Maintenance Of Cell Cultures Research Articles

Zebrafish as a Model Organism to Study Nanomaterial Toxicity

Recent developments in nanotechnology has increased the market value of nanoproducts in various industries. This has increased concerns associated with potential toxicity of nanoproducts to humans and the environments. Even though, green and biosynthesized nanoparticles are considered to be less toxic than chemically synthesized nanoparticles, they still possess some level of toxicity. Conventional toxicity assessments via human cells, live animals such as rat, frog or rabbit have several drawbacks including ethical issue and challenges involving the maintenance and development of cell cultures. Zebrafish (Danio rerio) is a transparent vertebrate fish that can reproduce rapidly. Its larvae develop in 5 days up to 3-5 cm long. It also possesses about 69% similar genetic profile, molecular mechanism, cell development and organ physiology as humans. Hence, it has the potential to be utilized as an alternative to humans or live animal models for initial drug screening and toxicity tests. European Union, USFDA and ICH have approved the use of zebrafish for toxicological evaluation of pharmaceutical products including nanomedicines. The article presents for the potential of zebrafish in preclinical evaluation of the toxicity of nanomaterials. It also discusses other potential applications, including medical imaging and environmental toxicity.

Breast tumour organoids: promising models for the genomic and functional characterisation of breast cancer.

Until recently, established cancer cell lines have been used extensively in breast cancer research, due largely to the difficulties associated with the manipulation and long-term maintenance in culture of primary tumour cells from patients. The recent development of organoid cultures has provided new opportunities to model and analyse patient samples, allowing the propagation of malignant cells under conditions that resemble the three-dimensional growth of breast tumours. They have proved efficacious in preserving the heterogeneity of primary samples and are emerging as a new model to further characterise the molecular features of breast cancer. Organoids formed from patient-derived cells are now in use for the evaluation of drug sensitivity and to validate disease-causing genomic variations. Here, the advantages and limitations of organoid cultures will be discussed and compared with the parallel development of other two- and three-dimensional culture strategies and with patient-derived xenografts. In particular, we will focus on the molecular characterisation of breast cancer organoids and provide some examples of how they have been used in functional studies.

In Vitro Culture and Characterization of Testis-Derived Cells from Clarias magur (Hamilton, 1822)

A cell-based model offers many potential applications in biology. In vitro culture of testis-originated cells of Clarias magur was carried out using explant method. The cells were grown in different media compositions (including fetal bovine serum), concentrations, and temperatures. The developed culture was grown up to five passages. Leibovitz-15 (L-15) media with 20% FBS was found suitable, and culture conditions between 25 and 28 °C temperatures supported cell growth. The study is further focused on evaluation of the requirement of the other supplements along with the favorable basal media for growth and maintenance of testis-derived cell culture of C. magur. Epithelial-like cells in morphology were observed which were further confirmed with cell-specific markers. Chromosome observation revealed diploid number of 50 chromosomes as reported for the species. The species specificity of cultured cells was confirmed by amplification of two universal markers COI and 16S rRNA. The present study can be useful in the development of testicular cell line of C. magur.

Reduced oxygen tension culturing conditionally alters toxicogenic response of differentiated H9c2 cardiomyoblasts to acrolein

Acrolein is a reactive electrophilic aldehyde known to cause mitochondrial dysfunction, oxidative stress, and dysregulation of signaling transduction in vitro. Most in vitro systems employ standard cell culture maintenance conditions of 95% air/5% CO2, translating to a culture oxygen tension of approximately 20%, far above most physiological tissues. The purpose of this investigation was to examine whether low-serum, retinoic acid differentiated H9c2 cells were less sensitive to acrolein insult when cultured under reduced oxygen tension. H9c2 cells were maintained separately in 20% and 5% oxygen, differentiated for 5 d, and then exposed to acrolein for 30 min in media containing varying concentrations of tricarboxylic acid and glycolytic substrates, followed by fresh medium replacement. Cells were then assessed for MTT reduction at 2 h and 24 h after acrolein insult. We showed that pyruvate supplementation in combination with lowered oxygen culturing significantly attenuated acrolein-induced viability loss at 24 h. Poly(ADP-ribose) polymerase inhibition and EGTA preferentially provided partial rescue to low oxygen cultures, but not for standard cultures. Collectively, these results offer evidence supporting altered toxicogenic response of H9c2 during physiologically relevant oxygen tension culturing.

