Showing papers on "Tissue culture published in 2014"

Addressing the Instability of DNA Nanostructures in Tissue Culture

Abstract: DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using in vitro tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced via the origami method to the in vitro tissue culture environment and identify solutions to prevent loss of nanostructure integrity. We examined whether the physiological cation concentrations of cell culture medium and the nucleases present in fetal bovine serum (FBS) used as a medium supplement result in denaturation and digestion, respectively. DNA nanostructure denaturation due to cation depletion was design- and time-dependent, with one of four tested designs remaining intact after 24 h at 37 °C. Adjustment of medium by addition of MgSO4 prevented denaturation. Digestion of nanostructures by FBS nucleases in Mg(2+)-adjusted medium did not appear design-dependent and became significant within 24 h and when medium was supplemented with greater than 5% FBS. We estimated that medium supplemented with 10% FBS contains greater than 256 U/L equivalent of DNase I activity in digestion of DNA nanostructures. Heat inactivation at 75 °C and inclusion of actin protein in medium inactivated and inhibited nuclease activity, respectively. We examined the impact of medium adjustments on cell growth, viability, and phenotype. Adjustment of Mg(2+) to 6 mM did not appear to have a detrimental impact on cells. Heat inactivation was found to be incompatible with in vitro tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two in vitro assays, immune cell activation and nanoparticle endocytosis, we show that using conditions compatible with cell phenotype and nanostructure integrity is critical for obtaining reliable experimental data. Our study thus describes considerations that are vital for researchers undertaking in vitro tissue culture studies with DNA nanostructures and some potential solutions for ensuring that nanostructure integrity and functions are maintained during experiments.

Inhibition of demethylases by GSK-J1/J4

Abstract: Arising from L. Kruidenier et al. 404–408 (2012); doi:10.1038/nature11262 The recent publication1 of the first highly potent and specific inhibitor GSK-J1/J4 of the H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A provides a potential tool compound for this histone demethylase subfamily1. This inhibitor was used in tissue culture assays to conclude that the catalytic activities of the KDM6 proteins are required in inflammatory responses1; the generation of the inhibitor is intriguing, because it provides a strategy for generating sub-type-specific inhibitors of the 27-member Jumonji family and for the future treatment of various types of disease2,3,4,5,6. Here we show that the inhibitor is not specific for the H3K27me3/me2-demethylase subfamily in vitro and in tissue culture assays. Thus, the inhibitor cannot be used alone for drawing conclusions regarding the specific role of H3K27me3/me2-demethylase activity in biological processes or disease. There is a Reply to this Brief Communications Arising by Kruidenier 514, http://dx.doi.org/10.1038/nature13689 (2014).

Plant tissue culture: applications and limitations

Abstract: Introduction. 1. The current status of plant tissue culture (Trevor A. Thorpe). 2. Organogenic differentiation (K. Tran Thanh Van and T.H. Trinh). 3. Somatic embryogenesis (M. Terzi and F. LoSchiavo). 4. Applications of somatic embryogenesis and embryo cloning (S.A. Merkle, W.A. Parrott and E.G. Williams). 5. Role of artificial seeds in alfalfa breeding (Keith Redenbaugh and Keith Walker). 6. Regulation of genes in differentiation (L.D. Owens and A.C. Smigocki). 7. Tissue culture in relation to ornamental plants (P. Debergh, J. Roggemans and R. Standaert-De Metsenaere). 8. Clonal multiplication of woody perennials (K. Paranjothy, S. Saxena, M. Banerjee, V.S. Jaiswal and S.S. Bhojwani). 9. Anther and pollen culture (R.S. Sangwan and B.S. Sangwan-Norreel). 10. In vitro gynogenesis (H.Y. Yang and C. Zhou). 11. Production of industrial compounds (Y. Fujita). 12. Cytogenetics of plant cell cultures (V. Nuti Ronchi). 13. Application of tissue culture variability to crop improvement (J. Semal and P. Lepoivre). 14. Somatic hybridization and cybridization (Y.Y. Gleba and L.R. Shlumukov). 15. Genetic engineering of crop plants (D.S. Brar and H. Uchimiya). 16. Zygotic embryo culture (M. Monnier). 17. In vitro conservation of germplasm (Brian W.W. Grout). 18. Expectations of plant breeders from tissue culture (G.S. Khush, S.S. Virmani and D.S. Brar). 19. Plant tissue culture in the twenty-first century (Kenneth L. Giles and David D. Songstad). Author Index.

