Preparation of single cells from aggregated Taxus suspension cultures for population analysis

A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism.     

Analysis of Nuclear DNA content in plant cells by Flow cytometry

Flow cytometry was used to analyse the DNA content of nuclei isolated from intact plant tissues and from callus and cell suspension cultures invitro. Cell nuclei were isolated either mechanically (chopping, syringing) or by a hypotonic lysis of isolated protoplasts. Although both methods gave similar results, a slight shift to lower ploidy levels was observed after protoplast isolation from intact tissues and calli. No differences were observed if the two methods were compared using cell suspension cultures. The results showed that flow cytometry is a rapid method of nuclear DNA content analysis in intact plant tissues and variousin vitro cultures.     

Applications of flow cytometry sorting in the pharmaceutical industry: A review

The article reviews applications of flow cytometry sorting in manufacturing of pharmaceuticals. Flow cytometry sorting is an extremely powerful tool for monitoring, screening and separating single cells based on any property that can be measured by flow cytometry. Different applications of flow cytometry sorting are classified into groups and discussed in separate sections as follows: (a) isolation of cell types, (b) high throughput screening, (c) cell surface display, (d) droplet fluorescent-activated cell sorting (FACS). Future opportunities are identified including: (a) sorting of particular fractions of the cell population based on a property of interest for generating inoculum that will result in improved outcomes of cell cultures and (b) the use of population balance models in combination with FACS to design and optimize cell cultures.     

流式细胞术的发展及在植物研究中的应用

流式细胞术是通过对液流中各种荧光标记的颗粒进行多参数快速高效的定性或定量测定的方法,在科学研究的多个领域发挥重要作用。然而,由于植物组织及细胞壁和次生代谢产物等细胞的特殊成分和结构,限制了其在植物研究领域的应用。本文在介绍流式细胞仪发展和组成分类的基础上,着重讨论了流式细胞术在植物领域的应用、研究进展及应用限制,进而展望该研究领域的发展趋势,为拓宽植物流式细胞术的潜在应用范围提供新的思考方向。     

Flow cytometric analysis of polysomaty and in vitro genetic instability in potato

The analysis of nuclear DNA contents in various tissues of potato genotypes showed that flow cytometry is a rapid method to characterize large populations of cells for polysomaty, that is, the occurrence of cells with normal DNA levels together with cells containing endoreduplicated nuclei. The proportion of endoreduplicated nuclei varied in different tissues and genotypes of potato. The analysis of callus and cell cultures showed that the temporal changes in nuclear DNA contents during in vitro growth can be followed and the degree of polyploidization quantified. It is concluded that flow cytometry is a highly suitable method to detect ploidy changes in differentiated plant tissues and calli which are often not amenable for chromosome number determination.     

Flow cytometric analysis of protein content in Taxus protoplasts and single cells as compared to aggregated suspension cultures

Plant suspension cultures are highly aggregated, preventing the direct application of flow cytometry for the study of population dynamics. The utility of single cells to accurately represent aggregated suspension cultures was tested through the analysis of total protein content. Specifically, protein content of two Taxus cuspidata suspension culture lines was studied using the Bradford assay for aggregated suspension cultures, and flow cytometry with fluorescein isothiocyanate staining for protoplasts and single cells. Taxus protein levels were measured at 75–160 mg per gram dry weight via the Bradford assay. Aggregated suspension cultures, protoplasts, and single cells predicted the same trend of protein content over the culture period (21 days). Normalized protein content of isolated single cells was statistically equivalent to aggregated suspensions for both cell lines. However, normalized protein content of isolated protoplasts showed significant differences from aggregated suspensions for one of the two cell lines. Elicitation with methyl jasmonate (MJ) is commonly utilized to increase paclitaxel accumulation in suspension cultures, and therefore the effect of MJ elicitation on protein content in aggregated suspensions, isolated single cells and protoplasts was assessed. Aggregated suspension cultures, protoplasts, and single cells did not show any change in total protein content following elicitation with MJ at 200 M on day 7. This study illustrates the usefulness of flow cytometry for obtaining culture population information and the value of using intact single cells for the study of plant metabolism.     

