Research on Electric Impedance Spectroscopy of Living Cell Suspensions by a Chip with Microelectrodes

A microfabricated electrical impedance spectroscopy (EIS) chip with microelectrodes was developed.The substrate and the electrodes of the chip were made of glass and gold,respectively.The experimental results demonstrated that the EIS-chip could distinguish different solutions (physiological saline,culture medium,living cell suspension etc.) by scanning from 10Hz to 45kHz.A 6-element circuit model was used for fitting the real part and the imaginary part admittance curves of the living cell suspension.An actual circuit was also built and tested to verify the 6-element circuit model proposed.The micro-EIS chip has several advantages including the use of small sample volumes,high resolution and ease of operation.It shows good application prospects in the areas of cellular electrophysioiogy,drug screening and bio-sensors etc.

Establishment of cell suspension line of Populus tomentosa Carr

Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L^-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subcultured into a MS liquid medium supplemented with 1.5 mg·L^-1 2,4-D. The best subculture medium was MS ＋ 0.8 mg＇L-1 2,4-D ＋ 30 g·L^-1 sucrose with a subculture cycle of seven days.

Somatic Embryogenesis from Cell Suspension Cultures of Aspen Clone

Suspension cultures initiated from callus derived from petiole explants of aspen hybrid （Populus tremuloides×P. tremula） produced somatic embryos. Callus was induced on a MS medium supplemented with 5mg·L^-1 2,4-D and 0.05mg·L^-1 zeatin under light conditions. Embryogenic calli were obtained when a subsequent subculture of calli was suspended in the same basal medium with 10mg·L^-1 2,4-D. The highest number of globular embryos were induced from embryogenic calli by cell suspension culture in a MS liquid medium supplemented with 10mg·L^-1 2,4-D. Genotype and 2,4-D concentration were vital to the induction of embryogenic calli producing competent cells. Embryogenic calli for each genotype were heterogeneous. Green calli with gel-like consistency could yield more competent cells than light yellow embryogenic calli. However, some globular embryos broke into slices and some developed abnormally after one month of culture under the same or other hormonal conditions.

Study on Cell Suspension Culture of Floribunda Rose

Friable callus was induced when immature seeds of floribunda rose were inoculated on MS medium supplemented with 2,4-D 3.0 mg.L^-1. When transfered onto subculture media, friable callus developed into embryogenic callus, which was used to establish cell suspension lines. Cell suspensions had to be subcultured at a interval of 4-5 days at the first several culture cycles. The best subculturing cycle for the stable cell suspensions was 8-10 days. The best inoculum quantity was 1 mL PCV（Packed Cell Volume） per 40 mL culture fluid.

Studies on Cell Suspension Culture of Ricinus communis L.

Ricinus communis L. is a new copper hyperaccumulator growing on Tonglushan copper mine in Hubei Province, China. This study aimed to establish a suspension cell line of R. communis L. with stable and rapid growth for further screening of heavy metal-resistant R. communis L. cells and breeding of hyperaccu- mulators. In this study, cell suspension culture conditions were optimized by using orthogonal experimental design with previously induced R. communis L. embyre- genic calluses as experimental materials, to establish the suspension cell line of R. communis L. Under the optimal conditions, growth curves of suspension cells and changes in pH of culture liquid were determined. The results showed that the optimal culture conditions for R. conmmnis L. suspension cell line were ： MS ＋ O. 5 rag/I, 6-BA ＋ O. 2 mg/L NAA ＋ 50 mg/L sucrose ＋ 350 mg/L casein hydrolysate as basic medium, with callus inoculation amount of 2.5 g/50 ml, dark culture at （26 ±2） ℃ with shaking at 110 r/min. Under these conditions, increment of fresh weight and dry weight ofR. commun/s L. suspension cells reached the maximum of 4.56 g/（50 ml 14 d） and 0.49 g/（50 ml 16 d）, respectively. Growth curves of R. communis L. suspension cells were basically in ＂S＂ shape. Each culture cycle lasted 16 d, and the rapid growth stage was from the 6th d to the 14th d. In a culture cycle, pH of the culture liquid declined first and then increased to the maximum and stabilized gradually.