In vitro culture: an epigenetic challenge for plants

Abstract: In vitro plant cell and tissue culture techniques are the basis of many micropropagation and breeding programs for scientific research. Plant tissue culture (PTC) involves organogenesis and embryogenesis, and the outcome depends on the different conditions to which the tissue is exposed. PTC is a stressful environment—high relative humidity, low ventilation rate, high concentrations of plant growth regulators, and low light availability—for plants that need to rapidly change their molecular regulation in order to respond fast and efficiently during cell division and growth. For instance, somatic embryogenesis (SE), which plays an important role in plant multiplication, requires complex cellular, biochemical and molecular processes for embryo formation and development. New data has come out about a connection between plant morphogenesis and epigenetics. Epigenetics is a very sensitive regulatory mechanism, which in most of cases is affected by the environment. Although it is known that, under plant morphogenesis, the genome has little or no change, DNA methylation and histone modifications are very susceptible to those in vitro environmental conditions. In the present review, we highlight the most used in vitro systems such as organogenesis and SE in plants and discuss how epigenetics plays a pivotal role in the phenotype outcome. Furthermore, we discuss the big role that the small RNAs have during cell division and propagation and propose different challenges and opportunities to study epigenetics in plant cell tissue and organ cultures.

Whole transcriptome profiling of maize during early somatic embryogenesis reveals altered expression of stress factors and embryogenesis-related genes.

Abstract: Embryogenic tissue culture systems are utilized in propagation and genetic engineering of crop plants, but applications are limited by genotype-dependent culture response. To date, few genes necessary for embryogenic callus formation have been identified or characterized. The goal of this research was to enhance our understanding of gene expression during maize embryogenic tissue culture initiation. In this study, we highlight the expression of candidate genes that have been previously regarded in the literature as having important roles in somatic embryogenesis. We utilized RNA based sequencing (RNA-seq) to characterize the transcriptome of immature embryo explants of the highly embryogenic and regenerable maize genotype A188 at 0, 24, 36, 48, and 72 hours after placement of explants on tissue culture initiation medium. Genes annotated as functioning in stress response, such as glutathione-S-transferases and germin-like proteins, and genes involved with hormone transport, such as PINFORMED, increased in expression over 8-fold in the study. Maize genes with high sequence similarity to genes previously described in the initiation of embryogenic cultures, such as transcription factors BABY BOOM, LEAFY COTYLEDON, and AGAMOUS, and important receptor-like kinases such as SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASES and CLAVATA, were also expressed in this time course study. By combining results from whole genome transcriptome analysis with an in depth review of key genes that play a role in the onset of embryogenesis, we propose a model of coordinated expression of somatic embryogenesis-related genes, providing an improved understanding of genomic factors involved in the early steps of embryogenic culture initiation in maize and other plant species.

Consistent and Heritable Alterations of DNA Methylation Are Induced by Tissue Culture in Maize

Abstract: Plants regenerated from tissue culture and their progenies are expected to be identical clones, but often display heritable molecular and phenotypic variation. We characterized DNA methylation patterns in callus, primary regenerants, and regenerant-derived progenies of maize using immunoprecipitation of methylated DNA (meDIP) to assess the genome-wide frequency, pattern, and heritability of DNA methylation changes. Although genome-wide DNA methylation levels remained similar following tissue culture, numerous regions exhibited altered DNA methylation levels. Hypomethylation events were observed more frequently than hypermethylation following tissue culture. Many of the hypomethylation events occur at the same genomic sites across independent regenerants and cell lines. The DNA methylation changes were often heritable in progenies produced from self-pollination of primary regenerants. Methylation changes were enriched in regions upstream of genes and loss of DNA methylation at promoters was associated with altered expression at a subset of loci. Differentially methylated regions (DMRs) found in tissue culture regenerants overlap with the position of naturally occurring DMRs more often than expected by chance with 8% of tissue culture hypomethylated DMRs overlapping with DMRs identified by profiling natural variation, consistent with the hypotheses that genomic stresses similar to those causing somaclonal variation may also occur in nature, and that certain loci are particularly susceptible to epigenetic change in response to these stresses. The consistency of methylation changes across regenerants from independent cultures suggests a mechanistic response to the culture environment as opposed to an overall loss of fidelity in the maintenance of epigenetic states.

