• OCT-4
• bone marrow cell culture
• de-differentiation
• macrophages
• mesangiogenic progenitor cells
• monocytes
• nanog
• trans-differentiation

• component processing
• human platelet lysate
• method development

• FBS-free
• animal component-free
• cell cultures
• chemically defined medium
• fetal bovine serum
• human platelet lysate
• multicellular models
• reproducibility crisis
• transition
• xeno-free

• Animals
• Cattle
• Cell Culture Techniques/methods
• Cell Differentiation
• Cell Proliferation
• Cells, Cultured
• Humans
• Mesenchymal Stem Cells
• Reproducibility of Results
• Trypsin

• myelofibrosis
• osteoblast
• osteosclerosis
• platelet
• secretome

• PRGF
• cell culture
• cell therapy
• platelet-rich plasma
• regenerative medicine
• stem cells
• xenogeneic supplements

Mesenchymal stem cell (MSC) culturing for cell therapies needs a step forward to be routinely used in clinical settings. Main concerns regard the use of animal origin reagents, in particular supplementing the culture medium with FBS. Lately, Human Platelet Lysate (HPL) has been proposed as animal-free alternative, described as an excellent supplement for culturing MSCs. The aim of this systematic review was to analyze the current literature on the effect of HPL and FBS on ASCs and BMSCs. The primary outcome was the proliferation rate of cells cultured with FBS and HPL. Differences in terms of doubling time (DT) and population doubling (PD) were evaluated by meta-analysis, subgrouping data according to the cell type. A total of 35 articles were included. BMSCs and ASCs were used in 65.7% (23) and 28.6% (10) studies, respectively. Only two studies included both cell types. Overall, 22 studies were eligible for the meta-analysis. Among them, 9 articles described ASCs and 13 BMSCs. The results showed that BMSCs and ASCs cultured with 10% HPL and 5% HPL have lower DT and higher PD compared to cells cultured with 10% FBS. A possible correlation between the DT decrease and the application of at least 3 freeze/thaw cycles to induce platelet lysis was found. Additionally, HPL increased VEGF secretion and maintained the immuno-modulatory abilities for both cell types. The clarification reported here of the higher efficiency of HPL compared to FBS can help the transition of the scientific community towards clinical-related procedures.

• Cell proliferation
• Fetal bovine serum (FBS)
• Freeze/thaw cycles
• Human platelet lysate (HPL)
• Mesenchymal stem cells (MSC)

• peptides
• recombinant peptide therapy
• cytokines
• expression systems

• Biomimetics
• Corynebacterium glutamicum
• Cytokines/metabolism
• Drug Design
• Intercellular Signaling Peptides and Proteins/metabolism
• Peptides/metabolism

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.

• mesenchymal stem cells
• optimization cultivation
• physiologic cultivation

• cell therapy
• clinical application
• growth factor
• platelet lysate
• regenerative medicine

Mesenchymal stromal cells (MSC) hold great promise for tissue engineering and cell-based therapies due to their multilineage differentiation potential and intrinsic immunomodulatory and trophic activities. Over the past years, increasing evidence has proposed extracellular vesicles (EVs) as mediators of many of the MSC-associated therapeutic features. EVs have emerged as mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. EVs are derived from cell membranes, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. In this work, EVs were produced by MSC isolated from different human tissue sources [bone marrow (BM), adipose tissue (AT), and umbilical cord matrix (UCM)]. A serum-/xeno-free microcarrier-based culture system was implemented in a Vertical-Wheel^TM bioreactor (VWBR), employing a human platelet lysate culture supplement (UltraGRO^TM-PURE), toward the scalable production of MSC-derived EVs (MSC-EVs). The morphology and structure of the manufactured EVs were assessed by atomic force microscopy, while EV protein markers were successfully identified in EVs by Western blot, and EV surface charge was maintained relatively constant (between −15.5 ± 1.6 mV and −19.4 ± 1.4 mV), as determined by zeta potential measurements. When compared to traditional culture systems under static conditions (T-flasks), the VWBR system allowed the production of EVs at higher concentration (i.e., EV concentration in the conditioned medium) (5.7-fold increase overall) and productivity (i.e., amount of EVs generated per cell) (3-fold increase overall). BM, AT and UCM MSC cultured in the VWBR system yielded an average of 2.8 ± 0.1 × 10¹¹, 3.1 ± 1.3 × 10¹¹, and 4.1 ± 1.7 × 10¹¹ EV particles (n = 3), respectively, in a 60 mL final volume. This bioreactor system also allowed to obtain a more robust MSC-EV production, regarding their purity, compared to static culture. Overall, we demonstrate that this scalable culture system can robustly manufacture EVs from MSC derived from different tissue sources, toward the development of novel therapeutic products.