Visual selection and maintenance of the cell lines with high plant regeneration ability and low ploidy level in Dianthus acicularis by monitoring with flow cytometry analysis

Efficient plant regeneration system from cell suspension cultures was established in D. acicularis (2n=90) by monitoring ploidy level and visual selection of the cultures. The ploidy level of the cell cultures closely related to the shoot regeneration ability. The cell lines comprising original ploidy levels (2C+4C cells corresponding to DNA contents of G1 and G2 cells of diploid plant, respectively) showed high regeneration ability, whereas those containing the cells with 8C or higher DNA C-values showed low or no regeneration ability. The highly regenerable cell lines thus selected consisted of compact cell clumps with yellowish color and relatively moderate growth, suggesting that it is possible to select visually the highly regenerable cell lines with the original ploidy level. All the regenerated plantlets from the highly regenerable cell cultures exhibited normal phenotypes and no variations in ploidy level were observed by flow cytometry (FCM) analysis.     

流式细胞仪检测高等植物细胞核DNA含量的方法

相对于动物和微生物而言,流式细胞术在植物科学上的应用会因植物组织与细胞(如细胞壁、中央液泡、特殊细胞器等)的特殊结构以及次生代谢产物等特殊成分,造成样品在前期处理、染色及测试等方面的困难,甚至导致检测失败或结果不准确。笔者在长期运用流式细胞仪测试工作中,积累了大量的植物样本检测经验,并参考国内外相关文献,总结出从植物取材、样品制备到植物细胞核DNA流式检测的方法和技巧,可为植物科学研究者及从事流式细胞检测的技术人员提供实验参考。     

Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence.

The expression of the nrd operon encoding ribonucleotide reductase in Escherichia coli has been shown to be cell cycle regulated. To identify the cis-acting elements required for the cell cycle regulation of the nrd promoter, different 5' deletions as well as site-directed mutations were translationally fused to a lacZ reporter gene. The expression of beta-galactosidase from these nrd-lacZ fusions in single-copy plasmids was determined with synchronously growing cultures obtained by repeated phosphate starvation as well as with exponentially growing cultures by flow cytometry analysis. Although Fis and DnaA, two regulatory proteins that bind at multiple sites on the E. coli chromosome, have been found to regulate the nrd promoter, the results in this study demonstrated that neither Fis nor DnaA was required for nrd cell cycle regulation. A cis-acting upstream AT-rich sequence was found to be required for the cell cycle regulation. This sequence could be replaced by a different sequence that maintained the AT richness. A flow cytometry analysis that combined specific immunofluorescent staining of beta-galactosidase with a DNA-specific stain was developed and employed to study the nrd promoter activity in cells at specific cell cycle positions. The results of the flow cytometry analysis confirmed the results obtained from studies with synchronized cells.     

Statistical analysis and biological interpretation of the flow cytometric heterogeneity observed in bacterial axenic cultures

Histogram comparison and meaningful statistics in flow cytometry is probably the most widely encountered mathematical problem in flow cytometry. Ideally, a test for determining the statistical equality or difference of flow cytometric distributions will identify the significant differences or similarities of the obtained histograms. This situation is of particular interest when flow cytometry is used to study the heterogeneity of axenic bacterial populations. We have statistically measured the heterogeneity of successive cytometric measures, the modifications produced after 20 transfers from the same culture, and the differences between 20 subcultures of identical origin. The heterogeneity of the bacterial populations and the similarity of the obtained 360 histograms were analysed by standard statistical methods. We have studied bacterial axenic cultures in order to detect, quantify and interpret their cytometric heterogeneity, and to assess intrinsic differences and differences produced by laboratory manipulations. We concluded that the standard axenic cultures have a considerable intrinsic cellular and molecular heterogeneity. We suggest that the heterogeneity we have detected basically has two origins: cell size diversity and cell cycle variations.     

Cell cycle analysis of Taxus suspension cultures at the single cell level as an indicator of culture heterogeneity

