Maintenance Of Cell Cultures Research Articles

Optimization of the Biogenic Synthesis of Silver Nanoparticles and their Application in the Degradation of Dyes

Using plant extracts for the green synthesis of silver nanoparticles (AgNPs) is being recognized as a promising and environmentally sustainable approach. Plant extracts in producing AgNPs obviate cell culture maintenance and allow scalability in a non-aseptic setting. This work aims to identify the categories of phytochemicals and to conduct the biosynthesis and characterization of AgNPs utilizing extracts from the leaves and stems of Clinacanthus nutans. The present work involved the synthesis of AgNPs using aqueous extracts derived from the leaves (L) and stems (S) of C. nutans. This synthesis approach was chosen due to its non-toxic nature, cost-effectiveness, and environmentally favorable characteristics. The Transmission Electron Microscopy (TEM) examination revealed the presence of silver nanoparticles, denoted as AgNP-L and AgNP-S, which exhibited size ranges of 20 to 200 nm and 25 to 250 nm, respectively. The average sizes of AgNP-L and AgNP-S were determined to be 99.38 nm and 68.47 nm, respectively. The AgNP has shown remarkable degrading efficiency towards commercial dyes, namely Nile Blue (NB), with a removal rate of 94% within 110 minutes, and Reactive Yellow 160 (RY160), with a removal rate of 82% within 110 minutes. The extract derived from the Clinacanthus nutans exhibited considerably elevated concentrations of biologically active chemicals (Flavonoid and Phenolic). The utilization of biogenic AgNPs presents a significant prospect for the remediation of water resources polluted with industrial dye, owing to their notable reusability, photocatalytic effectiveness, and compatibility with environmentally friendly synthesis methods.

An antibody-free evaluation of an mRNA COVID-19 vaccine

This manuscript describes the use of an analytical assay that combines transfection of mammalian cells and isotope dilution mass spectrometry (IDMS) for accurate quantification of antigen expression. Expired mRNA COVID-19 vaccine material was stored at 4 °C, room temperature (∼25 °C), and 56 °C over a period of 5 weeks. The same vaccine was also exposed to 5 freeze-thaw cycles. Every week, the spike protein antigenic expression in mammalian (BHK-21) cells was evaluated. Housekeeping proteins, β-actin and GAPDH, were simultaneously quantified to account for the variation in cell counts that occurs during maintenance and growth of cell cultures. Data show that vaccine stored at elevated temperatures results in reduced spike protein expression. Also, maintaining the vaccine in ultracold conditions or exposing the vaccine to freeze-thaw cycles had less effect on the vaccine's ability to produce the antigen in mammalian cells. We describe the use of IDMS as an antibody-free means to accurately quantify expressed protein from mammalian cells transfected with mRNA vaccine.

Automated cell counting for Trypan blue-stained cell cultures using machine learning.

Cell counting is a vital practice in the maintenance and manipulation of cell cultures. It is a crucial aspect of assessing cell viability and determining proliferation rates, which are integral to maintaining the health and functionality of a culture. Additionally, it is critical for establishing the time of infection in bioreactors and monitoring cell culture response to targeted infection over time. However, when cell counting is performed manually, the time involved can become substantial, particularly when multiple cultures need to be handled in parallel. Automated cell counters, which enable significant time reduction, are commercially available but remain relatively expensive. Here, we present a machine learning (ML) model based on YOLOv4 that is able to perform cell counts with a high accuracy (>95%) for Trypan blue-stained insect cells. Images of two distinctly different cell lines, Trichoplusia ni (High FiveTM; Hi5 cells) and Spodoptera frugiperda (Sf9), were used for training, validation, and testing of the model. The ML model yielded F1 scores of 0.97 and 0.96 for alive and dead cells, respectively, which represents a substantially improved performance over that of other cell counters. Furthermore, the ML model is versatile, as an F1 score of 0.96 was also obtained on images of Trypan blue-stained human embryonic kidney (HEK) cells that the model had not been trained on. Our implementation of the ML model comes with a straightforward user interface and can image in batches, which makes it highly suitable for the evaluation of multiple parallel cultures (e.g. in Design of Experiments). Overall, this approach for accurate classification of cells provides a fast, bias-free alternative to manual counting.

