Feeder cell density—A key parameter in human embryonic stem cell culture

Summary A key issue in human embryonic stem (ES) cell culture that has largely been ignored is the high degree of variability in the murine embryonic fibroblast (MEF) feeder cell density, which has been reported by different studies and protocols. Presumably, too low a feeder cell density would result in insufficient levels of secreted factors, extracellular matrix, and cellular contacts provided by the feeder cells for the maintenance of human ES cells in the undifferentiated state. Too high a feeder cell density, on the other hand, may result in a more rapid depletion of nutrients and oxygen within the in vitro culture milieu, as well as physically hinder the attachment and growth of ES colonies during serial passaging. Preliminary investigations by our group revealed that an elevated MEF cell density of 32,000 cells/cm², above the recommended value of 20,000 cells/cm², appeared to be highly detrimental to the attachment and growth of serially passaged ES colonies of the H9 line (WiCell Research Institute Inc., Wilmington, MA, USA). At the edge of ES colonies that have attached to the higher density feeder layer (32,000 cells/cm²), the ES cells appear to stack up to form a “bulge.” This was not observed under the recommended feeder cell density of 20,000 cells/cm². By contrast, other established ES cell lines are routinely propagated at much higher feeder densities of 60,000 to 70,000 cells/cm². This report briefly discusses the issue of MEF feeder cell density in relation to our preliminary observations, and the results of other studies.     

Artefacts in cell culture: α-Ketoglutarate can scavenge hydrogen peroxide generated by ascorbate and epigallocatechin gallate in cell culture media

Ascorbate and several phenolic compounds readily oxidise in cell culture media to generate hydrogen peroxide. However, addition of α-ketoglutarate, which is known to be released by several cell types, decreased the levels of H₂O₂, and the α-ketoglutarate was depleted and converted to succinate. These observations could account for previous reports of the protective effects of α-ketoglutarate in promoting the growth of cells in culture, and may contribute to explaining some of the variability in the literature in reported rates of H₂O₂ production from autoxidisable compounds in cell culture systems.     

Oxidative stress in cell culture: an under-appreciated problem?

Cell culture studies have given much valuable information about mechanisms of metabolism and signal transduction and of regulation of gene expression, proliferation, senescence, and death. However, cells in culture may behave differently from cells in vivo in many ways. One of these is that cell culture imposes a state of oxidative stress on cells. I argue that cells that survive and grow in culture might use ROS-dependent signal transduction pathways that rarely or never operate in vivo. A further problem is that cell culture media can catalyse the oxidation of compounds added to them, resulting in apparent cellular effects that are in fact due to oxidation products such as ROS. Such artefacts may have affected many studies on the effects of ascorbate, thiols, flavonoids and other polyphenolic compounds on cells in culture.     

Cereal β-glucan quantification with calcofluor-application to cell culture supernatants

The specific binding of the fluorescent dye calcofluor to cereal β-glucan results in increased fluorescence intensity of the formed complex and is in use for the quantification of β-glucan above a critical molecular weight (MW) by flow injection analysis. In this study, this method was applied in a fast and easy batch mode. In order to emphasize the spectral information of the emission spectra of the calcofluor/β-glucan complexes, derivative signals were calculated. A linear relationship was found between the amplitude of the second derivative signals and the β-glucan concentration between 0.1 and 0.4μg/mL. The low detection limit of this new method (0.045μg/mL) enabled its use to study the transport of cereal β-glucans over differentiated Caco-2 cell monolayers. Additionally, the method was applied to quantify β-glucan in arabinoxylan samples, which correlated well with data by an enzyme based method.     

Production of functional human α1-antitrypsin by plant cell culture

Recombinant human α₁-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice α-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6–5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins. Received: 18 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999     

Expression of α1-adrenergic receptor subtypes in heart cell culture

Three ₁AR subtypes have been cloned so far and are designated as _1a, _1b, and _1d. Organspecific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of ₁AR subtypes on RNA and proteinlevel in heart tissue, cultured cardiomyocytes and nonmyocytes of the rat. Each ₁ARsubtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the _1aAR subtype is preferentially expressed in adult cardiomyocytes, the _1bAR subtype was preferentially expressed in the nonmyocyte cell fraction. The RTPCR results were confirmed by Westernblotting (_1b) and immunocytochemical studies. Incubation with an ₁agonist (phenylephrine) for 72 h led to a significant reduction of the _1bAR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an ₁antagonist (prazosin) induced a 1.6 fold upregulation of the _1aAR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the ₁AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents.     

