والكتاب الامتنان توم روبنسون للقراءة دليل على المخطوطة، C. BÄRTSCHI وH. بنز لبناء نظام مراقبة ضغط مبنية خصيصا. نود أيضا أن نعترف استخدام مركز المجهر الضوئي (LMC)، وكلاهما في ETH زيورخ أول مرفق غرفة نظيفة و. وقد تم تمويل هذا العمل من قبل شركة ميرك سيرونو ومجلس البحوث الأوروبي (ERC) في إطار برنامج الإطار 7 (ERC ابتداء غرانت، المشروع رقم 203428، nμLIPIDs).

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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

وقد فتحت التكنولوجيا على microfluidics إمكانيات جديدة ورائعة للتحليل وحيدة الخلية. على وجه الخصوص، وإمكانية اعتراض وشل الخلايا بشكل فردي عن طريق أدوات ميكروفلويديك سمحت دراسات منهجية على المدى القصير والطويل على خصائص وحيدة الخلية والاستجابة لها. بالإضافة إلى ذلك، التغل...

وقد كشفت العديد من الدراسات في الماضي خلية الى خلية الخلافات بين السكان زنزانة كبيرة 1-3، ولا سيما إشارات العمليات 4، أو مبالغ الجزيئات الحيوية مثل البروتينات داخل الخلايا 5،6، الأيض، والعوامل المساعدة 7،8. وتعتبر هذه التغاير أن يكون أهمية أساسية للتكيف الخلايا والتطور 9، ولكن أيضا أن تلعب دورا رئيسيا في ظهور والعلاج من الأمراض مثل السرطان 10-13. وبالتالي، دراسات على مستوى خلية واحدة هي من الفائدة المرتفعة في الأبحاث البيولوجية والدوائية، وخاصة إذا تكشف هذه الدراسات ردود مختلفة من الخلايا بعد العلاج مع المواد الكيميائية النشطة بيولوجيا. في السنوات الأخيرة، وقد وضعت العديد من المنصات التحليلية التي تسهل تحليل الخلايا الحية واحدة أو التركيب الكيميائي لمحتوى الخلية. بينما الفلورسنت خلية تنشيط الفرز (FACS) هو الذهب الحادي وANDARD لتحليل الإنتاجية العالية جدا من الخلايا الحية واحدة، وطريقة لا يمكن أن تستخدم لتقدير حجم المركبات داخل الخلايا أو يفرز. وقد وعدت ظهور منصات ميكروفلويديك استراتيجيات تحليلية جديدة لتحديد المواقع، والمعالجة والمراقبة للخلايا واحدة. وتم التوصل إلى علامة فارقة في على microfluidics مع دمج PDMS مرن الصمامات التي حققتها الزلزال وزملاء العمل 14،15. هذه الصمامات هي مفيدة لأنها يمكن عزل مناطق في الرقاقة، مثل فصل ثقافتين 16. علاوة على ذلك، كانت قابلة للتطبيق وخاصة بالنسبة للتحليل خلية واحدة، وبالتالي يساعد على تقليل المشاكل التخفيف تحليلها. قوة هذا النهج للتحليل وحيدة الخلية وقد تجلى مؤخرا من قبل هانسن وزملاء العمل، الذي حلل التعبير الجيني من مئات الخلايا واحد في نفس الوقت 17. عند استهداف البروتينات ونواتج الأيض، وتحليل أمر صعب جدا نظرا لعدم وجود مناسبةطرق mplification، وعدد كبير من مركبات مختلفة الحاضر، والاختلافات في الطبيعة الكيميائية. علاوة على ذلك، من المتوقع أن تكون موجودة بأعداد نسخة منخفضة في حدود بضع عشرة آلاف الجزيئات الحيوية داخل الخلايا 18 الأكثر، وبالتالي الطريقة التحليلية