Single cell growth and division was measured via flow cytometry in order to characterize the metabolic variability of Taxus cuspidata suspension cultures, which produce the valuable secondary metabolite Taxol. Good agreement was observed between the cell cycle distribution and biomass accumulation over the batch culture period. Specific growth rates of 0.13 days(-1) by fresh weight and 0.15 days(-1) by dry weight were measured. Elicitation with methyl jasmonate (MJ) significantly decreased both cell cycle progression and biomass accumulation, as the specific growth rate decreased to 0.027 days(-1) by fresh and dry weight. Despite the decrease in biomass accumulation for MJ elicited cultures, sucrose utilization was not significantly different from control cultures. MJ elicitation also increased the accumulation of paclitaxel and other taxanes. The accumulation of upstream taxanes (baccatin III and 10-deactylbaccatin III) increased during exponential growth, reached a maximum around day 12, and then declined throughout the stationary phase. The paclitaxel concentration increased during both exponential growth and stationary phase, reaching a maximum around days 20-25. Throughout the culture period, greater than 70% of the cells were in G(0)/G(1) phase of the cell cycle. Studies using bromodeoxyuridine (BrdU) incorporation showed that approximately 65% of the Taxus cells are noncycling, even during exponential growth. Although the role of these cells is currently unknown, the presence of a large, noncycling subpopulation can have a significant impact on the utilization of plant cell culture technology for the large-scale production of paclitaxel. These results demonstrate that there is a high degree of metabolic heterogeneity in Taxus cuspidata suspension cultures. Understanding this heterogeneity is important for the optimization of plant cell cultures, particularly the reduction of production variability.     

Online flow cytometry for monitoring apoptosis in mammalian cell cultures as an application for process analytical technology

Apoptosis is the main driver of cell death in bioreactor suspension cell cultures during the production of biopharmaceuticals from animal cell lines. It is known that apoptosis also has an effect on the quality and quantity of the expressed recombinant protein. This has raised the importance of studying apoptosis for implementing culture optimization strategies. The work here describes a novel approach to obtain near real time data on proportion of viable, early apoptotic, late apoptotic and necrotic cell populations in a suspension CHO culture using automated sample preparation in conjunction with flow cytometry. The resultant online flow cytometry data can track the progression of apoptotic events in culture, aligning with analogous manual methodologies and giving similar results. The obtained near-real time apoptosis data are a significant improvement in monitoring capabilities and can lead to improved control strategies and research data on complex biological systems in bioreactor cultures in both academic and industrial settings focused on process analytical technology applications.     

Immune monitoring in xenotransplantation: the multiparameter flow cytometric mixed lymphocyte culture assay

Xenotransplantation requires monitoring of complex cellular interactions in vitro. A tool to monitor cell proliferation in detail would be instrumental in understanding these cellular interactions in heterogeneous xenogeneic lymphocyte cultures and in patients after xenotransplantation. To accomplish this, we used a fluorescent cell proliferation marker, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), in combination with flow cytometry. CFSE, a green fluorescent molecule, binds covalently to intracellular macromolecules. Each cell division reduces the fluorescent intensity per cell by half and shows a characteristic multipeak pattern in flow cytometric analysis. For this study, human lymphocytes were labeled with CFSE and cultured in the presence of irradiated porcine lymphocytes. Cell proliferation was detected in CFSE-labeled lymphocytes in both a single and a multiparameter flow cytometry setting. Concurrently, tritiated ((3)H) thymidine incorporation, a common method to measure gross cell proliferation, was assessed. The kinetics of CFSE-labeled cell proliferation correlated with (3)H-thymidine incorporation in that both methods showed a lag phase for days 1-3 and a log phase for days 4-7. Multiparameter flow cytometric monitoring of mixed lymphocyte cultures allowed phenotyping and assessment of viability of proliferating populations in heterogeneous xenogeneic stimulated human lymphocyte cultures and complemented the classical (3)H-thymidine incorporation assay. The use of this technique will allow a wide array of immunologic parameters to be measured in a heterogeneous xenogeneic mixed lymphocyte culture. The information gained from these assays is essential to understanding the biological significance of xenogeneic cellular interaction and for monitoring the immune status of the xenotransplanted patient.     

Somatic embryogenesis in oak (<Emphasis Type="Italic">Quercus</Emphasis> spp.)

