Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible...Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.展开更多
The three-dimensional(3D)cell culture system has garnered significant attention in recent years as a means of studying cell behavior and tissue development,as opposed to traditional two-dimensional cultures.These syst...The three-dimensional(3D)cell culture system has garnered significant attention in recent years as a means of studying cell behavior and tissue development,as opposed to traditional two-dimensional cultures.These systems can induce specific cell reactions,promote specific tissue functions,and serve as valuable tools for research in tissue engineering,regenerative medicine,and drug discovery.This paper discusses current developments in the field of three-dimensional cell culture and the potential applications of 3D type 1 collagen gels to enhance the growth and maturation of dendritic cells.展开更多
In view of the problems of the traditional cell incubator,such as the small range of cell culture types,the inability to adjust the internal space of the incubator according to needs,and the inconvenient sampling,this...In view of the problems of the traditional cell incubator,such as the small range of cell culture types,the inability to adjust the internal space of the incubator according to needs,and the inconvenient sampling,this study innovatively designed a cell incubator structure.It proposed a new design concept that can solve the above-mentioned shortcomings.The cell incubator after the new structural modification can adjust the internal space structure of cell culture by setting the bolt-fixed connection between the fixed plate and the vessel divider.It realizes the cultivation of various cells through refrigeration modules and heating modules.Through setting a sampling hole in the glass inner door,it is favorable for operators to take samples,making cell culture more convenient and efficient.展开更多
Summary: This study aimed to establish a new in vitro three-dimensional (3D) cell culture and use quantum dots (QDs) molecular imaging to examine the invasive behaviors of hepatocellular carcinoma (HCC) cells. ...Summary: This study aimed to establish a new in vitro three-dimensional (3D) cell culture and use quantum dots (QDs) molecular imaging to examine the invasive behaviors of hepatocellular carcinoma (HCC) cells. Each well of the 24-well cell culture plate was cover-slipped. Matrigel diluted with se- rum-free DMEM was added and HCCLM9 cells were cultured on the Matrigel. The cell morphological and cell growth characteristics were observed by inverted microscopy and laser confocal microscopy at different culture time. Cell invasive features were monitored by QDs-based real-time molecular imaging techniques. The results showed that on this 3D cell culture platform, HCCLM9 cells exhibited typical multi-step invasive behaviors, including reversion of cell senescence, active focal proliferation and dominant clones invasion. During the process, cells under 3D cell culture showed biological behaviors of spatio-temporal characteristics. Cells first merged on the surface of matrix, then gradually infiltrated and migrated into deep part of matrix, presenting polygonal morphology with stretched protrusions, forming tubular, annular and even network structure, which suggested that HCC cells have the morpho- logical basis for vasculogenic mimicry. In addition, small cell clones with their edges well-circumscribed in early stage, progressed into a large irregular clone with ill-defined edge, while the other cells developed invadopodia. And QDs probing showed MT1-MMP was strongly expressed in the invadopodia. These findings indicate that a novel 3D cell culture platform has been successfully estab- lished, which can mimic the in vivo tumor microenvironment, and when combined with QDs-based mo- lecular imaging, it can help to better investigate the invasive behaviors of HCC cells.展开更多
Some biological characteristics of human bone marrow mesenchymal stem cells (MSCs) cultured in vitro were observed. hMSCs were isolated from bone marrow and purified by density gradient centrifugation method, and then...Some biological characteristics of human bone marrow mesenchymal stem cells (MSCs) cultured in vitro were observed. hMSCs were isolated from bone marrow and purified by density gradient centrifugation method, and then cultured in vitro. The proliferation and growth characteristics of hMSCs were observed in primary and passage culture. MSCs of passage 3 were examined for the purify by positive rate of CD29 and CD44 through flow cytometry. Human bone marrow MSCs showed active proliferation capacity in vitro. The purify of MSCs separated by our method was higher than 90 %. It was concluded that hMSCs have been successfully cultured and expanded effectively. It provided a foundation for further investigation and application of MSCs.展开更多