The role of silicon in plant tissue culture

Abstract: Growth and morphogenesis of in vitro cultures of plant cells, tissues and organs are greatly influenced by the composition of the culture medium. Mineral nutrients are necessary for the growth and development of plants. Several morpho-physiological disorders such as hooked leaves, hyperhydricity, fasciation and shoot tip necrosis are often associated with the concentration of inorganic nutrient in the tissue culture medium. Silicon (Si) is the most abundant mineral element in the soil. The application of Si has been demonstrated to be beneficial for growth, development and yield of various plants and to alleviate various stresses including nutrient imbalance. Addition of Si to the tissue culture medium improves organogenesis, embryogenesis, growth traits, morphological, anatomical and physiological characteristics of leaves, enhances tolerance to low temperature and salinity, protects cells and against metal toxicity, prevents oxidative phenolic browning and reduces the incidence of hyperhydricity in various plants. Therefore, Si possesses considerable potential for application in a wide range of plant tissue culture studies such as cryopreservation, organogenesis, micropropagation, somatic embryogenesis and secondary metabolites production.

Isolation of Mitochondria from Tissue Culture Cells

Abstract: The number of mitochondria per cell varies substantially from cell line to cell line. For example, human HeLa cells contain at least twice as many mitochondria as smaller mouse L cells. This protocol starts with a washed cell pellet of 1-2 mL derived from ∼10⁹ cells grown in culture. The cells are swollen in a hypotonic buffer and ruptured with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and mitochondria are isolated by differential centrifugation.

Human Umbilical Cord-Derived Mesenchymal Stem Cells Do Not Undergo Malignant Transformation during Long-Term Culturing in Serum-Free Medium

Abstract: Background Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are in the foreground as a preferable application for treating diseases. However, the safety of hUC-MSCs after long-term culturing in vitro in serum-free medium remains unclear. Methods hUC-MSCs were separated by adherent tissue culture. hUC-MSCs were cultured in serum-free MesenCult-XF medium and FBS-bases DMEM complete medium. At the 1st, 3rd, 5th, 8th, 10th, and 15th passage, the differentiation of MSCs into osteogenic, chondrogenic, and adipogenic cells was detected, and MTT, surface antigens were measured. Tumorigenicity was analyzed at the 15th passage. Conventional karyotyping was performed at passage 0, 8, and 15. The telomerase activity of hUC-MSCs at passage 1–15 was analyzed. Results Flow cytometry analysis showed that very high expression was detected for CD105, CD73, and CD90 and very low expression for CD45, CD34, CD14, CD79a, and HLA-DR. MSCs could differentiate into osteocytes, chondrocytes, and adipocytes in vitro. There was no obvious chromosome elimination, displacement, or chromosomal imbalance as determined from the guidelines of the International System for Human Cytogenetic Nomenclature. Telomerase activity was down-regulated significantly when the culture time was prolonged. Further, no tumors formed in rats injected with hUC-MSCs (P15) cultured in serum-free and in serum-containing conditions. Conclusion Our data showed that hUC-MSCs met the International Society for Cellular Therapy standards for conditions of long-term in vitro culturing at P15. Since hUC-MSCs can be safely expanded in vitro and are not susceptible to malignant transformation in serum-free medium, these cells are suitable for cell therapy.

Morpho-histology and genotype dependence of in vitro morphogenesis in mature embryo cultures of wheat.

