Showing papers on "Tissue culture published in 2012"

Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro

Abstract: The fabrication of 3D tissues retaining the original functions of tissues/organs in vitro is crucial for optimal tissue engineering and regenerative medicine. The fabrication of 3D tissues also contributes to the establishment of in vitro tissue/organ models for drug screening. Our laboratory has developed a fabrication system for functional 3D tissues by stacking cell sheets of confluent cultured cells detached from a temperature-responsive culture dish. Here we describe the protocols for the fabrication of 3D tissues by cell sheet engineering. Three-dimensional cardiac tissues fabricated by stacking cardiac cell sheets pulsate spontaneously, synchronously and macroscopically. Via this protocol, it is also possible to fabricate other tissues, such as 3D tissue including capillary-like prevascular networks, from endothelial cells sandwiched between layered cell sheets. Cell sheet stacking technology promises to provide in vitro tissue/organ models and more effective therapies for curing tissue/organ failures.

Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential

Abstract: While traditional cell culture methods have relied on growing cells as monolayers, three-dimensional (3D) culture systems can provide a convenient in vitro model for the study of complex cell–cell and cell–matrix interactions in the absence of exogenous substrates and may benefit the development of regenerative medicine strategies. In this study, mesenchymal stem cell (MSC) spheroids, or “mesenspheres”, of different sizes, were formed using a forced aggregation technique and maintained in suspension culture for extended periods of time thereafter. Cell proliferation and differentiation potential within mesenspheres and dissociated cells retrieved from spheroids were compared to conventional adherent monolayer cultures. Mesenspheres maintained in growth medium exhibited no evidence of cell necrosis or differentiation, while mesenspheres in differentiation media exhibited differentiation similar to conventional 2D culture methods based on histological markers of osteogenic and adipogenic commitment. Furthermore, when plated onto tissue culture plates, cells that had been cultured within mesenspheres in growth medium recovered morphology typical of cells cultured continuously in adherent monolayers and retained their capacity for multi-lineage differentiation potential. In fact, more robust matrix mineralization and lipid vacuole content were evident in recovered MSCs when compared to monolayers, suggesting enhanced differentiation by cells cultured as 3D spheroids. Thus, this study demonstrates the development of a 3D culture system for mesenchymal stem cells that may circumvent limitations associated with conventional monolayer cultures and enhance the differentiation potential of multipotent cells.

Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications

Abstract: In vitro cell and tissue-based systems have tremendous potential in fundamental research and for commercial applications such as clonal propagation, genetic engineering and production of valuable metabolites. Since the invention of plant cell and tissue culture techniques more than half a century ago, scientists have been trying to understand the morphological, physiological, biochemical and molecular changes associated with tissue culture responses. Establishment of de novo developmental cell fate in vitro is governed by factors such as genetic make-up, stress and plant growth regulators. In vitro culture is believed to destabilize the genetic and epigenetic program of intact plant tissue and can lead to chromosomal and DNA sequence variations, methylation changes, transposon activation, and generation of somaclonal variants. In this review, we discuss the current status of understanding the genomic and epigenomic changes that take place under in vitro conditions. It is hoped that a precise and comprehensive knowledge of the molecular basis of these variations and acquisition of developmental cell fate would help to devise strategies to improve the totipotency and embryogenic capability in recalcitrant species and genotypes, and to address bottlenecks associated with clonal propagation.

Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues

Abstract: Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

A Peroxidase-Dependent Apoplastic Oxidative Burst in Cultured Arabidopsis Cells Functions in MAMP-Elicited Defense

Abstract: Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.

Plant Tissue Culture: Current Status and Opportunities

Abstract: Tissue culture is the in vitro aseptic culture of cells, tissues, organs or whole plant under controlled nutritional and environmental conditions [1] often to produce the clones of plants. The resultant clones are true-to type of the selected genotype. The controlled conditions provide the culture an environment conducive for their growth and multiplication. These conditions include proper supply of nutrients, pH medium, adequate temperature and proper gaseous and liquid environment.

Tissue Culture of the Nervous System

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Plaque assay for murine norovirus.

Abstract: Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3. Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4. In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Alteration of Cellular Behavior and Response to PI3K Pathway Inhibition by Culture in 3D Collagen Gels

Abstract: Most investigations into cancer cell drug response are performed with cells cultured on flat (2D) tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D) extracellular matrix (ECM) is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments.

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Abstract: Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3α mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.