In this study, using Taxus cuspidata as a raw material, we obtained stable high-yielding cell lines by subculturing and quantified paclitaxel content using ultra- sonic extraction combined with TLC-UV spectrophotom- e...In this study, using Taxus cuspidata as a raw material, we obtained stable high-yielding cell lines by subculturing and quantified paclitaxel content using ultra- sonic extraction combined with TLC-UV spectrophotom- etry. In single factor and multiple factors tests to optimize design and study the effects of elicitors, precursors, and metabolic inhibitors on paclitaxel production by Taxus cuspidata cells, paclitaxel production reached 4.32 mg/L when 100 μmol/L methyl jasmonate, 20 mg/L salicylic acid, 400 mg/L phenylalanine and 2 mg/L gibberellin (GA3) were added to the culture medium of suspension cells. When adding metabolic adjustment factors on the 7th day of culture, extra- and intracellular paclitaxel production was the highest at 4.855 mg/L, paclitaxel release rate was 10.48 %, fresh mass and paclitaxel production of cell increased, respectively, by 6.08 and 11.57 %. By controlling the anabolism of paclitaxel, paclitaxel yield was significantly improved.展开更多
Aim: To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. Methods: Tissue was obtained from patients undergoing therapeutic orchide...Aim: To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. Methods: Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Epithelial cell cultures were obtained from the caput, corpus and cauda epididymides. Enzymatic activity was measured in conditioned media by colorimetric methods in absence or presence of 1, 10 or 100 nrnoI.L^(-1) testosterone. The effect of 1 μmol.L^(-1) flutamide was also evaluated. Results: GPx activity was higher in cultures from corpus and cauda than caput epididymidis. The presence of different concentrations of testosterone increase enzyme activity in cell cultures from all epididymal regions. Addition of flutamide reverses the androgen dependent increase of GPx activity. Conclusion: GPx activity is secreted from human epididymal cells in a region dependent manner and is regulated by androgens.展开更多
Desmin and vimentin are major components of intermediate filament proteins in cardiac myocytes. We developed a primary cell culture method for cardiac myocytes of axolotl embryos. Cardiac myocytes of embryonic stage 3...Desmin and vimentin are major components of intermediate filament proteins in cardiac myocytes. We developed a primary cell culture method for cardiac myocytes of axolotl embryos. Cardiac myocytes of embryonic stage 39 were cultured for 1-14 days. Myocytes showed spontaneous contractions (15-30 beats/min) after 48-72 hours in culture, round shape and large irregular projections. Desmin and vimentin were observed in the cultured myocytes by means of immunofluorescent staining in combination with immunofluorescent microscopy. Immunofluorescent staining of the cultured cardiac myocytes after different lengths of time in culture(3,6,9 days) showed that vimentin staining was stronger than desmin staining during the early stages of culture (3 days). The myocytes exhibited various forms of staining, including parallel lines and interconnected networks. Some lines showed regular striation; most of the myofibrils were arranged in parallel arrays along the cell's long axis. Both desmin and vimentin in the cell appeared to encirele the Z lines and to link myofibrils laterally at the Z lines.展开更多
We describe a novel technique, low surface energy Gas Expansion Molding (GEM), to fabricate microbubble arrays in polydimethylsiloxane (PDMS) which are incorporated into parallel plate flow chambers and tested in ...We describe a novel technique, low surface energy Gas Expansion Molding (GEM), to fabricate microbubble arrays in polydimethylsiloxane (PDMS) which are incorporated into parallel plate flow chambers and tested in cell sorting and microcell cuTture applications. This architecture confers several operational advantages that distinguish this technology approach from currently used methods. Herein we describe the GEM process and the parameters that are used to control microbubble formation and a Vacuum-Assisted Coating (VAC) process developed to selectively and spatially alter the PDMS surface chemistry in the wells and on the microchannel surface. We describe results from microflow image visualization studies conducted to investigate fluid streams above and within microbubble wells and conclude with a discussion of cell culture studies in PDMS.展开更多
To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase(ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed th...To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase(ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbedwith a structure resembling the loose connective tissue morphology in a novel 3 D culture system. We confirmed that the 3 D system using CellbedTMaccurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3 D-cultured tongue cancer cell lines than in 2 D cultures. Typically, under conventional 2 D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells.However, in the 3 D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3 D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3 D culture systems using Cellbed? are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.展开更多
