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Quasi Vivo流动培养促进气液界面培养呼吸道上皮细胞分化

——提高AIL培养人支气管上皮细胞、人小气道上皮细胞分化程度、缩短分化时间

 

呼吸道上皮细胞气液界面培养是研究经空气传播的病原体,如SARS等的常用的模型。传统的培养方式是用TransWell在普通培养箱中静置培养。但是此种培养方式无法模拟培养过程中营养物质和代谢废物在组织内的运输,培养得到的模型通常有各种各样的缺陷,并且所需实验周期较长。

而Quasi Vivo流动培养系统可为细胞培养提供持久恒定的流动培养环境,最大限度模拟体内环境。研究发现,使用Quasi Vivo系统进行流动培养与静态培养相比,气液界面培养的呼吸道上皮细胞(正常人气管上皮细胞 Normal Human Bronchial Epithelial Cells,简称NHBE;小气道上皮细胞 Small Airway Epithelial Cells,简称SAE),发育分化速度更快,表现为纤毛分化度更高,纤毛运动更强、粘液产生和屏障功能更强。在灌注下加速分化后,将上皮细胞转移到静态条件下,并添加抗原呈递细胞(APC)以研究其在病原体感染后的功能。(Chandorkar P, et al., Fast-track development of an in vitro 3D lung/immune cell model to study Aspergillus infections. Sci Rep. 2017 7(1):11644. doi: 10.1038/s41598-017-11271-4.)
流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平
图1. 人体内所有的细胞都需要营养物质和代谢废物的流动


流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平 
图2. 肺部气管/支气管和小气道上皮结构精细,进行体外培养模拟体内环境,对呼吸道病原体的研究至关重要。
 
流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平
图3. 呼吸道上皮细胞的常规transwell静止培养方式

流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平
Quais Vivo(QV600)流动培养系统(含腔室+储液瓶+底座+管道 )

流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平
流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平
图4. 采用全新的流动培养方式培养呼吸道上皮细胞(采用QV600)

有研究显示,使用transwell静止培养(Static Conditions)Quasi Vivo流动培养系统(Perfused Conditions),呼吸道上皮细胞的生长和分化呈现更好状态:
流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平 
图5 电镜照片显示,采用流动培养方式(Perfused conditions)的呼吸道上皮细胞,分化程度更高。

流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平
图6 使用MUC5B染色可以发现,采用流动培养方式(Perfused conditions)的呼吸道上皮细胞,在培养的第7天即可分泌大量粘液。用OCCLUDIN染色可以发现,细胞间的紧密连接发育更完善。

流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平
图7. 使用WGA染色发现,采用流动培养方式(Perfused conditions)的呼吸道上皮细胞,纤毛分化度更高
 
流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平
图8. 测量TEER(经细胞电阻),采用流动培养方式(Perfused conditions)的呼吸道上皮细胞TEER值更大,代表得到的上皮细胞膜状结构更完整。

流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平
全球使用Kirkstall公司Quasi Vivo流动培养系统的学术及研究机构已达70+个,包括:
1. 美国
2. 英国
3. 法国
4. 瑞典
5. 奥地利
6. 意大利
7. 荷兰
8. 瑞士
9. 日本

目前Quasi Vivo流动培养系统已成功用于以下器官模型的培养:
1. 呼吸系统(培养的热点)
2. 肝脏
3. 肾脏
4. 心血管
5. 成纤维细胞
6. 糖尿病模型
7. 血脑屏障
8. 脑组织类器官


【不同型号Quasi Vivo系统选哪个?】
1、QV500:适用于单一细胞培养,所有腔室培养相同的细胞。
流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平

2、QV600:适用于多细胞共培养,每个腔室培养2种或以上细胞。
流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平

 

3、QV900:用于多种细胞共培养,可以使管路上游的细胞培养基成为下游细胞的条件培养基。
流动培养提高人支气管上皮细胞分化 人小气道上皮细胞分化 quasi vivo流动培养中国独家代理商北京泽平

 

Quasi Vivo流动培养系统,贴壁细胞培养实验指导

 

 