Abstract: Cellular totipotency is one of the basic principles of plant biotechnology. Currently, the success of the procedure used to produce transgenic plants is directly proportional to the successful insertion of foreign DNA into the genome of suitable target tissue/cells that are able to regenerate plants. The mature embryo (ME) is increasingly recognized as a valuable explant for developing regenerable cell lines in wheat biotechnology. We have previously developed a regeneration procedure based on fragmented ME in vitro culture. Before we can use this regeneration system as a model for molecular studies of the morphogenic pathway induced in vitro and investigate the functional links between regenerative capacity and transformation receptiveness, some questions need to be answered. Plant regeneration from cultured tissues is genetically controlled. Factors such as age/degree of differentiation and physiological conditions affect the response of explants to culture conditions. Plant regeneration in culture can be achieved through embryogenesis or organogenesis. In this paper, the suitability of ME tissues for tissue culture and the chronological series of morphological data observed at the macroscopic level are documented. Genetic variability at each step of the regeneration process was evaluated through a varietal comparison of several elite wheat cultivars. A detailed histological analysis of the chronological sequence of morphological events during ontogeny was conducted. Compared with cultures of immature zygotic embryos, we found that the embryogenic pathway occurs slightly earlier and is of a different origin in our model. Cytological, physiological, and some biochemical aspects of somatic embryo formation in wheat ME culture are discussed.

Purification of Mitochondria by Sucrose Step Density Gradient Centrifugation

Abstract: Mitochondrial fractions isolated from tissue culture cells or tissue such as liver after differential centrifugation can be purified further by density gradient centrifugation. Here we describe the use of sucrose for this purpose because it is commonly used and inexpensive and the resulting mitochondria preparations are useful for many purposes.

A Novel Axial-Stress Bioreactor System Combined with a Substance Exchanger for Tissue Engineering of 3D Constructs

Abstract: This study introduced a prototype of an axial-stress bioreactor system that supports long-term growth and development of engineered tissues. The main features of this bioreactor are an integrated substance exchanger and feedback control of pH and PO₂. A 21-day study was conducted to validate the system's ability to maintain a stable environment, while remaining sterile. Our results showed that the pH, PO₂, and nutrient (glucose) remained balanced at appropriate levels, while metabolic waste (lactic acid) was removed. No bacteria or fungi were detected in the system or tissue; thus, demonstrating that it was sterile. These data indicate the bioreactor's strong potential for long-term tissue culture. To explore this idea, the effect of dynamic culture, including cyclic compression and automatic substance exchange, on mouse bone-marrow mesenchymal stem cells (BMSCs) seeded in decalcified bone matrix was studied using the bioreactor prototype. Histological sections of the engineered tissues showed higher cell densities in scaffolds in dynamic culture compared to those in static culture, while cell cycle analysis showed that dynamic culture promoted BMSC proliferation (proliferation index, PI=34.02±1.77) more effectively than static culture (PI=26.66±1.81). The results from a methyl thiazolyl tetrazolium assay were consistent with the loading experimental data. Furthermore, elevated alkaline phosphatase activity and calcium content were observed in dynamic condition compared to static culture. In conclusion, this bioreactor system supplies a method of modulating the pH and PO₂ in defined ranges with only small fluctuations; it can be used as a physiological or pathological analog. Automatic control of the environment is a practical solution for long-term, steady-state culture for future commercialization.

Storage and qualification of viable intact human amniotic graft and technology transfer to a tissue bank.

Abstract: Human amniotic membrane (hAM) is known to have good potential to help the regeneration of tissue. It has been used for over 100 years in many medical disciplines because of its properties, namely a scaffold containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. In order to use this “boosted membrane” as an advanced therapeutic medicinal product for bone repair, we aimed to observe the influence of tissue culture and/or cryopreservation on cell viability and tissue structure, and secondly, to adapt to a tissue bank, identify easy processes to store hAM containing viable cells and to verify the quality of the graft before its release for use. To this end, we tested different published culture or cryopreservation storage conditions and cell viability assays. Tissue structure was evaluated by Giemsa staining and was compared to histological analysis. Preliminary results show no dramatic decrease in cell viability in cultured hAM as compared to cryopreserved hAM, but tissue structure alterations were observed with both storage conditions. Histological and immunohistochemical data highlight that tissue damage was associated with significantly modified protein expression, which could lead to a possible loss of differentiation potential. Finally, we report that trypan blue and Giemsa staining could constitute controls that are “materially and easily transferable” to a tissue bank.