Cytological, molecular, cytogenetic, and physiological characterization of a novel immortalized human enteric glial cell line.

Enteric glial cells (EGCs), the major components of the enteric nervous system (ENS), are implicated in the maintenance of gut homeostasis, thereby leading to severe pathological conditions when impaired. However, due to technical difficulties associated with EGCs isolation and cell culture maintenance that results in a lack of valuable in vitro models, their roles in physiological and pathological contexts have been poorly investigated so far. To this aim, we developed for the first time, a human immortalized EGC line (referred as ClK clone) through a validated lentiviral transgene protocol. As a result, ClK phenotypic glial features were confirmed by morphological and molecular evaluations, also providing the consensus karyotype and finely mapping the chromosomal rearrangements as well as HLA-related genotypes. Lastly, we investigated the ATP- and acetylcholine, serotonin and glutamate neurotransmitters mediated intracellular Ca2+ signaling activation and the response of EGCs markers (GFAP, SOX10, S100β, PLP1, and CCL2) upon inflammatory stimuli, further confirming the glial nature of the analyzed cells. Overall, this contribution provided a novel potential in vitro tool to finely characterize the EGCs behavior under physiological and pathological conditions in humans.

Short-term culture adaptation of Toxoplasma gondii archetypal II and III field isolates affects cystogenic capabilities and modifies virulence in mice

Most Toxoplasma gondii research has been carried out using strains maintained in the laboratory for long periods of time. Long-term passage in mice or cell culture influences T. gondii phenotypic traits such as the capability to produce oocysts in cats and virulence in mice. In this work, we investigated the effect of cell culture adaptation in the short term for recently obtained type II (TgShSp1 (Genotype ToxoDB#3), TgShSp2 (#1), TgShSp3 (#3) and TgShSp16 (#3)) and type III (#2) isolates (TgShSp24 and TgPigSp1). With this purpose, spontaneous and alkaline stress-induced cyst formation in Vero cells during 40 passages, from passage 10 (p10) to 50 (p50), and isolate virulence at p10 versus p50 were studied using a harmonized bioassay method in Swiss/CD1 mice. T. gondii cell culture maintenance showed a drastic loss of spontaneous and induced production of mature cysts after ≈25–30 passages. The TgShSp1, TgShSp16 and TgShSp24 isolates failed to generate spontaneously formed mature cysts at p50. Limited cyst formation was associated with an increase in parasite growth and a shorter lytic cycle. In vitro maintenance also modified T. gondii virulence in mice at p50 with events of exacerbation, increasing cumulative morbidity for TgShSp2 and TgShSp3 isolates and mortality for TgShSp24 and TgPigSp1 isolates, or attenuation, with absence of mortality and severe clinical signs for TgShSp16, and better control of the infection with the lowest parasite and cyst burdens in lungs and brain for the TgShSp1 isolate. The present findings show deep changes in relevant phenotypic traits in laboratory-adapted T. gondii isolates and open new discussion about their use for inferring keys to parasite biology and virulence.

Non-prophylactic resveratrol-mediated protection of neurite integrity under chronic hypoxia is associated with reduction of Cav1.2 channel expression and calcium overloading.