Summary The present review summarizes the factors involved in controlling the process of oak somatic embryogenesis as a method for vegetative plant propagation and includes also data on artificial seed production, cryopreservation and transformation. One major limitation, the inability to initiate embryogenic cultures from mature trees, has been recently overcome. Leaves from selected cork oak trees with an age of 50 yr and more have been used to initiale somatic embryogenesis (SE) with a frequency of up to 20%. These findings offer encouraging prospects for cloning proven superior plant material and to integrate this propagation system into tree improvement programs. Once the process of SE has been initiated, the multiplication cycle proceeds via secondary embryogenesis, which can be maintained indefinitely. Problems are reported by the formation of anomalous embryos. The mutability of somatic embryogenic cell lines of various oak species has been monitored by flow cytometry and molecular markers. No somaclonal variation was detected applying random amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers, whereas DNA-content measurements via flow cytometry revealed tetraploidy in some cell lines after several years of continuous subculture. Maturation and low germination frequencies are the main bottlenecks for a broader use of this technique. Recently attention has been on embryo quality and parameters for conversion capacity such as high endogenous cytokinin level and low abscisic acid (ABA) level. Although oak is probably the species that is the most well-developed system for a broadleaved forest tree, data on growth performances of somatic embryo-derived plants are rare.     

Increasing ploidy level in cell suspension cultures of Doritaenopsis by exogenous application of 2,4-dichlorophenoxyacetic acid

To clarify the causal factors for ploidy variation in plant cell culture, we attempted to alter ploidy distribution in cell cultures of a tetraploid cultivar of Doritaenopsis by changing the plant growth regulators (PGRs) in the culture medium. The original suspension cultured cells, which had been maintained in medium containing 0.1 mg l⁻¹ 1-naphthaleneacetic acid and 1 mg l⁻¹ benzyladenine, were transferred onto various gellan gum solidified media with a single application of PGRs, and the ploidy distributions of the cells were examined using flow cytometry analysis during 3 weeks of culture. Among the PGRs tested, 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid caused a drastic reduction in the 4C-cell proportion in cell cultures with an increased cell proportion of 8C or higher C-values. In the case of 2,4-D application, a reduction of cell viability was observed. A decreasing proportion was also observed in the 8C-cell population accumulated by 2,4-D treatment, following transfer back to the medium containing the standard PGR composition. These results suggest that the exogenous application of 2,4-D arrested the cell cycle at G2 phase in the Doritaenopsis cells, and the removal of 2,4-D might induce further endoreduplication or recover the mitotic cycle of the G2-arrested cells.     

Monitoring stem cell proliferation and differentiation in primary midgut cell cultures from Heliothis virescens larvae using flow cytometry

In the midgut of Heliothis virescens larvae, proliferation and differentiation of stem cell populations allow for midgut growth and regeneration. Basic epithelial regenerative function can be assessed in vitro by purifying these two cell type populations, yet efficient high throughput methods to monitor midgut stem cell proliferation and differentiation are not available. We describe a flow cytometry method to differentiate stem from mature midgut cells and use it to monitor proliferation, differentiation and death in primary midgut stem cell cultures from H. virescens larvae. Our method is based on differential light scattering and vital stain fluorescence properties to distinguish between stem and mature midgut cells. Using this method, we monitored proliferation and differentiation of H. virescens midgut cells cultured in the presence of fetal bovine serum (FBS) or AlbuMAX II. Supplementation with FBS resulted in increased stem cell differentiation after 5 days of culture, while AlbuMAX II-supplemented medium promoted stem cell proliferation. These data demonstrate utility of our flow cytometry method for studying stem cell-based epithelial regeneration, and indicate that AlbuMAX II-supplemented medium may be used to maintain pluripotency in primary midgut stem cell cultures.     

Statistical optimization of single-cell production from Taxus cuspidata plant cell aggregates

Flow-cytometric characterization of plant cell culture growth and metabolism at the single-cell level is a method superior to traditional culture average measurements for collecting population information. Investigation of culture heterogeneity and production variability by obtaining information about different culture subpopulations is crucial for optimizing bio-processes for enhanced productivity. Obtaining high yields of intact and viable single cells from aggregated plant cell cultures is an enabling criterion for their analysis and isolation using high-throughput flow cytometric methods. The critical parameters affecting the enzymatic isolation of single cells from aggregated Taxus cuspidata plant cell suspensions were optimized using response-surface methodology and factorial central composite design. Using a design of experiments approach, the output response single-cell yield (SCY, percentage of cell clusters containing only a single cell) was optimized. Optimal conditions were defined for the independent parameters cellulase concentration, pectolyase Y-23 concentration, and centrifugation speed to be 0.045% (w/v), 0.7% (w/v), and 1200?×?g, respectively. At these optimal conditions, the model predicted a maximum SCY of 48%. The experimental data exhibited a 72% increase over previously attained values and additionally validated the model predictions. More than 99% of the isolated cells were viable and suitable for rapid analysis through flow cytometry, thus enabling the collection of population information from cells that accurately represent aggregated suspensions. These isolated cells can be further studied to gain insight into both growth and secondary metabolite production, which can be used for bio-process optimization.     

