英國(guó)Kirkstall公司生產(chǎn)的創(chuàng)新性細(xì)胞培養(yǎng)產(chǎn)品,其Quasi Vivo灌流培養(yǎng)系統(tǒng)可為細(xì)胞培養(yǎng)提供持久恒定的流動(dòng)培養(yǎng)環(huán)境,zui大限度模擬體內(nèi)環(huán)境。相比各類(lèi)傳統(tǒng)靜態(tài)培養(yǎng)系統(tǒng),Quasi Vivo流動(dòng)培養(yǎng)系統(tǒng)更大限度模擬了細(xì)胞在體內(nèi)生長(zhǎng)環(huán)境,其流動(dòng)培養(yǎng)方式更利于培養(yǎng)基內(nèi)營(yíng)養(yǎng)物質(zhì)的擴(kuò)散和細(xì)胞代謝產(chǎn)物的運(yùn)輸,更有利于復(fù)雜細(xì)胞模型的構(gòu)建,尤其適合需要在氣液界面進(jìn)行分化的各類(lèi)呼吸道上皮細(xì)胞的生長(zhǎng)。

quan球使用Kirkstall公司Quasi Vivo灌流培養(yǎng)系統(tǒng)的學(xué)術(shù)及研究機(jī)構(gòu)已達(dá)70+個(gè),遍布美國(guó)、英國(guó)、法國(guó)、瑞典、奧地利、意大利、荷蘭、瑞士、日本等。目前Quasi Vivo灌流培養(yǎng)系統(tǒng)已成功用于以下器官模型的培養(yǎng):

呼吸道上皮細(xì)胞的氣液界面培養(yǎng)是研究經(jīng)空氣傳播的病原體,如SARS等的常用的模型。傳統(tǒng)的培養(yǎng)方式是用TransWell在普通培養(yǎng)箱中靜置培養(yǎng)。但是此種培養(yǎng)方式wu法模擬培養(yǎng)過(guò)程中營(yíng)養(yǎng)物質(zhì)和代謝廢物在組織內(nèi)的運(yùn)輸,培養(yǎng)得到的模型通常有各種各樣的缺陷,并且所需實(shí)驗(yàn)周期較長(zhǎng)。

Quais Vivo(QV600)灌流培養(yǎng)系統(tǒng)(腔室+儲(chǔ)液瓶+底座+管道+泵等)

而Quasi Vivo灌流培養(yǎng)系統(tǒng)可為細(xì)胞培養(yǎng)提供持久恒定的流動(dòng)培養(yǎng)環(huán)境,zui大限度模擬體內(nèi)環(huán)境。研究發(fā)現(xiàn),使用Quasi Vivo系統(tǒng)進(jìn)行灌流培養(yǎng)與靜態(tài)培養(yǎng)相比,氣液界面培養(yǎng)的呼吸道上皮細(xì)胞(正常人氣管上皮細(xì)胞 Normal Human Bronchial Epithelial Cells,簡(jiǎn)稱(chēng)NHBE;小氣道上皮細(xì)胞 Small Airway Epithelial Cells,簡(jiǎn)稱(chēng)SAE),發(fā)育分化速度更快,表現(xiàn)為纖毛分化度更高,纖毛運(yùn)動(dòng)更強(qiáng)、粘液產(chǎn)生和屏障功能更強(qiáng)。在灌注下加速分化后,將上皮細(xì)胞轉(zhuǎn)移到靜態(tài)條件下,并添加抗原呈遞細(xì)胞(APC)以研究其在病原體感染后的功能。(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.)

01、人體內(nèi)鎖有的細(xì)胞都需要營(yíng)養(yǎng)物質(zhì)和代謝廢物的流動(dòng)

02、肺部氣管/支氣管和小氣道上皮結(jié)構(gòu)精細(xì),進(jìn)行體外培養(yǎng)模擬體內(nèi)環(huán)境,對(duì)呼吸道病原體的研究至關(guān)重要

03、采用quan新的灌流培養(yǎng)方式培養(yǎng)呼吸道上皮細(xì)胞(采用QV600)

相比使用transwell靜止培養(yǎng)(Static Conditions),Quasi Vivo灌流培養(yǎng)系統(tǒng)(Perfused Conditions)中,呼吸道上皮細(xì)胞的生長(zhǎng)和分化呈現(xiàn)更好狀態(tài)