Tissue Culture of Epithelial Cells

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In Vitro Influences of TiO 2 Nanoparticles on Barley ( Hordeum vulgare L.) Tissue Culture

Abstract: In the last decades, extensive research on the effects of nano-TiO2 on plant systems and different microorganisms has confirmed its photocatalytic and antimicrobial activity. However, there is no report on its application in plant cell and tissue culture as well as its role in eliminating contaminating microorganisms in tissue culture. In this work, barley mature embryos were cultured in Murashige and Skoog medium with four concentrations (0, 10, 30, 60 μg/ml) of TiO2 suspension in four repetitions. Quantitative and qualitative characteristics of calli were analyzed after each subculture. Data analysis for calli number in the first culture and callus size in all three cultures showed that the effect of treatment was significant at p > 0.95. As a result, quantitative features such as callus color, shape, embryogenesis, etc. were completely similar in both control and TiO2 nanoparticle treatments; there is no doubt that TiO2 nanoparticles could dramatically increase callugenesis and the size of calli. As well, TiO2 nanoparticles are effective bactericides with an aseptic effect, causing no negative change in the quality of the callus. It is necessary to do more complementary works to identify mechanisms involved for the increased calli size and embryogenesis of explants in darkness.

Anti-inflammatory effect of quercetin in a whole orbital tissue culture of Graves' orbitopathy.

Abstract: Purpose The authors previously reported that quercetin significantly inhibits interleukin (IL)-1b-induced increases of proinflammatory cytokines in cultured primary orbital fibroblasts from Graves’ orbitopathy (GO). This study investigated the inhibitory effect of quercetin on inflammation in cultured whole orbital tissue. Methods Orbital fat tissues from GO and normal samples were cultured with or without non-toxic concentrations of quercetin. Lactate dehydrogenase (LDH) release was used to identify non-toxic concentrations of quercetin. IL-6, IL-8, IL-1a, IL-1b and tumour necrosis factor alpha (TNFa) proteins were measured in tissue culture supernatants by ELISA, and gene transcript levels were determined using quantitative PCR, expressed as relative fold changes of threshold cycle value relative to the control group. Results The maximal non-cytotoxic treatment of quercetin was 100 mM for 72 h, based on the considerably low LDH release with these conditions. IL-1b, IL-6 and TNFa protein levels corrected for tissue weight were significantly higher in supernatants of GO samples than normal controls (p<0.05). Quercetin reduced IL-6, IL-8 and TNFa protein production in supernatants of all GO samples (n¼4) in a dosedependent manner; however, only the reduction in IL-6 was statistically significant (p<0.05). Quercetin had a significant suppression of tissue IL-6, IL-8, IL-1b and TNFa mRNA expression in cultured orbital tissues from three GO samples relative to untreated control tissue (p<0.05). Conclusions Inhibition of proinflammatory cytokines by the natural product quercetin in both primary orbital fibroblasts and tissue culture provides the basis for its potential use as an anti-inflammatory agent in the treatment of GO.

The Prerequisite of the Success in Plant Tissue Culture: High Frequency Shoot Regeneration

Abstract: Plant tissue culture is a term containing techniques used to propagate plants vegetatively by using small parts of living tissues (explants) on artificial growth mediums under sterile conditions. Explants regenerate shoots and roots, and consequently whole fertile plants under certain cultural conditions. Micropropagation is the production of whole plants through tissue culture from small parts such as shoot and root tips, leaf tissues, anthers, nodes, meristems and embryos. Micropropagation is the vegetative (asexual) propagation of plants under in vitro conditions and is widely used for commercial purposes worldwide [1-3].

Comparison of in vitro- and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue.

Abstract: At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5-2.0×1.0-1.2×0.8-1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips.

A novel in vitro three-dimensional retinoblastoma model for evaluating chemotherapeutic drugs.