Cellular hypoxia is a major cause of oxidative stress, culminating in neuronal damage in neurodegenerative diseases. Numerous ex vivo studies have implicated that hypoxia episodes leading to disruption of Ca2+ homeostasis and redox status contribute to the progression of various neuropathologies and cell death. Isolation and maintenance of primary cell culture being cost-intensive, the details of the time course relationship between Ca2+ overload, L-type Ca2+ channel function, and neurite retraction under chronic and long-term hypoxia remain undefined. In order to explore the effect of oxidative stress and Ca2+ overload on neurite length, first, we developed a 5-day-long neurite outgrowth model using N2a cell line. Second, we propose a chronic hypoxia model to investigate the modulation of the L-type Ca2+ channel (Cav1.2) and oxidative resistance gene (OXR1) expression level during the process of neurite retraction and neuronal damage over 32h. Thirdly, we developed a framework for quantitative analysis of cytosolic Ca2+, superoxide formation, neurite length, and constriction formation in individual cells using live imaging that provides an understanding of molecular targets. Our findings suggest that an increase in cytosolic Ca2+ is a feature of an early phase of hypoxic stress. Further, we demonstrate that augmentation in the L-type channel leads to amplification in Ca2+ overload, ROS accumulation, and a reduction in neurite length during the late phase of hypoxic stress. Next, we demonstrated that non-prophylactic treatment of resveratrol leads to the reduction of calcium overloading under chronic hypoxia via lowering of L-type channel expression. Finally, we demonstrate that resveratrol-mediated reduction of Cav1.2 channel and STAT3 expression are associated with retention of neurite integrity. The proposed in vitro model assumes significance in the context of drug designing and testing that demands monitoring of neurite length and constriction formations by imaging before animal testing.

Identification of cell culture contamination by an unusual species of Mycoplasma related to the M. mycoides cluster.

Mycoplasma contamination is a significant problem in cell culture replication and maintenance. From more than 200 known species, a limited number of Mycoplasma species have been detected in cell cultures, representing new species or variants that can escape detection systems. A qPCR commercial kit was used for Mycoplasma detection in cell cultures. Furthermore, an amplified Mycoplasma species was sequenced and summited for sequence assembly, clustering, and evolutionary analysis study. Our work has identified a new and unusual variant or species of Mycoplasma that possesses a high degree of homology with species related with M. mycoides cluster. This variant is usually associated with cattle but has been detected contaminating a cell culture. Mycoplasma testing (even for unusual species) in cell cultures is essential to ensure the validity and reproducibility of research that uses cell cultures and to ensure the quality of cell line deposits in biobanks. For this reason, it is necessary to perform continuous checks for the absence of Mycoplasma in cell cultures and engage in the continuous adaptation of relevant detection systems.

2-3% fetal calf serum in cell media is sufficient for the development and growth of human mononuclear cell cultures

Aim: It is a common practice that 10-20% fetal calf serum (FCS) is being used in the cell culture media for the development and maintenance of human mononuclear cells as well as other cell cultures. FCS is produced through a painful process involving animals and is one of the major cost factors. The reduction in the volume of FCS can make it less unethical and reduce the costs strongly. Materials and Methods: Therefore 2-3 % FCS and 10% FCS were used in the development of different mononuclear cell cultures for making comparisons and different parameters like morphology, production of antibodies, isolation of DNA/RNA with mini-column and magnetic beads cell isolations along with the presence of human internal controls with real-time PCR were tested. Results: It was found that there was no difference between 2-3 % FCS and 10 % FCS containing media in the above-said parameters. More than a hundred such cell cultures were developed and many of them were maintained for more than 3 years. Conclusion: 2-3 % FCS is sufficient for the development and maintenance of human mononuclear cell cultures over a long period of time.

Impact of the redox environment on propagation of radiation bystander effects: The modulating effect of oxidative metabolism and oxygen partial pressure

