Conventional Cell Culture Media Research Articles

Influence of inflammation on the expression of microRNA-140 in extracellular vesicles from 2D and 3D culture models of synovial-membrane-derived stem cells.

In osteoarthritis (OA), articular homeostasis is regulated by microRNA-140 that inhibits ADAMTS-5, an enzyme that cleaves aggrecan and stimulates the synthesis of other inflammatory mediators. This study aims to evaluate the expression of microRNA-140 in extracellular vesicles (EVs) derived from equine synovial-membrane-derived mesenchymal stem cells (eqSMMSCs) cultured in monolayer (2D) and three-dimensional (3D) culture models under an in vitro inflammatory environment. Four experimental groups of eqSMMSC cultures were defined for isolation of the EVs. The 2D and 3D control groups were cultured in a conventional cell culture medium, while the 2D-OA and 3D-OA treatment groups were exposed to an OA-like medium containing IL-1β and TNFα. The culture media samples were collected at 24h, 72h, and 120h time points for EV isolation and characterization using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Reverse transcription quantitative polymerase chain reaction was employed to assess the expressions of microRNA-140 in both the cells and EVs. All statistical analyses were conducted at the 5% significance level. Encapsulation of the eqSMMSCs protected the cells from the inflammatory media compared to the monolayer cultures. EVs were found in higher concentrations in the 3D-OA cultures. Additionally, higher expressions of microRNA-140 were observed in the cells of the 3D-OA group at 24 and 72h, whereas microRNA-140 expressions in the EVs were higher in the 3D group at 72h and in the 2D-OA group at 120h (p < 0.001). However, the 3D-OA culture showed higher expression of the mRNA Adamts5 in the EVs at 120h. The responses of the eqSMMSCs to inflammatory stimuli involve intracellular expression of microRNA-140 and its subsequent transportation via the EVs, with quicker responses observed in the 3D than 2D cultures. This study sheds light on the behaviors of stem cells in restoring homeostasis in osteoarthritic joints.

ALL the comforts of homing: lymphoblasts find cerebrospinal fluid inhospitable without meningeal cell contact.

ALL the comforts of homing: lymphoblasts find cerebrospinal fluid inhospitable without meningeal cell contact.

Encapsulation of hydrophobic allergens into nanoparticles improves the in vitro immunological diagnosis of allergic contact dermatitis

The diagnosis of allergic contact dermatitis (ACD) relies on in vivo patch testing. In vitro immunological assays based on the characterization of circulating allergen-specific memory T cells represent a promising alternative to patch testing. However, their development is hampered by the technical challenge of assessing hydrophobic allergens in serum-based assays. In this study, we show that the encapsulation of fragrance mix 1 (FMI, a mixture of 8 hydrophobic allergens) into poly-ε-caprolactone nanoparticle (NP) vectors: (1) dramatically increases the solubilization of allergens in conventional cell culture media and (2) allows for a robust in vitro reactivation of allergen-specific T cells in large numbers of fragrance allergic patients. Therefore, the encapsulation of hydrophobic allergens into NP vectors opens new avenues to improve the in vitro immunobiological diagnosis of ACD. From the Clinical EditorAllergic Contact Dermatitis (ACD) is a delayed-type hypersensivity reaction prevalent in many individuals. Currently, skin patch testing has been the mainstay for diagnosis clinically. In this study, the authors described an improvement to in vitro immunological assays measuring circulating allergen-specific memory T cells, using nanoparticle vectors. The positive data might provide an exciting alternative to current practice of patch-testing.

Stability of prostaglandin E2 (PGE2) embedded in poly-d,l-lactide-co-glycolide microspheres: a pre-conditioning approach for tissue engineering applications

Prostaglandin E(2) (PGE(2)) is involved in angiogenesis, bone repair and cartilage metabolism. Thus, PGE(2) might represent a suitable signaling molecule in different tissue engineering applications. PGE(2) also has a short half-life time. Its incorporation into poly-D: ,L: -lactide-co-glycolide (PLGA) microspheres was demonstrated in a previous study. However, the stability of bioactive PGE(2) in these microspheres is unknown. With an adjusted mass spectrometry assay we investigated the amount of incorporated PGE(2) and the stability of PGE(2) in conventional cell culture medium and in PLGA microspheres. The stability of PGE(2) was closely pH dependent. Strong acidic or basic environments reduced the half-life from 300 h (pH 2.6-4.0) to below 50 h at pH 2.0 or pH 8.8. The half-life of PGE(2) incorporated into poly-D: ,L: -lactide-co-glycolide increased drastically to 70 days at 37 degrees C and to 300 days at 8 degrees C. Analysis with scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrated a distinct nanostructure of the polymeric phase and both nano- and microporosity.

