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3D-Printed electrochemical sensor-integrated transwell systems

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Microsystems & Nanoengineering 2020年第1期6卷 357-368页

作者： Pradeep Ramiah Rajasekaran Ashley Augustiny Chapin David N.Quan Jens Herberholz William E.Bentley Reza Ghodssi Institute for Systems Research University of MarylandCollege ParkMDUSA Fischell Department of Bioengineering University of MarylandCollege ParkMDUSA Department of Psychology and Neuroscience and Cognitive Science Program University of MarylandCollege ParkMDUSA Institute for Bioscience and Biotechnology Research University of MarylandCollege ParkMDUSA Robert E.Fischell Institute for Biomedical Devices University of MarylandCollege ParkMDUSA Department of Electrical and Computer Engineering University of MarylandCollege ParkMDUSA

This work presents a 3D-printed,modular,electrochemical sensor-integrated transwell system for monitoring cellular and molecular events in situ without sample extraction or microfluidics-assisted downstream omics.Simple additive manufacturing techniques such as 3D printing,shadow masking,and molding are used to fabricate this modular system,which is autoclavable,biocompatible,and designed to operate following standard operating protocols(SOPs)of cellular biology.Integral to the platform is a flexible porous membrane,which is used as a cell culture substrate similarly to a commercial transwell insert.Multimodal electrochemical sensors fabricated on the membrane allow direct access to cells and their products.A pair of gold electrodes on the top side of the membrane measures impedance over the course of cell attachment and growth,characterized by an exponential decrease(~160%at 10Hz)due to an increase in the double layer capacitance from secreted extracellular matrix(ECM)proteins.Cyclic voltammetry(CV)sensor electrodes,fabricated on the bottom side of the membrane,enable sensing of molecular release at the site of cell culture without the need for downstream fluidics.Real-time detection of ferrocene dimethanol injection across the membrane showed a three order-of-magnitude higher signal at the membrane than in the bulk media after reaching equilibrium.This modular sensor-integrated transwell system allows unprecedented direct,real-time,and noninvasive access to physical and biochemical information,which cannot be obtained in a conventional transwell system.

关键词： transwell electrochemical culture

In vitro study of the nephrotoxicity of total Dahuang(Radix Et Rhizoma Rhei Palmati) anthraquinones and emodin in monolayer human proximal tubular epithelial cells cultured in a transwell chamber

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Journal of Traditional Chinese Medicine 2019年第5期39卷 609-623页

作者： Cao Chunyu Hui Lianqiang Li Chun Yang Yifei Zhang Jiyuan Liu Ting Hao Ran Zhang Yi Safety Evaluation Center for Chinese Materia Medica Institute of Chinese Materia MedicaChina Academy of Chinese Medical SciencesBeijing 100700China Quality Standard Center for Chinese Materia Medica Institute of Chinese Materia MedicaChina Academy of Chinese Medical SciencesBeijing 100700China Department of Sciences and Education Institute of Chinese Materia MedicaChina Academy of Chinese Medical SciencesBeijing 100700China Pharmacology Research Center for Chinese Materia Medica Institute of Chinese Materia MedicaChina Academy of Chinese Medical SciencesBeijing 100700China