Redox modulated pathways play important roles in out-of-field effects of ionizing radiation. We investigated how the redox environment impacts the magnitude of propagation of stressful effects from irradiated to bystander cells. Normal human fibroblasts that have incorporated [3H]-thymidine were intimately co-cultured with bystander cells in a strategy that allowed isolation of bystander cells with high purity. The antioxidant glutathione peroxidase (GPX) was maintained either at wild-type conditions or overexpressed in the bystanders. Following 24 h of coculture, levels of stress-responsive p21Waf1, p-Hdm2, and connexin43 proteins were increased in bystander cells expressing wild-type GPX relative to respective controls. These levels were significantly attenuated when GPX was ectopically overexpressed, demonstrating by direct approach the involvement of a regulator of intracellular redox homeostasis. Evidence of participation of pro-oxidant compounds was generated by exposing confluent cell cultures to low fluences of 3.7 MeV α particles in presence or absence of t-butyl hydroperoxide. By 3 h post-exposure to fluences wherein only ∼2% of cells are traversed through the nucleus by a particle track, increases in chromosomal damage were greater than expected in absence of the drug (p < 0.001) and further enhanced in its presence (p < 0.05). While maintenance and irradiation of cell cultures at low oxygen pressure (pO2 3.8 mm Hg) to mimic in vivo still supported the participation of bystander cells in responses assessed by chromosomal damage and stress-responsive protein levels (p < 0.001), the effects were attenuated compared to ambient pO2 (155 mm Hg) (p < 0.05). Together, the results show that bystander effects are attenuated at below ambient pO2 and when metabolic oxidative stress is reduced but increased when the basal redox environment tilts towards oxidizing conditions. They are consistent with bystander effects being independent of radiation dose rate.

Evaluation of establishment and maintenance of primary cell cultures from several strains of common carp (Cyprinus carpio L.).

As a surrogate for the whole organism, primary cultures and cell lines serve as valuable tools for investigating exogenous and endogenous cytopathy. Studying cell responsiveness to diseases and contaminants is considered a less demanding and more readily accessible research approach that minimizes animal distress and provides more specific data. In the current work, the authors established primary cultures from several different organs and tissues of common carp (Cyprinus carpio L.) for subsequent use in other applications. They investigated the technical challenges in obtaining successful and durable carp-derived tissue cultures. The trials indicate that the type of tissue grown, carp strain and fish age impact equally upon culturing success, as do the cultivating conditions. Cells from gill epithelia, head and trunk kidneys, spleen, skin, gonads and ocular tissue were successfully established and maintained for further use in in-vitro testing. The primary cultures were, therefore, used to investigate and assess pathogens and pollutants emerging in carp's environment.

Cyclic and dimeric fibroblast growth factor 2 variants with high biomedical potential

Fibroblast growth factor 2 (FGF2) is a pleiotropic protein engaged in the regulation of key cellular processes in a wide spectrum of cells. FGF2 is an important object of basic research as well as a molecule used in regenerative medicine, in vitro cell culture maintenance, and as an anticancer drug carrier. However, the unsatisfactory stability and pleiotropic activities of the wild-type FGF2 largely limit its use as a medical product. To overcome these limitations, we have designed a set of FGF2-based macromolecules via sortase A-mediated cyclization and oligomerization. We obtained heparin-switchable FGF2 variants with enhanced stability and improved ability to stimulate cell proliferation and migration. We have shown that stimulation of glucose uptake by adipocytes is modulated by the architecture of FGF2 oligomers. Moreover, we used hyper-stable FGF2 variants for the construction of highly effective drug carriers for selective killing of FGFR1-overproducing cancer cells. The strategy for FGF2 engineering presented in this work provides novel insights into the design of growth factor variants for regenerative and anti-cancer precise medicine.

The Epigenetic Regulation in Plant Specialized Metabolism: DNA Methylation Limits Paclitaxel in vitro Biotechnological Production.