Abstract: Purpose: Novel strategies are being applied for creating better in vitro models that simulate in vivo conditions for testing the efficacy of anticancer drugs. In the present study we developed surface-engineered, large and porous, biodegradable, polymeric microparticles as a scaffold for three dimensional (3-D) growth of a Y79 retinoblastoma (RB) cell line. We evaluated the effect of three anticancer drugs in naive and nanoparticle-loaded forms on a 3-D versus a two-dimensional (2-D) model. We also studied the influence of microparticles on extracellular matrix (ECM) synthesis and whole genome miRNA-gene expression profiling to identify 3D-responsive genes that are implicated in oncogenesis in RB cells. Methods: Poly(D,L)-lactide-co-glycolide (PLGA) microparticles were prepared by the solvent evaporation method. RB cell line Y79 was grown alone or with PLGA–gelatin microparticles. Antiproliferative activity, drug diffusion, and cellular uptake were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay, fluorescent microscope, and flow cytometry. Extra cellular matrix (ECM) synthesis was observed by collagenase assay and whole genome miRNA-microarray profiling by using an Agilent chip. Results: With optimized composition of microparticles and cell culture conditions, an eightfold increase from the seeding density was achieved in 5 days of culture. The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC50 values. Using doxorubicin, the flow cytometry data demonstrated a 4.4 fold lower drug accumulation in the cells grown in the 3-D model at 4 h. The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion. The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects. Conclusions: Our 3-D retinoblastoma model could be used in developing effective drugs based on a better understanding of the role of chemical, biologic, and physical parameters in the process of drug diffusion through the tumor mass, drug retention, and therapeutic outcome. A suitable three dimensional (3-D) culture system provides a more physiologically relevant approach to the analysis of gene function, regulation, and cell phenotype ex vivo [1]. It has previously been shown that engineering the cell culture micro-environment to create growth conditions that more accurately mimic the in vivo behavior of cells is an essential step for improving the predictive accuracy of the drug discovery process [2]. Evidence suggests that modification of cell growth conditions can radically influence the behavior of cells in response to chemical reagents [3]. Many important studies have been done on cell proliferation, differentiation, and function in vitro; however, experience with cells in a flat tissue culture flask is different from the complexities of tissues within the body. In tissues, cells connect to each other as well as to the extracellular matrix

Human platelet lysate gel provides a novel three dimensional-matrix for enhanced culture expansion of mesenchymal stromal cells.

Abstract: Cell culture in regenerative medicine needs to facilitate efficient expansion according to good manufacturing practice requirements. Human platelet lysate (HPL) can be used as a substitute for fetal calf serum without the risk of xenogeneic immune reactions or transmission of bovine pathogens. Heparin needs to be added as anticoagulant before addition of HPL to culture medium; otherwise, HPL-medium forms a gel within 1 h. Here, we demonstrated that such HPL-gels provide a suitable 3D-matrix for cell culture that—apart from heparin—consists of the same components as the over-layered culture medium. Mesenchymal stromal cells (MSCs) grew in several layers at the interface between HPL-gel and HPL-medium without contact with any artificial biomaterials. Notably, proliferation of MSCs was much higher on HPL-gel compared with tissue culture plastic. Further, the frequency of initial fibroblastoid colony forming units (CFU-f) increased on HPL-gel. The viscous consistency of HPL-gel enabled passaging with a conven...

Antifibrotic Effects of Quercetin in Primary Orbital Fibroblasts and Orbital Fat Tissue Cultures of Graves' Orbitopathy

Abstract: Purpose We investigated the effects of quercetin on fibrotic markers and matrix metalloproteinases (MMPs) in primary cells and whole orbital tissues from Graves' orbitopathy (GO). Methods Orbital fat tissues were harvested from GO for primary cell and tissue cultures during orbital fat decompression. To determine noncytotoxic dose and time of quercetin treatment, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and LDH release assay were performed. The effects of quercetin on fibrosis were evaluated according to a scratch wound closure assay, and Western blotting for expression of fibronectin, collagen Iα, α-smooth muscle actin with or without TGF-β stimulation, and MMP-2, -7, -9, and tissue inhibitor of metalloproteinase-1 with or without IL-1β stimulation. The gelatinolytic activities of MMP-2 and MMP-9 were measured using gelatin zymography. In tissue cultures, MMP secretion and MMP and collagen Iα mRNA levels were determined by enzyme-linked immunosorbent assays and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Results Quercetin significantly inhibited cell migration at nontoxic concentrations. In primary cells, quercetin dose-dependently downregulated expression of TGF-β-stimulated fibronectin and collagen Iα, and IL-1β-enhanced MMP-2 and MMP-9. However, without IL-1β stimulation, 10-50 μM of quercetin increased MMP-2 expression and activity, but dose-dependently suppressed MMP-9 expression and activity. In tissue cultures, quercetin dose-dependently inhibited MMP-2 and -9 activity and secretion, but 30 and 50 μM of quercetin increased tissue MMP-2 mRNA. MMP-9 and collagen Iα mRNA levels were dose-dependently suppressed. Conclusions Quercetin inhibited fibrotic markers and affected MMP-2 and MMP-9 activities in primary cell and orbital fat tissue cultures from GO at nontoxic concentrations. Our results support the potential use of quercetin for active inflammation and treatment or prevention of chronic fibrosis in GO.

