Different Time Points Of Culture Research Articles

Physiological characteristics of Bacillus thuringiensis subspecies israelensis with double mutations of cwlE and sigK and its activity against larvae of Culex pipiens pallens

To investigate the physiological characteristics of Bacillus thuringiensis subspecies israelensis (Bti) with double mutations of cwlE and sigK genes and to assess the activity of Bti against larvae of Culex pipiens pallens under different external factors, so as to provide the theoretical evidence for the use of engineered bacteria of Bti for effective mosquito control. B. thuringiensis wild-type strain Bt-59 and Bt-59 strain with cwlE mutation [Bt-59 (ΔcwlE)] were cultured in nutrient broth media for 24 hours, and Bt-59 strains with sigK mutation [Bt-59 (ΔsigK)] and double mutations of cwlE and sigK [Bt-59 (ΔcwlE-sigK)] were cultured in nutrient broth media for 48 hours. Then, 5 μL of culture media were transferred to glass sides, and cell morphology and mother cell lysis were observed under an optical microscope. The optical densities of Bti strain culture media were measured at different time points of culture, and the growth curves of Bt-59, Bt-59 (ΔcwlE), Bt-59 (ΔsigK), and Bt-59 (ΔcwlE-sigK) strains were plotted. The differences in carbon source metabolism of four Bti strains were analyzed using the Biolog microplate culture method, and the metabolic activity of these strains was estimated with average well color development (AWCD). The fermentation media of these four Bti strains were diluted into final concentrations of 2.000, 1.000, 0.500, 0.250, and 0.125 μL/L, and the median lethal concentrations (LC50 values) of these four strains against the third instar larvae of Cx. pipiens pallens were determined. In addition, the fermentation media of Bti strains were processed as follows: pH adjusted to 5, 7 and 9; treated at 30, 40 ℃ and 50 ℃ for 12 hours; and exposed to irradiation with ultraviolet lights for 0 hour and 6 hours. Then, 20 third instar larvae of Cx. pipiens pallens were exposed to the above processed fermentation media at a final concentration of 1 μL/L in 200 mL of water at 26 ℃ for 24 hours, and the mosquito mortality was estimated to evaluate the effects of pH, temperature and ultraviolet irradiation on the larvicidal activity of four Bti strains. The growth curves of the Bt-59 strain and its mutants shared a similar changing trend, and both experienced a stable phase 6 hours post-culture. Both spores and crystal proteins were found in Bt-59 and Bt-59 (ΔcwlE) cells, and only crystal proteins were found in Bt-59 (ΔsigK) and Bt-59 (ΔcwlE-sigK) cells. No lysis was found in the cell wall of the Bt-59 (ΔcwlE-sigK) strain, and the crystal protein was embedded in the mother cell. Biolog microplate culture assay showed that the AWCD values of four Bti strains showed a similar changing trend over time, and 33 carbon sources were found to be metabolized by all of the four strains, including dextrin, D-maltose and D-trehalose. The LC50 values of the fermentation media of Bt-59, Bt-59 (ΔcwlE), Bt-59 (ΔsigK), and Bt-59 (ΔcwlE-sigK) strains were 0.60, 0.51, 0.70 μL/L and 0.72 μL/L against Cx. pipiens pallens, respectively. The adjusted mortality of larval Cx. pipiens pallens reduced by 76.60%, 76.00%, 66.67%, and 0 following exposure to the fermentation media of Bt-59, Bt-59 (ΔcwlE), Bt-59 (ΔsigK), and Bt-59 (ΔcwlE-sigK) strains at a pH of 5 relative to at a pH of 7, and the adjusted mortality reduced by 49.02%, 51.06%, 36.36%, and 4.44% following 6-hour exposure to ultraviolet irradiation relative to 0-hour exposure, while the adjusted mortality was 68.33% to 83.33% following treatment with the fermentation media of four Bti strains at different temperatures. Bt-59 (ΔcwlE-sigK) strains do not generate spores, and the absence of cwlE and sigK does not affect the growth, carbon source metabolism, and larvicidal activity of Bti strains against larval Cx. pipiens pallens. Cell wall embedding of Bt-59 (ΔcwlE-sigK) strains may protect larvicidal crystal proteins of Bti strains from external environmental factors, including ultraviolet irradiation, and pH alteration.