المستخدمة يجب أن يكون لديك حساسية عالية. فحوصات أكثر قوة مثل المناعية والمناعية انزيم مرتبط (ELISA) يصعب على الاندماج في الأجهزة ميكروفلويديك لأنها تتطلب العديد من الخطوات الغسيل والحضانة وكذلك تجميد السطح. نظرا لهذه التحديات، فإنه ليس من المستغرب أن تم الإبلاغ سوى أمثلة قليلة حيث تم كميا البروتينات او نواتج على مستوى خلية واحدة. على سبيل المثال، تم الإبلاغ عن دراسات على إفراز مركبات الفلورسنت 19،20. في الآونة الأخيرة، وقدم مع تنفيذ ELISA لتحليل يفرز (nonfluorescent) البروتينات من ثقافة الخلية (الخلايا THP-1) 21 واحد (طمناعية) الخلايا 10. استهداف البروتينات داخل الخلايا، شي وآخرون. تطوير جهاز ميكروفلويديك التي سهلت تحديد البروتينات داخل الخلايا لتحليل مسارات الإشارات في الخلايا السرطانية عن طريق والمناعية 11. ومع ذلك، تم تحديد كميات النسبية فقط من البروتينات، وكان يستخدم أي التضخيم الأنزيمية لزيادة إشارة للبروتينات وفرة منخفضة. في الآونة الأخيرة، كنا قادرين على الجمع بين محاصرة microdevice وحيدة الخلية مع المقايسات المناعية مضان 8 و 22 (الشكل 1). محاصرون الخلايا بشكل سلبي في الهياكل عقبة microsized، والتي تسمح العرض والصرف (السريع) من العوامل الكيميائية الأخرى المتوسطة ودون أي حركة الخلايا. صمام على شكل حلقة حول كل فخ يمكن عزل الخلية في حجم صغير جدا (&quot;وmicrochamber&quot;). وهذا صمام دفعتها مباشرة بعد إدخال عازلة الخلايا lysing (hypoosmolar)، وقال انهالامتحانات التنافسية الوطنية ومنع الجزيئات داخل الخلايا أو الجزيئات يفرز لنزع فتيل بعيدا. الأهم من ذلك، ونظرا لصغر حجم وحدة التخزين (625 رر) يتم تجنب تخفيف كبير من الجزيئات. علاوة على ذلك، منذ يتم تنفيذ تحلل والتحليل في في نفس الموقف في رقاقة، ليست هناك خسارة من analytes بسبب النقل. تصميم رقاقة الموصوفة هنا تضم ​​8 صفوف بالتناوب إما 7 أو 8 microchambers، بلغ مجموعها 60 microchambers. ودفعتها في غرف الصفوف، بحيث تنتفي صفة انتقال التلوث على طول الخط. منصة يمكن استخدامها في تركيبة مع المقايسات مضان فضلا عن المقايسات المناعية (الشكل 1D). لهذا الأخير، أنشأنا بروتوكولات لتجميد الأجسام المضادة، التي تتوافق مع إنتاج رقاقة وعملية التجميع. وبالتالي منصة يفتح الطريق لفحوصات حساسة وموثوقة وقابلة للقياس الكمي على مستوى خلية واحدة. حتى الآن، وقد استخدمنا جهاز لتحليل طالانزيمات ntracellular ويفرز (الكمي النسبي الذي المقايسات الأنزيمية)، العوامل المساعدة داخل الخلايا والبروتينات والجزيئات الصغيرة (الكمي المطلق من قبل المقايسات نقطة النهاية أو ELISA). في ما يلي، ونحن تصف عملية تصنيع رقاقة عن طريق الطباعة الحجرية الناعمة متعدد الطبقات وبروتوكولات لالزخرفة من الأجسام المضادة عن طريق microcontact الطباعة والكيمياء السطح. بالإضافة إلى ذلك، يتم إعطاء بعض الأمثلة على استخدام رقاقة والعمليات.

