Fish Chromosome Preparation: Air-Dried Displays of Cultured Ovarian Cells in Two Killifishes (Fundulus)

1970 ◽  
Vol 27 (1) ◽  
pp. 158-161 ◽  
Author(s):  
T. R. Chen
Keyword(s):  
Cell Culture ◽  
Cell Suspension ◽  
Ovarian Tissue ◽  
Single Cell Suspension ◽  
High Quality ◽  
Karyotypic Analysis ◽  
Ovarian Cells ◽  
Short Period ◽  
General Similarity

A relatively simple and reliable in vitro methodology for fish chromosome study is described. A single cell suspension obtained by trypsinizing ovarian tissue was cultured for 5–7 days at 31 C. The cells were treated with colchicine and hypotonic solutions then fixed in acetic-alcohol fixative. Air-dried preparations were made from these cells and were stained with aceto-orcein.Numerous metaphase plates unmarred by overlapping chromosomes were recovered and high quality karyograms were available for detailed karyotypic analysis. The general similarity of squash and air-dried karyotypes suggests that short-period cell culture preserves natural karyotypes.

scholarly journals The Influence of the Grapevine Bacterial and Fungal Endophytes on Biomass Accumulation and Stilbene Production by the In Vitro Cultivated Cells of Vitis amurensis Rupr.

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1276
Author(s):  
Olga A. Aleynova ◽  
Andrey R. Suprun ◽  
Nikolay N. Nityagovsky ◽  
Alexandra S. Dubrovina ◽  
Konstantin V. Kiselev
Keyword(s):  
Cell Culture ◽  
Cell Suspension ◽  
Endophytic Fungi ◽  
Endophytic Bacteria ◽  
Fungal Endophytes ◽  
Biomass Accumulation ◽  
Vitis Amurensis ◽  
Grape Cell ◽  
Stilbene Biosynthesis

Plant endophytes are known to alter the profile of secondary metabolites in plant hosts. In this study, we identified the main bacterial and fungal representatives of the wild grape Vitis amurensis Rupr. microbiome and investigated a cocultivation effect of the 14 endophytes and the V. amurensis cell suspension on biomass accumulation and stilbene biosynthesis. The cocultivation of the V. amurensis cell culture with the bacteria Agrobacterium sp., Bacillus sp., and Curtobacterium sp. for 2 weeks did not significantly affect the accumulation of cell culture fresh biomass. However, it was significantly inhibited by the bacteria Erwinia sp., Pantoea sp., Pseudomonas sp., and Xanthomonas sp. and fungi Alternaria sp., Biscogniauxia sp., Cladosporium sp., Didymella sp. 2, and Fusarium sp. Cocultivation of the grapevine cell suspension with the fungi Didymella sp. 1 and Trichoderma sp. resulted in cell death. The addition of endophytic bacteria increased the total stilbene content by 2.2–5.3 times, while the addition of endophytic fungi was more effective in inducing stilbene accumulation by 2.6–16.3 times. The highest content of stilbenes in the grapevine cells cocultured with endophytic fungi was 13.63 and 13.76 mg/g of the cell dry weight (DW) after cultivation with Biscogniauxia sp. and Didymella sp. 2, respectively. The highest content of stilbenes in the grapevine cells cocultured with endophytic bacteria was 4.49 mg/g DW after cultivation with Xanthomonas sp. The increase in stilbene production was due to a significant activation of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) gene expression. We also analyzed the sensitivity of the selected endophytes to eight antibiotics, fluconazole, and trans-resveratrol. The endophytic bacteria were sensitive to gentamicin and kanamycin, while all selected fungal strains were resistant to fluconazole with the exception of Cladosporium sp. All endophytes were tolerant of trans-resveratrol. This study showed that grape endophytes stimulate the production of stilbenes in grape cell suspension, which could further contribute to the generation of a new stimulator of stilbene biosynthesis in grapevine or grape cell cultures.

scholarly journals THE PROLONGED GROWTH AND SURVIVAL OF OVARIAN TISSUE OF THE PROMETHEA MOTH (CALLOSAMIA PROMETHEA) IN VITRO

1958 ◽  
Vol 41 (5) ◽  
pp. 1027-1034 ◽  
Author(s):  
T. D. C. Grace
Keyword(s):  
Cell Culture ◽  
Cell Migration ◽  
Cell Cultures ◽  
Ovarian Tissue ◽  
Explant Culture ◽  
Growth And Survival ◽  
Live Tissue ◽  
Cell Divisions ◽  
Callosamia Promethea