• bioreactors
• extracellular vesicles
• mesenchymal stromal cells (MSC)
• scalable production
• serum-/xenogeneic-free

• Adipose derived stem cells
• Cell therapy
• Foetal bovine serum
• Human platelet lysate
• Mesenchymal stem cells

• 3D cell culture
• Quantum Molecular Resonance
• esophagus
• mesenchymal stromal cells
• scaffold
• tissue engineering

• Chemical-defined medium
• MSCs
• Mesenchymal stem cells
• NBVbe medium

• Cell Differentiation
• Cell Proliferation
• Cells, Cultured
• Culture Media
• Humans
• Mesenchymal Stem Cells
• Umbilical Cord

• Natural Science Foundation of Shenzhen
• Medical Foundation of Guangdong

• cord blood
• hematology
• regeneration
• repair
• skin and wound care

Cellulose nanocrystals can bind different patterns of platelet lysate-derived protein in a surface sulfation dependent manner. The potential to direct stem cell fate by solid-phase presentation of defined protein coronas is demonstrated.

• Fetal bovine serum (FBS)
• Fibrinogen
• Mitotic bookmarking/transcription factors
• Pooled human platelet lysate (pHPL)
• Stromal cells

• adipogenesis
• adipose-derived stromal/stem cells
• chondrogenesis
• colony forming unit-fibroblast
• fetal bovine serum
• human platelet lysate
• mesenchymal stem cell
• osteogenesis
• regenerative medicine

• cell culture supplement
• cell therapy
• fetal bovine serum
• human platelet lysate
• pathogen reduction treatment

• Bioreactor
• Cell therapy
• Hematopoietic stem/progenitor cells (HSPC)
• Manufacturing
• Mesenchymal stem/stromal cells (MSC)
• Process engineering

• adipose-derived stem cells
• fat grafting
• platelet-rich plasma
• wound healing

Mesenchymal stem cells (MSCs) are heterogeneous multipotent stem cells that are involved in the development of mesenchyme-derived evolving structures and organs during ontogeny. In the adult organism, reservoirs of MSCs can be found in almost all tissues where MSCs contribute to the maintenance of organ integrity. The use of these different MSCs for cell-based therapies has been extensively studied over the past years, which highlights the use of MSCs as a promising option for the treatment of various diseases including autoimmune and cardiovascular disorders. However, the proportion of MSCs contained in primary isolates of adult tissue biopsies is rather low and, thus, vigorous ex vivo expansion is needed especially for therapies that may require extensive and repetitive cell substitution. Therefore, more easily and accessible sources of MSCs are needed. This review summarizes the current knowledge of the different strategies to generate human MSCs in vitro as an alternative method for their applications in regenerative therapy.

Recently, platelet‐derived growth factors present in lysates became an extreme interest in the field of regenerative medicine such as in wound healing and as substitutes to foetal bovine serum in xeno‐free cell culture systems. However, the generation of such platelet lysates completely depends on the availability of platelet donors. In this study, the possibility to use in vitro‐generated megakaryocytes derived from induced pluripotent stem cells (iPSCs) as a cell source for typical platelet growth factors was investigated. Therefore, the presence and levels of those factors were characterized in in vitro‐produced megakaryocytes. In comparison with platelets, in vitro‐generated megakaryocytes showed a multifold increased content in transcript and protein levels of typical platelet growth factors including platelet‐derived growth factors (PDGFs), transforming growth factor (TGF)‐1β, vascular endothelial cell factor (VEGF)‐A, epidermal growth factor (EGF), insulin‐like growth factor (IGF)‐1 and tissue factor (TF). Hence, iPSC‐derived megakaryocytes may serve as an efficient cell source for a donor‐independent generation of growth factor‐rich lysates with a broad application potential in innovative cell culture systems and regenerative therapies.