<table><tbody><tr><td>&lt;strong&gt;REAGENTS:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Name of the Reagent</td><td>Company</td><td>Catalogue Number</td><td>Comments (optional)</td></tr><tr><td>SU-8 2015</td><td>MicroChem Corp. (Netwon, MA)</td><td>n.a.</td><td></td></tr><tr><td>1H,1H,2H,2H-Perfluorodecyl-dimethylchloro-silane</td><td>ABCR (Karlsruhe, Germany)</td><td>AB103608</td><td></td></tr><tr><td>4-Methylumbelliferyl &amp;beta;-D-N,N&amp;prime;-diacetylchitobioside hydrate</td><td>Sigma Aldrich</td><td>M9763</td><td></td></tr><tr><td>Acetone</td><td>Merck VWR (Darmstadt, Germany)</td><td>100014</td><td></td></tr><tr><td>Avidin</td><td>AppliChem (Axon Lab AG)</td><td>A-2568</td><td></td></tr><tr><td>AZ 1518</td><td>AZ Electronic Materials (Wiesbaden, Germany)</td><td>n.a.</td><td></td></tr><tr><td>AZ 726 developer</td><td>AZ Electronic Materials (Wiesbaden, Germany)</td><td>n.a.</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A-4503</td><td></td></tr><tr><td>Bovine serum albumin, biotin</td><td>Sigma Aldrich</td><td>A-8549</td><td></td></tr><tr><td>Cell dissociation buffer</td><td>Invitrogen</td><td>13151-014</td><td></td></tr><tr><td>Hexamethyldisilazane (HDMS)</td><td>Sigma Aldrich</td><td>40215</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fluka</td><td>84422</td><td></td></tr><tr><td>Isopropanol</td><td>Merck VWR (Darmstadt, Germany)</td><td>109634</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Fluka</td><td>63068</td><td></td></tr><tr><td>MR developer 600</td><td>Microresist technology GmbH (Berlin, Germany)</td><td>n.a.</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>10010-031</td><td></td></tr><tr><td>PLL-g-PEG grafted</td><td>SuSoS, (D&amp;uuml;bendorf, Switzerland)</td><td>n.a.</td><td></td></tr><tr><td>PLL-g-PEG grafted biotin</td><td>SuSoS, (D&amp;uuml;bendorf, Switzerland)</td><td>n.a.</td><td></td></tr><tr><td>Potassium chloride</td><td>Fluka</td><td>60132</td><td></td></tr><tr><td>Protein G, biotin</td><td>Sigma Fine Chemicals</td><td>41624</td><td></td></tr><tr><td>Silicon wafer</td><td>Si-Mat (Kaufering, Germany)</td><td>n.a.</td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit (PDMS)</td><td>Dow Corning</td><td>39100000</td><td></td></tr><tr><td>Tris(hydroxymethyl)-aminomethan</td><td>Biorad</td><td>1610716</td><td></td></tr><tr><td>Tween 20</td><td>Biorad</td><td>1706531</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Material Name</td><td>Company</td><td>Catalogue Number</td><td>Comments (optional)</td></tr><tr><td>0.22 &amp;micro;m PES syringe filter</td><td>TRP</td><td>99722</td><td></td></tr><tr><td>1/1.5 mm biopsy puncher</td><td>Miltex, York PA</td><td>33-31AA/33-31A</td><td></td></tr><tr><td>Cell Trics filter 20 &amp;micro;m</td><td>Partec</td><td>04-004-2325</td><td></td></tr><tr><td>Centrifuge Sigma 3-18K</td><td>Kuehner</td><td>n.a.</td><td></td></tr><tr><td>Hotplate HP 160 III BM</td><td>Sawatec, Sax, Switzerland</td><td>n.a.</td><td></td></tr><tr><td>MA-6 mask aligner</td><td>Karl Suess</td><td>n.a.</td><td></td></tr><tr><td>Multizoom AZ100M microscope</td><td>Nikon Corporation</td><td>n.a.</td><td></td></tr><tr><td>Photomask</td><td>Microlitho, Essex, U.K.</td><td>n.a</td><td></td></tr><tr><td>Plasma Cleaner PDC-32G</td><td>Harrick</td><td>n.a.</td><td></td></tr><tr><td>Spin coater Modell WS-400 BZ-6NPP/LITE</td><td>Laurell</td><td>n.a.</td><td></td></tr><tr><td>Spin Modules SM 180 BM</td><td>Sawatec, Sax, Switzerland</td><td>n.a.</td><td></td></tr><tr><td>Step profiler Dektak XT Advanced</td><td>Bruker</td><td>n.a.</td><td></td></tr><tr><td>Syringe pump neMESYS</td><td>Cetoni</td><td>n.a.</td><td></td></tr></tbody></table>

