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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

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 approach.In 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 bank.Methods Fifty-two specimens were collected over a two-year period from people with breast cancer who needed surgical treatment in our hospital.Cells were isolated and used to establish successful cell culture.Cell activity and cell purity were measured before liquid nitrogen cryopreservation.Results(1)At the initial culture stage,cells grew with adherence.Cell multiplication could be seen after the cell medium was exchanged three times.Cell 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 cryopreserved.Conclusion(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

来源： cnki会议评论

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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 chicks.Therefore,in the present study,two experiments were conducted to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.In 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 staining.Experiment 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,respectively.The results from experiment 1 demonstrated that primary cultured tibial osteoblasts of broilers showed a spindle-shaped,triangular or polygonal morphology.More than 95%of the cells were stained blue-black after ALP staining,and mineralized nodules were formed after 4 days of continuous incubation.In experiment 2,lactate dehydrogenase(LDH)activity stayed at a relatively stabilized level although incubation time affected(P=0.0012)it during the whole culture period.Additionally,incubation time affected(P≤0.0001)the number and proportion of the area of mineralized nodules.They 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 stabilized vitality from 4 to 32 days of incubation;and t

关键词： broiler tibial osteoblast primary culture vitality mineralization

In vitro models of retinal diseases

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Annals of Eye Science 2022年第1期7卷 106-113页

作者： Martina Lucchesi Silvia Marracci Department of Biology University of PisaPisaItaly

Background:Continuous and primary in vitro cultures are largely used to study cellular mechanisms occurring in several pathologic-like or pathological conditions.Continuous cell lines allow to perform long-lasting experiments since they do not undergo senescence.Methods:The immortalized Moorfields/Institute of Ophtalmology-Müller 1(MIO-M1)cell type represents a valuable model to analyze the mechanistic pathways characterizing Müller glial cells,both in health and in disease.MIO-M1 can be used to dissect the response of these glial cells following treatments which mimic pathological condition.For instance,MIO-M1 are useful to study the response of this cell type to stress condition as the case of oxidative stress(OS)(cultured with hydrogen peroxide),pathological neovascularization(cultured with VEGF),hypoxic or hyperoxic condition(cultured in low or high oxygen chamber).On the other hand,primary cultures allow to specifically analyze cellular responses without the interference of the whole organ,although the experimental treatment is performed in vivo.primary Müller cells can be used to perform electrophysiological analyses of different cell sites.Discussion:We describe how to manage MIO-M1 cells and how to analyze their response to different stress conditions;moreover,we report how to isolate and identify primary Müller cells and how to perform patch clamp and single cell recordings on them.

关键词： Müller cells Moorfields/Institute of Ophtalmology-Müller 1(MIO-M1) primary culture

Differential development and electrophysiological activity in cultured cortical neurons from the mouse and cynomolgus monkey

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Neural Regeneration Research 2021年第12期16卷 2446-2452页

作者： Xue-Yan Zhang Jun Li Cai-Juan Li Ying-Qi Lin Chun-Hui Huang Xiao Zheng Xi-Chen Song Zhu-Chi Tu Xiao-Jiang Li Sen Yan Guangdong Key Laboratory of Non-Human Primate Models Guangdong-Hongkong-Macao Institute of CNS RegenerationJinan UniversityGuangzhouGuangdong ProvinceChina Key Laboratory of CNS Regeneration Ministry of EducationJinan UniversityGuangzhouGuangdong ProvinceChina

In vitro cultures of primary cortical neurons are widely used to investigate neuronal function.However,it has yet to be fully investigated whether there are significant differences in development and function between cultured rodent and primate cortical neurons,and whether these differences influence the utilization of cultured cortical neurons to model pathological conditions.Using in vitro culture techniques combined with immunofluorescence and electrophysiological methods,our study found that the development and maturation of primary cerebral cortical neurons from cynomolgus monkeys were slower than those from mice.We used a microelectrode array technique to compare the electrophysiological differences in cortical neurons,and found that primary cortical neurons from the mouse brain began to show electrical activity earlier than those from the cynomolgus monkey.Although cultured monkey cortical neurons developed slowly in vitro,they exhibited typical pathological features-revealed by immunofluorescent staining-when infected with adeno-associated viral vectors expressing mutant huntingtin(HTT),the Huntington’s disease protein.A quantitative analysis of the cultured monkey cortical neurons also confirmed that mutant HTT significantly reduced the length of neurites.Therefore,compared with the primary cortical neurons of mice,cultured monkey cortical neurons have longer developmental and survival times and greater sustained physiological activity,such as electrophysiological activity.Our findings also suggest that primary cynomolgus monkey neurons cultured in vitro can simulate a cell model of human neurodegenerative disease,and may be useful for investigating time-dependent neuronal death as well as treatment via neuronal regeneration.All mouse experiments and protocols were approved by the Animal Care and Use Committee of Jinan University of China(IACUC Approval No.20200512-04)on May 12,2020.All monkey experiments were approved by the IACUC protocol(IACUC Approval No.LDACU 20190820-01)on August 23,2019 for animal