BACKGROUND: Previous studies have used many methods for in vitro Schwann cells (SCs) cultures and purification, such as single cell suspension and cytosine arabinoside. However, it has been difficult to obtain suff...BACKGROUND: Previous studies have used many methods for in vitro Schwann cells (SCs) cultures and purification, such as single cell suspension and cytosine arabinoside. However, it has been difficult to obtain sufficient cellular density, and the procedures have been quite tedious. OBJECTIVE: To investigate the feasibility of culturing high-density SCs using fetal human dorsal root ganglion tissue explants. DESIGN, TIME AND SETTING: Cell culture and irnmunohistochernistry were performed at the Central Laboratory of Kunrning General Hospital of Chinese PLA between March 2001 and October 2008. MATERIALS: Culture media containing 10% fetal bovine serum, as well as 0.2% collagenase and 0.25% trypsin were purchased from Gibco, USA; mouse anti-human S-100 monoclonal antibody and goat anti-mouse IgG labeled with horseradish peroxidase were provided by Beijing Institute of Biological Products, China. METHODS: Primarily cultured SCs were dissociated from dorsal root ganglia of human aborted fetuses at 4 6 months pregnancy. Following removal of the dorsal root ganglion perineurium, the ganglia were dissected into tiny pieces and digested with 0.2% collagenase and 0.25% trypsin (volume ratio 1:1), then explanted and cultured. SC purification was performed with 5 rnL 10% fetal bovine serum added to the culture media, followed by differential adhesion. MAIN OUTCOME MEASURES: SCs morphology was observed under inverted phase contrast light microscopy. SC purity was evaluated according to percentage of S-100 immunostained cells. RESULTS: SCs were primarily cultured for 5 6 days and then subcultured for 4 5 passages. The highly enriched SC population reached 〉 95% purity and presented with normal morphology. CONCLUSION: A high purity of SCs was obtained with culture methods using human fetal dorsal root ganglion tissue explants.展开更多
In vitro tissue culture of hard woody, endangered, medicinal plant Coscinium fenestratum is most challenging to plant tissue culturists. In the present study, petiole and leaf explants of Coscinium fenestratum were in...In vitro tissue culture of hard woody, endangered, medicinal plant Coscinium fenestratum is most challenging to plant tissue culturists. In the present study, petiole and leaf explants of Coscinium fenestratum were induced to form callus when cultured on vermicompost extract media along with coelomic fluid. Suspension medium was developed using vermicompost extract and coelomic fluid in 3:1 ratio. Phytochemical analysis of the alkaloid berberine was confirmed from callus, suspension cell culture and suspension medium by Thin Layer Chromatography and High Performance Liquid Chromatography. Vermicompost and its extracts with coelomic fluid have shown maximum (100 per cent) response of callus induction. Callus mass enlarged with increasing concentration of coelomic fluid and callus growth was assessed from the biomass. Incubation of culture tubes in dark supported callus development significantly. The Rf value of 0.36 confirmed the presence of berberine by Thin Layer Chromatography. Qualitative analysis confirmed the presence of alkaloid berberine with the retention time of 2.8 minutes similar to that of standard reference sample from Sigma chemicals, USA. The suspension medium turned deep yellow because of the release of the alkaloid. Vermicompost and its extracts along with coelomic fluid have shown the economical approach for micropropagation of economically and medicinally important plants.展开更多
Plant cell culture technology is a technology that applies the research results of cell engineering to produce plant biological products at the cellular level.In recent years,the secondary metabolites of plants have a...Plant cell culture technology is a technology that applies the research results of cell engineering to produce plant biological products at the cellular level.In recent years,the secondary metabolites of plants have attracted more and more attention.The use of plant cell culture technology is a fast and efficient method of producing secondary metabolites.展开更多
This short review is to list pros and cons which are based on the literature and personal experience in cell culture studies related to possible commercial production of artificial meat as functional food. The general...This short review is to list pros and cons which are based on the literature and personal experience in cell culture studies related to possible commercial production of artificial meat as functional food. The general view of muscle composition and determinants of meat quality are shortly described. Principles of muscle cell propagation in culture and mutual relationships between different cell types present in this organ are briefly discussed. Additionally, the effects of some cytokines and growth factors for muscle cell growth and muscle tissue development are indicated. Finally, conclusion remarks related to detrimental consequences of meat production to natural environment as well as personal opinion of author on the prospects of artificial meat production are declared.展开更多
Deacetoxy-13-oxo sinenxan A (1) was converted to 9a-hydroxy-13-oxo-2a, 5a, 10b-triacetoxy-4(20),11-taxadiene (2) and 10b-hydroxy-13-oxo-2a,5a,9a-triacetoxy- 4(20), 11- taxadiene (3) by Ginkgo cell suspension cultures ...Deacetoxy-13-oxo sinenxan A (1) was converted to 9a-hydroxy-13-oxo-2a, 5a, 10b-triacetoxy-4(20),11-taxadiene (2) and 10b-hydroxy-13-oxo-2a,5a,9a-triacetoxy- 4(20), 11- taxadiene (3) by Ginkgo cell suspension cultures in 45% and 15% yields, respectively.展开更多