OBJECTIVE: To examine changes in the morphology and physiological functions of human proximal tubular epithelial cells (HK-2 cells) caused by total Dahuang (Radix Et Rhizoma Rhei Palmati) anthraquinones (TDA) and emodin. METHODS: HK-2 cells were cultured on polycarbonate (PCF) membranes to form a complete monolayer of cells. A fluorescein isothiocyanate- dextran (FITC) permeability assay was conducted and secretion of γ-glutamyltranspeptidase (GGT), lactate dehydrogenase (LDH), N-acetyl-β-D-glucosaminidase (NAG) and kidney injury molecule 1 (KIM-1) was examined. The reabsorption of glucose and the excretion of para-aminohippuric acid (PAH) by HK-2 cells were also examined. The morphology of HK-2 cells was observed using optical microscopy and scanning electron microscopy. The cytoskeleton of HK-2 cells was observed under a fluorescence microscope. RESULTS: Compared with the results for the dimethyl sulfoxide group, treatment of cells with TDA and emodin showed statistically significant differences in the FITC leakage rate, the apical / basolateral ratio of LDH and GGT, and the secretion of GGT, LDH, NAG and KIM-1. At 64 μg/mL, TDA markedly inhibited blood glucose reabsorption and remarkably suppressed PAH excretion by HK-2 cells. Both TDA and emodin caused various degrees of damage to the morphology and cytoskeleton of HK-2 cells with the degree of damage correlating positively with the dosage of the tested substances.CONCLUSION: Both TDA and emodin caused damage to human renal proximal tubular epithelial cells at certain dosages. At the same dosage, TDA caused more severe damage than emodin to the HK-2 cells.

关键词： Anthraquinones Rheum Emodin Nephrotoxicity Human proximal tubular epithelial cell transwell chamber

transwell 3-D联合共培养促使人羊水克隆样细胞来源干细胞向肝样细胞分化的研究

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中华实验外科杂志 2015年第6期32卷 1323-1328页

作者：张胜利范应中郑州大学第一附属医院小儿外科 450052

目的探讨transwell 3-D联合共培养促使人羊水克隆样细胞来源干细胞向肝样细胞分化的可能性.方法利用克隆杯,从人孕中期羊水标本中分离获得人羊水克隆样细胞来源干细胞,培养扩增后,通过流式细胞仪检测表面抗原进行鉴定.利用transwell... 详细信息

目的探讨transwell 3-D联合共培养促使人羊水克隆样细胞来源干细胞向肝样细胞分化的可能性.方法利用克隆杯,从人孕中期羊水标本中分离获得人羊水克隆样细胞来源干细胞,培养扩增后,通过流式细胞仪检测表面抗原进行鉴定.利用transwell小室联合共培养肝细胞诱导其向肝样细胞分化,观察诱导后细胞形态的变化,利用细胞免疫荧光检测甲胎蛋白（AFP）、白蛋白（ALB）和细胞角蛋白-18（CK-18）在诱导后细胞中的表达,反转录-聚合酶链反应（RT-PCR）检测肝细胞相关基因的表达.结果利用克隆杯分离出的人羊水克隆样细胞来源干细胞CD44和CD90表达阳性,CD10、CD14、CD34、CD45和人类白细胞抗原DR基因（HLA-DR）表达阴性.诱导2～4周后,肝样细胞出现,检测证实这些细胞表达AFP、ALB、CK-18、GATA结合蛋白-4（GATA4）、肝细胞核因子（HNF）-1α和细胞色素20A（CYP20A）等肝细胞相关标志和基因.结论 transwell 3-D联合共培养可以促使人羊水克隆样细胞来源干细胞向肝样细胞分化.

关键词：人羊水克隆样细胞来源干细胞 transwell 3-D联合共培养肝样细胞

transwell和Wound healing在细胞迁移中的应用比较

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中国临床解剖学杂志 2007年第6期25卷 687-689页

作者：唐杰陈龙菊王军王配军陈道帮四川大学华西基础与法医学院人体解剖学教研室成都610041 郧阳医学院解剖教研室湖北十堰442000

目的:比较分析transwell和划痕法(Wound healing)检测细胞迁移能力的差异,探讨两种方法在血清饥饿调节的血管平滑肌细胞迁移中的应用。方法:用DMEM和含10%小牛血清的DMEM分别作用于体外培养的大鼠血管平滑肌细胞24h,采用transwell迁移... 详细信息