Full-Thickness Skin with Mature Hair Follicles Generated from Tissue Culture Expanded Human Cells

Abstract: The goal of regenerative medicine is to reconstruct fully functional organs from tissue culture expanded human cells. In this study, we report a method for human reconstructed skin (hRSK) when starting with human cells. We implanted tissue culture expanded human epidermal and dermal cells into an excision wound on the back of immunodeficient mice. Pigmented skin covered the wound 4 weeks after implantation. Hair shafts were visible at 12 weeks and prominent at 14 weeks. Histologically, the hRSK comprises an intact epidermis and dermis with mature hair follicles, sebaceous glands and most notably, and unique to this system, subcutis. Morphogenesis, differentiation, and maturation of the hRSK mirror the human fetal process. Human antigen markers demonstrate that the constituent cells are of human origin for at least 6 months. The degree of new skin formation is most complete when using tissue culture expanded cells from fetal skin, but it also occurs with expanded newborn and adult cells; however, no appendages formed when we grafted both adult dermal and epidermal cells. The hRSK system promises to be valuable as a laboratory model for studying biological, pathological, and pharmaceutical problems of human skin.

Profiling microRNAs in Eucalyptus grandis reveals no mutual relationship between alterations in miR156 and miR172 expression and adventitious root induction during development.

Abstract: The change from juvenile to mature phase in woody plants is often accompanied by a gradual loss of rooting ability, as well as by reduced microRNA (miR) 156 and increased miR172 expression. We characterized the population of miRNAs of Eucalyptus grandis and compared the gradual reduction in miR156 and increase in miR172 expression during development to the loss of rooting ability. Forty known and eight novel miRNAs were discovered and their predicted targets are listed. The expression pattern of nine miRNAs was determined during adventitious root formation in juvenile and mature cuttings. While the expression levels of miR156 and miR172 were inverse in juvenile and mature tissues, no mutual relationship was found between high miR156 expression and rooting ability, or high miR172 expression and loss of rooting ability. This is shown both in E. grandis and in E. brachyphylla, in which explants that underwent rejuvenation in tissue culture conditions were also examined. It is suggested that in these Eucalyptus species, there is no correlation between the switch of miR156 with miR172 expression in the stems and the loss of rooting ability.

Genetic transformation of wheat via Agrobacterium-mediated DNA delivery.

Abstract: The method described involves an initial incubation of wheat immature embryos in a liquid culture of Agrobacterium tumefaciens. The Agrobacterium strain is engineered to contain a binary vector with a gene of interest and a selectable marker gene placed between the T-DNA borders; the T-DNA is the region transferred to the plant cells, thus harnessing the bacterium's natural ability to deliver specific DNA into host cells. Following the initial inoculation with the Agrobacterium, the embryos are co-cultivated for several days after which the Agrobacterium is selectively destroyed using an antibiotic. Tissue culture of the embryos on plant media with a correct balance of hormones allows embryogenic callus formation followed by regeneration of plantlets, and in the later stages of tissue culture a selectable marker (herbicide) is included to minimize the incidence of non-transformed plants. This protocol has been used successfully to generate transformed plants of a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.).

Extended metAFLP approach in studies of tissue culture induced variation (TCIV) in triticale

Abstract: We present the development of the theoretical background of the metAFLP approach which allows for partition of complex variation into sequence changes, de novo methylation and demethylation of the regenerants derived via in vitro tissue culture methods in the case of triticale. It was demonstrated that, independent of whether andro- or embryogenesis was used for plant regeneration, the level of sequence changes identified between regenerants is about 10 %. Moreover, DNA demethylation prevails over de novo methylation of the regenerants compared to the donor plant. The metAFLP approach allows for the evaluation of numerous quantitative characteristics. For instance, one may quantify the number of sites unaffected by tissue culture approaches, global site DNA methylation etc. It is suggested that the approach could be useful for breeders in order to control plant material uniformity or for the evaluation of modified in vitro tissue culture approaches allowing for control of the (epi)mutation level. The extended metAFLP approach presented here delivers sufficient background for the evaluation of software that could facilitate analyses of the tissue culture induced variation.