1. The ovarian tissues from diapausing pupae of the promethea moth (Callosamia promethea) have survived and grown for 186 days under in vitro conditions. There was continual cell migration and multiplication for a period of 53 days, followed by a period of 47 days during which no cells migrated from the tissues. Between the 100th and 105th days after setting up the cultures, cell migration was resumed, and by the 111th day 250 cells were present in the medium. A few cell divisions were observed between the 126th and 136th days. After the tissues were subcultured on the 140th day, the explant culture continued to survive, but the cell culture died 3 days later. 2. The tissues were subcultured a total of 6 times during the 186 days. By the introduction of a piece of live tissue into the cell cultures, the growth and survival of the cells were increased from 8 days to about 20 days. 3. It is possible that the tissues had become adapted to the medium during their long survival, as the cells which migrated from them after 100 days showed considerably longer survival than those in earlier cultures.

scholarly journals Identification and quantitative determination of pinoresinol in Taxus ×media Rehder needles, cell suspension and shoot cultures

2015 ◽  
Vol 84 (1) ◽  
pp. 125-132 ◽  
Author(s):  
Paulina Mistrzak ◽  
Hanna Celejewska-Marciniak ◽  
Wojciech J. Szypuła ◽  
Olga Olszowska ◽  
Anna K. Kiss
Keyword(s):  
Cell Culture ◽  
Suspension Culture ◽  
Quantitative Determination ◽  
Cell Suspension ◽  
Dry Weight ◽  
Shoot Cultures ◽  
Taxus Media ◽  
In Vitro Shoot Cultures

The aim of our study was to investigate the presence and quantitative contents of lignans in the tissues of <em>Taxus</em> ×<em>media</em>. The presence of the lignans: pinoresinol, matairesinol and secoisolariciresinol was assessed in needles, shoots cultures and suspension culture. Pinoresinol was the only lignan found in the tissue of <em>T.</em> ×<em>media</em>. The total pinoresinol content in the needles and in the shoots was 1.24 mg/g dry weight (dw) and 0.69 mg/g dw, respectively. Most of the pinoresinol identified was appeared glycosidically bound. In needles, the amount of glycosidically bound pinoresinol (0.81 mg/g dw) was about twice as high as that of free pinoresinol (0.43 mg/g dw). The content of free and glycosidically bound pinoresinol showed the level of 0.18 mg/g dw and 0.51 mg/g dw, respectively in the in vitro shoot cultures. In the cell culture, no pinoresinol was found.

scholarly journals Techniques for RNA extraction from cells cultured in starPEG–heparin hydrogels

Open Biology ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 200388
Author(s):  
Anna Jaeschke ◽  
Nicholas R. Harvey ◽  
Mikhail Tsurkan ◽  
Carsten Werner ◽  
Lyn R. Griffiths ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Biomechanical Properties ◽  
High Quality ◽  
Gene Expression Analyses

Three-dimensional (3D) cell culture models that provide a biologically relevant microenvironment are imperative to investigate cell–cell and cell–matrix interactions in vitro . Semi-synthetic star-shaped poly(ethylene glycol) (starPEG)–heparin hydrogels are widely used for 3D cell culture due to their highly tuneable biochemical and biomechanical properties. Changes in gene expression levels are commonly used as a measure of cellular responses. However, the isolation of high-quality RNA presents a challenge as contamination of the RNA with hydrogel residue, such as polymer or glycosaminoglycan fragments, can impact template quality and quantity, limiting effective gene expression analyses. Here, we compare two protocols for the extraction of high-quality RNA from starPEG–heparin hydrogels and assess three subsequent purification techniques. Removal of hydrogel residue by centrifugation was found to be essential for obtaining high-quality RNA in both isolation methods. However, purification of the RNA did not result in further improvements in RNA quality. Furthermore, we show the suitability of the extracted RNA for cDNA synthesis of three endogenous control genes confirmed via quantitative polymerase chain reaction (qPCR). The methods and techniques shown can be tailored for other hydrogel models based on natural or semi-synthetic materials to provide robust templates for all gene expression analyses.