Quasi Vivo参考文献

1.Tommaso S. et al., 2011. Engineering Quasi-Vivo in vitro organ models. Advances in Experimental Medicine and Biology Volume: 745, pp 138-153.
2.Patricia M. et al., 2018. A novel dynamic multicellular co-culture system for studying individual blood-brain barrier cell types in brain diseases and cytotoxicity testing. Scientific Reports Volume: 8, Issue: 1, pp 8784.
3.Basma E. et al. 2020. A dynamic perfusion based blood-brain barrier model for cytotoxicity testing and drug permeation. Scientific Reports Volume: 10, Issue: 1, pp 3788.
4.Miranda A. et al., 2016. A three dimensional (3D) human in vitro blood-brain barrier (BBB). Heart Volume: 102.
5.Buesch S. et al., 2018. A Novel In Vitro Liver Cell Culture Flow System Allowing Long-Term Metabolism and Hepatotoxicity Studies. Applied In Vitro Toxicology Volume: 4, Issue: 3, pp 232-237.
6.Alec O. et al., 2019. Development of an in vitro media perfusion model of Leishmania major macrophage infection. 2019 PLOS ONE Volume: 14, Issue: 7.
7.Sean M. et al., 2017. In-silico Characterisation of the Kirkstall QV900 In-Vitro System for Advanced Cell Culture. 5th International Conference on Computational and Mathematical Biomedical Engineering pp 1174-1177.
8.Ahluwalia A. et al., 2011. Hepatotoxicity of diclofenac in a Quasi-Vivo™ multicompartment bioreactor. oxicology Letters Volume: 205. 
9.Tomlinson, L. et al., 2019. In vitro liver zonation of primary rat hepatocytes.Front. Bioeng. Biotechnol., 7(17). 
10.Elbakary, B. and Badhan R. K. S, 2020. A dynamic perfusion based blood brain barrier model for cytotoxicity testing and drug permeation. Scientific Reports, 10(1),3788. 
11.O’Keefe, A. et al., 2019. Development of an in vitro media perfusion model of Leishmania major macrophage infection. Plos One, 14(7).  
12.Miranda-Azpiazu, P. et al., 2018. A novel dynamic multicellular co-culture system for studying individual blood-brain barrier cell types in brain diseases. Scientific Reports, 8, 8784. 
13.Chandorkar, P. et al., 2017. Fast-track development of an in vitro 3D lung/immune cell model to study Aspergillus infections. Scientific Reports, 7, 11644. 
14.Iori, E. et al., 2012. Glucose and fatty acid metabolism in a 3 tissue in-vitro model challenged with normo- and hyperglycaemia. PLoS ONE, 7(4).  
15.Mattei, G., Giusti, S. & Ahluwalia, A., 2014. Design Criteria for Generating Physiologically Relevant In Vitro Models in Bioreactors. Processes, 2(3).   
16.Mazzei, D. et al., 2010. A low shear stress modular bioreactor for connected cell culture under high flow rates. Biotechnology and Bioengineering, 106.  
17.Nithiananthan, S. et al., 2016. Physiological Fluid Flow Moderates Fibroblast Responses to TGF-β1. Journal of Cellular Biochemistry, 13.   
18.Ramachandran, S.D. et al., 2015. In vitro generation of functional liver organoid-like structures using adult human cells. PLoS ONE, 10(10).  
19.Rashidi, H. et al., 2016. Fluid shear stress modulation of hepatocyte like cell function. Archives of Toxicology, 90, 7.  
20.Vinci, B. et al., 2012. An in vitro model of glucose and lipid metabolism in a multicompartmental bioreactor. Biotechnology Journal, 7. 
21.Iori, E. et al., 2012. Glucose and fatty acid metabolism in a 3 tissue in-vitro model challenged with normo- and hyperglycaemia. PLoS ONE, 7(4), pp.1–9. 
22.Mattei, G., Giusti, S. & Ahluwalia, A., 2014. Design Criteria for Generating Physiologically Relevant In Vitro Models in Bioreactors. Processes, 2, pp.548–569.  
23.Mazzei, D. et al., 2010. A low shear stress modular bioreactor for connected cell culture under high flow rates. Biotechnology and Bioengineering, 106, pp.127–137. 
24.Nithiananthan, S. et al., 2016. Physiological Fluid Flow Moderates Fibroblast Responses to TGF-β1. Journal of cellular biochemistry, 13(October), pp.1–13. Available at: 
25.Ramachandran, S.D. et al., 2015. In vitro generation of functional liver organoid-like structures using adult human cells. PLoS ONE, 10(10), pp.1–14. 
26.Rashidi, H. et al., 2016. Fluid shear stress modulation of hepatocytelike cell function. Archives of Toxicology, pp.3–7. 
27.Iori, E. et al., 2012. Glucose and fatty acid metabolism in a 3 tissue in-vitro model challenged with normo- and hyperglycaemia. PLoS ONE, 7(4).  
28.Vinci, B. et al., 2011. Modular bioreactor for primary human hepatocyte culture: Medium flow stimulates expression and activity of detoxification genes. Biotechnology Journal, 6, pp.554–564. 
29.Tommaso S. et al., 2011. Engineering Quasi-Vivo in vitro organ models. Advances in Experimental Medicine and Biology Volume: 745, pp 138-153.
30.Ahluwalia A. et al., 2011. Hepatotoxicity of diclofenac in a Quasi-Vivo™ multicompartment bioreactor. oxicology Letters Volume: 205. 

 

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-流动培养用于糖尿病模型cross-talk研究

 

LONZA人原代细胞相关产品

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——Quasi Vivo中国独家代理商,北京泽平

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