The development of large-scale cell cultivation and non-invasive cell harvesting is highly desired in various fields,including biological regeneration and pharmaceutical research.When using traditional microcarriers f...The development of large-scale cell cultivation and non-invasive cell harvesting is highly desired in various fields,including biological regeneration and pharmaceutical research.When using traditional microcarriers for cell culture,trypsinization is often necessary during cell collection,leading to partial cells damage.In this work,we developed a thermoresponsive glass microcarrier modified with poly(γ-propargyl-L-glutamate)(PPLG)and poly(N-isopropylacrylamide)(PNIPAM).We utilized these microcarriers for three-dimensional cell culture and enzyme-free cell harvesting,and the results indicated that the prepared microcarriers exhibited excellent non-invasive cell culture performance.展开更多
There is no clear consensus regarding how cells respond to hydrostatic pressure. This is largely attributable to the high heterogeneity among cell types and the diverse custom-made devices used in previous studies. Th...There is no clear consensus regarding how cells respond to hydrostatic pressure. This is largely attributable to the high heterogeneity among cell types and the diverse custom-made devices used in previous studies. The aim of this work was to develop a facile device that could mimic various pressure environments and then delineate the cellular response to pressure stimulus. The device described here achieved both stable and periodic pressurization without oxygen deprivation. The biological utility of the device was assessed using human umbilical vein endothelial cells. We found more stereoscopic nuclear morphology and re-distribution of lamin A/C under high hydrostatic pressure compared to control cells. Mass spectrometry-based proteomics analysis showed significant changes in mitochondria-related pathways. Western blot analysis confirmed that high hydrostatic pressure induced a tendency toward mitochondrial fusion. Increased mitochondrial activity was observed as well. In conclusion, this device can be readily applied in biological research and extend our understanding of cellular mechano-sensation and the associated changes in mitochondrial behaviors.展开更多
Surface topographies of cell culture substrates can be used to generate in vitro cell culture environments similar to the in vivo cell niches. In vivo, the physical properties of the extracellular matrix (ECM), such a...Surface topographies of cell culture substrates can be used to generate in vitro cell culture environments similar to the in vivo cell niches. In vivo, the physical properties of the extracellular matrix (ECM), such as its topography, provide physical cues that play an important role in modulating cell function. Mimicking these properties remains a challenge to provide in vitro realistic environments for cells. Artificially generated substrates’ topographies were used extensively to explore this important surface cue. More recently, the replication of natural surface topographies has been enabling to exploration of characteristics such as hierarchy and size scales relevant for cells as advanced biomimetic substrates. These substrates offer more realistic and mimetic environments regarding the topographies found in vivo. This review will highlight the use of natural surface topographies as a template to generate substrates for in-vitro cell culture. This review starts with an analysis of the main cell functions that can be regulated by the substrate’s surface topography through cell-substrate interactions. Then, we will discuss research works wherein substrates for cell biology decorated with natural surface topographies were used and investigated regarding their influence on cellular performance. At the end of this review, we will highlight the advantages and challenges of the use of natural surface topographies as a template for the generation of advanced substrates for cell culture.展开更多
BACKGROUND Efficient extraction of nucleic acids and proteins(ENAP)from cells is a prerequisite for precise annotation of gene function,and has become laboratory routine for revealing the mysteries of life.However,cel...BACKGROUND Efficient extraction of nucleic acids and proteins(ENAP)from cells is a prerequisite for precise annotation of gene function,and has become laboratory routine for revealing the mysteries of life.However,cell samples are often from different culture dishes,resulting in inevitable experimental errors and sometimes poor repeatability.AIM To explore a method to improve the efficiency of ENAP,minimizing errors in ENAP processes,enhancing the reliability and repeatability of subsequent experimental results.METHODS A protocol for the sequential isolation of RNA,DNA,and proteins from the same cultured HepG2 cells using RNAzol reagent is presented here.The first step involves culturing HepG2 cells to the exponential phase,followed by the sequential isolation of RNA,DNA,and proteins from the same cultured cells in the second step.The yield of nucleic acids and proteins is detected in the third step,and their purity and integrity are verified in the last step.RESULTS The procedure takes as few as 3-4 d from the start to quality verification and is highly efficient.In contrast to the existing kits and reagents,which are primarily based on independent isolation,this RNAzol reagent-based method is characterized by the sequential isolation of RNA,DNA,and proteins from the same cells,and therefore saves time,and has low cost and high efficiency.CONCLUSION The RNA,DNA,and proteins isolated using this method can be used for reverse transcription-polymerase chain reaction,polymerase chain reaction,and western blotting,respectively.展开更多
基金support from the National Key Research and Development Program of China(Grant No.2017YFA0700501),and the Innovation Fund of WNLO.