Ex vivo optical metabolic measurements from cultured tissue reflect in vivo tissue status

Abstract: Optical measurements of metabolism are ideally acquired in vivo; however, intravital measurements are often impractical. Accurate ex vivo assessments would greatly broaden the applicability of optical measurements of metabolism. We investigate the use of live tissue culture experiments to serve as a surrogate for in vivo metabolic measurements. To validate this approach, NADH and FAD fluorescence intensity and lifetime images were acquired with a two-photon microscope from hamster cheek pouch epithelia in vivo, from biopsies maintained in live tissue culture up to 48 h, and from flash-frozen and thawed biopsies. We found that the optical redox ratio (fluorescence intensity of NADH/FAD) of the cultured biopsy was statistically identical to the in vivo measurement until 24 h, while the redox ratio of the frozen-thawed samples decreased by 15% (p 0.05) during the first 8 h of tissue culture, while the NADH τm of frozen-thawed samples increased by 13% (p 0.05). All results were consistent across multiple depth layers in this stratified squamous epithelial tissue. Histological markers for proliferation and apoptosis also confirm the viability of tissues maintained in culture. This study suggests that short-term ex vivo tissue culture may be more appropriate than frozen-thawed tissue for optical metabolic and morphologic measurements that approximate in vivo status.

Patterns of Amino Acid Metabolism by Proliferating Human Mesenchymal Stem Cells

Abstract: The nutritional requirements of stem cells have not been determined; in particular, the amino acid metabolism of stem cells is largely unknown. In this study, we investigated the amino acid metabolism of human mesenchymal stem cells (hMSCs), with focus on two questions: Which amino acids are consumed and/or secreted by hMSCs and at what rates? To answer these questions, hMSCs were cultured on tissue culture plastic and in a bioreactor, and their amino acid profile was analyzed. The results showed that the kinetics of hMSCs growth and amino acid metabolism were significantly higher for hMSCs in tissue culture plastic than in the bioreactor. Despite differences in culture conditions, 8 essential and 6 nonessential amino acids were consumed by hMSCs in both tissue culture plastic and bioreactor cultures. Glutamine was the most consumed amino acid with significantly higher rates than for any other amino acid. The metabolism of nonessential amino acids by hMSCs deviated significantly from that of other cell lines. The secretion of alanine, glycine, glutamate, and ornithine by hMSCs showed that there is a strong overflow metabolism that can be due to the high concentrations of amino acids provided in the medium. In addition, the data showed that there is a metabolic pattern for proliferating hMSCs, which can contribute to the design of medium without animal serum for stem cells. Further, this study shows how to implement amino acid rates and metabolic principles in three-dimensional stem cell biology.

Introduction to cell culture.

Abstract: The basics of cell culture as applied to human cells are discussed Biosafety when working with human tissue, which is often pathogenic, is important The requirements for a tissue culture laboratory are described, particularly the range of equipment needed to carry out cell isolation, purification, and culture Steps must be taken to maintain aseptic conditions to prevent contamination of cultures with micro-organisms Basic cell-handling techniques are discussed, including choice of media, primary culture, and cryopreservation of cells so they can be stored for future use Common assays which are used to determine cell viability and activity are considered

Variations in leaf morphology and DNA methylation following in vitro culture of Malus xiaojinensis

Abstract: Juvenile trees of Malus xiaojinensis produce leaves with three to five lobes; however, adult trees do not. Thus, M. xiaojinensis serves as a model plant for studies on rejuvenation of woody perennials. To better understand effects of in vitro culture, number of passages, and plant growth regulators on observed somaclonal variations in leaf morphology, total levels of DNA methylation and expression of associated genes, including DRM2 and MET1, of apical shoot meristems of M. xiaojinensis, were evaluated. Following 17 passages of nodal stem segment culture and four passages of callus culture, levels of DNA methylation and expression of DRM2 and MET1 were determined, and found to decrease from the 11th passage of nodal stem segment culture or adventitious bud culture and from the first passage of callus culture. Levels of DNA methylation increased with increasing 2,4-dichlorophenoxyacetic acid concentrations, but decreased significantly with increasing 6-benzyladenine (BA) concentrations. Expression of DRM2 and MET1 in regenerated plantlets were negatively correlated with BA concentrations in the culture medium. Overall, less than 11 passages of nodal stem segment or adventitious bud culture did not contribute to incidence of somaclonal variation, even in the presence of low concentrations of BA (<0.5 mg/L) in the culture medium. Moreover, when nodal stem segment cultures were maintained for up to 17 passages, complete rejuvenation was observed. However, epigenetic changes were detected following the first passage of callus cultures.

Decellularized extracellular matrix

Abstract: Methods for producing compositions of decellularized extracellular matrix (DM) tissue culture are described. The compositions can be used for coating supports such as tissue culture substrates, osteogenic gels, and medical devices.