04、電鏡照片顯示,采用灌流培養(yǎng)方式(Perfused conditions)的呼吸道上皮細(xì)胞,分化程度更高

05、使用MUC5B染色可以發(fā)現(xiàn),采用灌流培養(yǎng)方式(Perfused conditions)的呼吸道上皮細(xì)胞,在培養(yǎng)的弟7天即可分泌大量粘液。用OCCLUDIN染色可以發(fā)現(xiàn),細(xì)胞間的緊密連接發(fā)育更完善

06、使用WGA染色發(fā)現(xiàn),采用灌流培養(yǎng)方式(Perfused conditions)的呼吸道上皮細(xì)胞,纖毛分化度更高

07、測(cè)量TEER(經(jīng)細(xì)胞電阻),采用灌流培養(yǎng)方式(Perfused conditions)的呼吸道上皮細(xì)胞TEER值更大,代表得到的上皮細(xì)胞膜狀結(jié)構(gòu)更完整

:使管路上游的細(xì)胞培養(yǎng)基成為下游細(xì)胞的條件培養(yǎng)基。

流動(dòng)培養(yǎng)形成含血管的3D心臟組織 | 再生醫(yī)學(xué)

在再生醫(yī)學(xué)領(lǐng)域,怎樣培養(yǎng)出含血管的組織,是未來(lái)應(yīng)用能否成功的關(guān)鍵之一。早期的臨床試驗(yàn)采用生長(zhǎng)因子或細(xì)胞注射的方法來(lái)修補(bǔ)損傷的心臟,但由于注射細(xì)胞造成的炎癥反應(yīng)和局部缺血會(huì)在體內(nèi)造成低氧環(huán)境,使得注射的細(xì)胞定植率低而死亡率高,不能有效地修復(fù)損傷的心臟功能。

Quasi Vivo QV500流動(dòng)培養(yǎng)系統(tǒng)為接種在明膠支架上的人間充質(zhì)干細(xì)胞(hMSCs)和人心肌祖細(xì)胞(hCMPC)提供充足的氧氣,促進(jìn)細(xì)胞和營(yíng)養(yǎng)物質(zhì)向支架核心內(nèi)擴(kuò)散,并能快速有效地排除組織內(nèi)的代謝廢物,促進(jìn)血管生成,從而形成由血管樣和心臟樣細(xì)胞組成的組織結(jié)構(gòu)密集的適于體內(nèi)移植的原組織。(Pagliari S, et al. A multistep procedure to prepare pre-vascularized cardiac tissue constructs using adult stem cells, dynamic cell cultures, and porous scaffolds. Frontiers in Physiology. 2014; 5: 210)

Quasi-Vivo流動(dòng)培養(yǎng)系統(tǒng) (QV500型)的蠕動(dòng)泵將培養(yǎng)基從儲(chǔ)液瓶泵到兩個(gè)串聯(lián)的培養(yǎng)腔室內(nèi),并能保持恒定流速(200μl/min),保證多孔明膠支架內(nèi)層的培養(yǎng)基流動(dòng)。

構(gòu)建含血管的3D心臟的實(shí)驗(yàn)方案示意圖。明膠多孔支架被浸入稀釋的Matrigel中,然后轉(zhuǎn)移至內(nèi)皮分化培養(yǎng)基中。之后將人間充質(zhì)干細(xì)胞接種在支架上,使人間充質(zhì)干細(xì)胞定植在支架培養(yǎng)上并向內(nèi)皮進(jìn)行分化,96小時(shí)后,將在聚苯乙烯細(xì)胞培養(yǎng)板用心臟分化培養(yǎng)基預(yù)先定型2周的心臟TNT-GFP人心肌祖細(xì)胞接種于血管化的支架上,用QV500流動(dòng)培養(yǎng)系統(tǒng)在心臟分化培養(yǎng)基中培養(yǎng)7天。

采用上述實(shí)驗(yàn)方案,對(duì)用QV500培養(yǎng)一周后的共培養(yǎng)結(jié)構(gòu)進(jìn)行檢測(cè),發(fā)現(xiàn)在支架上有大量細(xì)胞定殖。

QV500流動(dòng)培養(yǎng)條件下支架內(nèi)部浸潤(rùn)了大量的血管樣細(xì)胞(紅色)和人心肌前體細(xì)胞(hCMPC)衍生的心肌細(xì)胞(綠色),而靜態(tài)培養(yǎng)條件下,細(xì)胞大部分分布在支架表面。