Genome-wide transcriptome profiling of ex-vivo precision-cut slices from human pancreatic ductal adenocarcinoma

Ex-vivo tumor tissue culture systems are used as models to test specific anti-cancer drugs. Their main advantage is that they are closely comparable with the in vivo tumor in their host organism. We previously reported that precision-cut organotypic tissue slices of pancreatic ductal adenocarcinoma (PDAC) can be successfully cultured ex-vivo for at least 4 days. In order to study how culturing might affect transcription patterns, we now performed genome-wide transcriptome profiling of both baseline (0 h) and explanted tumors at daily intervals (24, 48 and 72 h) after start of culturing. The total-RNA from five samples of surgically resected human PDAC tumors at baseline and at different time points in culture was sequenced. Differential gene expression analysis of the whole transcriptome, testing 58,713 genes and over 206,000 transcripts, found that only a small number of genes showed significant changes in expression between baseline and cultured samples. The cultured tumor slices showed upregulation of a median of 12, 10 and 15 genes and downregulation of a median of 15, 12 and 25 genes at 24, 48 and 72 h in culture, respectively. One sample had morphologically increasing loss of tissue viability (range 0–18%). The vascular endothelial growth factor A (VEGFA) was significantly upregulated during the entire culture period in this case. Pathway over-representation analysis suggested that VEGFA together with the PTGS2 gene were upregulated at the same time as HIF-1-triggered cell apoptosis via NF-ĸB and the AP-1 activating factor was induced. Indeed, increased areas of apoptotic lesions were visible in this sample after 24 hours of culture. In conclusion, genome-wide transcriptome analysis supports that ex-vivo cultured tissue slices of PDAC may be a representative model of the original tumor. Transcriptome analysis was found to be a valuable complement to morphology for evaluation of ex-vivo cultures of PDAC.

An RNA-seq based transcriptomic investigation into the productivity and growth variants with Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are widely used in producing monoclonal antibodies (mAbs), however, these cell lines can show variants associated with productivity and growth. Fundamental understanding of cellular mechanisms can benefit high mAbs production while maintaining robust cell growth. In this study, RNA sequencing (RNA-seq) based approach was used to study the transcriptomic space among three mAb-producing CHO cell lines. A phenotypic contrast of higher productivity but lower growth was found with one cell line versus the other two cell lines. The messenger RNAs (mRNAs) from those cells at two different time points in culture were sequenced to seek the gene functions possibly associated with the phenotypic differences. It was found that the mAb transcripts from each cell line were correlated with the mAb production level, indicating a strong determination of production by the expression level of gene of interest (GOI). Further exploration in the global transcriptome showed that the high producers had more expression with genes related with secretion and protein transportation. In the meantime, the slower growth of the high producers was linked to higher regulation to cell growth as well as lower gene expression on biosynthesis, central metabolism and nutrient transport.

Switch From Fetal to Adult SCN5A Isoform in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Unmasks the Cellular Phenotype of a Conduction Disease-Causing Mutation.