Analysis of axonal growth and cell migration in 3D hydrogel cultures of embryonic mouse CNS tissue

Abstract: Analysis of axonal growth and cell migration in 3D hydrogel cultures of embryonic mouse CNS tissue

Pathogen and Biological Contamination Management in Plant Tissue Culture: Phytopathogens, Vitro Pathogens, and Vitro Pests

Abstract: The ability to establish and grow plant cell, organ, and tissue cultures has been widely exploited for basic and applied research, and for the commercial production of plants (micro-propagation). Regardless of whether the application is for research or commerce, it is essential that the cultures be established in vitro free of biological contamination and be maintained as aseptic cultures during manipulation, growth, and storage. The risks from microbial contamination are spurious experimental results due to the effects of latent contaminants or losses of valuable experimental or commercial cultures. Much of the emphasis in culture contamination management historically focussed on the elimination of phytopathogens and the maintenance of cultures free from laboratory contamination by environmental bacteria, fungi (collectively referred to as "vitro pathogens", i.e. pathogens or environmental micro-organisms which cause culture losses), and micro-arthropods ("vitro pests"). Microbial contamination of plant tissue cultures is due to the high nutrient availability in the almost universally used Murashige and Skoog (Physiol Plant 15:473-497, 1962) basal medium or variants of it. In recent years, it has been shown that many plants, especially perennials, are at least locally endophytically colonized intercellularly by bacteria. The latter, and intracellular pathogenic bacteria and viruses/viroids, may pass latently into culture and be spread horizontally and vertically in cultures. Growth of some potentially cultivable endophytes may be suppressed by the high salt and sugar content of the Murashige and Skoog basal medium and suboptimal temperatures for their growth in plant tissue growth rooms. The management of contamination in tissue culture involves three stages: disease screening (syn. disease indexing) of the stock plants with disease and endophyte elimination where detected; establishment and pathogen and contaminant screening of established initial cultures; observation, random sampling, and culture screening for micro-organism in multiplication and stored cultures. The increasing accessibility of both broad-spectrum and specific molecular diagnostics has resulted in advances in multiple pathogen and latent contaminant detection. The hazard analysis critical control point management strategy for tissue culture laboratories is underpinned by staff training in aseptic technique and good laboratory practice.

Somatic embryogenesis, plant regeneration, and cryopreservation for Torreya taxifolia , a highly endangered coniferous species

Abstract: Torreya taxifolia Arn., an ancient evergreen tree, is on the brink of extinction from attack by a fungal disease, recently reported to be caused by a novel isolate of Fusarium. We report the development of a somatic embryogenesis tissue culture system that can be used for cryogenic storage of T. taxifolia cultures and subsequent plant regeneration. Initiation of embryogenic tissue from immature zygotic embryos occurred on a conifer tissue culture medium containing 0.25 % activated charcoal, 43.8 mM maltose, 0.5 mM 2,4-dichlorophenoxacetic acid, 0.2 mM 6-benzylaminopurine, 0.2 mM kinetin, 0.1 μM brassinolide, 3.8 μM abscisic acid, 20.5 μM biotin, 1.13 μM folic acid, 1.28 mM 2(n-morpholino)ethanesulfonic acid and 0.69 mM pyruvic acid. Embryo induction ranged from 60 to 100 % across six seed sources. Somatic embryo development occurred on a medium containing 43.8 mM maltose, 1 % activated charcoal, 37.8 μM abscisic acid, 20.5 μM biotin, 0.1 μM brassinolide, 0.205 mM folic acid, 1.28 mM 2(n-morpholino)ethanesulfonic acid and 0.69 mM pyruvic acid. Germination of somatic embryos ranged from 64 to 82 %. Embryogenic tissue cultures from 30 genotypes representing seed from six mother trees were cryopreserved, and culture recovery was demonstrated after freezing. In contrast to many other coniferous tree seeds, the measured water potential (−MPa) of T. taxifolia megagametophyte tissue rose greatly during seed after-ripening. Duplication of this rise in vitro allowed development of somatic embryos to the cotyledonary stage.

Plant Tissue Culture Media

Abstract: Optimal growth and morphogenesis of tissues may vary for different plants according to their nutritional requirements Moreover, tissues from different parts of plants may also have different requirements for satisfactory growth [1] Tissue culture media were first developed from nutrient solutions used for culturing whole plants eg root culture medium of White and callus culture medium of Gautheret White’s medium was based on Uspenski and Uspenska’s medium for algae, Gautheret’s medium was based on Knop’s salt solution [2] Basic media that are frequently used include Murashige and Skoog (MS) medium [1], Linsmaier and Skoog (LS) medium [3], Gamborg (B5) medium [4] and Nitsch and Nitsch (NN) medium [5]