Combination of quantification and observation methods for study of "Side Population" cells in their "in vitro" microenvironment.

BACKGROUND: Qualitative and quantitative analyses of the rare phenotypic variants in in vitro culture systems is necessary for the understanding of cell differentiation in cell culture of primary cells or cell lines. Slide-based cytometry combines image acquisition and data treatment, and associates the power of flow cytometry (FCM) and the resolution of the microscopic studies making it suitable for the analysis of cells with rare phenotype. In this paper we develop a method that applies these principles to a particularly hot problem in cell biology, the study of stem cell like cells in cultures of primary cells, cancer cells, and various cell lines. METHODS: The adherent cells were labeled by the fluorescent dye Hoechst 33342. The images of cell populations were collected by a two-photon microscope and processed by a software developed by us. The software allows the automated segmentation of the nuclei in a very dense cell environment, the measurement of the fluorescence intensity of each nucleus and the recording of their position in the plate. The cells with a given fluorescence intensity can then be located easily on the recorded image of the culture plate for further analysis. RESULTS: The potential of our method is illustrated by the identification and localization of SP cells in the cultures of the C2C12 cell line. Although these cells represent only about 1% of the total population as calculated by flow cytometry, they can be identified in the culture plate with high precision by microscopy. CONCLUSION: Cells with the rare stem-cell like phenotype can be efficiently identified in the undisturbed cultures. Since the fluorescence intensity of rare events and the position of thousands of surrounding cells are recorded at the same time, the method associates the advantage of the FCM analysis and the microscopic observation.     

LL-37, HNP-1, and HBD2/3 modulate the secretion of cytokines TNF-α, IL-6, IFN-γ, IL-10 and MMP1 in human primary cell cultures

The aim of this study was to evaluate the effects of the LL-37, HNP-1 and HBD2/3 peptides on cytokine andMMPproduction in human polymorphonuclear cells, mononuclear cells and chondrocytes. The levels of cytokines in supernatants from mononuclear and polymorphonuclear cell cultures were measured with a cytometric bead array by flow cytometry. Likewise, the levels of metalloproteinase/MMP-1, 3, and 13 were measured in supernatants from chondrocyte cultures using an ELISA. The expression of RANKL on lymphocytes was analyzed by flow cytometry.We observed increased levels of TNF-α, IL-6 and IL-10 in mononuclear and polymorphonuclear cell cultures stimulated with HBD-2/3.We also observed increased levels of IFN-γ, IL-10, and IL-6 in mononuclear cell cultures stimulated with HNP-1, and increased IL-6 levels were observed in polymorphonuclear cell cultures exposed to HNP-1. We also found that the MMP-1 level increased in the chondrocyte cultures stimulated with HBD-3, whereas the MMP-1 level was decreased in cultures exposed to LL-37. The present report is the first study to determine that HNP-1and HBD2/3 promote the secretion of pro-inflammatory cytokines by polymorphonuclear and mononuclear cells and the secretion of MMP by chondrocytes, whereas LL-37 diminishes MMP1 secretion. Our results suggest that HBD-2/3 and HNP1 might play a pathological role in rheumatoid arthritis, while LL-37 might have a protective role.     

Effect of 17-alpha-ethynylestradiol on germ cell proliferation in organ and primary culture of medaka (Oryzias latipes) testis

Organ cultures and primary cell cultures of medaka (Oryzias latipes) testis were compared with respect to cell viability and cell proliferation. The analysis by fluorescence microscopy and flow cytometry showed that in both cultures, the cells remained viable for at least 1 day and cell proliferation could be analyzed reliably by BrdU incorporation. The proliferating cells were mostly spermatogonia located at the periphery of the testis in tissue sections. Both culture systems were used to study the effect of 17-alpha-ethynylestradiol on cell proliferation. The results obtained with organ and primary cultures were consistent: low concentrations (0.01 and 1 nm) of synthetic estrogen stimulated cell proliferation slightly, while a higher concentration (100 nm) had an inhibitory effect. Both culture methods are suitable for the analysis of substances that might interfere with germ cell proliferation or other functions in spermatogenesis.