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primary culture of Bovine Mammary Epithelial Cells

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Agricultural Science & Technology 2009年第1期10卷 119-123页

作者：吴娟王凤龙王申元内蒙古农业大学动物科学与医学学院病理研究室

[ Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [ Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tissue explant method in order to investigate the optimal culture conditions. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, Giemsa staining and cytokeratin immunohistochemistry. [ Result] Observed with inverted microscope, most of the bovine mammary epithelial cells were polygonal and displayed typical slabstone-like appearance. As it can be seen from cell staining results, the cell body was big and the nucleus was stained dark blue and was round or oval in shape, with clearly visible nucleoli, generally 2 -4 nucleoli. The tissue-specific expression of cytokeratin 14 and cytokeratin 18 genes in mammary epithelial cells was identified by cytokeratin immunohistochemistry. [ Conclusion] primary bovine mammary epithelial cells were successfully cultured in biochemical incubator.

关键词： Bovine mammary epithelial cells primary culture Cells growing on cover slip Immunohistochemistry

primary culture and identify of cerebral cortical astrocytes from Sprague-Dawley rats

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中国药理学与毒理学杂志 2017年第5期31卷 486-486页

作者： ZHANG Hai-wei WU Chun-yang ZHANG Dan-shen Department of Phrmacy Hebei North University Hebei University of Science and Technology

OBJECTIVE To establish a method for primary cultured and iden-tified Sprague-Dawley(SD) rats cerebral cortical astrocytes. METHODS Cerebral co-rtex of SD neonatal rats within 24 h was taken with stereo microscope and was cut topieces(1 mm^3),digested by Accutase and 0.1% DNAase(37℃,15 min),anddispersed cell suspension was made by mechanical method and filtered. The fibroblast cells and microglia were removed through differential adhesion and sha-ke. Passaged cells were identified by immunofluorescent with anti-Glial fibrillaryacidic protein(GFAP) antibody. RESULTS The astrocytes of rats cerebral cortex were cultured in this method,which had a large number of cells,good activity,high purity,abundant and elongated cell processes,and were interwoven into a network,showing a typical and good growth state. The third generation of the cel s comprised >95% astrocytes. CONCLUSION This simple and reliable cultivation method of astrocyte from rat cerebral tissue is established with high purity,and in a good growth condition.

关键词： astrocytes primary culture identify

An improved primary culture method for hippocampal neurons in fetal rats and MAP2 identification

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TMR Integrative Medicine 2018年第1期2卷 1-6页

作者： Yi-Ran Shao Yong Chang DIWU Tao Yue First Clinical Medical College of Shaanxi University of Chinese Medicine Xianyang 712046 China

Objective： To establish a simple, effective and high-purity primary culture method for fetal rat hippocampalneurons. Methods： Wistar rats of gestational age 18 days were taken and the brain tissue was separated under themicroscope. Single neuronal cells were obtained by digestion with Brain Dissociation Kit, and then were seeded incell plates to observe the basic morphologic structure after 24h, 3d, and 5d. Immunofluorescence of microtubuleassociated protein 2 was applied to assess cell purity of the culture. Results： The hippocampal neurons obtained inthis culture method are in good condition and grow vigorously. On the 7th day after culture, the purity of neuronswas up to 99.62%. Conclusion： The method is simple and effective for obtaining the high-purity and stableneurons.

关键词： Hippocampal neurons Fetal rats primary culture

The study of selective primary culture and determination of a breast cancer cell line in vitro

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Oncology and Translational Medicine 2020年第2期6卷 68-71页

作者： Meng Ren Huixia Xu Xiangji Lu Bingping Wang Rina Su Hao Zhang Song Jiang Fengying Gao Yanwei Gao Department of Abdominal Neoplasms Surgical Inner Mongolia People’s HospitalHohhot 010017China Department of Hematology Inner Mongolia People’s HospitalHohhot 010017China Department of Surgical 2 Armed Police General Hospital Inner MongoliaHohhot 010000China Inner Mongolia Institute for Cancer Research Inner Mongolia People’s HospitalHohhot 010017China Graduate School Inner Mongolia Medical UniversityHohhot 010000China

Objective The successful establishment of a tumor cell bank is based on the premise that the target cells can be cultured by a legitimate *** this experiment,we used primary culture to select and detect breast cancer cells in vitro,which can provide experimental ideas and methods for the establishment of a living tumor tissue cell *** Fifty-two specimens were collected over a two-year period from people with breast cancer who needed surgical treatment in our *** were isolated and used to establish successful cell *** activity and cell purity were measured before liquid nitrogen ***(1)At the initial culture stage,cells grew with *** multiplication could be seen after the cell medium was exchanged three *** viability was above 86%,while the viability of the target cells was above 75%,as detected by hematoxylin and eosin(HE)staining.(2)The number of breast cancer cells decreased,while the number of fibroblasts increased after five rounds of passage.(3)The success rate was 73.08%,which did not include polluted cells and those that were not successfully ***(1)breast cancer cells could be selected from primary culture in vitro through an appropriate method.(2)Exchange of the cell medium and further cell passage improved cell multiplication.(3)The experimental results could be monitored using trypan blue and HE staining.(4)The success of breast cancer cell culture in vitro could be used as a reference for other cell culture,so as to establish a tumor tissue cell bank.