A Non-Frozen Living Tissue Bank for Allotransplantation Using Green Tea Polyphenols

Generally, mammalian cells and living tissues can be cryopreserved in a frozen state at very low temperatures over a long storage term. The survival rate of cell suspensions is often acceptable however, living tissues suffer a variety of injuries. In this paper, it was demonstrated that the addition of polyphenols extracted from green tea to conventional cell culture medium and tissue compatible liquid, can control cell proliferation and also preserve tissues for several months at ordinary room temperature, including such tissues as blood vessels, cartilage, islet cells and corneas. This protocol allows a non-frozen living tissue bank to be established using the preservation fluid described.

Total Anaerobiosis during In vitro Culture in Conventional Cell Culture Media Is Required for Retaining Infectivity of Trichinella spiralis L1 Larvae

Total Anaerobiosis during In vitro Culture in Conventional Cell Culture Media Is Required for Retaining Infectivity of Trichinella spiralis L1 Larvae

Effect of selenium supplementation on the activities of glutathione metabolizing enzymes in human hepatoma Hep G2 cell line

Cell culture is an important tool for studying injury to cells exposed to oxidative stress. The human hepatoblastoma derived Hep G2 cells retain their morphology and most of their function in culture and are therefore widely used as an in vitro model of human hepatocytes. Conventional cell culture media are deficient in selenium, which is essential for activation of glutathione peroxidase (GPx), a key enzyme in the defense against oxidative stress. Supplementation of the culture media with 1 μM sodium selenite increased the activities of total GPx by threefold and the selenium-dependent GPx by fourfold as compared to cells cultured in control media. The non-selenium-dependent GPx activity was unchanged. The activities of the other glutathione (GSH)-related enzymes were practically unchanged despite a tendency toward elevation. The activities of oxidized glutathione (GSSG) reductase and catalase increased by 22.4 and 27.4%, respectively. These relatively small increases did not carry statistical significance. Supplementation of tissue culture media with selenium may prove important, particularly for cell protection against oxidative stress.

Cell viability improves following inhibition of cryopreservation-induced apoptosis.

A new concept in cryopreservation solution design was developed that focuses on the use of an intracellular-type, hypothermic maintenance medium coupled with additives that inhibit cryopreservation-induced apoptosis. HypoThermosol' (HTS), a hypothermic (4 degrees C) maintenance medium utilized in the long-term storage of cell, tissue, and organ systems, was tested for cryoprotective capability on a renal cell line (Madin-Darby Canine Kidney cells). HTS and HTS derivatives were tested against conventional cell culture medium (Dulbecco's Minimal Essential medium, DME) as the cryoprotectant carrier solution because (1) cells are exposed to an extended state of hypothermia during the freeze-thaw process, and (2) HTS is designed to protect cells exposed to a hypothermic state. Cells separately cryopreserved in either HTS or DME + 5% dimethyl sulfoxide (DMSO) yielded equivalent 24-h postthaw survival (approximately 30%) and 5-d recovery (approximately 90%). Cells cryopreserved in CryoStor CS 5, a HTS derivative containing 5% DMSO, yielded approximately 75% 24-h postthaw survival and recovery to 100% within 3 d. DNA gel electrophoresis was performed to determine the mechanisms of cell death contributing to cryopreservation failure. Cells preserved in DME (DMSO-free) died primarily through necrosis, whereas cells preserved in either DME + 5% DMSO, HTS, or CryoStor CS 5 died through a combination of apoptosis and necrosis. This observation led to the inclusion of an apoptotic inhibitor designed to improve cryopreservation outcome. MDCK cells cryopreserved in CryoStor CS 5 supplemented with an apoptotic inhibitor (Caspase I Inhibitor V), hereafter termed CryoStor CS 5N, resulted in a 24-h postthaw survival and recovery rate exceeding that of any other cryoprotective solution tested (85%). We conclude that: (1) the use of HTS (a dextran-based, intracellular-type solution) without DMSO can yield postthaw viability equivalent to that of standard DMSO-based cryopreservation methods, (2) postthaw viability can be significantly increased through the use of an intracellular-type solution in conjunction with DMSO, (3) the use of HTS allows for cryopreservation to be accomplished with reduced levels of cryoprotectants, and (4) the regulation of apoptosis is essential for the improvement of cryopreservation outcome.