BackgroundHuman induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) can recapitulate features of ion channel mutations causing inherited rhythm disease. However, the lack of maturity of these cells is considered a significant limitation of the model. Prolonged culture of hiPSC‐CMs promotes maturation of these cells. We studied the electrophysiological effects of the I230T mutation in the sodium channel gene SCN5A in hiPSC‐CMs generated from a homozygous (I230Thomo) and a heterozygous (I230Thet) individual from a family with recessive cardiac conduction disease. Since the I230T mutation occurs in the developmentally regulated “adult” isoform of SCN5A, we investigated the relationship between the expression fraction of the adult SCN5A isoform and the electrophysiological phenotype at different time points in culture.Methods and ResultsAfter a culture period of 20 days, sodium current (IN a) was mildly reduced in I230Thomo hiPSC‐CMs compared with control hiPSC‐CMs, while I230Thet hiPSC‐CMs displayed no reduction in IN a. This coincided with a relatively high expression fraction of the “fetal” SCN5A isoform compared with the adult isoform as measured by quantitative polymerase chain reaction. Following prolonged culture to 66 days, the fraction of adult SCN5A isoform increased; this was paralleled by a marked decrease in IN a in I230Thomo hiPSC‐CMs, in line with the severe clinical phenotype in homozygous patients. At this time in culture, I230Thet hiPSC‐CMs displayed an intermediate loss of IN a, compatible with a gene dosage effect.ConclusionsProlonged culture of hiPSC‐CMs leads to an increased expression fraction of the adult sodium channel isoform. This new aspect of electrophysiological immaturity should be taken into account in studies that focus on the effects of SCN5A mutations in hiPSC‐CMs.

Expression and Promoter CpG Island Methylation Status of miR-34b in Leukemia Cell Lines and Their Clinical Significance

To explore the expression and promoter CpG island methylation status of miR-34b in leukemia cell lines and their clinical significance. A total of 10 cases of non-hematologic diseases were selected as control group, and the bone marrow cells of control group and HL-60, K562 cells were selected; the relative expression of miR-34b was detected in bone marrow cells, HL-60 and K562 cell lines by fluorescence quantitative PCR, and the MiR-34b methylation status was detected by methylation-specific PCR, the HL-60 and K562 cell lines were treated with decitabine, and the expression levels and methylation status of miR-34b in the 2 cell lines were detected by the same method. Has-miR-34b was transfected into K562 cells, which were divided into non-transfection group, negative control group and Has-miR-34b transfection group; if the transfection was successful, the cell proliferation should be recorded at different time points of culture, and the proliferation inhibition rate should be calculated. The relative expression level of miR-34b in the control group was (5.23 ± 0.75), in HL-60 was (0.05 ± 0.01) and in K562 was (0.04 ± 0.01). The difference between 3 groups was statistically significant (F = 44.812, P < 0.01). The promoter regions of CpG island in HL-60 and K562 cell lines were methylated, while the bone marrow cells were not methylated in 10 cases of non hematologic diseases children.Through miR-34b expression levels of HL-60 and K562 cell lines significantly increased by decitabine treatment (P < 0.05), and the methylation of leukemia cell line promoter region CpG island was found before and after decitabine treatment, but after administration of decitabine the methylation significantly decreased, suggesting that decitabine has an inhibitory effect on methylation of promoter region CpG island. After being cultured for 48, 72, 96 and 120 hrs, the cell proliferation in Has-miR-34b transfection group reached to 24.8%, 46.7%, 33.6% and 4.7%, repectively, and significantly lower than that in non transfection group (P < 0.05). CpG island methylation of miR-34b promoter region in leukemia cell lines can decrease the expression levels of miR-34b, which is also the reason why miR-34b can reduce the inhibition of cell proliferation, thus miR-34b might be a tumor suppressor gene involved in the regulation of leukemia.

In vitro investigations of bone remodeling on a transparent hydroxyapatite ceramic

The light microscopic examination of cells directly on bioceramic materials in the transmission mode is impossible because many of these materials are opaque. In order to enable direct viewing of living cells and to perform time-lapse studies, nearly transparent bioceramic materials were developed. A dense and fine-grained transparent hydroxyapatite (tHA) was processed by a gel-casting route followed by low-temperature sintering (1000 °C). By virtue of its transparency, direct visualization of cellular events on this material is possible in transmitted light. In this study, the interaction of different bone cell types with the tHA ceramic was envisaged. Investigation of rat calvaria osteoblasts (RCO) cultured on tHA by means of transmission light microscopy indicated good cytocompatibility of tHA. Microscopic analysis of osteogenic-induced human bone marrow stromal cells (hBMSC) on tHA and quantitative analysis of their lactate dehydrogenase (LDH) activity at different time points of culture revealed favorable proliferation as well. An increase of the alkaline phosphatase (ALP) activity indicated the differentiation of osteogenic-induced hBMSC towards the osteoblastic lineage. In addition, the differentiation of human monocytes to osteoclast-like cells could also be demonstrated on tHA and was confirmed by fluorescent microscopy imaging of multinucleated cells on the transparent material.