Cell replacement with stem cell-derived retinal ganglion cells from different protocols

Glaucoma,characterized by a degenerative loss of retinal ganglion cells,is the second leading cause of blindness worldwide.There is currently no cure for vision loss in glaucoma because retinal ganglion cells do not regenerate and are not replaced after injury.Human stem cell-derived retinal ganglion cell transplant is a potential therapeutic strategy for retinal ganglion cell degenerative diseases.In this review,we first discuss a 2D protocol for retinal ganglion cell differentiation from human stem cell culture,including a rapid protocol that can generate retinal ganglion cells in less than two weeks and focus on their transplantation outcomes.Next,we discuss using 3D retinal organoids for retinal ganglion cell transplantation,comparing cell suspensions and clusters.This review provides insight into current knowledge on human stem cell-derived retinal ganglion cell differentiation and transplantation,with an impact on the field of regenerative medicine and especially retinal ganglion cell degenerative diseases such as glaucoma and other optic neuropathies.

Acaricidal activities of whole cell suspension,cell-free supernatant,and crude cell extract of Xenorhabdus stokiae against mushroom mite (Luciaphorus sp.)

Xenorhabdus bacterium has been used as a biological control agent against Luciaphorus sp.,a mushroom mite endemic in Thailand.To develop an effective formulation of Xenorhabdus stokiae,treatments using different parts of X.stokiae isolate PB09 culture,including whole cell suspension,cell-free supernatant,and crude cell extract,were performed.The results show that different parts of X.stokiae isolate PB09 culture could induce variable effects on mite mortality and fecundity.Application with cell-free supernatant of X.stokiae culture resulted in both the highest mite mortality rate [(89.00±3.60)%] and the lowest mite fecundity [(41.33±23.69) eggs/gravid female].Whole cell suspen-sion of X.stokiae isolate PB09 culture was found to be slightly less effective than its cell-free supernatant,suggesting that X.stokiae was more likely to release its metabolites with acaricidal activities to the surrounding culture media.Crude cell extract of X.stokiae was not effective against mites.Cell-free supernatant of X.stokiae isolate PB09 was the most effective biological control agent and it could be conveniently used in future formulations instead of live bacteria.

Influences of Plant Growth Regulators， Basal Media and Carbohydrate Levels on Cell Suspension Culture of Panax ginseng

A cell suspension culture of Panax ginseng which may be continuously subcultured has been established. Embryogenic callus derived from clutured young leaves was used to initiate the culture.Plant growth regulators, basal medium formula and carbohydrate levels were examined to determine their various effects on suspension culture cell growth and development. The best selection of plant growth regulator, hasal medium and carbohydrate level is 2 mg / L 2,4-D: 0.5 mg / L KT,MS and 3% sucrose respectively.

Cell Suspension Culture of Neem Tree

The establishment of suspension culture system for neem (Azadirachta indica A. Juss) cells and the suspension culture condition was studied. It shows that the neem cell suspension culture system was best in B5 liquid medium, 2.0~4.0mg/L NAA with direct spill method. Based on the integrated analysis of cell biomass, Azadirachtin content and productivity, the optimum culture conditions were B5 liquid medium, 2.0-4.0 mg/L NAA, 3% sucrose at 25 ℃. The optimum rotating speed of the shaker and broth content d...

Ce^(4+)-Induced Apoptosis of Taxus cuspidata Cells in Suspension Culture

The standard detection hallmarks of apoptosis of Taxus cuspidata cells in suspension culture with Ce 4+ were studied. The condensation and margination of chromatin were observed under the electron microscopy. DNA fragmentation ranged 'DNA ladder' on agarose gel electrophoresis. TdT mediated dUTP nick end labeling (TUNEL) analysis of the cells reveals that the nuclear DNA strand breaks can be identified by labeling free 3′ OH termini. These results suggest that Ce 4+ can induce apoptosis of Taxus cuspidata cells and also indicate that there is a certain relationship between apoptosis and secondary metabolite product Taxol.