Initial steps in reconstruction of the human ovary: survival of pre-antral stage follicles in a decellularized human ovarian scaffold

2019 ◽  
Vol 34 (8) ◽  
pp. 1523-1535 ◽  
Author(s):  
S E Pors ◽  
M Ramløse ◽  
D Nikiforov ◽  
K Lundsgaard ◽  
J Cheng ◽  
...  
Keyword(s):  
European Union ◽  
Ovarian Tissue ◽  
The European Union ◽  
Antral Follicles ◽  
Human Ovarian Tissue ◽  
Ovarian Cells ◽  
Pas Staining ◽  
Cryopreserved Ovarian Tissue ◽  
Decellularized Scaffolds

Abstract STUDY QUESTION Can a reconstructed ovary using decellularized human ovarian tissue (DCT) support survival of pre-antral stage follicles? SUMMARY ANSWER We have demonstrated an effective protocol for decellularization of human ovarian tissues and successful recellularization with isolated human ovarian cells and pre-antral follicles. WHAT IS KNOWN ALREADY Survivors of leukemia or ovarian cancer run a risk of reintroducing malignancy when cryopreserved ovarian tissue is transplanted to restore fertility. A reconstructed ovary free of malignant cells could provide a safe alternative. Decellularization of ovarian tissue removes all cells from the extracellular matrix (ECM) including possible malignancies and leaves behind a physiological scaffold. The ECM offers the complex milieu that facilitates the necessary interaction between ovarian follicles and their surroundings to ensure their growth and development. Previous studies have shown that decellularized bovine ovarian scaffolds supported murine follicle growth and restoration of ovarian function in ovariectomized mice. STUDY DESIGN, SIZE, DURATION Optimizing a decellularization protocol for human ovarian tissues and testing biofunctionality of the decellularized scaffolds in vitro and in vivo by reseeding with both murine and human pre-antral follicles and ovarian cells. PARTICIPANTS/MATERIALS, SETTING, METHODS Donated human ovarian tissue and isolated pre-antral follicles were obtained from women undergoing ovarian tissue cryopreservation for fertility preservation. Ovarian cortical and medullary tissues were decellularized using 0.1% sodium dodecyl sulfate (SDS) for 3, 6, 18 and 24 hours followed by 24 hours of 1 mg/mL DNase treatment and washing. Decellularization of ovarian tissues and preservation of ECM were characterized by morphological evaluation using Periodic Acid–Schiff (PAS) staining, DNA quantification, histochemical quantification of collagen content and immunofluorescence analysis for collagen IA, laminin, fibronectin and DNA. Human ovarian stromal cells and isolated human pre-antral follicles were reseeded on the DCT and cultured in vitro. Isolated murine (N = 241) and human (N = 20) pre-antral follicles were reseeded on decellularized scaffolds and grafted subcutaneously to immunodeficient mice for 3 weeks. MAIN RESULTS AND THE ROLE OF CHANCE Incubation in 0.1% SDS for 18–24 hours adequately decellularized both human ovarian medullary and cortical tissue by eliminating all cells and leaving the ECM intact. DNA content in DCT was decreased by >90% compared to native tissue samples. Histological examination using PAS staining confirmed that the cortical and medullary tissues were completely decellularized, and no visible nuclear material was found within the decellularized sections. DCT also stained positive for collagen I and collagen quantities in DCT constituted 88–98% of the individual baselines for native samples. Human ovarian stroma cells were able to recellularize the DCT and isolated human pre-antral follicles remained viable in co-culture. Xenotransplantation of DCT reseeded with human or murine pre-antral follicles showed, that the DCT was able to support survival of human follicles and growth of murine follicles, of which 39% grew to antral stages. The follicular recovery rates after three weeks grafting were low but similar for both human (25%) and murine follicles (21%). LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Further studies are needed to increase recovery and survival of the reseeded follicles. Longer grafting periods should be evaluated to determine the developmental potential of human follicles. Survival of the follicles might be impaired by the lack of stroma cells. WIDER IMPLICATIONS OF THE FINDINGS This is the first time that isolated human follicles have survived in a decellularized human scaffold. Therefore, this proof-of-concept could be a potential new strategy to eliminate the risk of malignant cell re-occurrence in former cancer patients having cryopreserved ovarian tissue transplanted for fertility restoration. STUDY FUNDING/COMPETING INTEREST(S) This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. Furthermore, Project ITN REP-BIOTECH 675526 funded by the European Union, European Joint Doctorate in Biology and Technology of the Reproductive Health, the Research Pools of Rigshospitalet, the Danish Cancer Foundation and Dagmar Marshalls Foundation are thanked for having funded this study. The funders had no role in the study design, data collection and interpretation, or in the decision to submit the work for publication.