Environmental conditions are key factors in the modulation of the epigenetic mechanisms regulating gene expression in plants. Specifically, the maintenance of cell cultures in optimal in vitro conditions alters methylation patterns and, consequently, their genetic transcription and metabolism. Paclitaxel production in Taxus x media cell cultures is reduced during its maintenance in in vitro conditions, compromising the biotechnological production of this valuable anticancer agent. To understand how DNA methylation influences taxane production, the promoters of three genes (GGPPS, TXS, and DBTNBT) involved in taxane biosynthesis have been studied, comparing the methylation patterns between a new line and one of ~14 years old. Our work revealed that while the central promoter of the GGPPS gene is protected from cytosine methylation accumulation, TXS and DBTNBT promoters accumulate methylation at different levels. The DBTNBT promoter of the old line is the most affected, showing a 200 bp regulatory region where all the cytosines were methylated. This evidence the existence of specific epigenetic regulatory mechanisms affecting the last steps of the pathway, such as the DBTNBT promoter. Interestingly, the GGPPS promoter, a regulatory sequence of a non-specific taxane biosynthetic gene, was not affected by this mechanism. In addition, the relationship between the detected methylation points and the predicted transcription factor binding sites (TFBS) showed that the action of TFs would be compromised in the old line, giving a further explanation for the production reduction in in vitro cell cultures. This knowledge could help in designing novel strategies to enhance the biotechnological production of taxanes over time.

Generation of Human Pluripotent Stem Cell-Derived Polarized Hepatocytes.

Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are valuable tools to study liver biology. HLCs, however, lack certain key in vivo characteristics relevant to their physiological function. One such characteristic is cellular polarity, which is critical to hepatocyte counter-current flow systems involving canalicular bile secretion and sinusoidal secretion of large quantities of serum proteins into blood. Model systems using non-polarized hepatocytes, therefore, cannot recapitulate this physiological function of hepatocytes. Here, we describe a stepwise protocol to generate hPSC-derived polarized HLCs (pol-HLCs), which feature clearly defined basolateral and apical membranes separated by tight junctions. Pol-HLCs not only display many hepatic functions but are also capable of directional cargo secretion, mimicking the counter-current flow systems. We describe protocols for stem cell culture maintenance and for differentiating hPSCs into pol-HLCs. In addition, we describe protocols to assay the pol-HLCs for basic hepatic functions and polarized hepatic characteristics. Once successfully differentiated, these pol-HLCs can be used as an in vitro model system to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism. The establishment of hepatic polarity from non-polarized hPSCs also provides a useful tool to study the development and maintenance of hepatic polarity. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Maintenance of hPSCs Basic Protocol 2: Differentiation of hPSCs to pol-HLCs Basic Protocol 3: Assaying pol-HLCs for basic hepatic functions Support Protocol 1: Assessment of pol-HLC monolayer tightness Support Protocol 2: Assessment of pol-HLC polarity.

IPSC-Derived Micro-Heart Muscle for Medium-Throughput Pharmacology and Pharmacogenomic Studies.

Micro-heart muscle arrays enable medium-throughput experiments to model the cardiac response to a variety of environmental and pharmaceutical effects. Here, we describe stem cell culture maintenance, methods for successful cardiac differentiation, and formation of micro-heart muscle arrays for electrophysiology and molecular biology assays.

Green Synthesis of Silver Nano Particles Derieved from Leaf Extract of Syzygiumcuminii (SNSC) – to Evaluate Antibacterial Activity

Synthesis of metals nanoparticles from the plant or plant extracts has emerged as an important alternative to the chemical method. The biological approach to the synthesis of nanoparticles has many advantages such as non-elaborative process, no multiple purification steps, no need of intracellular synthesis and dosenot require maintenance of microbial cell cultures. Medicinal plants and green synthesis of silver nanoparticles (AgNPs) have proven to be good sources of agents effective in the treatment of various diseases. The present study focuses on the green synthesis of SNSC, silverna noparticles (AgNPs) from leaf extract of Syzygiumcuminii in order to evaluate the antibacterial properties of this extract and synthesized AgNPs. The characterization of the Synthesized nanoparticles (SNSC) was determined using UV-VIS spectroscopy, TEM,X ray diffraction and FTRI studies, silver nanoparticles (AgNPs) showed absorption peak at 470 nm in aqueous medium in UV-VIS spectrum. TEM analysis shows themorphology of AgNPs as a hexagonal matrix with average particle size of about 50 nm. XRD analysis displays the crystalline structure of AgNPs.FTIR analysis shows that amide groups present in proteins are dominant reducing agents and play an important role in the bio reduction of Ag+ ions to Ag0. The synthesized silver nanoparticles from leaf extract of Syzygiumcuminii(SNSC) showed antibacterial activity against common clinical pathogens. Owing to the remarkable potential antibacterial activity against common pathogenic microorganisms, the synthesized AgNPs derieved from SNSCcan have potential for development in medical applications in the future..