Qu-2,a robust poplar suspension cell line for molecular biology

Populus spp.have long been used as model woody plant species for molecular biology research.However,tissues of poplar are often recalcitrant to experimental procedures for molecular studies.We generated a hormone autotrophic poplar suspension cell line from a hybrid of Populus alba×P.berolinensis‘Yinzhong’,named Qu-2.Qu-2 cells are suitable as a model biological system for studying woody plants.Qu-2 cells have many advantages over suspension cell lines derived so far from any other woody plants.Qu-2 cells are very easy to cultivate and can grow on several common plant culture media without the addition of any plant hormone.They show exceptionally high growth rates,reaching an approximately 150-fold increase in biomass after one week of culturing.Another important unique characteristic of Qu-2 cells is that they can be cryopreserved and readily reactivated.Qu-2 cells are suitable for molecular manipulations such as protoplast production,transient transformation,and RNA-seq analysis.Therefore,Qu-2 cells have the great potential to be an excellent model cell line in tree molecular biological research,ranging from physiology to gene function.The Qu-2 cells will be made available to the plant community for research.

Plant regeneration of protoplasts isolated from suspension cells derived from leaf blale of Oryza sative

We used the leaf blade of rice (cultivars were Nonghu 6, Sugeng 2, Huyou 2 and Hanfeng) as initial material for protoplast culture, and a great number of regenerated plants were obtained. Rice seeds were sterilized and germinated. The immature leaves were cut into 3-5 mm pieces when the third or forth leaf appeared. Leaf pieces were inoculated on MS medium with 2,4-D 4 mg/1, NAA 2mg/1 and IAA Img/1. After 2 wk culture, calli were induced and subcultured once or twice for multiplication. 3-5 g calli were transferred to the modified MS liquid medium with 2,4-D 2 mg/1 and KT 0.5mg/1 for suspension culture. Embryogenic cell suspension was established after 2 mo culture. The effect of the growth period of suspension cells on the

Coscinium fenestratum: Callus and Suspension Cell Culture of the Endangered Medicinal Plant Using Vermicompost Extract and Coelomic Fluid as Plant Tissue Culture Media

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.

Effect of La,Ce on Taxus Cuspidata Cell Growth,Biosynthesis and Release of Taxol

The effect of La, Ce was firstly tested on growth of the Taxus(T.) cuspidata cell suspensions, biosynthesis and release of taxol. The results indicate that the growth pattern of T.cuspidata cells is altered significantly by adding high concentration of rare earth in the medium. The lag and exponential phases of cell growth are shortened, the stationary phase disappears and the biomass fluctuates periodically during the decline phase. The rare earth compounds added in the exponential phase obviously increase the taxol biosyntheis and release yields of T.cuspidata cells, and the supplement of carbon source in the medium containing rare earth is also favorable to taxol biosynthesis.

Synaptic development in the injured spinal cord cavity following co-transplantation of fetal spinal cord cells and autologous activated Schwann cells

Transplantation of activated transgenic Schwann cells or a fetal spinal cord cell suspension has been widely used to treat spinal cord injury. However, little is known regarding the effects of co-transplantation. In the present study, autologous Schwann cells in combination with a fetal spinal cord cell suspension were transplanted into adult Wistar rats with spinal cord injury, and newly generated axonal connections were observed ultrastructurally. Transmission electron microscopic observations showed that the neuroblast first presented cytoplasmic processes, followed by pre- and postsynaptic membranes with low electron density forming a dense projection. The number and types of synaptic vesicles were increased. Synaptic connections developed from single cell body-dendritic synapses into multiple cell body-dendritic and dendrite-dendritic synapses. In addition, the cell organs of the transplanted neuroblast, oligodendroblast and astroblast matured gradually. The blood-brain barrier appeared subsequently. Moreover, neurofilament, histamine, calcitonin-gene-related peptides, and glial fibrillary acidic protein positive fibers were observed in the transplant region. These findings demonstrate that fetal spinal cord cells in the presence of autologous activated Schwann cells can develop into mature synapses in the cavity of injured spinal cords, suggesting the possibility of information exchange through the reconstructed synapse between fetal spinal cord cells and the host.

Biotransformation of 14-Deacetoxy-13-oxo sinenxan A by Ginkgo Cell 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.

