25 CRYOPRESERVATION AND IN VITRO CULTURE AFFECT HISTONE ACETYLATION LEVELS OF BOVINE FIBROBLASTS

2008 ◽  
Vol 20 (1) ◽  
pp. 93 ◽  
Author(s):  
L. Chacón ◽  
M. C. Gómez ◽  
J. A. Jenkins ◽  
G. Wirtu ◽  
B. L. Dresser ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Cell Line ◽  
Cell Lines ◽  
Chromatin Structure ◽  
Donor Cell ◽  
Epigenetic Changes ◽  
Blastocyst Development ◽  
Histone 3 ◽  
Donor Cells

Long-term culture of bovine fibroblasts increases histone acetylation levels (Enright et al. 2003 Biol. Reprod. 69, 1525–1530), and blastocyst development and pregnancy rates are enhanced by using donor cells having high levels of histone acetylation (Yang et al. 2007 Reproduction 133, 219–230; Yang et al. 2006 Biol. Reprod. 76, 36–42). When histone acetylation levels are increased in amino acid residues, the nucleosome is less tightly bound to DNA and, as a consequence, relaxation of the chromatin structure occurs (Zlatanova et al. 2000 FASEB J. 14, 1697–1704). Exposure of the donor cell with relaxed chromatin structure to oocyte cytoplasm factors after nuclear transfer (NT) may be effective for nuclear reprogramming (Tóth et al. 2004 J. Cell Sci. 117, 4277–4287). Bovine embryos can be reconstructed with donor cells thawed immediately before NT, but it is not known if epigenetic changes arise during cell cryopreservation, and if NT efficiency is affected. The acetylated form of histone H3-lysine 9-lysine 14 (acH3K9/14) is associated with active chromatin configuration (Rice and Allis 2001 Curr. Opin. Cell Biol. 13, 263–273) and active transcription (Fuks 2005 Curr. Opin. Genet. Dev. 15, 490–495) and, thus, can be used as a marker to determine epigenetic changes in somatic cells. The purpose of our study was to evaluate histone acetylation levels of cultured and cryopreserved bovine fibroblasts. Cells were derived from skin of three adult cows and cultured in GMEM for 15 days. Fibroblasts from each of the three cell lines were analyzed at passage 1 (P1), 2 (P2), and 10 (P10). At each of the three passages, cells were cultured until reaching 100% confluence, followed by an additional 3 days in culture during which time acetylation levels were measured in fresh and frozen cells. For cryopreservation, cells at P1, P2, and P10 were disaggregated with accutase, resuspended in CryoStor™ (CS10; BioLife Solutions, Bothell, WA, USA), and cooled at 1.0°C min–1 to –80°C prior to storage in liquid nitrogen. Cells were fixed with ethanol for 12 h and incubated for 30 min with antibody directed against acetylated lysine 9 on histone 3 (H3K9). Then, cells were incubated with a fluorescein isothiocyanate (FITC) conjugated secondary antibody and DNA stain and evaluated by flow cytometry. Overall, histone acetylation levels in frozen cells (70%) were lower than levels in fresh cells (86%; P < 0.05), and all cell lines, whether fresh or frozen, showed lower acetylation levels at P1 (61%) than at P2 and P10 (88% and 85%, respectively; P < 0.001). No significant differences were observed between individual cell lines for fresh and frozen treatments. However, frozen cells from cell line 3 showed lower histone acetylation levels than fresh cells from cell line 3 and cell lines 1 and 2 at each passage. In summary, histone acetylation levels were lower in cryopreserved bovine fibroblasts and were higher in cell lines cultured for longer times. Table 1. Flow cytometrically detected acetylation levels of lysine 9 on histone 3 in cultured or cryopreserved bovine fibroblast cell lines passed for 1, 2, or 10 times

400 KARYOTYPIC ANALYSIS AND EPIGENETIC MODIFICATIONS OF CULTURED PORCINE ADIPOSE TISSUE-DERIVED STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 356
Author(s):  
K. J. Williams ◽  
K. R. Bondioli ◽  
R. A. Godke
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Chromatin Remodeling ◽  
Primary Cultures ◽  
Donor Cell ◽  
Global Methylation ◽  
Histone 3 ◽  
Donor Cells