Mutual interactions between telomere heterogeneity and cell culture growth dynamics shape stochasticity of cell aging.

Mathematical modeling and computational simulations are often used to explain the stochastic nature of cell aging. The models published thus far are based on the molecular mechanisms of telomere biology and how they dictate the dynamics of cell culture proliferation. However, the influence of cell growth conditions on telomere dynamics has been widely overlooked. These conditions include interactions with surrounding cells through contact inhibition, gradual accumulation of non-dividing cells, culture propagation and other cell culture maintenance factors. In order to follow the intrinsic growth dynamics of normal human fibroblasts we employed the fluorescent dye DiI and FACS analysis which can distinguish cells that undergo different numbers of divisions within culture. We observed rapid generation of cell subpopulations undergoing from 0 to 9 divisions within growing cultures at each passage analyzed. These large differences in number of divisions among individual cells guarantee a strong impact on generation of telomere length heterogeneity in normal cell cultures and suggest that culture conditions should be included in future modeling of cell senescence.

Developing a Continuous Bioprocessing Approach to Stromal Cell Manufacture.

To this day, the concept of continuous bioprocessing has been applied mostly to the manufacture of molecular biologics such as proteins, growth factors, and secondary metabolites with biopharmaceutical uses. The present work now sets to explore the potential application of continuous bioprocess methods to source large numbers of human adherent cells with potential therapeutic value. To this purpose, we developed a smart multifunctional surface coating capable of controlling the attachment, proliferation, and subsequent self-detachment of human corneal stromal cells. This system allowed the maintenance of cell cultures under steady-state growth conditions, where self-detaching cells were continuously replenished by the proliferation of those remaining attached. This facilitated a closed, continuous bioprocessing platform with recovery of approximately 1% of the total adherent cells per hour, a yield rate that was maintained for 1 month. Moreover, both attached and self-detached cells were shown to retain their original phenotype. Together, these results represent the proof-of-concept for a new high-throughput, high-standard, and low-cost biomanufacturing strategy with multiple potentials and important downstream applications.

In Vitro Monitoring of a Cultured Human Retinal Pigment Epithelium Using 1375-nm Spectral-Domain Optical Coherence Tomography

Measurement of cell number is important in cell-based in vitro screenings for therapeutic drugs and tissue engineering such as cell culture maintenance, cell plating, and cell growth, as well as monitoring of cell viability, proliferation, and cytotoxicity. We performed cell counting using intensity-based in vitro spectral-domain optical coherence tomography (SD-OCT). Adult retinal pigment epithelium cells were cultured on a glass dish to prevent diffuse reflection from the surface, and the dish was tilted by 15° during OCT imaging to reduce coherence noise from specular reflection. Rasterized scanning was performed to generate a three-dimensional volume image with an en face image of the retinal pigment epithelium cell layers. Cell counting was achieved by measuring the density of bright spots after layer extraction and thresholding. Two other cell counting methods were also performed for the purpose of comparison, one using a hemocytometer and the other using water-soluble tetrazolium-1; cell counting by in vitro OCT yielded results better than those from the hemocytometer. Our results showed that in vitro OCT systems can be a powerful tool for estimating and analyzing cell density in a cultured sample without the need for dyeing the sample or causing cell death.

Establishing and maintaining primary cell cultures derived from the ctenophore Mnemiopsis leidyi.

We have developed an efficient method for the preparation and maintenance of primary cell cultures isolated from adult Mnemiopsis leidyi, a lobate ctenophore. Our primary cell cultures are derived from tissue explants or enzymatically dissociated cells, and maintained in a complex undefined ctenophore mesogleal serum. These methods can be used to isolate, maintain and visually monitor ctenophore cells to assess proliferation, cellular morphology and cell differentiation in future studies. Exemplar cell types that can be easily isolated from primary cultures include proliferative ectodermal and endodermal cells, motile amebocyte-like cells, and giant smooth muscle cells that exhibit inducible contractile properties. We have also derived 'tissue envelopes' containing sections of endodermal canal surrounded by mesoglea and ectoderm that can be used to monitor targeted cell types in an in vivo context. Access to efficient and reliably generated primary cell cultures will facilitate the analysis of ctenophore development, physiology and morphology from a cell biological perspective.