目的:比较分析transwell和划痕法(Wound healing)检测细胞迁移能力的差异,探讨两种方法在血清饥饿调节的血管平滑肌细胞迁移中的应用。方法:用DMEM和含10%小牛血清的DMEM分别作用于体外培养的大鼠血管平滑肌细胞24h,采用transwell迁移小室和划痕方法检测血清饥饿(serum starvation)和有血清条件下平滑肌细胞的迁移能力。结果:transwell法和划痕法分别显示了VSMC在血清饥饿组的迁移数目和距离明显比10%血清刺激组少和短。结论:两种方法都能较好地反映细胞的迁移能力,即血清饥饿组明显低于10%血清刺激组。但划痕法更适用于本实验。

关键词： transwell 划痕血管平滑肌细胞血清饥饿

transwell培养体系中人AGM区基质细胞支持脐血CD34^+细胞造血

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中山大学学报（医学科学版） 2005年第2期26卷 231-235,239页

作者：张绪超陈惠芹黄绍良吴燕峰包蓉吴北燕周敦华中山大学附属第二医院干细胞研究中心广东广州510120

【目的】探讨transwell系统中人胚主动脉-性腺-中肾(AGM)区基质细胞对脐带血造血干/祖细胞的造血能力长期维持及扩增的作用。【方法】采用免疫磁珠方法分离人脐带血CD34+细胞,接种于底层铺有人AGM区基质细胞的transwell培养板的Inserts... 详细信息

【目的】探讨transwell系统中人胚主动脉-性腺-中肾(AGM)区基质细胞对脐带血造血干/祖细胞的造血能力长期维持及扩增的作用。【方法】采用免疫磁珠方法分离人脐带血CD34+细胞,接种于底层铺有人AGM区基质细胞的transwell培养板的Inserts中,非接触共培养7-35 d,每星期取样检测细胞总数,用半同体培养基分析CFU-C及HPP-CFU集落形成数,流式细胞术检测CD34+、CD34+CD38-细胞百分率。【结果】在transwell中非接触共培养条件下,人AGM区基质细胞培养体系较胚胎躯干基质细胞和无饲养层培养体系对有核细胞总数、CFC和CD34+细胞均具有明显的扩增作用,共培养14 d的CD34+、CD34+CD38-造血干/祖细胞均获得峰值扩增(2.62±0.8和2.15±1.04,P<0.05),而MNC总数和CFC均在21 d获得最大扩增(32.5±4.3和4.2±0.6倍,P<0.05).克隆分析发现CFU-Mix、CFU-GM、BFU-E在共培养4-5星期后均仍然可见。原始祖细胞HPP-CFC在3星期也得到2.23倍的扩增,较空白及hFT对照组均有显著性差异(P<0.05),并在共培养35 d 后仍可见HPP-CFU集落形成。【结论】人AGM区基质细胞hAGM-S3/hAGM-S4均具有造血支持作用,在非接触共培养条件下中可长期维持脐血中造血干/祖细胞的多系造血能力和高增殖潜能达21～35 d,对脐血CD34+/

关键词： transwell 主动脉-性腺-中肾基质细胞 CD34^+细胞

<i>In Vitro</i>Study of the Nephrotoxicity of Tripterygium Tablet Extract and Triptolide in Monolayer HK-2 Cells Cultured in a transwell Chamber

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Chinese Medicine 2018年第1期9卷 34-54页

作者： Ran Hao Lianqiang Hui Chun Li Chunyu Cao Yifei Yang Jiyuan Zhang Ting Liu Yi Zhang Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences Beijing China