The introduction of genetic modifications in donor cells for NT requires a significant number of population doublings (PD), and the deleterious effects, which may be attributed to aneuploidy or changes in DNA methylation and histone acetylation, are difficult at this time to circumvent. We hypothesize that the identification of a donor cell that is genetically stable for a long period of time in vitro such as somatic stem cells or those cells that demonstrate stem-like characteristics may be reprogrammed more completely, thus providing the key to increasing the efficiency of NT. Regulators of development in undifferentiated cells are suggested to be silenced by the presence of a bivalent domain modification pattern in which a large region of repressive histone 3 lysine 27 trimethylation (H3K27me3) contains smaller regions of activating histone 3 lysine 4 trimethylation (H3K4me3).The dual marks work to silence developmental genes in embryonic stem cells while simultaneously keeping them receptive to activation. The objectives of the current study were to determine the chromosomal stability of porcine adipose tissue-derived adult stem cells (pASC) through in vitro culture, to analyze pASC alongside fetal porcine fibroblasts (FPF) for gene expression profiles of chromatin remodeling proteins and global methylation and acetylation patterns, and to determine the presence of a co-enrichment of H3K27me3 and H3K4me3 within the promoter regions of developmentally important transcription factors. Metaphase spreads were prepared, and the presence of H3K27me3 and H3K4me3 was investigated in each of 3 individual pASC primary cultures for each analysis; whereas, gene expression and global methylation and acetylation were analyzed in each of 4 individual pASC and FPF primary cultures. Of 714 metaphases analyzed, 509 (71.3%) were aneuploid and only 205 (28.7%) were normal diploid porcine cells. For each cell population, we found a remarkable percentage of aneuploidies (43.7, 48.9, and 47.3, with a 46.6 ± 1.5 average) present immediately after the cultures were established. Chi-square analysis indicated that the percent of aneuploid cells during PD 1-10 was significantly less than that for PD 11-20 and PD 21-30. Also, porcine ASC demonstrated a consistently lower level of DNA methylation and histone acetylation through passages 2 through 7; whereas, the patterns for FPF varied. The expression levels of chromatin remodeling transcripts remained lower in pASC throughout culture when compared with FPF. Finally, porcine ASC possess a co-enrichment of H3K27me3 and H3K4me3 on the promoter region of the developmentally important transcription factor OCT-4. In vitro-cultured porcine ASC used as donor cells for NT should be chosen from early PD because of increased levels of aneuploidy at later PD. With a more complete characterization of porcine ASC, a donor cell population that can be more efficiently reprogrammed following fusion with the oocyte might be identified.

54 IN VITRO AND IN VIVO DEVELOPMENT OF FLAT-HEADED CAT (PRIONAILURUS PLANICEPS) CLONED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 127
Author(s):  
A. Thongphakdee ◽  
S. Manee-in ◽  
N. Klincumhom ◽  
B. Siriaroonrat ◽  
S. Kamolnorranarth ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Individual Cell ◽  
Research Unit ◽  
Domestic Cat ◽  
Control Group ◽  
Donor Cells ◽  
Chi Square Analysis