Estrogen signaling in the proliferative endometrium: implications in endometriosis.

Even though the physiological role of estrogen in the female reproductive cycle and endometrial proliferative phase is well established, the signaling pathways by which estrogen exerts its action in the endometrial tissue are still little known. In this regard, advancements in cell culture techniques and maintenance of endometrial cells in cultures enabled the discovery of new signaling mechanisms activated by estrogen in the normal endometrium and in endometriosis. This review aims to present the recent findings in the genomic and non-genomic estrogen signaling pathways in the proliferative human endometrium specifically associated with the pathogenesis and development of endometriosis.

ChemInform Abstract: Green Synthesis and Biocompatibility of Titanium Nanoparticles

AbstractReview: 56 refs.

Induced pluripotent stem cells for modeling neurological disorders.

Several diseases have been successfully modeled since the development of induced pluripotent stem cell (iPSC) technology in 2006. Since then, methods for increased reprogramming efficiency and cell culture maintenance have been optimized and many protocols for differentiating stem cell lines have been successfully developed, allowing the generation of several cellular subtypes in vitro. Gene editing technologies have also greatly advanced lately, enhancing disease-specific phenotypes by creating isogenic cell lines, allowing mutations to be corrected in affected samples or inserted in control lines. Neurological disorders have benefited the most from iPSC-disease modeling for its capability for generating disease-relevant cell types in vitro from the central nervous system, such as neurons and glial cells, otherwise only available from post-mortem samples. Patient-specific iPSC-derived neural cells can recapitulate the phenotypes of these diseases and therefore, considerably enrich our understanding of pathogenesis, disease mechanism and facilitate the development of drug screening platforms for novel therapeutic targets. Here, we review the accomplishments and the current progress in human neurological disorders by using iPSC modeling for Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal muscular atrophy, amyotrophic lateral sclerosis, duchenne muscular dystrophy, schizophrenia and autism spectrum disorders, which include Timothy syndrome, Fragile X syndrome, Angelman syndrome, Prader-Willi syndrome, Phelan-McDermid, Rett syndrome as well as Nonsyndromic Autism.

Derivation of T cells in vitro from mouse embryonic stem cells.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells <em>In Vitro</em> from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Maintenance of primary cell cultures of immunocytes from Cacopsylla spp. psyllids: a new in vitro tool for the study of crop pest insects.

Primary cell cultures of immunocytes have been developed from the three psyllid species Cacopsylla melanoneura, Cacopsylla pyri (vectors of 'Candidatus Phytoplasma mali' and 'Candidatus Phytoplasma pyri', respectively) and Cacopsylla crataegi. The medium most suitable of those evaluated was Hert-Hunter 70 (HH70) psyllid medium. In fact, good survival and proliferation of the Cacopsylla immunocytes for over 60 d were observed, with mitosis activities starting at 15-d post culture. Moreover, adhesion and phagocytosis activities were confirmed for all the psyllid cell cultures by functionality tests. Morphological examination of cultured immunocytes revealed the presence of different cell types in all the three psyllid species in accordance to published data about insect immunocytes. The in vitro maintenance of psyllid immunocytes represents a powerful tool for a wide range of applications, especially for psyllid cell biology. In particular, in-depth studies on the biology of psyllids as vector insects as well as analyses to understand the mechanisms behind the interactions with pathogens and symbionts are now possible. These cultures can be used as an in vitro model to study psyllid humoral immune responses, which also will allow in-depth investigations on the abilities of psyllids as vectors of phytoplasmas. All these applications provide new opportunities to develop more focused and specific pest control strategies.