We established a monolayer polarized cell model using human kidney 2 (HK-2) cells cultured in a transwell chamber to examine the changes in the morphology and physiological functions of human-derived renal proximal tubular epithelial cells caused by tripterygium tablet extract (TTE) and triptolide. HK-2 cells were cultured on PCF membranes to form a complete monolayer of cells. A MTT assay was used to select 10, 40, 160, 640 μg·ml-1 TTE or 4, 16, 64, 256 ng·ml-1 triptolide to treat HK-2 monolayer cells. After 24 hours, a FITC permeability assay was performed;GGT, LDH and NAG secretion on the apical (AP) and basolateral (BL) sides of the cells by HK-2 cells were examined. The morphology and the monolayer structure of HK-2 cells was observed via optical microscope and scanning electron microscope, respectively. The effect on the cytoskeleton of HK-2 cells was observed under a fluorescence microscope. The IC50 of TTE was 277.122 μg·ml-1, and the IC50 of triptolide was 148.035 ng·ml-1. Compared with the DMSO group, the FITC leakage rate with TTE 160, 640 μg·ml-1 treated group and 4 - 256 ng·ml-1 triptolide dose group exhibited statistically significant increase. TTE significantly increased secretion of GGT and LDH at 160, 640 μg·ml-1, meanwhile, dramatically increased the AP/BL ratio of LDH at 160 μg·ml-1;triptolide significantly increased secretion and AP/BL ratio of GGT and LDH at 256 ng·ml-1. The morphological observations via optical and electron microscope indicated various degrees of damage to HK-2 cells by TTE and triptolide, and the degree of damage correlated positively with the dosage of the tested articles. Compared with DMSO group, the cellular damage degrees at TTE dosages of 40 - 640 μg·ml-1 and triptolide dose group at 16, 256 ng·ml-1 exhibited statistically significant differences via observation under optical microscope. Both TTE and triptolide caused various degrees of shortening and thickening of intracellular F-actin bundles of HK-2 cells;aggravation of these changes was observed with increasing d

关键词： Nephrotoxicity Tripterygium Tablet Extract Triptolide HK-2 Cell transwell Chamber

Suppression of matrix metalloproteinase-2 via RNA interference inhibits pancreatic carcinoma cell invasiveness and adhesion

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World Journal of Gastroenterology 2009年第9期15卷 1072-1078页

作者： Ying-Hui Zhi Mao-Min Song Pi-Lin Wang Tie Zhang Zi-Yi Yin Department of General Surgery Affiliated Beijing Tiantan Hospital Capital Medical University Beijing 100050 China

AIM:To investigate the inhibitory effects of RNA interference (RNAi) on expression of matrix metalloproteinase-2 (MMP-2) gene and invasiveness and adhesion of human pancreatic cancer cell line,BxPC-3.METHODS:RNAi was performed using the vector (pGPU6)-based small interference RNA (siRNA) plasmid gene silence system to specifically knock down MMP-2 expression in pancreatic cancer cell line,BxPC-3. Four groups of different specific target sequence in coding region of MMP-2 and one non-specific sequence were chosen to construct four experimental siRNA plasmids of pGPU6-1,pGPU6-2,pGPU6-3 and pGPU6-4,and one negative control siRNA plasmid of pGPU6 (-). MMP-2 expression was measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation and apoptosis were examined by methyl thiazolyl tetrazolium (MTT) and flow cytometry,respectively. The abilities of adhesion and invasion were detected by cell adhesion assay and cell invasion assay using transwell chambers.RESULTS:The expression of MMP-2 was inhibited and the inhibitory effects of different sequence varied. pGPU6-1 group had the most efficient inhibitory effect,followed by pGPU6-2 and pGPU6-3 groups.Invasiveness and adhesion were more significantly reduced in pGPU6-1,pGPU6-2 and pGPU6-3 groups as compared with pGPU6 (-) and blank control groups. However,no difference concerning cell proliferation and apoptosis was observed after transfection between experiment groups and control groups.CONCLUSION:RNAi against MMP-2 successfully inhibited the mRNA and protein expression of MMP-2 in the pancreatic cancer cell line,BxPC-3,leading to a potent suppression of tumor cell adhesion and invasion without affecting cell proliferation and apoptosis. These findings suggest that the RNAi approach towards MMP-2 may be an effective therapeutic strategy for the clinical management of pancreatic tumor.