The objectives of the study were to investigate (1) the effect of individual cell line and gender of donor cells on flat-headed cat (FC) cloned embryo production (Study I) and (2) pregnancy establishment of recipients receiving cloned FC embryos with or without domestic cat (DC) IVF embryo co-transfer. The DC IVF embryos were used as a control (Study II). Study I Three cell lines of FC fibroblasts (passage 3–5) collected from 2 females (L1 and L2; biopsied from muscle and skin, respectively) and a male (L3; biopsied from skin) were used as donor cells for nuclear transfer. Donor cells were fused with enucleated in vitro matured DC oocytes. Fused couplets were induced by electrical pulses and subsequently incubated in activation medium containing 10 μg mL–1 cycloheximide and 5 μg mL–1 cytochalasin B for 4 h. Reconstructed embryos were cultured in SOFaa medium supplemented with 5% fetal bovine serum (FBS) at 38.5°C in air, and monitored for 7 days. Differences in the percentages of fusion and embryo development to a particular stage between cell lines and genders of donor cells were determined by chi-square analysis. Variations of fusion efficiency and embryo developmental success were observed between the cell lines. Greater cleavage number (P < 0.05) was observed when L1 was used as donor cells than that of L2 and L3. Developmental success to morula stage of embryo reconstructed from L1 was greater (P < 0.05) than that of L3 but not L2 (P > 0.05). However, there was no difference in the blastocyst formation success among cell lines. The development of the embryos derived from female and male donor cells at subsequent stages was not different. Study II Estrus and ovulation were induced in 15 DC recipients using 100 to 150 IU of pregnant mare serum gonadotropin (PMSG) and 100 IU of hCG (subcutaneous injection). Recipients were divided into 3 groups; (1) cloned group (n = 5) receiving FC cloned embryos (mean 41.4 ± 13), (2) co-transferred group (n = 4) receiving FC cloned and DC IVF embryos (mean 55 ± 15; 43.3 ± 15 of FC cloned and 10.8 ± 1.5 of DC IVF embryos), and (3) IVF/control group (n = 6) receiving only DC IVF embryos (mean 25 ± 9). Control DC IVF embryos were produced by co-incubation of DC oocytes with fresh DC semen for 18 h. Day 1 embryos were transferred into oviducts of recipients. Pregnancy evaluation using ultrasonography at Day 30 post-transfer demonstrated that pregnancy was not observed in any recipients in cloned group. One recipient from co-transferred group became pregnant and delivered DC IVF stillbirths (n = 2) and live kittens (n = 6). All recipients in IVF group became pregnant and 3 recipients delivered 5 DC kittens. These results indicate that (1) the individual cell line but not the gender of donor cells influences the development of FC cloned embryos and (2) with or without co-transfer of FC cloned and DC IVF embryos, FC cloned offspring was not able to be produced in the study. Table 1.Developmental success of FC cloned embryos This study was supported by the Zoological Park Organization under the Royal Patronage of H.M. the King, and the Reproductive Biotechnology Research Unit, Chulalongkorn University. A. Thongphakdee is supported by the Royal Golden Jubilee Ph.D. Program, and the Thailand Research Fund.

29 BIRTH OF A FOAL CLONED BY ADULT SOMATIC CELL NUCLEAR TRANSFER WITH ROSCOVITINE-TREATED DONOR CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
Y. H. Choi ◽  
Y. G. Chung ◽  
D. D. Varner ◽  
K. Hinrichs
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Direct Injection ◽  
Donor Cell ◽  
Mixed Gas ◽  
Blastocyst Development ◽  
Donor Cells ◽  
Significant Difference

Only one horse foal produced from adult somatic cell nuclear transfer has been reported in the scientific literature (Galli et al. 2003 Nature 425, 680); a second foal from the same laboratory was reported in the popular press in 2005. In these reports, the blastocyst rates were 3 and 17%, and efficiency to birth of a live foal from total reconstructed oocytes was 0.1 and 0.5%, respectively. In cattle, roscovitine treatment of donor cells has been associated with a decrease in blastocyst development, but an increase in live births (Gibbons et al. 2002 Biol. Reprod. 66, 895-900). The present study was performed to determine the effect of roscovitine treatment of donor cells on blastocyst production after equine nuclear transfer and to evaluate the viability of pregnancies established via this treatment. In Experiment 1, fibroblasts were either grown to confluence or treated with 15 �g/mL roscovitine, for 24 h. Enucleated in vitro-matured oocytes were reconstructed by direct injection of fibroblasts using a piezo drill. Recombined oocytes were activated by injection of stallion sperm extract, followed by culture in the presence of 2 mM 6-DMAP for 4 h. They were then placed in culture in DMEM/F-12 with 10% fetal bovine serum (FBS) under mixed gas for 8 days and evaluated for blastocyst development. In Experiment 2, oocytes recombined with either confluent or roscovitine-treated donor cells were activated as above either alone or with the addition of 10 �g/mL cycloheximide at the time of 6-DMAP treatment. Resulting blastocysts from Experiment 2 were transferred transcervically to the uteri of recipient mares. One embryo was transferred per mare. In Experiment 1, there was no difference in rates of cleavage (73-19%) or blastocyst development between confluence and roscovitine treatments (2/55, 3.6% vs. 2/56, 3.6%, respectively). In Experiment 2, there was no significant difference in rates of cleavage (78-18%) or blastocyst development (0-1%; 4/105, 0/104, 0/106, 2/108) among donor cell or activation treatments. Six blastocysts were transferred to mares: two from confluent donor cells and four from roscovitine-treated donor cells. One mare, which received an embryo from the roscovitine donor/6-DMAP treatment, established pregnancy after transfer. The pregnancy continued normally and the mare delivered a colt with minimal assistance on Day 389. Typing for 13 equine microsatellites confirmed that the colt was of the same DNA type as the donor fibroblasts. The colt has grown and developed normally. Results of these studies show that roscovitine treatment of equine donor cells does not negatively affect the proportion of recombined oocytes that progress to the blastocyst stage. A viable colt resulted from an embryo produced with roscovitine-treated donor cells. More work is needed on methods to increase blastocyst rates after nuclear transfer in this species. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.

