Name: Microfluidic Shear Apparatus Setup for Single-Cell Fluid Shear Assay
Uploaded: 2023-05-19T13:30:00
Description: Watch this Scientific Journal Video about Microfluidic Shear Apparatus Setup for Single-Cell Fluid Shear Assay at JoVE.com

microfluidic shear apparatus setup for single-cell fluid shear assay

एकल-कोशिका यांत्रिकी की मात्रा निर्धारित करके लक्षित कैंसर निदान को बढ़ाना

Studying the biophysical differences between cancerous and non-cancerous cells allows for novel diagnostic and therapeutic opportunities1. Understanding how differences in biomechanics/mechanobiology contribute to tumor progression and treatment resistance will reveal new avenues for targeted therapy and early diagnosis2.
While it is known that cancer cell mechanical properties differ from normal cells (e.g., viscoelasticity of the plasma membrane and nuclear envelope)3,4,5, robust and reproducible methods for measuring these properties in live cells are lacking6. The shear assay method is used to quantify the mechanical properties of cells by subjecting single cells to fluid shear stress and analyzing their individual responses and resistance to the applied stress3,4,5,7,8,9. Although several methods and techniques have been used to characterize the mechanical properties of single cells, these tend to affect cell material properties by i) perforating/damaging the cell membrane due to the indentation depth, complex tip geometries, or substrate stiffening associated with atomic force microscopy (AFM)10,11, ii) inducing cellular photodamage during optical trapping12,13, or iii) inducing complex stress states associated with micropipette aspiration14,15. These external effects are associated with significant uncertainties in the accuracy of cell viscoelasticity measurements6,16,17.
To address these limitations, the shear assay method described here provides a highly controllable and simple approach to simulate physiological flow in the body without affecting cellular material properties in the process. Fluid shear stresses in this assay represent mechanical stresses experienced by cells in the body either by fluids within the tumor interstitium or in the blood during circulation18,19,20. Further, these fluid stresses promote various malignant behaviors in cancer cells, including progression, migration, metastasis, and cell death19,21,22,23&#160;which vary between tumorigenic and non-tumorigenic cells. Moreover, the altered mechanical features of cancer cells (i.e., they are often &#34;softer&#34; than normal cells found within the same organ) allow them to persist in hostile tumor microenvironments, invade surrounding normal tissues, and metastasize to distant sites24,25,26. By creating a pseudo-biological environment where cells experience physiological levels of fluid shear stress, a process that is physiologically relevant and not destructive to the cell is achieved. The cellular responses to these applied fluid shear stresses allow us to characterize cell mechanical properties.
This paper provides a shear assay protocol for the extensive study of the mechanical properties and behavior of cancerous and non-cancerous cells under applied shear stress. Cells respond to external forces in an elastic and viscous manner and can therefore be idealized as a viscoelastic material3. This technique is categorized into: (i) cell culture of dispersed single cells, (ii) controlled application of fluid shear stress, (iii) in situ imaging and observation of cellular behavior (including resistance to stress and deformation), (iv) strain analysis of cells to determine the extent of deformation, and (v) characterization of the viscoelastic properties of single cells. By interrogating these mechanical properties and behaviors, complex cellular mechanobiology can be distilled to quantifiable data. A protocol outlining this method allows for the cataloging of and comparison between various malignant and non-malignant cell types. Quantifying these differences has the potential to establish diagnostic and therapeutic biomarkers.