关键词： breast cancer primary culture Trypan blue staining hematoxylin and eosin(HE)staining

Establishment and Characterization of primary Astrocyte culture from Adult Mouse Brain

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Establishment and Characterization of Primary Astrocyte Cult...

Establishment and evaluation of the primary cultured tibial osteoblast model of broiler chicks

中国神经科学学会第十二届全国学术会议

作者： Xiu Sun Xin Hu Dan Wang Yimin Yuan Shangyao Qin Zijian Tan Yakun Gu Xiao Huang Cheng He Zhida Su Institute of Neuroscience Key Laboratory of Molecular Neurobiology of Ministry of Education Second Military Medical University

As a major class of glial cells, astrocytes have been indicated to play multiroles in physiological and pathological brain. Astrocyte cultures derived from postnatal mouse brains have been extensively used to characterize their biological properties. However, the inability to culture adult mouse primary astrocytes has long stymied studies of function in adult brain. Here, we developed a protocol to successfully establish highly enriched astrocyte cultures from the brains of adult mouse. Cortical tissues were collected to prepare cell suspension by enzymatic digestion and mechanical dissociation, and then plated onto vessels pre-coated with gelatin and matrigel and cultured in DMEM medium containing 20% fetal bovine serum（FBS）. Forskolin（FSK） and glial-derived neurotrophic factor（GDNF） were used to promote astrocyte proliferation and survival respectively. These adult astrocyte cultures were identified by immunocytochemical, immunoblotingand PCR analysis. Furthermore, biological and functional analysis indicated that they possess the biochemical and physiological properties of astrocytes, suggestive of a useful cell model for astroglial studies in adult brain.

关键词： astrocyte primary culture adult mouse biological property

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Journal of Integrative Agriculture 2023年第2期22卷 551-558页

作者： CAO Su-mei LI Ting-ting SHAO Yu-xin ZHAO Yu-zhen ZHANG Li-yang LU Lin ZHANG Ri-jun HOU Shui-sheng LIAO Xiu-dong LUO Xu-gang WANG Run-lian Poultry Mineral Nutrition Laboratory College of Animal Science and TechnologyYangzhou UniversityYangzhou 225000P.R.China Mineral Nutrition Research Division State Key Laboratory of Animal NutritionInstitute of Animal SciencesChinese Academy of Agricultural SciencesBeijing 100193P.R.China Laboratory of Feed Biotechnology State Key Laboratory of Animal NutritionCollege of Animal Science and TechnologyChina Agricultural UniversityBeijing 100193P.R.China Department of Animal Science Guangdong Ocean UniversityZhanjiang 524088P.R.China

Osteoblasts are considered as a major factor contributing to bone development and mineralization,however,few studies have been done to establish and evaluate the primary cultured tibial osteoblast model of broiler ***,in the present study,two experiments were conducted to establish and evaluate the primary cultured tibial osteoblast model of broiler *** experiment 1,osteoblasts were isolated from the tibia of one-day-old Arbor Acre male broiler chicks using the explant method and identified through the cell morphology,alkaline phosphatase(ALP)and alizarin red *** 2 was carried out to evaluate the vitality and mineralization of primary cultured tibial osteoblasts of broilers on days 4,8,12,16,20,24,28 and 32 after incubation,*** results from experiment 1 demonstrated that primary cultured tibial osteoblasts of broilers showed a spindle-shaped,triangular or polygonal *** than 95%of the cells were stained blue-black after ALP staining,and mineralized nodules were formed after 4 days of continuous *** experiment 2,lactate dehydrogenase(LDH)activity stayed at a relatively stabilized level although incubation time affected(P=0.0012)it during the whole culture ***,incubation time affected(P≤0.0001)the number and proportion of the area of mineralized *** increased linearly and quadratically(Pprimary cultured tibial osteoblast model of broilers has been established successfully by the explant method,and it showed typical osteoblast morphology and characteristics of ALP activity and mineralization,and could maintain a relatively stabilize

关键词： broiler tibial osteoblast primary culture vitality mineralization

Establishment of primary cultures of craniopharyngioma cells

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Neural Regeneration Research 2012年第8期7卷 601-605页

作者： Hao Liu Liang Liu Zhiyong Liu Qiang Li Chao You Jianguo Xu Department of Neurosurgery West China Hospital Sichuan University Chengdu 610041 Sichuan Province China Department of Neurosurgery Affiliated Hospital of Luzhou Medical College Luzhou 646000 Sichuan Province China

Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positively expressed in the cytomembrane and cytoplasm of craniopharyngioma cells at 6-8 passages, confirming that all cultured cells were squamous epithelial cells. The doubling time of craniopharyngioma cells was 3 days, as confirmed by the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay. In this study, craniopharyngioma cells cultured in vitro were established; however, establishment of immortalized craniopharyngioma cell lines requires further research.