Ultrastructural and Physiological Characterization of YELP, a Novel, Yellow, Chlorophyll-Deficient Cell Mutant Line That Develops Etioplast-Like Plastids in the Light

In this work, we present an ultrastructural and physiological description of a novel chlorophyll-deficient, yellow cell line of the grass Bouteloua gracilis that develops etioplast-like plastids in presence of light (YELP). These mutant cells were compared to the parental, wild-type, highly chlorophyllous cells from which they were isolated. Growth analysis, based on fresh and dry weights, indicated that YELP accumulates biomass at a slower rate than the parental, green cells. Besides, YELP developed very low levels of photosynthetic pigments, reaching only 9.3% and 38.4% of chlorophyll a and chlorophyll b, respectively, developed by the wild-type cells. Likewise, the accessory pigments, carotenes and xanthophylls, were only synthesized at 8.0% and 5.4%, respectively, of the levels reached by the green cells. Electron microscopy revealed remarkable differences in plastid ultrastructure between the wild-type and mutant cells. Plastids of YELP were heterogeneous and smaller than those found in wild-type cells. YELP plastids were abnormal with poorly developed membrane systems that prevented the accumulation of chlorophyll and accessory pigments in the mutant cell line. We expect this novel, mutant cell line will provide new tools for studying plastid development and differentiation.

Alcohol Dehydrogenase Activity in Cultured Cells from Different Tobacco (Nicotiana tabacum L.) Varieties

The activity of alcohol dehydrogenase (ADH) in cultured cells of various tobacco was determined. It was found that significant differences existed in cells of different varieties cultured under normal conditions and as well after treated with exogenous ethanol. The ADH activity had positive relation with the ability of the cells to catabolize exogenous ethanol, indicating that the main function of the ADH in tobacco cells was in the direction of converting ethanol to acetaldehyde.

A cyclic effect of cAMP and calcium signaling contributes to jujube growth and development

3´,5´-Cyclic adenosine monophosphate(cAMP)is an important metabolite that is specifically enriched in jujube.However,the effect of cAMP on jujube cellular responses has not been comprehensively studied.Here,we established jujube cell suspension cultures and investigated the calcium influx in response to cAMP treatment through protoplast isolation and fluorescence intensity.Firstly,cAMP treatment could promote jujube growth and increase the content of endogenous cAMP.Using transcriptome analysis with transgenic Arabidopsis plants overexpressing adenylate cyclase(ZjAC)as a positive control,we identified 60 calcium-related differential expressed genes(DEGs)that contributed to the calcium signaling and inter-or intra-cellular responses.Pharmacological treatments such as cAMP and the calcium ionophore A23187 could induce ZjAC expression,the accumulation of cAMP and calcium influx in jujube cells,while ethylene glycol tetraacetic acid(EGTA)or bithionol treatment inhibited these changes.Moreover,the calcium channels and transporters in calcium influx,such as the ZjCNGC2 channel and the mitogen activated protein(MAP)kinase pathway,could be activated by cAMP treatment.In summary,our findings demonstrated that cAMP biosynthesis is dependent on calcium influx and the amplifying effect between calcium and cAMP may be involved in intracellular signal induction,which might contribute to the growth and development of jujube.

Efficient gene transfection of suspension cells by highly branched poly(β-amino ester)

Suspension cells play a crucial role in many biological processes. However, compared to adherent cells, it is particularly challenging to introduce exogenous genes into suspension cells to regulate their biological functions with non-viral gene vectors, mainly due to the low cellular uptake and endosomal escape of polyplexes. Herein, to improve the interactions of polyplexes with cellular membranes, we design and synthesize highly branched poly(β-amino ester)(HPAE) via an “A2 + B4 + C2” Michael addition strategy.Results show that branching significantly increases DNA condensation of HPAE, cellular uptake and endosomal escape of HPAE/DNA polyplexes. In mast cells(MCs), HPAE exhibits up to 80-fold higher gene transfection efficiency compared to the corresponding linear poly(β-amino ester)(LPAE) and the leading commercial gene transfection reagents PEI25k, jetPEI, and Lipofectamine 3000, without causing obvious cytotoxicity. Our study establishes a reliable non-viral platform for efficient gene transfection of suspension cells.