免疫組化結(jié)果顯示通過(guò)QV500動(dòng)態(tài)培養(yǎng)可以促進(jìn)心肌樣細(xì)胞(GFP,綠色)和內(nèi)皮樣細(xì)胞(VCAM-1陽(yáng)性細(xì)胞,紅色)向支架內(nèi)部浸潤(rùn)。

(A) 切片顯示QV500流動(dòng)培養(yǎng)的內(nèi)皮樣細(xì)胞(VCAM-1陽(yáng)性細(xì)胞,紅色)排列成孔狀,形成管狀結(jié)構(gòu),并與心肌樣細(xì)胞(GFP,綠色)接觸。(B)QV500流動(dòng)培養(yǎng)條件下,支架內(nèi)廣泛的細(xì)胞分布導(dǎo)致形成密集組裝的多細(xì)胞組織,該組織衍生自所用的人間充質(zhì)干細(xì)胞(hMSCs)和人心肌前體細(xì)胞(hCMPC)。

總結(jié):在本文中使用的QV500流動(dòng)培養(yǎng)系統(tǒng),能增強(qiáng)氧氣與營(yíng)養(yǎng)物質(zhì)的運(yùn)輸,進(jìn)而增強(qiáng)工程化心血管組織的活性和功能。

與眾不同的Quasi Vivo流動(dòng)培養(yǎng)系統(tǒng),讓日、美、英、法、瑞士、瑞典等quan球70多個(gè)研究機(jī)構(gòu)獲得了更強(qiáng)大的細(xì)胞培養(yǎng)工具,在包括呼吸系統(tǒng)、心血管系統(tǒng)、肝臟、腎臟、腸道、腦組織類(lèi)器官,以及糖尿病的研究上更進(jìn)一步。

流動(dòng)培養(yǎng)實(shí)現(xiàn)血腦屏障三種細(xì)胞共培養(yǎng) | 阿爾茨海默病新模型

血腦屏障(blood-brain barrier, BBB)在中樞神經(jīng)系統(tǒng)(CNS)的生理和病理中都起著重要的作用。血腦屏障功能異常會(huì)引起包括阿爾茨海默癥(AD)等許多神經(jīng)退行性疾病。組成血腦屏障的毛細(xì)血管內(nèi)皮細(xì)胞(capillary endothelial cells)、周細(xì)胞(pericytes)以及星形膠質(zhì)細(xì)胞(astrocytes)間的復(fù)雜的相互作用使得很難在體內(nèi)確定這三種細(xì)胞對(duì)神經(jīng)毒性各自的貢獻(xiàn)。

而Quasi Vivo流動(dòng)培養(yǎng)系統(tǒng)可為體外培養(yǎng)這三種細(xì)胞提供在不形成屏障的情況下維持細(xì)胞間通訊的zui佳培養(yǎng)環(huán)境。Quasi Vivo流動(dòng)培養(yǎng)系統(tǒng)為未來(lái)研究不同類(lèi)型的血腦屏障細(xì)胞在中樞神經(jīng)系統(tǒng)疾病和細(xì)胞毒性試驗(yàn)中的te殊作用提供一個(gè)有價(jià)值的工具。(Miranda-Azpiazu P, et al. A novel dynamic multicellular co-culture system for studying individual blood-brain barrier cell types in brain diseases and cytotoxicity testing. Sci Rep. 2018; 8(1): 1-10.)

圖 1. 單du培養(yǎng)的人星形膠質(zhì)細(xì)胞(A,GFAP陽(yáng)性)、周細(xì)胞(B,α-actin陽(yáng)性)、血管內(nèi)皮細(xì)胞(C,CD31陽(yáng)性)以及血管內(nèi)皮細(xì)胞形成的緊密連接(D,ZO1陽(yáng)性)。

圖 2 用Quasi-Vivo QV500培養(yǎng)共享相同的培養(yǎng)基的星形膠質(zhì)細(xì)胞、周細(xì)胞和血管內(nèi)皮細(xì)胞的示意圖(A),R為儲(chǔ)液瓶,P為蠕動(dòng)泵。連接培養(yǎng)基存儲(chǔ)瓶的一個(gè)Quasi-Vivo QV500流動(dòng)培養(yǎng)系統(tǒng)的細(xì)胞培養(yǎng)腔室(B)。

圖 3 Quasi-Vivo QV500流動(dòng)培養(yǎng)系統(tǒng)建立的能同時(shí)培養(yǎng)三種不同細(xì)胞的多細(xì)胞共培養(yǎng)體系。