Estrogen receptor subtypes localization shifts in cultured mouse ovarian follicles

Estrogens play an important role in the growth, differentiation, and function of female reproductive tissues. Estrogen signals through estrogen receptors (ERs), members of the nuclear receptor superfamily. The two major forms, ERalpha and ERbeta, are expressed in the mouse ovary, where ERbeta is predominantly expressed in granulosa cells, and ERalpha in theca cells. In this study, we determined the expression pattern of ER subtypes within mouse follicles cultured from the early preantral stage up to the preovulatory stage and after an ovulatory stimulus in different culture conditions. Immunohistochemical studies performed at different time points of culture revealed that ERbeta was found exclusively in granulosa cell nuclei regardless of follicular growth stage or culture conditions. In contrast, ERalpha was found in oocyte, granulose, and theca cells, and its subcellular localization differed between follicular growth stages and culture conditions. A shift from a predominant cytoplasmic to a predominant nuclear immunolocalization was observed in granulosa cells as follicles reached the antral growth phase, and was postponed in culture conditions with minimal growth factor supplementation. In response to hCG, ERbeta protein levels in luteinized granulosa cells spectacularly declined to undetectable levels, while ERalpha immunostaining again shifted to cytoplasmic regions, but not in theca cells.

Human Bone Marrow-Derived Mesenchymal Stem Cells Do Not Undergo Transformation after Long-Term In Vitro Culture and Do Not Exhibit Telomere Maintenance Mechanisms.

Mesenchymal sem cells (MSCs) are multipotent progenitors with the ability to differentiate along multiple cell lineages and are today considered a useful tool for cell therapy and tissue engineering approaches. Concerns that adult human MSCs may undergo malignant transformation have been recently raised. In fact, human adipose tissue-derived MSCs have been shown to spontaneously transform after long-term in vitro culture. Aim of this study was to investigate the susceptibility to transformation of human bone marrow (BM)-derived MSCs at different time points of in vitro culture. MSCs were isolated from BM of 10 healthy donors and propagated continuously in vitro until reaching a senescence phase or passage (P) 25. MSCs in the senescence phase were closely monitored for 8–12 weeks, to look for the appearance of a crisis phase. MSCs were characterized by morphology, differentiation capacity and immunophenotype at different time-points in culture. The genetic characterization of MSCs was investigated through array comparative genomic hybridization (array-CGH), classical cytogenetics and subtelomeric FISH analysis both before and after prolonged in vitro culture. MSCs were tested for the expression of telomerase activity (TA), hTERT transcripts and alternative mechanisms of telomere lengthening (ALT) at different time-points in culture. MSCs from 5 donors were also analyzed for telomere length at P3 and P15. p53 gene status was also analyzed in MSCs from donors #1 and #2 that rapidly reached senescence in culture. A huge variability between donors was noted in terms of proliferative capacity and in vitro life-span of MSCs. All MSCs maintained the typical spindle-shaped morphology, phenotype and ability to differentiate into osteoblasts and adipocytes throughout the culture period. All MSCs displayed a progressive decrease in the proliferative capacity until reaching senescence, not followed by any further growth. Array-CGH, karyotype and subtelomeric FISH analyses demonstrated that MSCs expanded in vitro did not show chromosomal rearrangements, also after long term culture. TA was not evidenced in the samples tested, including a post-senescence culture. hTERT transcripts (full-length and the additional splice variants a−, b−, a−b−) were found not to be expressed in any of the examined cultures. Telomeres shortened during the culture period. ALT were not evidenced in the MSCs tested, as indicated by the lack of ALT-associated promyelocytic leukemia bodies. Direct DNA sequencing of exons 2–11 in pre-senescence cultures from donors #1 and #2 did not evidence the presence of mutation in the p53 gene. Human BM-derived MSCs do not display an aptitude for spontaneous transformation and can be safely expanded in vitro without any sign of immortalization or development of chromosomal abnormalities. Our results provide support to the concept that the biological properties of human BM-derived MSCs after ex vivo expansion remain suitable for use in cell-therapy approaches; however, it is strongly recommended that phenotype, functional and genetic characteristics of MSCs after in vitro culture and before in vivo infusion are tested, to further guarantee safety for the patient.