Formation of hair follicles from a single-cell suspension of embryonic rat skin by a two-step procedure in vitro

1991 ◽  
Vol 266 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Setsunosuke Ihara ◽  
Mariko Watanabe ◽  
Eriko Nagao ◽  
Nobuyuki Shioya
Keyword(s):  
Cell Suspension ◽  
Single Cell ◽  
Hair Follicles ◽  
Single Cell Suspension ◽  
Rat Skin ◽  
Step Procedure

Evaluation of the developmental potential of metaphase I oocytes from stimulated intracytoplasmic sperm injection cycles

2011 ◽  
Vol 23 (3) ◽  
pp. 433 ◽  
Author(s):  
Mei Li ◽  
Yuan Li ◽  
Shui-Ying Ma ◽  
Huai-Liang Feng ◽  
Hui-Jun Yang ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Control Group ◽  
High Quality ◽  
Developmental Potential ◽  
Short Period ◽  
Group A ◽  
Group B ◽  
Group D ◽  
Metaphase I

The objective of the present study was to evaluate the developmental potential and clinical application value of metaphase I (MI) oocytes obtained from stimulated intracytoplasmic sperm injection (ICSI) cycles. ICSI was performed on MI oocytes immediately after denudation (Group A), or on in vitro-matured (IVM) oocytes following culture; oocytes in culture were further divided into two groups, being cultured for either 3–5 h (Group B) or 24–28 h (Group C). Metaphase II oocytes from the same cycle(s) isolated for ICSI served as the control group (Group D). The rates of normal fertilisation, cleavage and high-quality embryos were compared among the four groups. High-quality embryos were transferred whenever possible, and pregnancy rates were evaluated. Results showed that normal fertilisation rates for Groups B, C and D were significantly higher than that of Group A (68.6%, 57.8%, 74.5% and 30.1%, respectively; P < 0.01). The rate of high-quality embryos in Group B was comparable with Group D; the rate for Group C was significantly lower than that of the other groups (P < 0.05). Two clinical pregnancies were achieved after transfer of embryos from IVM oocytes. In vitro maturation of MI oocytes for a short period of time may increase the number of available embryos; however, overnight in vitro culture of MI oocytes did not improve results.

scholarly journals Protoplast Isolation, Fusion, Culture and Transformation in the Woody Plant Jasminum spp.

Agriculture ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 699
Author(s):  
Mohamed A. A. Ahmed ◽  
Miao Miao ◽  
Emmanouil D. Pratsinakis ◽  
Hongliang Zhang ◽  
Wei Wang ◽  
...  
Keyword(s):  
Cell Culture ◽  
Protoplast Fusion ◽  
Fusion Rate ◽  
Protoplast Isolation ◽  
Stem Explants ◽  
Peg 4000 ◽  
Short Period ◽  
Potential Applications ◽  
Exogenous Plasmid

Plant protoplasts are significant for plant cell culture, somatic cell fusion, genetics, and breeding studies. In addition, in vitro plant regeneration has great importance for developmental biology, manifesting potential applications in agriculture and biotechnology. In this regard, we present a well-established protocol regarding protoplast isolation, cell culture and protoplast fusion of Jasminum spp. In particular, different tissues of Jasminum samab L. and Jasminum mesnyi were employed for protoplast isolation, and stem explants provided a high callus induction rate in a short period of time. The best source for protoplast isolation was calli tissues. The optimized isolation protocol consisted of digesting callus in an enzyme solution containing 0.4 M mannitol, 0.2 M MES, 1 M CaCl2, 0.2 M KCL and 1 M NaH2PO4, 1.5% Cellulases onozuka R-10, 0.4% Macerozyme R-10 and 0.8% Pectinase for 4 h at 26 °C in the dark, providing a yield of 23.8 × 106 Protoplast/gFW with 88% viability. Protoplasts were cultured both in liquid and agarose medium under optimum conditions, leading to microcalli formation after eight weeks. A 5% protoplast-fusion rate can be achieved when cultured in 40% (w/v) PEG-MW6000 supplemented with 0.1 M CaCl2, 0.1 M sorbitol and 1 M Tris for 20 min. Furthermore, we developed an efficient PEG-mediated transformation protocol for jasmine protoplasts. The best results regarding protoplast transformation were obtained when the protoplast concentration was 4 × 105 cells/mL and the exogenous plasmid DNA added had a concentration of 10 µg DNA/100 µL protoplast solution, followed by the application of 40% PEG-4000 for 10 min.

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