3‐Dimensional Tissue Is Formed From Cancer Cells In Vitro on Gelfoam®, But Not on MatrigelTM

Abstract: Cell and tissue culture can be performed on different substrates such as on plastic, in Matrigel™, and on Gelfoam(®), a sponge matrix. Each of these substrates consists of a very different surface, ranging from hard and inflexible, a gel, and a sponge-matrix, respectively. Folkman and Moscona found that cell shape was tightly coupled to DNA synthesis and cell growth. Therefore, the flexibility of a substrate is important for cells to maintain their optimal shape. Human osteosarcoma cells, stably expressing a fusion protein of α(v) integrin and green fluorescent protein (GFP), grew as a simple monolayer without any structure formation on the surface of a plastic dish. When the osteosarcoma cells were cultured within Matrigel™, the cancer cells formed colonies but no other structures. When the cancer cells were seeded on Gelfoam(®), the cells formed three-dimensional tissue-like structures. The behavior of 143B osteosarcoma cells on Gelfoam(®) in culture is remarkably different from those of these cells in monolayer culture or in Matrigel™. Tissue-like structures were observed only in Gelfoam(®) culture. The data in this report suggest a flexible structural substrate such as Gelfoam(®) provides a more in vivo-like culture condition than monolayer culture or Matrigel(TM) and that Matrigel(TM) does not result in actual three-dimensional culture.

Feet on the ground: Physical support of the inner retina is a strong determinant for cell survival and structural preservation in vitro.

Abstract: Purpose: The purpose of this study was to explore the importance of local physical tissue support for homeostasis in the isolated retina. Methods: Full-thickness retinal sheets were isolated from adult porcine eyes. Retinas were cultured for 5 or 10 days using the previously established explant protocol with photoreceptors positioned against the culture membrane (porous polycarbonate) or the Muller cell endfeet and inner limiting membrane (ILM) apposed against the membrane. The explants were analyzed morphologically using hematoxylin and eosin staining, immunohistochemistry, TUNEL labeling, and transmission electron microscopy (TEM). Results: Standard cultures displayed a progressive loss of retinal lamination and extensive cell death, with activated, hypertrophic Muller cells. In contrast, explants cultured with the ILM facing the membrane displayed a maintenance of the retinal laminar architecture, and a statistically significant attenuation of photoreceptor and ganglion cell death. TEM revealed intact synapses as well as preservation of normal cellular membrane structures. Immunohistochemistry showed no signs of Muller cell activation (GFAP), with maintained expression of important metabolic markers (GS, bFGF). Conclusion: Providing physical support to the inner but not the outer retina appears to prevent the tissue collapse resulting from perturbation of the normal biomechanical milieu in the isolated retinal sheet. Using this novel paradigm, gliotic reactions are attenuated, and metabolic processes vital for tissue health are preserved which significantly increases neuronal cell survival. This finding opens up new avenues of adult retinal tissue culture research, and increases our understanding of pathological reactions in biomechanically related conditions in vivo.

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

Abstract: Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.

Chemically defined serum-free and xeno-free media for multiple cell lineages.

Abstract: Cell culture is one of the most common methods used to recapitulate a human disease environment in a laboratory setting. Cell culture techniques are used to grow and maintain cells of various types including those derived from primary tissues, such as stem cells and cancer tumors. However, a major confounding factor with cell culture is the use of serum and animal (xeno) products in the media. The addition of animal products introduces batch and lot variations that lead to experimental variability, confounds studies with therapeutic outcomes for cultured cells, and represents a major cost associated with cell culture. Here we report a commercially available serum-free, albumin-free, and xeno free (XF) media (Neuro-Pure TM ) that is more cost-effective than other commercial medias. Neuro-Pure was used to maintain and differentiate various cells of neuronal lineages, fibroblasts, as well as specific cancer cell lines; without the use of contaminants such serum, albumin, and animal products. Neuro-Pure allows for a controlled and reproducible cell culture environment that is applicable to translational medicine and general tissue culture.