关键词：基质金属蛋白酶-2 人胰腺癌细胞系肿瘤细胞粘附 RNAi技术癌细胞侵袭干扰抑制逆转录聚合酶链反应 transwell

Neural cell injury microenvironment induces neural differentiation of human umbilical cord mesenchymal stem cells

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Neural Regeneration Research 2012年第34期7卷 2689-2697页

作者： Jin Zhou Guoping Tia Jinge Wang Xiaoguang Luo Siyang Zhang Jianping Li Li Li Bing Xu FengZhu Xia Wang Chunhong Jia Weijin Zhao Danyang Zhao Aihua Xu Department of Neurology First People's Hospital of Shenyang Shenyang 110041 Liaoning Province China First Affiliated Hospital of China Medical University Shenyang 110001 Liaoning Province China College of Basic Medical Sciences China Medical University Shenyang 110001 Liaoning Province China Liaoning Provincial Blood Center Shenyang 110044 Liaoning Province China

This study aimed to investigate the neural differentiation of human umbilical cord mesenchymal stem cells （hUCMSCs） under the induction of injured neural cells. After in vitro isolation and culture, passage 5 hUCMSCs were used for experimentation, hUCMSCs were co-cultured with normal or AI31.4o-injured PC12 cells, PC12 cell supernatant or PC12 cell lysate in a transwell co-culture system. Western blot analysis and flow cytometry results showed that choline acetyltransferase and microtubule-associated protein 2, a specific marker for neural cells, were expressed in hUCMSCs under various culture conditions, and highest expression was observed in the hUCMSCs co-cultured with injured PC12 cells. Choline acetyltransferase and microtubule-associated protein 2 were not expressed in hUCMSCs cultured alone （no treatment）. Cell Counting Kit-8 assay results showed that hUCMSCs under co-culture conditions promoted the proliferation of injured PC12 cells. These findings suggest that the microenvironment during neural tissue injury can effectively induce neural cell differentiation of hUCMSCs. These differentiated hUCMSCs likely accelerate the repair of injured neural ceils.

关键词： stem ceil umbilical cord mesenchymal stem cell co-culture induction differentiation neural cell microtubule-associated protein 2 injured cell transwell neural regeneration regeneration

Puerarin exhibits greater distribution and longer retention time in neurons than astrocytes in a co-cultured system

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Neural Regeneration Research 2015年第4期10卷 605-609页

作者： Shu-Yong Wei Jie Tong Qiang Xue Fang-hong Shang Yan-jun Li Yang Liu Bin-bin Feng Xiao-yu Xu Pharmaceutical Sciences College & Chinese Medicine College of Southwest University Chongqing Engineering Research Center for Pharmacodynamics Evaluation Rongchang Campus of Southwest University Chongqing Three Gorges Medical College

The phytoestrogen puerarin has been shown to protect neurons and astrocytes in the brain, and is therefore an attractive drug in the treatment of Alzheimer’s disease, Parkinson’s disease and cerebral ischemia. Whether puerarin exhibits the same biological processes in neurons and astro-cytesin vitro has rarely been reported. In this study, cortical neurons and astrocytes of newborn Sprague-Dawley rats were separated, identiifed and co-cultured in a system based on transwell membranes. The retention time and distribution of puerarin in each cell type was detected by lfuorescence spectrophotometry and lfuorescence microscope. The concentration of puerarin in both co-cultured and separately cultured neurons was greater than that of astrocytes. Puerarin concentration reached a maximum 20 minutes after it was added. At 60 minutes after its addi-tion, a scant amount of drug was detected in astrocytes; however in both separately cultured and co-cultured neurons, the concentration of puerarin achieved a stable level of about 12.8 ng/mL. The results indicate that puerarin had a higher concentration and longer retention time in neu-rons than that observed in astrocytes.

关键词： nerve regeneration puerarin in vitro experiments co-culture neurons astrocytes transwell neonatal rats neural regeneration