圖4 幾種流動(dòng)培養(yǎng)方式示意圖:A圖為單du星形角質(zhì)細(xì)胞流動(dòng)培養(yǎng),B圖為單du周細(xì)胞流動(dòng)培養(yǎng),C圖為單du血管內(nèi)皮細(xì)胞流動(dòng)培養(yǎng),D圖為三種細(xì)胞組合后一起流動(dòng)培養(yǎng)。

圖5 用MTT法測(cè)細(xì)胞活力,與靜態(tài)培養(yǎng)相比,采用Quasi-Vivo QV500流動(dòng)培養(yǎng)系統(tǒng)對(duì)單du培養(yǎng)血管內(nèi)皮細(xì)胞(HBECs)、周細(xì)胞(HBVPs)、星形角質(zhì)細(xì)胞(HAs)(A)或三種細(xì)胞共培養(yǎng)(B)的血管內(nèi)皮細(xì)胞的細(xì)胞活力有明顯升高。

圖6 用MTT法測(cè)細(xì)胞活力,與靜態(tài)培養(yǎng)(Static)相比,流動(dòng)培養(yǎng)(Dynamic)的周細(xì)胞(HBVPs)會(huì)更早受到Aβ25-35(淀粉樣蛋白β肽的Aβ25-35片段,用于阿爾茨海默病的造模)的毒害。

總結(jié):本文中研究者利用Quasi-Vivo QV500流動(dòng)培養(yǎng)系統(tǒng)建立了三種細(xì)胞的共培養(yǎng)。這些細(xì)胞不接觸,通過(guò)共享培養(yǎng)基實(shí)現(xiàn)細(xì)胞間的通信,不形成屏障能更好的研究這些細(xì)胞類(lèi)型單du對(duì)不同化合物的響應(yīng)情況。并且研究者還發(fā)現(xiàn)共享相同培養(yǎng)基的星形膠質(zhì)細(xì)胞、周細(xì)胞和血管內(nèi)皮細(xì)胞的zui適流速為50 μl/min。

作為創(chuàng)新的細(xì)胞培養(yǎng)方法,Quasi Vivo流動(dòng)培養(yǎng)已經(jīng)quan球70余家專(zhuān)業(yè)機(jī)構(gòu)使用驗(yàn)證,獲得了令人側(cè)目的培養(yǎng)效果,在美、英、法、日等多國(guó)開(kāi)展了頗具新意的細(xì)胞研究,涉及呼吸系統(tǒng)、肝臟、腎臟、心血管、成纖維細(xì)胞、糖尿病模型、腦組織類(lèi)器官等。

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應(yīng)用:

器官型模型(骨重建,腫瘤微環(huán)境)

3D細(xì)胞擴(kuò)增和分化

細(xì)胞 - 支架相互作用的研究

細(xì)胞外基質(zhì)相互作用的研究

生成適合臨床前實(shí)驗(yàn)的3D細(xì)胞支架結(jié)構(gòu)

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The Ucup perfusion bioreactor is a user-friendly tool to establish and control your 3D cell and tissue cultures.