Conventional cell culture media do not adequately supply cells with antioxidants and thus facilitate peroxide-induced genotoxicity

Commercially available calf serum did not supply the cultured murine fibroblast cell line L929 with amounts of selenium and α-tocopherol sufficient to protect against peroxide damage. Supplementation of the culture medium with 30μM α-tocopherol or 50 nM sodium selenite led to a substantial increase of cellular α-tocopherol concentrations from 18 ± 3.0 to 3179 ±93.0 pmol/10 6 cells or cellular selenium concentrations from 0.17 ±0.02 to 1.75 ±0.16 ng/10 6 cells, respectively. L929 fibroblasts grown in selenite-containing medium also had markedly raised activities of both cytosolic glutathione peroxidase (from 11 ±0.9 to 67.2 ±4.2 mU/10 7 cells) and phospholipid hydroperoxide glutathione peroxidase (from 0.2 to 9.5 ±0.9 mU/10 7 cells). Supplementation with α-tocopherol inhibited single-strand breaks induced by low concentrations of H 2O 2 only, whereas an adequate selenium supply almost completely inhibited single-strand breaks induced by up to 30μM H 2O 2 and also significantly reduced H 2O 2induced cell death. An inadequate selenium supply and corresponding increase of GPx activity upon selenite supplementation was also observed with other cell lines, for instance, D10N, ECV-304, HepG2, and THP-1. Our data strengthen the relevance of standardized and adequate supplementation of tissue culture media with antioxidants to improve viability and genetic stability of cultured cells in general and in particular, if they are oxidatively challenged.

An energetically structured model of mammalian cell metabolism. 1. Model development and application to steady-state hybridoma cell growth in continuous culture.

Incomplete understanding of mammalian cell culture kinetics hinders the ability of the biochemical engineer or biologist to design and control mammalian cell culture systems and to develop operating strategies. To address this problem, a mechanistic, structured mathematical model has been developed to simulate mammalian cell culture kinetics under a variety of bioreactor operating conditions. An important feature of in vitro mammalian cell metabolism in conventional cell culture media is the partial substitutability of the substrates glucose and glutamine for provision of energy in the cell. The utilization of glucose and glutamine by cells can therefore vary substantially, and these changes can profoundly affect the culture behavior. The model developed here specifically addresses the dynamics of substrate consumption and energy metabolism in mammalian cell culture. This energetically structured (ES) model is also distinguished by the consideration of changes in the specific cell mass with specific growth rate and the consideration of essential amino acids as potentially growth rate limiting. The model is applied here to simulate literature data on the growth and metabolism of a murine hybridoma in continuous culture.

Comparative Thermal Resistance of Human and Simian Rotaviruses Assayed on Cells Grown in a Serum-Free Medium

Thermal resistance of the Wa strain of human rotavirus was compared with that of SA-11 simian rotavirus in a plaque assay with MA-104 cells grown in conventional cell culture medium supplemented with filtrate from reconstituted and acid-precipitated nonfat dry milk. Both viruses produced two-component survival curves at 56 and 60°C. Average D-values (min) for the Wa human virus were 306.3 at 56°C, 47.8 at 60°C, 12.3 at 62.8°C and 2.4 at 65°C, with a z-value of 4.33°C. For the SA-11 simian virus, the average D-values were 890.5 at 56°C, with a z-value of 4.35°C. These values indicate that low levels of rotavirus should be inactivated by pasteurization processes.