Hydrogen peroxide. Ubiquitous in cell culture and in vivo?

Hydrogen peroxide (H2O2) is widely regarded as a cytotoxic agent whose levels must be minimized by the action of antioxidant defence enzymes. In fact, H2O2 is poorly reactive in the absence of transition metal ions. Exposure of certain human tissues to H2O2 may be greater than is commonly supposed; levels of H2O2 in the human body may be controlled not only by catabolism but also by excretion, and H2O2 could play a role in the regulation of renal function and as an antibacterial agent in the urine. Cell culture is a widely used method for the investigation of "physiological" processes such as signal transduction and regulation of gene expression, but chemical reactions involving cell culture media are rarely considered. Addition of reducing agents to commonly used cell-culture media can lead to generation of substantial amounts of H2O2. Some or all of the reported effects of ascorbic acid and polyphenolic compounds (e.g., quercetin, catechin, epigallocatechin, epigallocatechin gallate) on cells in culture may be due to H2O2 generation by interaction of these compounds with cell culture media.     

Abeta mediated diminution of MTT reduction--an artefact of single cell culture?

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) reduction assay is a frequently used and easily reproducible method to measure beta-amyloid (Abeta) toxicity in different types of single cell culture. To our knowledge, the influence of Abeta on MTT reduction has never been tested in more complex tissue. Initially, we reproduced the disturbed MTT reduction in neuron and astroglia primary cell cultures from rats as well as in the BV2 microglia cell line, utilizing four different Abeta species, namely freshly dissolved Abeta (25-35), fibrillar Abeta (1-40), oligomeric Abeta (1-42) and oligomeric Abeta (1-40). In contrast to the findings in single cell cultures, none of these Abeta species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Abeta to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Abeta also did not influence the MTT reduction in the respective tissue. Failure of Abeta penetration into the tissue cannot explain the differences between single cells and the more complex brain tissue. Thus electrophysiological investigations disclosed an impairment of long-term potentiation (LTP) in the CA1 region of hippocampal slices from rat by application of oligomeric Abeta (1-40), but not by freshly dissolved Abeta (25-35) or fibrillar Abeta (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Abeta species on MTT reduction. Particularly, the differential effect of oligomeric versus other Abeta forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Abeta oligomer effect on synaptic function reflected by LTP impairment precedes changes in formazane formation rate or that cells embedded in a more natural environment in the tissue are less susceptible to damage by Abeta, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies.     

The cell culture medium — a functional extracellular compartment of suspension-cultured cells

The spent medium of suspension-cultured cells of Lupinus polyphyllus was analyzed by capillary GC and GC-MS and shown to contain ethanol (up to 160 mmol l^-1), organic acids (lactate, benzoate, succinate, fumarate, malate), amino acids (main components: alanine, glycine, serine, aspartate, ornithine, glutamate), and quinolizidine alkaloids (lupanine and an uncharacterized malonylderivative). In addition, cells obviously secrete polysaccharides and enzymes (acid phosphatase, phosphodiesterase, DNAse, esterase, -mannosidase, -galactosidase, -glucosidase, lipase, protease and peroxidase) into the medium. Typically these enzymes are localized in the vacuole of intact cells. Cytosolic enzymes, such as glutamate dehydrogenase and malate dehydrogenase were retained by the cells. Peroxidase is overexpressed in suspension-cultured lupin cells but only one basic isoenzyme is secreted, whereas the others are retained in the vacuole. In lupin leaves this isoenzyme is sequestered in the vacuole, implying that secretion is selective and needs a change in the sorting signals of the peroxidase protein. The cell culture medium shares many features of the vacuole. We assume therefore that the medium functions as a lytic compartment. In addition it provides a sink-source system for nutrients and metabolites.Abbreviations ADH alcohol dehydrogenase - FW fresh weight - GC gas chromatography - GC-MS gas chromatography — mass spectrometry - POD peroxidase - QA quinolizidine alkaloids     

Pinoresinol and matairesinol accumulation in a Forsythia × intermedia cell suspension culture

A newly established Forsythia × intermedia cell suspension culture was shown to accumulate (+)- and (–)-pinoresinol as well as matairesinol. The influence of the sucrose content of the culture medium and of the cultivation time on pinoresinol and matairesinol accumulation was evaluated. The highest pinoresinol yield was achieved from cells grown in medium containing 6% sucrose for 12 ± 2 days with levels of 0.6–0.8 mg g^–1 dry weight and an average enantiomeric composition of 75 ± 5% (+)-pinoresinol. The highest matairesinol amount was reached in the same medium at the 14th ± 2 culture day with levels of 1.0–2.7 mg g^–1 dry weight. To our knowledge, this is the first report on pinoresinol accumulation in Forsythia × intermedia plants or cell suspension cultures.     