Characterization of primary rat proximal tubular cells by gene expression analysis

The kidney plays a major role in excretory and reabsorptive processes. The kidney cortex consists primarily of proximal tubular cells, which are epithelial cells that are often involved in the induction and progression of various kidney diseases. Therefore primary proximal tubular cells are widely used as a renal cell model. To further characterize this kidney in vitro model different time points in culture after isolation of the cells were compared to the cortex in vivo using gene expression analysis based on microarrays. This study revealed that many metabolic pathways and some kidney-specific functions are lacking in the in vitro model. Furthermore genes involved in RNA and protein synthesis, intracellular transport, extracellular matrix and cytoskeletal organization were upregulated in culture compared to in vivo, indicating proliferation of the cells and differentiation into a cell culture phenotype. The data represented here may help to evaluate the in vivo relevance of results obtained with this in vitro model.

Study on biological characters of SGC7901 gastric cancer cell-dendritic cell fusion vaccines

To detect the biological characters of the SGC7901 gastric cancer cell-dendritic cell fusion vaccines. The suspending living SGC7901 gastric cancer cells and dendritic cells were induced to be fusioned by polyethylene glycol. Pure fusion cells were obtained by selective culture with the HAT/HT culture systems. The fusion cells were counted at different time points of culture and their growth curves were drawn to reflect their proliferative activities. The fusion cells were also cultured in culture medium to investigate whether they could grow into cell clones. MTT method was used to test the stimulating abilities of the fusion cells on T lymphocytes' proliferations. Moreover, the fusion cells were planted into nude mice to observe whether they could grow into new planted tumors in this kind of immunodeficiency animals. The fusion cells had weaker proliferative activity and clone abilities than their parental cells. When they were cultured, the counts of cells did not increase remarkably, nor could they grow into cell clones in culture medium. The fusion cells could not grow into new planted tumors after planted into nude mice. The stimulating abilities of the fusion cells on T lymphocytes' proliferations were remarkably increased than their parental dendritic cells. The SGC7901 gastric cancer cell-dendritic cell fusion vaccines have much weaker proliferative abilities than their parental cells, but they keep strong abilities to irritate the T lymphocytes and have no abilities to grow into new planted tumors in immunodeficiency animals. These are the biological basis for their anti-tumor biotherapies.

Derivation of Lymphoid Lineage Cells from Human Embryonic Stem Cells on Co-Culture in OP9 and OP9DL1 Stromal Cells.