लेखक स्पॉटलाइट: एकल सेल सामग्री गुणों के निर्धारण के लिए कतरनी परख प्रोटोकॉल  

एकल-सेल सामग्री गुणों के निर्धारण के लिए कतरनी परख प्रोटोकॉल

The physical hallmarks of cancer are relatively new and exciting topics in cancer research. Our research seeks to specifically characterize the mechanical properties of both cancers and non-cancer cells to better understand the physical mechanisms by which cancer cells survive shear forces in the body and invade the immune system. Recent development in my period of research is the use of microfluidic devices and high-throughput techniques to understudy in a larger scale, cellular deformability and the viscoelastic properties of cells, and also in the identification of novel biomarkers such as the regulation of cytoskeletal proteins and extracellular matrix proteins for early cancer diagnosis.Current experimental challenges involved in this field are the complexity of the tumor microenvironment, scaling throughput, clinical translation, standardization and reproducibility, and integration of multimodal data. So, some of the significant findings I've established in this field from my past research and from my current research is that cancer cells can be distinguished from normal cells based on their responses to mechanical stressors. And the extent to which resist mechanical stressors is dependent on structural integrity, which is dictated to a reasonable extent by the presence of cytoskeletal proteins like the filamentous actin and other structural proteins within the cell.Our protocol offers a simple, repeatable, and non-destructive approach to characterize cultured cells mechanically. This is in contrast to other techniques like AFM and optical tweezers, which require a certain level of technical expertise in order to perform an experiment in a relatively low throughput. Within the use of the shear acid technique, will greatly advance the single cell mechanical characterization, and will also aid in many different cross-sectional areas such as rare disease diagnosis, personalized diagnosis and care, and monitoring single-cell drug interactions.

Watch this Scientific Journal Video about Microfluidic Shear Apparatus Setup for Single-Cell Fluid Shear Assay at JoVE.com

Microfluidic Shear Apparatus Setup for Single-Cell Fluid Shear Assay  

एकल-सेल द्रव कतरनी परख के लिए माइक्रोफ्लुइडिक कतरनी उपकरण सेटअप

कतरनी परख चिपचिपा प्रवाह मीडिया तैयार करके प्रोटोकॉल शुरू करें। ऐसा करने के लिए, चुंबकीय हलचल या गर्म प्लेट का उपयोग करके 60 से 70 डिग्री सेल्सियस पर लगभग 10 से 20 मिनट के लिए बेस कल्चर मीडिया को प्रीहीट करें। मीडिया को लगातार हिलाते हुए, धीरे से इसमें मिथाइलसेल्यूलोज जोड़ें ताकि मिथाइलसेल्यूलोज कणों को जल्दी से फैलने में मदद मिल सके।मीडिया और सेल्यूलोज के स्पष्ट समरूप मिश्रण को सुनिश्चित करने के लिए इस प्रक्रिया को लगभग 15 से 24 घंटे तक जारी रखने की अनुमति दें। कतरनी तंत्र स्थापित करने के लिए, प्रवाह कक्ष और पेट्री डिश के बीच संबंध को बांधने के लिए प्रवाह पथ पर एक रबर गैसकेट संलग्न करें, और एकल कोशिकाओं के नियंत्रित समान प्रवाह को सुनिश्चित करें। रबर गैसकेट वांछित प्रवाह प्रोफ़ाइल और अवलोकन के क्षेत्र के आधार पर विभिन्न आकारों में आता है।इसके बाद, चिपचिपा कल्चर मीडिया के जलसेक और वापसी के लिए प्रोग्राम करने योग्य सिरिंज पंप से जुड़े 60 या 100 मिलीलीटर दोहरी सिरिंज से युक्त कतरनी परख प्रणाली स्थापित करें। तैयार चिपचिपा प्रवाह मीडिया के साथ एक सिरिंज भरें और भरे हुए सिरिंज को सिरिंज पंप पर अपने संबंधित स्थान पर संलग्न करें। फिर, सिरिंज पंप पर अन्य सिरिंज स्थान पर एक खाली सिरिंज संलग्न करें।1/16 इंच टयूबिंग और ट्यूबिंग कनेक्टर का उपयोग करके, दोनों सिरिंज को प्रवाह कक्ष से कनेक्ट करें। पंप को एक निर्दिष्ट दर पर तरल पदार्थ की एक निश्चित मात्रा को डालने और वापस लेने के लिए प्रोग्राम करें, और संबंधित सिरिंज का चयन करें। स्क्रीन पर एक उदाहरण प्रोग्राम दिखाया गया है।कतरनी की तैयारी के लिए पेट्री डिश से सेल कल्चर मीडिया को एस्पिरेट करें। इसके बाद, संलग्न कोशिकाओं वाले पेट्री डिश पर रबर गैसकेट के साथ प्रवाह कक्ष डालें और ठीक करें। डिश में कोशिकाओं के साथ फिट किए गए माइक्रोफ्लुइडिक फ्लो चैंबर को डिस्प्ले मॉनिटर से जुड़े उल्टे माइक्रोस्कोप पर रखें।सेटअप अब एक एकल कोशिका पर द्रव कतरनी तनाव लागू करने और परिणामस्वरूप सेलुलर विरूपण की ऑप्टिकल रूप से निगरानी करने के लिए तैयार है।

Research