Perfusion mammalian cell culture for recombinant protein manufacturing – A critical review

The manufacturing of recombinant protein is traditionally divided in two main steps: upstream (cell culture and synthesis of the target protein) and downstream (purification and formulation of the protein into a drug substance or drug product). Today, cost pressure, market uncertainty and market growth, challenge the existing manufacturing technologies. Leaders in the field are active in designing the process of the future and continuous manufacturing is recurrently mentioned as a potential solution to address some of the current limitations. This review focuses on the application of continuous processing to the first step of the manufacturing process. Enabling technologies and operation modes are described in the first part. In the second part, recent advances in the field that have the potential to support its successful future development are critically discussed.     

Induction of a photomixotrophic plant cell culture of Helianthus annuus and optimization of culture conditions for improved α-tocopherol production

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, which are synthesized only by photosynthetic organisms. Due to their enormous potential to protect cells from oxidative damage, tocopherols are used, e.g., as nutraceuticals and additives in pharmaceuticals. The most biologically active form of vitamin E is α-tocopherol. Most tocopherols are currently produced via chemical synthesis. Nevertheless, this always results in a racemic mixture of different and less effective stereoisomers because the natural isomer has the highest biological activity. Therefore, tocopherols synthesized in natural sources are preferred for medical purposes. The annual sunflower (Helianthus annuus L.) is a well-known source for α-tocopherol. Within the presented work, sunflower callus and suspension cultures were established growing under photomixotrophic conditions to enhance α-tocopherol yield. The most efficient callus induction was achieved with sunflower stems cultivated on solid Murashige and Skoog medium supplemented with 30 g l^?1 sucrose, 0.5 mg l^?1 of the auxin 1-naphthalene acetic acid, and 0.5 mg l^?1 of the cytokinin 6-benzylaminopurine. Photomixotrophic sunflower suspension cultures were induced by transferring previously established callus into liquid medium. The effects of light intensity, sugar concentration, and culture age on growth rate and α-tocopherol synthesis rate were characterized. A considerable increase (max. 230 %) of α-tocopherol production in the cells was obtained within the photomixotrophic cell culture compared to a heterotrophic cell culture. These results will be useful for improving α-tocopherol yields of plant in vitro cultures.     

A xeno-free culture method that enhances Wharton's jelly mesenchymal stromal cell culture efficiency over traditional animal serum–supplemented cultures

Background aimsMesenchymal stromal cell (MSC) transplantation holds great promise for use in medical therapies. Several key features of MSCs, including efficient cell growth, generation of sufficient cell numbers and safety, as determined by teratoma formation, make MSCs an ideal candidate for clinical use. However, MSCs derived under standard culture conditions, co-cultured with animal by-products, are inappropriate for therapy because of the risks of graft rejection and animal virus transmission to humans. Alternative serum sources have been sought for stem cell production.MethodsWe demonstrate for the first time that human serum from umbilical cord blood (hUCS) is an effective co-culture reagent for MSC production from Wharton's jelly MSCs (WJMSCs). Ten umbilical cords were used to generate parallel cultures of WJMSC lines under medium supplemented with hUCS and embryonic stem cell-qualified fetal bovine serum. The WJMSC lines from each medium were analyzed and compared with regard to cell line derivation, proliferation ability and characteristic stability.ResultsThe phenotypic characteristics of WJMSC derived under either medium showed no differences. WJMSC lines derived under hUCS medium displayed comparable primary culture cell outgrowth, lineage differentiation capacity and cell recovery after cryopreservation compared with supplementation with embryonic stem cell-qualified fetal bovine serum medium. However, superior cell proliferation rates and retention of in vitro propagation (>22 passages) were observed in WJMSC cultures supplemented with hUCS. Additionally, more robust population doubling times were observed in hUCS-supplemented cultures.ConclusionsWe conclude that hUCS is an efficient and effective serum source for animal product–free WJMSC line production and can generate MSC lines that may be appropriate for therapeutic use.     