Ucup has been specifically designed to be used by any scientist or lab technician working in the life science related field, without necessarily requiring any previous experience with the use of bioreactors. Applications
Organotypic models (Bone remodeling, Tumor microenvironment)
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Investigation of cell-scaffold interactions
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Apply instantly your current cell culture concepts and simply let Ucup further extend them by performing the seamless transition to the 3D context.
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Direct perfusion Unifrom cell seeding Uniform tissue
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Supple scaffold adaptors Compatible with a wide spectrum of 3D porous scaffolds of various composition, architecture and sizes
Access to cell culture medium through valves Suitable to seed and co-culture several cell types, even at different culture time points Possibility to investigate complex cell-cell and cell-extracellular matrix interaction
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Product Configuration
1x syringe pump
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The driving force of the system. It generates the oscillating fluid flow of the cell/medium suspension. It cannot be purchased separately. A rotating rack for easy and correct positioning of Ucup disposable bioreactors. It can also be purchased separately. The central core of the system. It is disposable and it comes with 10x adaptors to fit the specific size of your scaffolds. Scaffolds can also be purchased separately. It provides all what you need to start your 3D cell cultures. Additional accessories (e.g. forceps, syringes) and testing units are also included.
The performance of Ucup has been extensively validated and certified by scientific publications in peer-review journals.
If you are convinced of the benefits that a 3D culture environment can provide,the Ucup bioreactor is the essential tool to conduct with your experiments.
For assistance and advice to set up your experiment, do not hesitate to contact CELLEC’s expert team to address your questions.
應(yīng)用文獻(xiàn):
Boccardo and Gaudiello 2016 In this paper, the perfusion-based bioreactor is used for the generation of an adipose mesenchymal stromal cells -based engineered constructs (Title: Engineered mesenchymal cell-based patches as controlled VEGF delivery systems to induce extrinsic angiogenesis, Acta Biomaterials)
Cerino 2016 presents an application for engineering an in vitro 3D multi-cellular muscle-like tissue model (Title: Three-dimensional multi-cellular muscle-like tissue engineering in perfusion-based bioreactors, Biotechnology and Bioengineering)
Hirt and Papadimitropoulos 2015 demonstrates the importance of perfusion flow in 3D cultures of tumor cells to efficiently mimic functional features observed “in vivo” and to test anticancer compounds (Title: Bioreactor-engineered cancer tissue-like structures mimic phenotypes, gene expression profiles and drug resistance patterns observed in vivo,Biomaterials)
Centola 2015 In this study, the perfusion-based bioreactor system is used to improve cartilage digestion, resulting in higher and more reproducible yield of cell populations with high proliferation and chondrogenic capacity (Title: An improved cartilage digestion method for research and clinical applications, Tissue Engineering Part C, Methods)
Bao 2015 presents a humanized in vitro model that reduces the need for experimental animal models, while recapitulating key biological events in a periodontal pocket (Title: Establishment of an oral infection model resembling the periodontal pocket in a perfusion bioreactor system, Virulence)
Papadimitropoulos 2014 presents an efficient expansion method of mesenchymal stromal cells by direct seeding and culturing fresh bone marrow preparation within the pores of 3D porous scaffold (Title: Expansion of human mesenchymal stromal cells from fresh bone marrow in a 3D scaffold-based system under direct perfusion, PLoS One)
Hirt 2014 highlights the potential of perfusion-based models to create 3D tumour microenvironment for cancer immunobiology studies and pre-clinical assessment of innovative treatments (Title: In vitro 3D models of tumor-immune system interaction, Advance Drug Delivery Review)
Papadimitropoulos 2013 presents an application/method for seeding open porous rapid prototyped polymeric scaffolds (Title: A collagen network phase improves cell seeding of open-pore structure scaffolds under perfusion, Journal of Tissue Engineering and Regenerative Medicine)
Sadr 2012 presents an application/method to generate a decellularized cell-laid extacellular matrix which enhances the biological performance of polymeric materials (Title: Enhancing the biological performance of synthetic polymeric materials by decoration with engineered, decellularized extracellular matrix, Biomaterials)
Gueven 2011 presents an application for upscaling osteogenic and vasculogenic grafts (Title: Engineering of large osteogenic grafts with rapid engraftment capacity using mesenchymal and endothelial progenitors from human adipose tissue, Biomaterials)
Papadimitropoulos 2011 presents an application for engineering an in vitro bone organ model (Title: A 3D in vitro bone organ model using human progenitor cells, European Cell & Materials)
Di Maggio 2011 a review for our approaches to engineering in 3D vitro niches (Title: Toward modeling the bone marrow niche using scaffold-based 3D culture systems, Biomaterials)
Santoro 2010 presents an application for upscaling cartilaginous grafts (Title: Bioreactor based engineering of large-scale human cartilage grafts for joint resurfacing, Biomaterials)
Scherberich 2007 presents an application for generating osteogenic and vasculogenic grafts (Title: Three-dimensional perfusion culture of human adipose tissue-derived endothelial and osteoblastic progenitors generates osteogenic constructs with intrinsic vascularization capacity, Stem Cells)
Wendt 2006 describes the system for maintaining living uniform tissues in the scaffolds (Title: Uniform tissues engineered by seeding and culturing cells in 3D scaffolds under perfusion at defined oxygen tensions, Biorheology)
Braccini 2005 presents an application for generating osteogenic grafts (Title: Three-dimensional perfusion culture of human bone marrow cells and generation of osteoinductive grafts, Stem Cells)
Wendt 2003 describes the principle of the Ucup and its impact on cell seeding (Title: Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity, Biotechnology and Bioengineering)

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