• growth factors
• megakaryocytes
• platelet lysate
• regenerative medicine

Mesenchymal stem cell (MSC) therapy is being increasingly used to treat dogs and horses with naturally-occurring diseases. However these animals also serve as critical large animal models for ongoing translation of cell therapy products to the human market. MSC manufacture for clinical use mandates improvement in cell culture systems to meet demands for higher MSC numbers and removal of xeno-proteins (i.e. fetal bovine serum, FBS). While serum-free media (SFM) is commercially available, its affects on MSC phenotype and immunomodulatory functions are not fully known. The objective of this study was to determine if specific MSC culture conditions, MSC expansion in HYPERFlasks® or MSC expansion in a commercially available SFM, would alter MSC proliferation, phenotype or immunomodulatory properties in vitro. MSCs cultured in HYPERFlasks® were similar in phenotype, proliferative capacity and immunomodulatory functions to MSCs grown in standard flasks however MSC yield was markedly increased. HYPERFlasks® therefore provide a viable option to generate greater cell numbers in a streamlined manner. Canine and equine MSCs expanded in SFM displayed similar proliferation, surface phenotype and inhibitory effect on lymphocyte proliferation in vitro. However, MSCs cultured in the absence of FBS secreted significantly less PGE₂, and were significantly less able to inhibit IFNγ secretion by activated T-cells. Immunomodulatory functions altered by expansion in SFM were species dependent. Unlike equine MSCs, in canine adipose-derived MSCs, the inhibition of lymphocyte proliferation was not principally modulated by PGE₂. The removal of FBS from both canine and equine MSC culture systems resulted in altered immunomodulatory properties in vitro and warrants further investigation prior to moving towards FBS-free culture conditions.

• Adipose tissue
• Bone marrow
• Canine
• Cell culture
• Equine
• Immunomodulation
• Lymphocyte
• Mesenchymal stem cells
• Proliferation
• Surface protein phenotype

• Cell engraftment
• Cell therapy
• Coagulation
• Complement
• Cryopreservation
• Cryopreservation-induced cell death
• Freeze injury
• Freeze-thawing
• Mesenchymal stromal cell
• T cells

• good manufacturing practices
• human serum
• mesenchymal stem cells
• platelet lysate
• platelet releasate
• platelet-rich plasma
• serum-free media

• DMSO-free cryopreservation
• Human adipose-derived mesenchymal stromal cells
• Platelet lysate
• Sucrose pretreatment
• Xeno-free expansion

The use of fetal bovine serum (FBS) as a cell culture supplement is discouraged by regulatory authorities to limit the risk of zoonoses and xenogeneic immune reactions in the transplanted host. Additionally, FBS production came under scrutiny due to animal welfare concerns. Platelet derivatives have been proposed as FBS substitutes for the ex-vivo expansion of mesenchymal stem/stromal cells (MSCs) since platelet-derived growth factors can promote MSC ex-vivo expansion. Platelet-derived growth factors are present in platelet lysate (PL) obtained after repeated freezing–thawing cycles of the platelet-rich plasma or by applying physiological stimuli such as thrombin or CaCl₂.

PL-expanded MSCs have been used already in the clinic, taking advantage of their faster proliferation compared with FBS-expanded preparations. Should PL be applied to other biopharmaceutical products, its demand is likely to increase dramatically. The use of fresh platelet units for the production of PL raises concerns due to limited availability of platelet donors. Expired units might represent an alternative, but further data are needed to define safety, including pathogen reduction, and functionality of the obtained PL. In addition, relevant questions concerning the definition of PL release criteria, including concentration ranges of specific growth factors in PL batches for various clinical indications, also need to be addressed. We are still far from a common definition of PL and standardized PL manufacture due to our limited knowledge of the mechanisms that mediate PL-promoting cell growth. Here, we concisely discuss aspects of PL as MSC culture supplement as a preliminary step towards an agreed definition of the required characteristics of PL for the requirements of manufacturers and users.

• Fetal bovine serum
• Manufacturing
• Mesenchymal stem/stromal cells
• Pathogen reduction
• Platelet lysate
• Platelet releasate

• Bone regeneration
• Mesenchymal stem cells
• Platelet lysate

The administration of human adipose-derived stem cells (ASCs) represents a promising regenerative therapy for the treatment of orthopedic injuries. While ASCs can be easily isolated from liposuction-derived adipose tissue, most clinical applications will likely require in vitro culture expansion of these cells using nonxenogeneic components. In this study, platelet releasate was generated using a novel rapid thrombin activation method (tPR). ASCs grown in media supplemented with tPR proliferated much faster than ASCs grown in media supplemented with 10% fetal bovine serum. The cells also retained the ability to differentiate along chondrogenic, adipogenic, and osteogenic lineages. The tPR cultured ASCs displayed elevated expression of BMP-4 (5.7 ± 0.97-fold increase) and BMP-2 (4.7 ± 1.3-fold increase) and decreased expression of PDGF-B (4.0 ± 1.4-fold decrease) and FGF-2 (33 ± 9.0-fold decrease). No significant changes in expression were seen with TGF-β and VEGF. This pattern of gene expression was consistent across different allogeneic tPR samples and different ASC lines. The use of allogeneic rapidly activated tPR to culture ASCs is associated with both an increased cell yield and a defined gene expression profile making it an attractive option for cell expansion prior to cell-based therapy for orthopedic applications.