关键词： Axion-MEA electrical activities human disease model Huntington’s disease HTT monkey neuron morphometric analysis mouse neuron neurodegenerative diseases primary culture

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)status.Translational research has few available in vitro models with which to study the different pathophysiological behavior of OSCCs.The 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 cells.Methods:We compared the phenotypic and genetic features of HPV-positive and HPV-negative OSCC cell lines cultured on common monolayer supports and on scaffolds.We also explored cancer cell adaptation to the 3 D microenvironment and its impact on the efficacy of drugs tested on cell lines and primary cultures.Results:HPV-positive and HPV-negative cell lines were successfully grown in the 3 D model and displayed different collagen fiber organization.The 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 xenografts.The expression of hypoxia-inducible factor 1α(1α)and glycolysis markers were indicative of the development of a hypoxic microenvironment inside the scaffold area.Furthermore,the 3 D cultures activated drug-resistance signaling pathways in both cell lines and primary cultures.Conclusions:Our results suggest that collagen-based scaffolds could be a suitable model for the reproduction of the pathophysiological features of OSCCs.Moreover,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

Identification of potential candidate proteins for reprogramming spinal cord-derived astrocytes into neurons:a proteomic analysis

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Neural Regeneration Research 2021年第11期16卷 2257-2263页

作者： Wen-Hao Chen Yu-Xiang Lin Ling Lin Bao-Quan Zhang Shu-Xia Xu Wei Wang Department of Pediatric Surgery Fujian Maternity and Child Health HospitalAffiliated Hospital of Fujian Medical UniversityFuzhouFujian ProvinceChina Department of Breast Surgery Affiliated Union HospitalFujian Medical UniversityFuzhouFujian ProvinceChina Institutes of Biomedical Sciences of Shanghai Medical School Fudan UniversityShanghaiChina Department of Neonatology Fujian Maternity and Child Health HospitalAffiliated Hospital of Fujian Medical UniversityFuzhouFujian ProvinceChina Department of Pathology Fujian Maternity and Child Health HospitalAffiliated Hospital of Fujian Medical UniversityFuzhouFujian ProvinceChina Department of Anatomy and Histoembryology Fujian Medical UniversityFuzhouFujian ProvinceChina

Our previous study has confirmed that astrocytes overexpressing neurogenic differentiation factor 1(NEUROD1)in the spinal cord can be reprogrammed into neurons under in vivo conditions.However,whether they can also be reprogrammed into neurons under in vitro conditions remains unclear,and the mechanisms of programmed conversion from astrocytes to neurons have not yet been clarified.In the present study,we prepared reactive astrocytes from newborn rat spinal cord astrocytes using the scratch method and infected them with lentivirus carrying NEUROD1.The results showed that NEUROD1 overexpression reprogrammed the cultured reactive astrocytes into neurons in vitro with an efficiency of 13.4%.Using proteomic and bioinformatic analyses,1952 proteins were identified,of which 92 were differentially expressed.Among these proteins,11 were identified as candidate proteins in the process of reprogramming based on their biological functions and fold-changes in the bioinformatic analysis.Furthermore,western blot assay revealed that casein kinase II subunit alpha(CSNK2A2)and pinin(PNN)expression in NEUROD1-overexpressing reactive astrocytes was significantly increased,suggesting that NEUROD1 can directly reprogram spinal cord-derived reactive astrocytes into neurons in vitro,and that the NEUROD1-CSNK2A2-PNN pathway is involved in this process.This study was approved by the Animal Ethics Committee of Fujian Medical University,China(approval No.2016-05)on April 18,2016.

关键词： astrocytes glial cells label-free proteomic analysis mechanism neurons primary culture reprogramming spinal cord injury

Which form of collagen is suitable for nerve cell culture?

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Neural Regeneration Research 2013年第23期8卷 2165-2170页

作者： Mohsen Fathi Najafi Saber Zahri Fatemeh Vahedi Leila Esmaililian Toosi Nazila Ariaee Razi Vaccine and Serum Research Institute Departamnt of Biology University of Mohaghegh Ardebili Inflammation and Inflammatory Diseases Research Center Mashhad University of Medical Science

In this study, we investigated the effects of hydrolyzed and non-hydrolyzed collagen and two-di- mensional and three-dimensional collagen matrices on cell survival, attachment and neurite out- growth of primary cultured nerve cells using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide （MTT） colorimetric assay and inverted microscopy. Hydrolyzed collagen facilitated nerve cell survival and neurite outgrowth, but it had no obvious influences on cell attachment. In contrast, non-hydrolyzed two-dimensional collagen matrix had no obvious effects on neurite outgrowth. These findings suggest that hydrolyzed collagen is an ideal nerve cell culture media.

关键词： neural regeneration nerve cells collagen primary culture survival cell attachment growth matrix grants-supported paper neuroregeneration