The Influence of Risk Perception on Biosafety Level-2 Laboratory Workers’ Hand-To-Face Contact Behaviors

Pathogen transmission in the laboratory is thought to occur primarily through inhalation of infectious aerosols or by direct contact with mucous membranes on the face. While significant research has focused on controlling inhalation exposures, little has been written about hand contamination and subsequent hand-to-face contact (HFC) transmission. HFC may present a significant risk to workers in biosafety level-2 (BSL-2) laboratories where there is typically no barrier between the workers’ hands and face. The purpose of this study was to measure the frequency and location of HFC among BSL-2 workers, and to identify psychosocial factors that influence the behavior. Research workers (N = 93) from 21 BSL-2 laboratories consented to participate in the study. Two study personnel measured workers’ HFC behaviors by direct observation during activities related to cell culture maintenance, cell infection, virus harvesting, reagent and media preparation, and tissue processing. Following observations, a survey measuring 11 psychosocial predictors of HFC was administered to participants. Study personnel recorded 396 touches to the face over the course of the study (mean = 2.6 HFCs/hr). Of the 93 subjects, 67 (72%) touched their face at least once, ranging from 0.2–16.0 HFCs/hr. Among those who touched their face, contact with the nose was most common (44.9%), followed by contact with the forehead (36.9%), cheek/chin (12.5%), mouth (4.0%), and eye (1.7%). HFC rates were significantly different across laboratories F(20, 72) = 1.85, p = 0.03. Perceived severity of infection predicted lower rates of HFC (p = 0.03). For every one-point increase in the severity scale, workers had 0.41 fewer HFCs/hr (r = −.27, P < 0.05). This study suggests HFC is common among BSL-2 laboratory workers, but largely overlooked as a major route of exposure. Workers’ risk perceptions had a modest impact on their HFC behaviors, but other factors not considered in this study, including social modeling and work intensity, may play a stronger role in predicting the behavior. Mucous membrane protection should be considered as part of the BSL-2 PPE ensemble to prevent HFC.

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson's disease. Acute activation of Gαi/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of Gαi/o-linked receptors including D2 dopamine and μ opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gbγ signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384 well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e. CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (>20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Chromatin structure, pluripotency and differentiation

The state of cell differentiation in adult tissues was once thought to be permanent and irreversible. Since Dolly's cloning and, more recently, the generation of induced pluripotent stem cells (iPSCs) from differentiated cells, the traditional paradigm of cell identity has been reexamined. Much effort has been directed toward understanding how cellular identity is achieved and maintained, and studies are ongoing to investigate how cellular identity can be changed. Cell-specific transcription patterns can be altered by modulating the expression of a few transcription factors, which are known as master regulators of cell fate. Epigenetics also plays a major role in cell type specification because the differentiation process is accompanied by major chromatin remodeling. Moreover, whole-genome analyses reveal that nuclear architecture, as defined by the establishment of chromatin domains, regulates gene interactions in a cell-type-specific manner. In this paper, we review the current knowledge of chromatin states that are relevant to both pluripotency and gene expression during differentiation. Information about the epigenetic regulation of gene expression in iPSCs or naïve embryonic stem cells, compared with their differentiated derivatives, will be important as a practical consideration in the long-term maintenance of pluripotent cell cultures for therapeutic purposes.