• differentiation
• fetal calf serum
• platelet lysate
• platelet-poor plasma
• proliferation
• stem progenitor cells

• Adult
• Adult Stem Cells/metabolism
• Animals
• Becaplermin
• Blood Platelets/metabolism
• Cattle
• Cell Culture Techniques
• Cell Differentiation
• Cell Extracts/pharmacology
• Cell Line
• Cell Proliferation/drug effects
• Chondrocytes/metabolism
• Culture Media, Serum-Free/chemistry
• Culture Media, Serum-Free/metabolism
• Humans
• Mesenchymal Stem Cells/cytology
• Osteoblasts/metabolism
• Plasma/cytology
• Proto-Oncogene Proteins c-sis/metabolism
• T-Lymphocytes/metabolism
• Vascular Endothelial Growth Factor A/metabolism

Pooled human platelet lysate (pHPL) is an efficient alternative to xenogenic supplements for ex vivo expansion of mesenchymal stem cells (MSCs) in clinical studies. Currently, porcine heparin is used in pHPL-supplemented medium to prevent clotting due to plasmatic coagulation factors. We therefore searched for an efficient and reproducible medium preparation method that avoids clot formation while omitting animal-derived heparin.

We established a protocol to deplete fibrinogen by clotting of pHPL in medium, subsequent mechanical hydrogel disruption and removal of the fibrin pellet. After primary culture, bone-marrow and umbilical cord derived MSCs were tested for surface markers by flow cytometry and for trilineage differentiation capacity. Proliferation and clonogenicity were analyzed for three passages.

The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained. All MSC types were propagated in standard and fibrinogen-depleted medium. Flow cytometric phenotype profiles and adipogenic, osteogenic and chondrogenic differentiation potential in vitro were independent of MSC-source or medium type. Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium. Interestingly, this proliferative response to heparin was not detected after an initial contact with fibrinogen during the isolation procedure.

Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.

The online version of this article (doi:10.1186/s12967-015-0717-4) contains supplementary material, which is available to authorized users.

• Cell therapy
• Endothelial colony-forming progenitor cells
• Human platelet lysate
• Mesenchymal stem cells
• Platelet-rich plasma
• Tissue engineering

Mesenchymal stem cells (MSCs) are a promising source of cells for regenerative therapies. Although they can be isolated easily from several tissues, cell expansion is limited since their properties are lost with successive passages. Hence, pluripotent derived MSCs (PD-MSCs) arise as a suitable alternative for MSC production. Nevertheless, at present, PD-MSC derivation protocols are either expensive or not suitable for clinical purposes.

In this work we present a therapy-grade, inexpensive and simple protocol to derive MSCs from pluripotent stem cells (PSCs) based on the use of platelet lysate (PL) as medium supplement.

We showed that the PD-MSC_PL expressed multiple MSC markers, including CD90, CD73, CD105, CD166, and CD271, among others. These cells also show multilineage differentiation ability and immunomodulatory effects on pre-stimulated lymphocytes. Thorough characterization of these cells showed that a PD-MSC_PL resembles an umbilical cord (UC) MSC and differs from a PSC in surface marker and extracellular matrix proteins and integrin expression. Moreover, the OCT-4 promoter is re-methylated with mesenchymal differentiation comparable with the methylation levels of UC-MSCs and fibroblasts. Lastly, the use of PL-supplemented medium generates significantly more MSCs than the use of fetal bovine serum.

This protocol can be used to generate a large amount of PD-MSCs with low cost and is compatible with clinical therapies.

The online version of this article (doi:10.1186/scrt540) contains supplementary material, which is available to authorized users.

• Blood Platelets/cytology
• Blood Platelets/drug effects
• Cell Culture Techniques/methods
• Cell Culture Techniques/standards
• Cell Proliferation
• Culture Media, Serum-Free
• Humans
• Platelet-Derived Growth Factor/pharmacology
• Stem Cells/cytology