文摘Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.
文摘The three-dimensional(3D)cell culture system has garnered significant attention in recent years as a means of studying cell behavior and tissue development,as opposed to traditional two-dimensional cultures.These systems can induce specific cell reactions,promote specific tissue functions,and serve as valuable tools for research in tissue engineering,regenerative medicine,and drug discovery.This paper discusses current developments in the field of three-dimensional cell culture and the potential applications of 3D type 1 collagen gels to enhance the growth and maturation of dendritic cells.
基金Supported by Young and Middle-aged Teacher Education Research Project of Fujian Province(Science and Technology Category:JAT210477)College Student Innovation and Entrepreneurship Training Program of Xiamen Medical College(X202112631068)。
文摘In view of the problems of the traditional cell incubator,such as the small range of cell culture types,the inability to adjust the internal space of the incubator according to needs,and the inconvenient sampling,this study innovatively designed a cell incubator structure.It proposed a new design concept that can solve the above-mentioned shortcomings.The cell incubator after the new structural modification can adjust the internal space structure of cell culture by setting the bolt-fixed connection between the fixed plate and the vessel divider.It realizes the cultivation of various cells through refrigeration modules and heating modules.Through setting a sampling hole in the glass inner door,it is favorable for operators to take samples,making cell culture more convenient and efficient.
基金supported by grants from National Natural Science Foundation of China(No.81171396)Creative Research Groups of the National Natural Science Foundation of China(No.20921062)+1 种基金National Science and Technology Major Project(No.2012ZX10002012-12)National University Students Innovation Training Project of China(No.111048673)
文摘Summary: This study aimed to establish a new in vitro three-dimensional (3D) cell culture and use quantum dots (QDs) molecular imaging to examine the invasive behaviors of hepatocellular carcinoma (HCC) cells. Each well of the 24-well cell culture plate was cover-slipped. Matrigel diluted with se- rum-free DMEM was added and HCCLM9 cells were cultured on the Matrigel. The cell morphological and cell growth characteristics were observed by inverted microscopy and laser confocal microscopy at different culture time. Cell invasive features were monitored by QDs-based real-time molecular imaging techniques. The results showed that on this 3D cell culture platform, HCCLM9 cells exhibited typical multi-step invasive behaviors, including reversion of cell senescence, active focal proliferation and dominant clones invasion. During the process, cells under 3D cell culture showed biological behaviors of spatio-temporal characteristics. Cells first merged on the surface of matrix, then gradually infiltrated and migrated into deep part of matrix, presenting polygonal morphology with stretched protrusions, forming tubular, annular and even network structure, which suggested that HCC cells have the morpho- logical basis for vasculogenic mimicry. In addition, small cell clones with their edges well-circumscribed in early stage, progressed into a large irregular clone with ill-defined edge, while the other cells developed invadopodia. And QDs probing showed MT1-MMP was strongly expressed in the invadopodia. These findings indicate that a novel 3D cell culture platform has been successfully estab- lished, which can mimic the in vivo tumor microenvironment, and when combined with QDs-based mo- lecular imaging, it can help to better investigate the invasive behaviors of HCC cells.
文摘Some biological characteristics of human bone marrow mesenchymal stem cells (MSCs) cultured in vitro were observed. hMSCs were isolated from bone marrow and purified by density gradient centrifugation method, and then cultured in vitro. The proliferation and growth characteristics of hMSCs were observed in primary and passage culture. MSCs of passage 3 were examined for the purify by positive rate of CD29 and CD44 through flow cytometry. Human bone marrow MSCs showed active proliferation capacity in vitro. The purify of MSCs separated by our method was higher than 90 %. It was concluded that hMSCs have been successfully cultured and expanded effectively. It provided a foundation for further investigation and application of MSCs.