a microfluidic chip for the versatile chemical analysis of single cells

نقدم جهاز ميكروفلويديك التي تمكن من تحديد كمية الجزيئات الحيوية داخل الخلايا في الخلايا وحيدة متعددة في نفس الوقت. لهذا الغرض، محاصرون الخلايا بشكل سلبي في منتصف microchamber. عند تفعيل طبقة تحكم، يتم عزل الخلية من حجم المحيطة في غرفة صغيرة. ويمكن بعد ذلك يتم تبادلها حجم المحيطة دون التأثير على الخلايا المعزولة. ومع ذلك، عند فتح وإغلاق قصيرة للغرفة، والحل في غرفة يمكن استبدالها في غضون بضع مئات من ميلي ثانية. ويرجع ذلك إلى العودة إلى الوراء من الغرف، والخلايا يمكن أن يتعرض لها حلول مختلفة بالتتابع بطريقة التحكم الى حد كبير، على سبيل المثال للحضانة، والغسيل، وأخيرا، تحلل الخلية. وmicrochambers مغلقة بإحكام تمكين الاحتفاظ المحللة، وتقليل والسيطرة على التخفيف بعد تحلل الخلية. منذ تحلل والتحليل تحدث في نفس المكان، ويتم الاحتفاظ حساسية عالية لأنه لا يوجد مزيد من دilution أو فقدان التحاليل يحدث أثناء النقل. وبالتالي فإن تصميم microchamber يتيح تحليل موثوق بها وقابلة للتكرار من الأرقام نسخة صغيرة جدا من الجزيئات داخل الخلايا (attomoles، zeptomoles) صدر من الخلايا الفردية. علاوة على ذلك، يمكن ترتيب العديد من microchambers في شكل مجموعة، والسماح للتحليل العديد من الخلايا في آن واحد، نظرا إلى أن تستخدم الأجهزة البصرية مناسبة للرصد. وقد استخدمنا بالفعل منصة لإثبات صحة مفهوم الدراسات لتحليل البروتينات داخل الخلايا، والإنزيمات، العوامل المساعدة والمرسلين الثاني بطريقة قابلة للقياس الكمي إما النسبية أو المطلقة.

برنامجنا هو قادرا على تحليل مجموعة متنوعة من الخلايا وكذلك جزيئات يفرزها موجودة في أو التي تنتجها الخلايا واحد. هنا، نود أن تقديم دراسات سبيل المثال مختلفة للتأكيد على مجموعة متنوعة من فحوصات ممكن. وسنقدم مثالا للانزيم يفرز (الشكل 5A)، وكذلك انزيم داخل الخلا?...

Watch this Scientific Journal Video about A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells at JoVE.com

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

في هذه المقالة نقدم رقاقة ميكروفلويديك لتحليل خلية واحدة. أنه يسمح للالكمي للبروتينات الخلايا، والإنزيمات، العوامل المساعدة، ورسله الثاني عن طريق فحوصات الفلورسنت أو المناعية.

رقاقة ميكروفلويديك للتحليل الكيميائي للخلايا متعددة الاستخدامات واحدة

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

a-microfluidic-chip-for-versatile-chemical-analysis-single

Research

JoVE Journal

Bioengineering

14.8K Views.  ETH Zurich, Switzerland.  في هذه المقالة نقدم رقاقة ميكروفلويديك لتحليل خلية واحدة. أنه يسمح للالكمي للبروتينات الخلايا، والإنزيمات، العوامل المساعدة، ورسله الثاني عن طريق فحوصات الفلورسنت أو المناعية. 

Video: A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

عد البروتينات في الخلايا المفردة مع الحبرية عنونة [ميكروارس]

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

Cancer Research

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

طرق الكشف عن منصات مضان الحبرية ميكروفلويديك

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Millifluidics لتخليق المواد الكيميائية والدراسات الميكنة الوقت لحلها

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

وبيوشيب الكهروكيميائية استنادا ميكروفلويديك لتسمية خالية تحليل الحمض النووي التهجين

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

رقاقة ميكروفلويديك لICPMS مقدمة عينة

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

ومنصة ميكروفلويديك لعزل وحيدة الخلية عالية الإنتاجية والثقافة

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

تصميم الأجهزة ميكروفلويديك لدراسة الردود الخليوي تحت واحدة أو تتعايش الكيميائية / الكهربائية / إجهاد القص المحفزات

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

منصة ميكروفلويديك مع تعدد كشف الالكترونية لتتبع المكانية من الجسيمات

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

إنشاء الثقافات المشتركة القائمة علي خليه واحده بطريقه قطعيه مع رقاقه ميكروفلويدريك

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

توليد الظروف البيئية الديناميكية باستخدام جهاز Microfluidic عالي الإنتاجية

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...