Employment of Cassia angustifolia leaf extract for zinc nanoparticles fabrication and their antibacterial and cytotoxicity.

The plant Cassia angustifolia belongs to Saudi Arabia, which is one of the native places and now cultured throughout the global countries. Medical care in the Arab world is an essential outlet for medicinal plants, both because they are crucial elements for prophetic medicine and due to their lengthy background in the Middle East. C.angustifolia is one of the medicinal plants used in the Saudi Arabia. The usage of plant extracts for synthesizing nanoparticles is conducive to other biological material, since it avoids the lengthy phase of cell culture maintenance. Silver nanoparticles attract further attention due to their strong conductivity, stability and antimicrobial activity across different metal nanoparticles. The present study was designed in the Saudi C. angustifolia leaves with the zinc synthesis of nanoparticles and its antibacterial ability. The plant extracts of C. angustifolia was used for synthesis of zinc nanoparticles, antimicrobial activities against bacterial strains have been tested along with transmission electron microscope (TEM), UV spectroscopy and antimicrobial activities have been conducted. This study showed that silver ions may be transferred from the plant extract to silver nanoparticles. AgNPs biogenic capacity to antibacterial with lovo cell with IC50 ranged from 33.5 ± 0.2 μg/mL demonstrated strong antibacterial capacity to antibody. The overall absorption value for the extract was between 420 and 440 nm and the color transition to green was the plasma absorption of the AgNPs. TEM results was showed in 200,000 magnification. The uniqueness of the current study is that Cassia angustifolia leaf extract from Saudi Arabia was used to prepare the metallic nanoparticles. Additionally, ZnCl2 may also be used as nanoparticles of mineral salt and zinc, which, since their application has been confirmed, are antimicrobial.

Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension.

Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In this regard, human embryonic kidney 293 (HEK293) cells constitute one of the main standard lab-scale mammalian hosts for recombinant protein production since these cells are relatively easy to handle, scale-up, and transfect. Here, we present a detailed protocol for the cost-effective, reproducible, and scalable implementation of HEK293 cell cultures in suspension (suitable for commercially available HEK293 cells, HEK293-F) for high-quantity recombinant production of secreted soluble multi-domain proteins. In addition, the protocol is optimized for a Monday-to-Friday maintenance schedule, thus simplifying and streamlining the work of operators responsible for cell culture maintenance. Graphic abstract: Schematic overview of the workflow described in this protocol.

Towards Reduction or Substitution of Cytotoxic DMSO in Biobanking of Functional Bioengineered Megakaryocytes.

Donor platelet transfusion is currently the only efficient treatment of life-threatening thrombocytopenia, but it is highly challenged by immunological, quality, and contamination issues, as well as short shelf life of the donor material. Ex vivo produced megakaryocytes and platelets represent a promising alternative strategy to the conventional platelet transfusion. However, practical implementation of such strategy demands availability of reliable biobanking techniques, which would permit eliminating continuous cell culture maintenance, ensure time for quality testing, enable stock management and logistics, as well as availability in a ready-to-use manner. At the same time, protocols applying DMSO-based cryopreservation media were associated with increased risks of adverse long-term side effects after patient use. Here, we show the possibility to develop cryopreservation techniques for iPSC-derived megakaryocytes under defined xeno-free conditions with significant reduction or complete elimination of DMSO. Comprehensive phenotypic and functional in vitro characterization of megakaryocytes has been performed before and after cryopreservation. Megakaryocytes cryopreserved DMSO-free, or using low DMSO concentrations, showed the capability to produce platelets in vivo after transfusion in a mouse model. These findings propose biobanking approaches essential for development of megakaryocyte-based replacement and regenerative therapies.