基金supported by development plan project during ‘‘the 12th Five Year Plan’’ Nation Science and Technology in rural area(No.2012AA10A506-04 and No.2013AA103005-04)Changchun City science and technology development program(No.2014174)Changchun City science and technology support program(No.2014NK002)
文摘In this study, using Taxus cuspidata as a raw material, we obtained stable high-yielding cell lines by subculturing and quantified paclitaxel content using ultra- sonic extraction combined with TLC-UV spectrophotom- etry. In single factor and multiple factors tests to optimize design and study the effects of elicitors, precursors, and metabolic inhibitors on paclitaxel production by Taxus cuspidata cells, paclitaxel production reached 4.32 mg/L when 100 μmol/L methyl jasmonate, 20 mg/L salicylic acid, 400 mg/L phenylalanine and 2 mg/L gibberellin (GA3) were added to the culture medium of suspension cells. When adding metabolic adjustment factors on the 7th day of culture, extra- and intracellular paclitaxel production was the highest at 4.855 mg/L, paclitaxel release rate was 10.48 %, fresh mass and paclitaxel production of cell increased, respectively, by 6.08 and 11.57 %. By controlling the anabolism of paclitaxel, paclitaxel yield was significantly improved.
文摘Aim: To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. Methods: Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Epithelial cell cultures were obtained from the caput, corpus and cauda epididymides. Enzymatic activity was measured in conditioned media by colorimetric methods in absence or presence of 1, 10 or 100 nrnoI.L^(-1) testosterone. The effect of 1 μmol.L^(-1) flutamide was also evaluated. Results: GPx activity was higher in cultures from corpus and cauda than caput epididymidis. The presence of different concentrations of testosterone increase enzyme activity in cell cultures from all epididymal regions. Addition of flutamide reverses the androgen dependent increase of GPx activity. Conclusion: GPx activity is secreted from human epididymal cells in a region dependent manner and is regulated by androgens.
文摘Desmin and vimentin are major components of intermediate filament proteins in cardiac myocytes. We developed a primary cell culture method for cardiac myocytes of axolotl embryos. Cardiac myocytes of embryonic stage 39 were cultured for 1-14 days. Myocytes showed spontaneous contractions (15-30 beats/min) after 48-72 hours in culture, round shape and large irregular projections. Desmin and vimentin were observed in the cultured myocytes by means of immunofluorescent staining in combination with immunofluorescent microscopy. Immunofluorescent staining of the cultured cardiac myocytes after different lengths of time in culture(3,6,9 days) showed that vimentin staining was stronger than desmin staining during the early stages of culture (3 days). The myocytes exhibited various forms of staining, including parallel lines and interconnected networks. Some lines showed regular striation; most of the myofibrils were arranged in parallel arrays along the cell's long axis. Both desmin and vimentin in the cell appeared to encirele the Z lines and to link myofibrils laterally at the Z lines.
基金supported by grants fromthe NIH/NIAID 5K25AI060884 to Lisa A.
文摘We describe a novel technique, low surface energy Gas Expansion Molding (GEM), to fabricate microbubble arrays in polydimethylsiloxane (PDMS) which are incorporated into parallel plate flow chambers and tested in cell sorting and microcell cuTture applications. This architecture confers several operational advantages that distinguish this technology approach from currently used methods. Herein we describe the GEM process and the parameters that are used to control microbubble formation and a Vacuum-Assisted Coating (VAC) process developed to selectively and spatially alter the PDMS surface chemistry in the wells and on the microchannel surface. We describe results from microflow image visualization studies conducted to investigate fluid streams above and within microbubble wells and conclude with a discussion of cell culture studies in PDMS.
文摘To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase(ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbedwith a structure resembling the loose connective tissue morphology in a novel 3 D culture system. We confirmed that the 3 D system using CellbedTMaccurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3 D-cultured tongue cancer cell lines than in 2 D cultures. Typically, under conventional 2 D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells.However, in the 3 D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3 D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3 D culture systems using Cellbed? are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.