We have been able to direct the differentiation of CD34pos cells derived from H9 and H7 hESCs toward lymphoid lineages using OP9 or OP9DL1 feeder layers. OP9 and OP9DL1 stromal cell lines are consistently better than S17 or OP9-jagged in inducing hESC-derived CD34pos cells at different time points in culture. By day 14 in culture, about 8.7% and 7.9% of differentiated H9 hES cells harvested from co-cultures in OP9 or OP9DL1 respectively were CD34pos. The CD34pos cells in these cultures were enriched for by magnetic sorting. The clonogenic potential of differentiated hES cell suspensions and of sorted CD34pos cells were determined using methylcellulose colony assays. The highest clonogenic potential was noted after 18 days in co-culture when we obtained 142 +/− 31 and 218 +/− 38 colonies per million cells plated from OP9 and OP9DL1. Selection for CD34pos cells enriched the clonogenic activity 27-fold. Morphologic analyses of representative colonies from the methylcellulose cultures showed cells of the erythroid, myeloid, lymphoid and megakaryocytic lineages were generated. Directed differentiation of CD34pos cells towards lymphoid lineages was initiated by replating the sorted CD34pos cells onto irradiated OP9 or OP9DL1 stromal cells supplemented with stem cell factor (SCF), IL3, IL7 and Flt3L. Under these conditions, B lineage (CD19pos) and myeloid lineage (CD14pos or CD15pos) cells were observed when CD34pos cells were grown in OP9, and T lineage cells were observed when CD34pos cells were grown in OP9DL1. CD4posCD8pos cells were evident after 12 days of secondary culture in OP9DL1 and were prominent by day 20. Only a few of the CD4pos, CD8pos and CD4posCD8pos cells, however, were noted to be CD3pos. Addition of SCF + IL3, SCF + Flt3L, or SCF + Flt3L + IL7 at initiation of hESC differentiation increased the percentage of CD34pos cells and/ or increased the hematopoietic (clonogenic) potential of sorted CD34pos cells. CD34pos cells derived from hESCs grown in OP9DL1 for 14 days and replated in OP9DL1 supplemented with Flt3L + IL7 +/− SCF generated the highest percentages of CD4posCD8pos cells. Additional phenotypic analyses and functional studies of sorted CD4pos, CD8pos and CD4posCD8pos cells generated from these cultures are needed to further characterize their lymphoid properties.

Factors Modulating p63 Expression in Cultured Limbal Epithelial Cells

The expression pattern of p63, a homologue of the transcription factor p53, and whether it can be used as a corneal epithelial stem cell specific marker remain controversial. We investigated the p63 expression pattern in cultured limbal epithelial cells at different time points in culture, in sparse and confluent cultures, after growth factor starvation, and in single-cell-derived colonies. Harvested limbal epithelial cells were plated at 2.5 (sparse) or 5 (dense) x 10 cells/cm and evaluated for p63 expression at day 1, day 4, day 7, after starvation for 72 hours, or in colonies derived from single cells. Expression of corneal lineage specific differentiation marker keratin 3 (K3) was correlated with p63 expression. Results were compared by 1-way ANOVA. More than 85% (85%-90%) of cells expressed p63 on day 1 regardless of cell plating density. On day 4, sparsely plated cultures were subconfluent and demonstrated high p63 expression (87.4%), whereas densely plated cells were confluent and had markedly reduced p63 expression (16.9%). Starvation of subconfluent cultures arrested cell division but did not decrease p63 expression. High-p63-expressing cultures expressed low levels of K3, and this trend was reversed in confluent cultures. Most cells in all colonies derived from single cells expressed p63. The majority of corneal limbal epithelial cells express p63 in colonies derived from single cells and in subconfluent cultures regardless of time in culture or continuance of cell division. This suggests that p63 expression in culture cannot be used as a marker for stem cells. Significantly reduced number of cells express p63 in confluent cultures, associated with increased cell-cell contact. It is notable that these cells continue to express p63 amid areas of increased cell-cell contact several days after cultures have attained full confluency. This may represent a unique subpopulation of cells that retain proliferative potential in a confluent culture and may be analogous to a subpopulation of stem cells present in vivo.

Phosphodiesterase in human colon carcinoma cell line CaCo-2 in culture

In a series of in vitro experiments we characterised the relationship between DNA distribution in the G1, S and G2/M phases of cell cycle and PDE and GST activity in CaCo-2 cells. The DNA distribution in CaCo-2 cells, was assessed by flow cytometry, with fluorescent dyes at different time points of culture. The exponential increase in cell number continued until day 10 when there was cell saturation. The effect of medium replacement on PDE activity was assayed in the first 10 h after medium replacement. The 6 th hour is the time at which PDE activity was found to be highest. We have assayed the PDE enzyme with cGMP and cAMP as substrates. Only cAMP was consumed from this enzyme. We found a very close correlation between the DNA distribution in the various phases of the cell cycle and the PDE activity. PDE activity was very high during the active replication phase, whereas GST activity was high after confluency.