PLANT TISSUE CULTURE IN BIOTECHNOLOGY: RECENT ADVANCES IN TRANSFORMATION THROUGH SOMATIC EMBRYOGENESIS

118 The development of cell, tissue and organ culture methods was rapidly accelerated in the second half of the previous century after establishing robust cell culture techniques and media formulation for in vitro growth of plant material [1–8]. A stunning number of articles have been published on in vitro induction and maintenance of non-differentiated cells and the regeneration the plants from them whether through organogenesis or somatic embryogenesis. Fine protocols were established for the culture of enzymatically isolated single cells and protoplasts [9], which were able to regenerate into plants [10–12]. In vitro culture methods became an essential part of many micropropagation protocols. Culture of plant cells and organs in bioreactors were used for the production of different secondary metabolites and pharmaceuticals. In vitro techniques were used for production of mutants, haploids, virus-free material, and also for maintenance and preservation of rare genotypes and specific cell cultures [13–15]. The first positive results on Agro bac te rium-mediated transformation in plants were reported in 1983 [16–18]. A simple method for transferring genes into plants through the inoculation of leaf discs with Agrobacterium tumefaciens followed by in vitro culture and regeneration of whole plants was reported by Horsch et al. [19]. Different methods for DNA delivery into plant cell, including electroporation [20], PEG treatment [21, 22], microinjection [23], sonication [24], biolistics or particle bombardment [25], silicon carbide Whiskers treatment [26, 27], were used for specific transformation purposes and different types of cells and genotypes. However, Agrobac terium-mediated transformation became a preferred method. Agrobacterium tumefaciens is a natural vector system for transgenes delivery into a wide range of plants species, providing an efficient and «clean» insertion of DNA into the plant genome and deserved to be called the «tzar of genetic engineering» [28]. In the past 30 years the discovery and application of new transformation technologies essentially sped up the improvement of major cultivated crops. The first, really commercially grown plants hit the market in the mid — 1990s. Practically all transformation systems were based on in vitro culture methods. It was impossible to transform whole plant organism at once. All techniques were based on transforming single cells of callus, leaves, pollen, roots or other organs and than regeneration of plants through somatic embryogenesis or organogenesis. Routine and highly efficient transformation methods for many important crop and particular genotypes were implemented in many biotechnology companies. UDK 575327/275.854

Relation between Amino Acids Profiles and Recalcitrancy of Cell Growth or Salt Tolerance in Tissue and Protoplast Cultures of Three Mangrove Species, &amp;lt;i&amp;gt;Avicennia alba&amp;lt;/i&amp;gt;, &amp;lt;i&amp;gt;Bruguiera sexangula&amp;lt;/i&amp;gt;, and &amp;lt;i&amp;gt;Sonneratia alba&amp;lt;/i&amp;gt;

Amino acids profiles were investigated in tissues, cultured cells, i.e. callus or suspension cells, and their protoplasts of three mangrove species, Avicennia alba, Bruguiera sexangula, and Sonneratia alba. Original tissues of cultured cells of three mangrove species were cotyledons and hypocotyls, leaves, and cotyledons, respectively. In protoplasts isolated from cultured cells, glutamine and alanine were the major amino acids. Different contents of glycine, proline and serine were observed among protoplasts of three mangrove species. Large differences in the major amino acids were found among cultured cells and their protoplasts while no difference was found between callus and suspension cells independent of additional salt in culture medium. Protoplasts of original tissues, young leaves and cotyledons, contained alanine and glutamine and/or asparagine. In suspension cells of B. sexangula, total contents of amino acids were low while their protoplasts showed similar value as of other samples. Protoplasts of leaf and cotyledons of A. alba and cotyledons of A. lanata, A. marina and S. alba were also investigated. The total contents of amino acids and their profiles might be related to the recalcitrance for the growth and salt tolerance or halophilic nature of cells and basal media used for the maintenance of cell cultures or protoplast cultures of the mangrove species. This is the first report on callus induction from hypocotyls of A. alba.

Hormone-autonomous cell culture from cotydelonary tissue of the marine plant Cymodocea nodosa

Abstract In vitro germinated seedlings of ­Cymodocea nodosa were used as a source of young embryonic tissue for cell culture. Histological study of the cotyledon revealed the presence of meristematic tissue that gave rise to the first leaf of the plantlet, which released large numbers of cells after enzymatic digestion and thus indicated great growth potential. An effective enzymatic digestion protocol for C. nodosa meristem tissue was developed; this consisted of a 24-h digestion period at 28°C using a 0.6-m enzymatic solution (plasmolysis medium containing 1% (w:v) cellulase and hemicellulase). It was necessary to add citric acid (0.02 m) as an antioxidant to the enzymatic solution to obtain the highest cell yield and viability (mean±SE: 371.6±30.1×105 cells g-1 fw and 97.8±0.6%, respectively). NaCl and other salt components of the seawater were key factors in the establishment of axenic cell cultures and their further progress; their absence in the medium inhibited contaminant overgrowth. Nevertheless, cells maintained growth for 90 days, even without hormones and deprived of salts, although these salts might be necessary to allow the development of morphogenetic programmes desired for plant production. In this manner, we generated a basic tool for the future of C. nodosa in vitro culture that will allow the establishment and maintenance of hormone-autonomous cell cultures with strong potential in future biotechnology for marine higher plants.