基金the Clinic New & High Technology Program of Chinese PLA(Major Program),No.01MB039
文摘BACKGROUND: Previous studies have used many methods for in vitro Schwann cells (SCs) cultures and purification, such as single cell suspension and cytosine arabinoside. However, it has been difficult to obtain sufficient cellular density, and the procedures have been quite tedious. OBJECTIVE: To investigate the feasibility of culturing high-density SCs using fetal human dorsal root ganglion tissue explants. DESIGN, TIME AND SETTING: Cell culture and irnmunohistochernistry were performed at the Central Laboratory of Kunrning General Hospital of Chinese PLA between March 2001 and October 2008. MATERIALS: Culture media containing 10% fetal bovine serum, as well as 0.2% collagenase and 0.25% trypsin were purchased from Gibco, USA; mouse anti-human S-100 monoclonal antibody and goat anti-mouse IgG labeled with horseradish peroxidase were provided by Beijing Institute of Biological Products, China. METHODS: Primarily cultured SCs were dissociated from dorsal root ganglia of human aborted fetuses at 4 6 months pregnancy. Following removal of the dorsal root ganglion perineurium, the ganglia were dissected into tiny pieces and digested with 0.2% collagenase and 0.25% trypsin (volume ratio 1:1), then explanted and cultured. SC purification was performed with 5 rnL 10% fetal bovine serum added to the culture media, followed by differential adhesion. MAIN OUTCOME MEASURES: SCs morphology was observed under inverted phase contrast light microscopy. SC purity was evaluated according to percentage of S-100 immunostained cells. RESULTS: SCs were primarily cultured for 5 6 days and then subcultured for 4 5 passages. The highly enriched SC population reached 〉 95% purity and presented with normal morphology. CONCLUSION: A high purity of SCs was obtained with culture methods using human fetal dorsal root ganglion tissue explants.
文摘In vitro tissue culture of hard woody, endangered, medicinal plant Coscinium fenestratum is most challenging to plant tissue culturists. In the present study, petiole and leaf explants of Coscinium fenestratum were induced to form callus when cultured on vermicompost extract media along with coelomic fluid. Suspension medium was developed using vermicompost extract and coelomic fluid in 3:1 ratio. Phytochemical analysis of the alkaloid berberine was confirmed from callus, suspension cell culture and suspension medium by Thin Layer Chromatography and High Performance Liquid Chromatography. Vermicompost and its extracts with coelomic fluid have shown maximum (100 per cent) response of callus induction. Callus mass enlarged with increasing concentration of coelomic fluid and callus growth was assessed from the biomass. Incubation of culture tubes in dark supported callus development significantly. The Rf value of 0.36 confirmed the presence of berberine by Thin Layer Chromatography. Qualitative analysis confirmed the presence of alkaloid berberine with the retention time of 2.8 minutes similar to that of standard reference sample from Sigma chemicals, USA. The suspension medium turned deep yellow because of the release of the alkaloid. Vermicompost and its extracts along with coelomic fluid have shown the economical approach for micropropagation of economically and medicinally important plants.
文摘Plant cell culture technology is a technology that applies the research results of cell engineering to produce plant biological products at the cellular level.In recent years,the secondary metabolites of plants have attracted more and more attention.The use of plant cell culture technology is a fast and efficient method of producing secondary metabolites.
基金provided by grant from the National Science Centre in Poland (UMO-2013/11/B/NZ5/03106)
文摘This short review is to list pros and cons which are based on the literature and personal experience in cell culture studies related to possible commercial production of artificial meat as functional food. The general view of muscle composition and determinants of meat quality are shortly described. Principles of muscle cell propagation in culture and mutual relationships between different cell types present in this organ are briefly discussed. Additionally, the effects of some cytokines and growth factors for muscle cell growth and muscle tissue development are indicated. Finally, conclusion remarks related to detrimental consequences of meat production to natural environment as well as personal opinion of author on the prospects of artificial meat production are declared.
基金This work is supported by the National Natural Science Foundation of China(to Jungui Dai,No.30100230).
文摘Deacetoxy-13-oxo sinenxan A (1) was converted to 9a-hydroxy-13-oxo-2a, 5a, 10b-triacetoxy-4(20),11-taxadiene (2) and 10b-hydroxy-13-oxo-2a,5a,9a-triacetoxy- 4(20), 11- taxadiene (3) by Ginkgo cell suspension cultures in 45% and 15% yields, respectively.
基金supported by the name of National Key Research and Development Program of China(No.2021YFB3800900)the National Natural Science Foundation of China(Nos.51925305,51873208,1833010,51973217,52373161)Jilin Province Science and Technology Development Program(No.20200201075JC)。
文摘The development of large-scale cell cultivation and non-invasive cell harvesting is highly desired in various fields,including biological regeneration and pharmaceutical research.When using traditional microcarriers for cell culture,trypsinization is often necessary during cell collection,leading to partial cells damage.In this work,we developed a thermoresponsive glass microcarrier modified with poly(γ-propargyl-L-glutamate)(PPLG)and poly(N-isopropylacrylamide)(PNIPAM).We utilized these microcarriers for three-dimensional cell culture and enzyme-free cell harvesting,and the results indicated that the prepared microcarriers exhibited excellent non-invasive cell culture performance.