Plant cell culture technology–harnessing a biological approach for competitive cyclotides production

Cyclotides are naturally occurring mini-proteins that have a cyclic backbone and a knotted arrangement of three disulfide bonds. They are remarkably stable and have a diverse range of therapeutically useful biological activities, including antimicrobial and anti-HIV activity, although their natural function appears to be plant defence agents. Cyclotides are amenable to chemical synthesis; however currently most bioactivity studies have involved the use of peptides extracted from plants. Plant cell culture technology shows promise towards the goal of producing therapeutically active cyclotides in qualities and quantities required for drug development.     

Hydroxylation and glycosylation of Δ9-tetrahydrocannabinol by Catharanthus roseus cell suspension culture

Δ⁹-tetrahydrocannabinol is the active constituent in Cannabis sativa, with reported analgesic, anti-emetic, anti-oxidative, neuroprotective, and anti-inflammatory activities. Δ⁹-THC has been used to treat a number of disease states including pain, anxiety, asthma, glaucoma, and hypertension. Poor water solubility of Δ⁹-THC greatly reduces its clinical effectiveness. Consequently, there is a need to modify the compound to increase its polarity and pharmaceutical efficacy. The aim of this study was to test the capability of Catharanthus roseus suspension cultured cells to convert Δ⁹-THC into more polar derivatives. The transformed metabolites were analyzed and isolated by HPLC. Structures of some new derivatives were proposed on the basis of molecular ion peaks and fragmentation patterns obtained from LC-MS and UV spectra obtained by HPLC, respectively. Δ⁹-THC was rapidly absorbed by Catharanthus roseus cultured cells and upon biotransformation new glycosylated and hydroxylated derivatives were isolated by preparative HPLC. In addition, cannabinol was detected as degradation product, including its glycosylated derivative. Based on these results, it is concluded that Catharanthus cultured cells have great potential to transform Δ⁹-THC into more polar derivatives and can be used for the large scale production of new cannabinoids, which can be a source of new compounds with interesting pharmacological profiles.     

Prothymosin-α interacts with mutant huntingtin and suppresses its cytotoxicity in cell culture

Huntington disease (HD), a fatal neurodegenerative disorder, is caused by a lengthening of the polyglutamine tract in the huntingtin (Htt) protein. Despite considerable effort, thus far there is no cure or treatment available for the disorder. Using the approach of tandem affinity purification we recently discovered that prothymosin-α (ProTα), a small highly acidic protein, interacts with mutant Htt (mHtt). This was confirmed by co-immunoprecipitation and a glutathione S-transferase (GST) pull-down assay. Overexpression of ProTα remarkably reduced mHtt-induced cytotoxicity in both non-neuronal and neuronal cell models expressing N-terminal mHtt fragments, whereas knockdown of ProTα expression in the cells enhanced mHtt-caused cell death. Deletion of the central acidic domain of ProTα abolished not only its interaction with mHtt but also its protective effect on mHtt-caused cytotoxicity. Additionally, overexpression of ProTα inhibited caspase-3 activation but enhanced aggregation of mHtt. Furthermore, when added to cultured cells expressing mHtt, the purified recombinant ProTα protein not only entered the cells but it also significantly suppressed the mHtt-caused cytotoxicity. Taken together, these data suggest that ProTα might be a novel therapeutic target for treating HD and other polyglutamine expansion disorders.     

What if cell culture media do not mimic in vivo redox settings?

Here, I address the topic of suitability for redox research of common settings in cell cultures. This is done through the prism of in vitro anticancer effects of vitamin C. Cell culture media show lower concentrations of iron and a higher level of oxygen compared to interstitial fluid. Such a setup promotes ascorbate-mediated production and accumulation of hydrogen peroxide, which efficiently kills a variety of cancer cell lines. However, the anticancer effects are annihilated if the iron level is corrected to mimic in vivo concentrations. It appears that the potential benefits of application of vitamin C in cancer treatment have been significantly overestimated. This might be true for other pro-oxidative agents as well, such as some (poly)phenols. We urgently need to establish medium formula and culture maintenance settings that are optimal for redox research.