Renal Progenitor Cells Have Higher Genetic Stability and Lower Oxidative Stress than Mesenchymal Stem Cells during In Vitro Expansion.

In vitro senescence of multipotent cells has been commonly associated with DNA damage induced by oxidative stress. These changes may vary according to the sources of production and the studied lineages, which raises questions about the effect of growing time on genetic stability. This study is aimed at evaluating the evolution of genetic stability, viability, and oxidative stress of bone marrow mesenchymal stem cells (MSCBMsu) and renal progenitor cells of the renal cortex (RPCsu) of swine (Sus scrofa domesticus) in culture passages. P2, P5, and P9 were used for MSCBMsu and P1, P2, and P3 for RPCsu obtained by thawing. The experimental groups were submitted to MTT, apoptosis and necrosis assays, comet test, and reactive substance measurements of thiobarbituric acid (TBARS), nitrite, reduced glutathione (GSH), and catalase. The MTT test curve showed a mean viability of 1.14 ± 0.62 and 1.12 ± 0.54, respectively, for MSCBMsu and RPCsu. The percentages of MSCBMsu and RPCsu were presented, respectively, for apoptosis, an irregular and descending behavior, and necrosis, ascending and irregular. The DNA damage index showed higher intensity among the MSCBMsu in the P5 and P9 passages (p < 0.05). In the TBARS evaluation, there was variation among the lines of RPCsu and MSCBMsu, presenting the last most significant variations (p < 0.05). In the nitrite values, we identified only among the lines, in the passages P1 and P2, with the highest averages displayed by the MSCBMsu lineage (p < 0.05). The measurement of antioxidant system activity showed high standards, identifying differences only for GSH values, in the RPCsu lineage, in P3 (p < 0.05). This study suggests that the maintenance of cell culture in the long term induces lower regulation of oxidative stress, and RPCsu presents higher genetic stability and lower oxidative stress than MSCBMsu during in vitro expansion.

Development of standard protocol for the cGMP production of human mesenchymal stem/stromal cells from different source tissues

Background & Aim Mesenchymal stem/stromal cells (MSC) hold great potential for tissue engineering and regenerative medicine because of their ability for self-renewal and differentiation into tissue-specific cells such as osteoblasts, chondrocytes, and adipocytes. MSCs are important pieces in the process of tissue development, maintenance and repair, producing secretory factors that support engraftment and trophic functions. For that reason, they are currently being used in hundreds of clinical trials as a promising treatment option for a variety of clinical conditions, with an increasing evidence of therapeutic efficacy. MSC constitute a heterogeneous subset of stromal regenerative cells which can be harvested from several adult tissues. Initially MSC were identified in bone marrow but currently they can be harvested from several autologous and allogeneic sources, including adipose tissue, peripheral blood, lungs, marrow spaces of long bone, synovial fluids, periodontal ligament, dental pulp and muscle. In addition, MSCs are also obtained from placenta, umbilical cord and cord blood. Notably, MSCs obtained from various sources differ in their biological characteristics, CFU-F efficiency, specific markers, multi-lineage differentiation as well as paracrine functions which may determine their different clinical applications. The use of MSC from different sources together with the fact that most laboratories use their own standardized protocols for cell expansion results in a clear variability in cell preparations between labs which can lead to variability in the outcome of experiments and clinical trials across different laboratories and institutions. Investigations have attempted to address this issue of unpredictable outcomes by seeking to establish standard practices for the isolation, characterization, and maintenance of cells in culture. The development of uniform protocols for the expansion and preparation of MSCs is crucial to improve comparison of results, for the exploitation of MSC clinical potential and for a successful translation of approaches to the clinic. Methods, Results & Conclusion Here we will present the development of a standardized protocol for a consistent and efficient cGMP production of clinically relevant numbers of MSCs obtained from different source tissues, using MSC NutriStem® XF Basal Medium supplemented with PLTGold®, a fully xenogeneic free human platelet lysate.