minimal criteria proposed by the International Society for Cell and Gene Therapy for defining multipotent mesenchymal stromal cells (MSC), human leukocyte antigen (HLA)-DR expression is largely unpredictable in ex vivo-expanded clinical-grade cultures. Although activation of MSC in culture does not appear to affect their functionality, a large study investigating the impact of HLA-DR expression on cell identity and potency is still missing in the literature. Methods: A retrospective analysis of HLA-DR expression in 130 clinical batches of bone marrow (BM)-MSC from two independent Good Manufacturing Practice-compliant production facilities was performed in order to identify the consequences on critical quality attributes as well as potential activation cues and dynamics of MSC activation in culture. Results: HLA-DR cells in culture were confirmed to maintain fibroblastic morphology, mesenchymal phenotype identity, multipotency in vitro, and immunomodulatory capacity. Interestingly, the use of either human sera or platelet lysate supplements resulted in similar results. Conclusions: HLA-DR expression should be considered informative rather than as a criterion to define MSC. Further work is still required to understand the impact of HLA-DR expression in the context of product specifications on BM-MSC qualities for clinical use in specific indications.

Off-the-shelf availability of human adipose-derived mesenchymal stromal cells (ASCs) for regenerative medicine application requires the development of nontoxic, safe, and efficient protocols for cryopreservation. Favorably, such cell processing protocols should not contain xenogeneic or toxic components, such as fetal bovine serum (FS) and dimethyl sulfoxide (DMSO). The objective of the study was to assess the sensitivity of ASCs to DMSO-free cryopreservation protocol depending on their expansion conditions: conventional, based on the application of FS or xeno-free, using PL as a medium supplement. ASCs expansion was carried out in α-MEM supplemented either with FS or PL. For DMSO- and xeno-free cryopreservation ASCs were pretreated with different concentrations of sucrose during 24 h of culture. Pretreated ASCs were cryopreserved in α-MEM containing 100–300 mM of sucrose with the cooling rate of 1 degree/min. ASCs were tested for survival (Trypan Blue test), viability (MTT test), recovery (Alamar Blue test), proliferation and ability to multilineage differentiation. The optimal concentrations of sucrose for ASCs pretreatment and as an additive in cryoprotective solution, which provided highest cell survival, comprised 100 and 200 mM, correspondingly. Survival and recovery rates of platelet lysate (PL)-expanded ASCs after DMSO-free cryopreservation comprised 59 and 51%, and were higher than in FS-cultured cells. After DMSO-free cryopreservation PL-processed ASCs had a shorter population doubling time and higher capacity for osteogenic differentiation than FS-processed cultures. The described DMSO- and xeno-free processing may form the basis for the development of safe and efficient protocols for manufacturing and banking of ASCs, providing their off-the-shelf availability for regenerative medicine applications.

Polyvalent immunoglobulins, platelet lysate and lenalidomide: cocktail for polyfunctional NK cells expansion for multiple myeloma