scholarly journals The Alkylating Histone Deacetylase Inhibitor Fusion Molecule Edo-S101 Displays Full Bi-Functional Properties in Preclinical Models of Hematological Malignancies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2100-2100 ◽  
Author(s):  
Ana Alicia López-Iglesias ◽  
Laura San-Segundo ◽  
Lorena González-Méndez ◽  
Susana Hernández-García ◽  
Daniel Primo ◽  
...  
Keyword(s):  
Single Dose ◽  
Cell Line ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Parp Cleavage ◽  
Hdac Inhibition ◽  
Histone 3 ◽  
Xenograft Models

Abstract Background Alkylating histone deacetylase inhibitors (HDACi) enhance the anticancer efficacy of alkylators by increasing chromatin accessibility and also down regulating DNA repair. EDO-S101 is a first-in-class fusion molecule that combines DNA damaging effect of bendamustine with the pan-HDACi vorinostat. Objectives To study the bi-functional properties of EDO-S101 as an alkylating agent and a pan-HDACi in various in vitro and in vivo xenograft models of hematological malignancies. Methods In vitro inhibition of HDAC Class I and II enzymes by EDO-S101 and vorinostat was tested using an recombinant human enzymatic assay (BPS Bioscience, Enzo Life Science) and in vivo in rat peripheral blood mononuclear cells (PBMCs). The degree of inhibition was measured 1 hour following a single dose of 10–50 mg/kg i.v. and duration of inhibition over 24 hours after a single i.v. dose of EDO-S101 of 25 mg/kg. HDAC inhibition, alkylation and apoptotic activity were evaluated in vitro in myeloid (HL60 AML cell line) and lymphoid cell lines, including Daudi Burkitt’s lymphoma (BL) and a panel of 6 MM cell lines (MM1S, MM1R, RPMI-8226, RPMI-LR5, U266, U266-LR7). In vivo intra-tumor effects were analyzed after short courses of treatment with EDO-S101 in MM1S human plasmacytoma (PC) and BL xenograft models. Changes in pathway activation, protein expression and activities influencing the cell cycle were measured by Western blot and immunohistochemistry. Anti-tumor activity in vitro was measured by MTT and in vivo using a caliper to assess tumor size at regular intervals. Results In vitro, EDO-S101’s pan-HDACi activity, at nanomolar concentrations in Class I and II recombinant enzymes, was similar to vorinostat. In vivo, in intact rat PBMCs, HDAC inhibition was maximal at 1 hour after a single dose of 10 mg/kg i.v.–the dose where antitumor activity starts. HDAC inhibition did not increase with doses up to 50 mg/kg, recovery began within 3 hours and was nearly complete at 16 hours. In the AML HL60 cell line in vitro, hyperacetylation of lysine residues K9, K14, K23 and K56 on histone 3 was found after exposure to 2–4 µM of EDO-S101. Histone 3 and 4 hyperacetylation was also demonstrated in MM cell lines at 1–5 µM concentrations. In xenograft models of human plasmacytoma and BL, EDO-S101 induced histone 3 hyperacetylation, indicating an HDACi effect in vivo. Alkylating activity was demonstrated in vitro in HL60 and MM cell lines by DNA cross-linking and double strand break formation in the comet assay by immunofluorescence. In vivo, in xenograft models of human plasmacytoma (60 mg/kg d 1, 8, 15) and BL (40 and 80mg/kg d1) exposure to EDO-S101 caused a strong DNA-repair response shown by activation of pH2AX and p53 (PC and BL) followed by an increase of DNA damage check point proteins pCHK1 (PC) and even more prominent pCHK2 (PC and BL). The kinetics of this effect, studied in vivo in BL tumors, showed that the pH2AX response fell at Day 8 after dosing while the p53 response lasted, particularly in the group treated with 80mg/kg. In Daudi-bearing mice tumors, p-ATR was completely suppressed at Day 8 after treatment, which was not clear in the PC tumors. EDO-S101 triggered apoptosis in vitro and in vivo, resulting in strong antitumor activity in HL60, Daudi and the panel of six MM cell lines. Initial in vitro experiments in HL60 cells showed an activation of the intrinsic pathway of apoptosis with cleavage of caspases 3, 9 and PARP and a marked reduction of anti-apoptotic proteins XIAP and Mcl-1. In the MM cell line, MM1S activation of the intrinsic and extrinsic pathways of apoptosis (C 8, 9, 3, 7 and PARP cleavage) was seen with a loss of mitochondrial membrane potential by DiOC6. Tumors of human plasmacytoma and BL in vivo were rapidly shrinking or completely eradicated after i.v. administration of EDO-S101. A decrease in proliferation (Ki67) and slight PARP cleavage was found in the tumor tissue (PC), and evidence of activation of apoptosis by cleavage of caspases 7 and 9 at Day 4 and caspase 8 and PARP at Day 8 after treatment in BL tumors. The level of caspase 3, different to MM, remained unchanged. Importantly, EDO-S101 induced a rapid and dose-dependent strong decrease of XIAP and Mcl-1 which lasted until Day 8. Conclusions This study demonstrates the bi-functional mechanism of ED0-S101 in both myeloid and lymphoid hematological malignancies. The data support the clinical investigation of EDO-S101 in treating hematological malignancies. Disclosures Ocio: Mundipharma: Honoraria, Research Funding. Mehrling:Mundipharma: Employment.

Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

Zygote ◽  
2010 ◽  
Vol 19 (3) ◽  
pp. 255-264 ◽  
Author(s):  
Liliana Chacón ◽  
Martha C. Gómez ◽  
Jill A. Jenkins ◽  
Staley P. Leibo ◽  
Gemechu Wirtu ◽  
...  
Keyword(s):  
Donor Cell ◽  
Blastocyst Stage ◽  
Cell Passage ◽  
Bovine Embryos ◽  
Cell Numbers ◽  
Histone 3 ◽  
Donor Cells ◽  
Genetic Source ◽  
Vitro Development

SummaryIn this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers.

ANTITUMOR EFFECT OF SUNITINIB AND BORTEZOMIB ON BREAST CANCER CELL LINE IN VITRO

2017 ◽  
Vol 63 (1) ◽  
pp. 141-145
Author(s):  
Yuliya Khochenkova ◽  
Eliso Solomko ◽  
Oksana Ryabaya ◽  
Yevgeniya Stepanova ◽  
Dmitriy Khochenkov
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Antitumor Effect ◽  
Cancer Cell Lines ◽  
Breast Cancer Cell Lines ◽  
Actual Problem

The discovery for effective combinations of anticancer drugs for treatment for breast cancer is the actual problem in the experimental chemotherapy. In this paper we conducted a study of antitumor effect of the combination of sunitinib and bortezomib against MDA-MB-231 and SKBR-3 breast cancer cell lines in vitro. We found that bortezomib in non-toxic concentrations can potentiate the antitumor activity of sunitinib. MDA-MB-231 cell line has showed great sensitivity to the combination of bortezomib and sunitinib in vitro. Bortezomib and sunitinib caused reduced expression of receptor tyrosine kinases VEGFR1, VEGFR2, PDGFRa, PDGFRß and c-Kit on HER2- and HER2+ breast cancer cell lines

Design, Synthesis, Molecular Docking, and Anticancer Evaluation of Pyrazole Linked Pyrazoline Derivatives with Carbothioamide Tail as EGFR Kinase Inhibitors

2020 ◽  
Vol 21 (1) ◽  
pp. 42-60
Author(s):  
Farah Nawaz ◽  
Ozair Alam ◽  
Ahmad Perwez ◽  
Moshahid A. Rizvi ◽  
Mohd. Javed Naim ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Cancer Cell Lines ◽  
Kinase Assay ◽  
Cervix Uteri ◽  
Egfr Kinase