关键词： craniopharyngioma cytokeratin-7 primary culture culture in vitro cell line

Nutrients Modulate T1r2 Transcript Levels in MIN 6 and primary cultured Taste Buds Cells under High Glucose Condition

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Food and Nutrition Sciences 2016年第4期7卷 312-319页

作者： Shin-ichi Nakmura Tetsuya Ookura Division of Food Function National Food Research Institute National Agricultural and Food Research Organization Tsukuba Japan

The sweet taste receptors comprised of T1r2 and T1r3, sense glucose concentrations in the gastrointestine. While hyperglycemia was reported to decrease the T1R2 and T1R3 tanscript levels in healthy subjects, no change was observed in type 2 diabetes patients. We investigated which glucose level and nutrients affect those transcript levels in MIN 6 and primary cultured taste buds cells using quantitative Reverse Trancription Polymerase Chain Reaction. High glucose diminished T1r2 transcript levels in MIN 6 and primary cultured taste buds cells. Resveratrol and its analogue augmented transcript levels of T1r1 and T1r2 above normal levels in MIN 6 cells in the medium with 25 mM glucose. Adenine, but not guanine, augmented T1r2 transcript levels of MIN 6 cells in the medium with 25 mM glucose. These results imply that nutrients in meals could affect sweet taste sensitivity by modulating T1r2 transcript levels in response to blood glucose levels.

关键词： Glucose T1r2 MIN6 primary culture Taste Buds

Three-dimensional collagen-based scaffold model to study the microenvironment and drug-resistance mechanisms of oropharyngeal squamous cell carcinomas

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Cancer Biology & Medicine 2021年第2期18卷 502-516页

作者： Giacomo Miserocchi Claudia Cocchi Alessandro De Vita Chiara Liverani Chiara Spadazzi Sebastiano Calpona Giandomenico Di Menna Massimo Bassi Giuseppe Meccariello Giovanni De Luca Angelo Campobassi Maria Maddalena Tumedei Alberto Bongiovanni Valentina Fausti Franco Cotelli Toni Ibrahim Laura Mercatali Osteoncology and Rare Tumors Center IRCCS Istituto Romagnolo per lo Studio dei Tumori(IRST)“Dino Amadori”Meldola 47014Italy Maxillofacial Surgery Unit Bufalini HospitalCesena 47521Italy Department of Head-Neck Surgery OtolaryngologyHead-Neck and Oral Surgery UnitMorgagni Pierantoni HospitalForlì47121Italy Pathology Unit “Bufalini”HospitalAUSL RomagnaCesena 47521Italy Biosciences Laboratory IRCCS Istituto Romagnolo per lo Studio dei Tumori(IRST)“Dino Amadori”Meldola 47014Italy Department of Biosciences Universitàdegli Studi di MilanoMilan 20133Italy

Objective:Squamous cell carcinoma(SCC)represents the most common histotype of all head and neck malignancies and includes oropharyngeal squamous cell carcinoma(OSCC),a tumor associated with different clinical outcomes and linked to human papilloma virus(HPV)*** research has few available in vitro models with which to study the different pathophysiological behavior of *** present study proposes a 3-dimensional(3 D)biomimetic collagen-based scaffold to mimic the tumor microenvironment and the crosstalk between the extracellular matrix(ECM)and cancer ***:We compared the phenotypic and genetic features of HPV-positive and HPV-negative OSCC cell lines cultured on common monolayer supports and on *** also explored cancer cell adaptation to the 3 D microenvironment and its impact on the efficacy of drugs tested on cell lines and primary ***:HPV-positive and HPV-negative cell lines were successfully grown in the 3 D model and displayed different collagen fiber *** 3 D cultures induced an increased expression of markers related to epithelial–mesenchymal transition(EMT)and to matrix interactions and showed different migration behavior,as confirmed by zebrafish embryo *** expression of hypoxia-inducible factor 1α(1α)and glycolysis markers were indicative of the development of a hypoxic microenvironment inside the scaffold ***,the 3 D cultures activated drug-resistance signaling pathways in both cell lines and primary ***:Our results suggest that collagen-based scaffolds could be a suitable model for the reproduction of the pathophysiological features of ***,3 D architecture appears capable of inducing drug-resistance processes that can be studied to better our understanding of the different clinical outcomes of HPV-positive and HPV-negative patients with OSCCs.

关键词： Oropharyngeal squamous cell carcinoma collagen biomimetic scaffold zebrafish drug-resistance primary culture