基金supported by grants from the National Key R&D Program of China(No.2018YFC1005002)the National Natural Science Foundation of China(Nos.82070482,81772007,21734003 and 51927805)+1 种基金the Shanghai Municipal Science and Technology Major Project(No.2017SHZDZX01)the Shanghai Municipal Education Commission(Innovation Program No.2017-01-07-00-07E00027)。
文摘There is no clear consensus regarding how cells respond to hydrostatic pressure. This is largely attributable to the high heterogeneity among cell types and the diverse custom-made devices used in previous studies. The aim of this work was to develop a facile device that could mimic various pressure environments and then delineate the cellular response to pressure stimulus. The device described here achieved both stable and periodic pressurization without oxygen deprivation. The biological utility of the device was assessed using human umbilical vein endothelial cells. We found more stereoscopic nuclear morphology and re-distribution of lamin A/C under high hydrostatic pressure compared to control cells. Mass spectrometry-based proteomics analysis showed significant changes in mitochondria-related pathways. Western blot analysis confirmed that high hydrostatic pressure induced a tendency toward mitochondrial fusion. Increased mitochondrial activity was observed as well. In conclusion, this device can be readily applied in biological research and extend our understanding of cellular mechano-sensation and the associated changes in mitochondrial behaviors.
基金This work is a result of the project FROnTHERA(NORTE-01-0145-FEDER-000023)supported by Norte Portugal Regional Operational Programme(NORTE 2020)+3 种基金under the PORTUGAL 2020 Partnership Agreement,through the European Regional Development Fund(ERDF)Portuguese Foundation for Science and Technology under the doctoral programme in Tissue Engineering,Regenerative Medicine and Stem Cells(PD/59/2013)(PD/BD/128087/2016)by the project Cells4_IDs(PTDC/BTM-SAL/28882/2017).
文摘Surface topographies of cell culture substrates can be used to generate in vitro cell culture environments similar to the in vivo cell niches. In vivo, the physical properties of the extracellular matrix (ECM), such as its topography, provide physical cues that play an important role in modulating cell function. Mimicking these properties remains a challenge to provide in vitro realistic environments for cells. Artificially generated substrates’ topographies were used extensively to explore this important surface cue. More recently, the replication of natural surface topographies has been enabling to exploration of characteristics such as hierarchy and size scales relevant for cells as advanced biomimetic substrates. These substrates offer more realistic and mimetic environments regarding the topographies found in vivo. This review will highlight the use of natural surface topographies as a template to generate substrates for in-vitro cell culture. This review starts with an analysis of the main cell functions that can be regulated by the substrate’s surface topography through cell-substrate interactions. Then, we will discuss research works wherein substrates for cell biology decorated with natural surface topographies were used and investigated regarding their influence on cellular performance. At the end of this review, we will highlight the advantages and challenges of the use of natural surface topographies as a template for the generation of advanced substrates for cell culture.
基金Supported by the Postdoctoral Science Foundation of China,No.2005038300and the National Natural Science Foundation of China,No.30671028.
文摘BACKGROUND Efficient extraction of nucleic acids and proteins(ENAP)from cells is a prerequisite for precise annotation of gene function,and has become laboratory routine for revealing the mysteries of life.However,cell samples are often from different culture dishes,resulting in inevitable experimental errors and sometimes poor repeatability.AIM To explore a method to improve the efficiency of ENAP,minimizing errors in ENAP processes,enhancing the reliability and repeatability of subsequent experimental results.METHODS A protocol for the sequential isolation of RNA,DNA,and proteins from the same cultured HepG2 cells using RNAzol reagent is presented here.The first step involves culturing HepG2 cells to the exponential phase,followed by the sequential isolation of RNA,DNA,and proteins from the same cultured cells in the second step.The yield of nucleic acids and proteins is detected in the third step,and their purity and integrity are verified in the last step.RESULTS The procedure takes as few as 3-4 d from the start to quality verification and is highly efficient.In contrast to the existing kits and reagents,which are primarily based on independent isolation,this RNAzol reagent-based method is characterized by the sequential isolation of RNA,DNA,and proteins from the same cells,and therefore saves time,and has low cost and high efficiency.CONCLUSION The RNA,DNA,and proteins isolated using this method can be used for reverse transcription-polymerase chain reaction,polymerase chain reaction,and western blotting,respectively.