Background: The Epidermal Growth Factor Receptor (known as EGFR) induces cell differentiation and proliferation upon activation through the binding of its ligands. Since EGFR is thought to be involved in the development of cancer, the identification of new target inhibitors is the most viable approach, which recently gained momentum as a potential anticancer therapy. Objective: To assess various pyrazole linked pyrazoline derivatives with carbothioamide for EGFR kinase inhibitory as well as anti-proliferative activity against human cancer cell lines viz. A549 (non-small cell lung tumor), MCF-7 (breast cancer cell line), SiHa (cancerous tissues of the cervix uteri), and HCT-116 (colon cancer cell line). Methods: In vitro EGFR kinase assay, in vitro MTT assay, Lactate dehydrogenase release, nuclear staining (DAPI), and flow cytometry cell analysis. Results: Compounds 6h and 6j inhibited EGFR kinase at concentrations of 1.66μM and 1.9μM, respectively. Furthermore, compounds 6h and 6j showed the most potent anti-proliferative results against the A549 KRAS mutation cell line (IC50 = 9.3 & 10.2μM). Through DAPI staining and phase contrast microscopy, it was established that compounds 6h and 6j also induced apoptotic activity in A549 cells. This activity was further confirmed by FACS using Annexin-V-FITC and Propidium Iodide (PI) labeling. Molecular docking studies performed on 6h and 6j suggested that the compounds can bind to the hinge region of ATP binding site of EGFR tyrosine kinase in a similar pose as that of the standard drug gefitinib. Conclusion: The potential anticancer activity of compounds 6h and 6j was confirmed and need further exploration in cancer cell lines of different tissue origin and signaling pathways, as well as in animal models of cancer development.

Survival and Function of Fibroblasts Stored as Stock Cultures at a Non-freezing Temperature

1993 ◽  
Vol 21 (2) ◽  
pp. 206-209
Author(s):  
Anders H. G. Andrén ◽  
Anders P. Wieslander
Keyword(s):  
Cell Growth ◽  
Cell Line ◽  
Cell Lines ◽  
Fibroblast Cell ◽  
Freezing Temperature ◽  
Mouse Fibroblast ◽  
Toxic Response ◽  
Normal Manner ◽  
And Function

Cytotoxicity, measured as inhibition of cell growth of cultured cell lines, is a widely used method for testing the safety of biomaterials and chemicals. One major technical disadvantage with this method is the continuous routine maintenance of the cell lines. We decided to investigate the possibility of storing stock cultures of fibroblasts (L-929) in an ordinary refrigerator as a means of reducing the routine workload. Stock cultures of the mouse fibroblast cell line L-929 were prepared in plastic vials with Eagle's minimum essential medium. The vials were stored in a refrigerator at 4–10°C for periods of 7–31 days. The condition of the cells after storage was determined as cell viability, cell growth and the toxic response to acrylamide, measured as cell growth inhibition. We found that the L-929 cell line can be stored for 2–3, weeks with a viabilty > 90% and a cell growth of about 95%, compared to L-929 cells grown and subcultured in the normal manner. The results also show that the toxic response to acrylamide, using refrigerator stored L-929 cells, corresponds to that of control L-929 cells. We concluded that it is possible to store L-929 cells in a refrigerator for periods of up to 3 weeks and still use the cells for in vitro cytotoxic assays.

scholarly journals Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

1983 ◽  
Vol 96 (1) ◽  
pp. 37-50 ◽  
Author(s):  
E Schmid ◽  
DL Schiller ◽  
C Grund ◽  
J Stadler ◽  
WW Franke
Keyword(s):  
Mammary Gland ◽  
Epithelial Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Intermediate Filament ◽  
Striking Difference ◽  
Intermediate Filament Proteins ◽  
Cell Clones ◽  
Vimentin Filaments

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow's udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.

scholarly journals Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 219-230 ◽  
Author(s):  
Feikun Yang ◽  
Ru Hao ◽  
Barbara Kessler ◽  
Gottfried Brem ◽  
Eckhard Wolf ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Epigenetic Mark ◽  
Cell Type ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Donor Cell Type

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres fromin vivofertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF,P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that inin vivofertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres fromin vivoderived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

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