Name: Preparation of the Microfluidic Device for Electrodeformation of RBCs
Uploaded: 2023-10-13T14:00:00
Description: Watch this Scientific Journal Video about Preparation of the Microfluidic Device for Electrodeformation of RBCs at JoVE.com

preparation of the microfluidic device for electrodeformation of rbcs

Mekanisk udmattelsestest af røde blodlegemer ved amplitudemoduleret elektrodeformation

Red blood cells (RBCs) are the most deformable cells in the human body1. Their deformability is directly related to their oxygen-carrying functionality. Reduced deformability in RBCs has been found to correlate with the pathogenesis of several RBC disorders2. Deformability measurements have led us to a better understanding of RBC-related diseases3. The normal lifespan of RBCs can vary from 70 to 140 day4. Therefore, it is important to measure how their deformability decreases along with the aging process, e.g., their fatigue behavior due to cyclic shear stresses3.
Measuring RBC deformability at high throughput is challenging because of the piconewton scale forces (~10-12 N) that are applied to the individual cells. Over the past decade, many technologies have been developed to measure cell deformability5. Deformation measurements of RBCs at the single-cell level can be performed by pipette aspiration and optical tweezers, while bulk analyses are done by osmotic gradient ektacytometry. Ektacytometry analyses provide an abundance of data, which provides an opportunity to diagnose blood disorders6,7. The deformability of RBCs can also be analyzed using the viscoelastic theory by colloid probe atomic force microscopy. In this method, computational analysis is applied to estimate the elastic modulus of RBCs, considering both time-dependent and steady-state responses. The deformability of individual RBCs can be measured by using the single-cell microchamber array method. This method analyzes each cell through the membrane and cytosolic fluorescent markers to provide information for RBC deformability and the distribution of cellular characteristics in complex RBC populations to detect hematologic disorders8.
Fatigue is a key factor in the degradation of properties of engineered materials and biomaterials. Fatigue testing enables a quantitative analysis of the integrity and longevity of a structure subjected to cyclic loading. Analysis of fatigue in biological cells has long been hampered by the lack of a general, readily applicable, high throughput, and quantitative method for the implementation of cyclic deformation in cell membranes. This is possible with the utilization of electrical signal modulation and electrodeformation techniques implemented in a microfluidic setting. The amplitude shift keying (ASK) technique as a digital modulation is applied through On-Off keying (OOK) modulation in this article. The concept of keying refers to the transmission of digital signals over the channel, which requires a sine wave carrier signal to function9. The ON and OFF times can be set equal. Under ON-keying, RBCs enter a deformed state while exposed to an external electrodeformation force (Fdep)10 created by the nonuniform electric field. Under OFF-keying, RBCs are in their relaxed state. We observe the fatigue of RBCs, namely a progressive degradation in their ability to stretch with increasing loading cycles. The fatigue-induced deformability loss in RBCs can provide insights into the accumulated membrane damage during blood circulation, enabling us to further investigate the connections between cell fatigue and disease states.
Here we provide step-by-step procedures on how fatigue testing of RBCs is implemented in a microfluidic device via ASK-modulated electrodeformation and the system settings such as microfluidic device, mechanical loading, and microscopic imagining for the characterization of the gradual degradation in mechanical deformability of RBCs.

Forfatter Spotlight: Undersøgelse af biomekanik af cirkulerende celler ved at modulere deres elektrodeformationsadfærd 

Amplitudemoduleret elektrodeformation til evaluering af mekanisk træthed af biologiske celler

Our research is focused on the study of cell biomechanics of circulating cells, such as human red blood cells. We leverage electrokinetics, microfluidics, and material science to understand the mechanical origins of damage in cell membranes, as well as the mechanisms underlying the shortened lifespan of blood cells in certain diseases. Fatigue study of biological cells is challenging.It request application of cyclic loads to cell membranes and tracking the deformation in individual cells. Using amplitude sheet key for ASK to modulate electrode deformation behavior of red blood cells, we could quantify how cell deformability changes aligned with the loading cycles. We demonstrated for the first time that red blood cells'membranes can be degraded by cyclic stretching alone.Our protocol utilizes dielectrophoresis to move cells to the electrode edges for electrode deformation measurement. It does not require any stabilization technique and can be used for cell suspension directly. Using interdigitated microelectrodes allows us to measure tens of cells in a single field of view.

Watch this Scientific Journal Video about Preparation of the Microfluidic Device for Electrodeformation of RBCs at JoVE.com

Preparation of the Microfluidic Device for Electrodeformation of RBCs  

Fremstilling af den mikrofluidiske anordning til elektrodeformation af RBC'er

Til at begynde med skal du tape SU8 master siliciumskiven til det mikrofluidiske kanaldesign på indersiden af en plastik 14 centimeter petriskål og rengøre den med nitrogengas. Der vejes passende mængder polydimethylsiloxan eller PDMS-base og PDMS-hærdningsmiddel i en papirkop. Bland de to ved hjælp af en træspatel, indtil blandingen bliver overskyet hvid i farven.Hæld PDMS-blandingen i petriskålen af plast, der indeholder siliciumskiven. Anbring derefter petriskålen i en vakuumekssikkator udstyret med en trevejs stophane. Drej stophanens ventil for at forbinde vakuumet til ekssikkatorkammeret for at fjerne luftbobler fra blandingen.Når alle luftbobler er fjernet fra kanalernes funktioner, skal du placere petriskålen inde i en ovn ved 70 grader Celsius i fire timer. Når petriskålen er afkølet til stuetemperatur, skal du placere den på en skæremåtte. Brug en skalpel til at skære den del af PDMS ud over siliciumskiven.Placer den udskårne PDMS mellem to ark af laboratorieindpakningsfilmen. Spalten mellem mikrokanalens indrykning og filmen hjælper med at lokalisere indløbet og udløbet af den mikrofluidiske kanal. Brug derefter et barberblad til at skære en individuel kanal ud af det store PDMS og med respektive biopsistansninger lave passende indløbs- og udløbshuller i kanalen.Placer den hullede kanal med kanalsiden opad på en ren glasrutsjebane. Anbring et glassubstrat indeholdende tyndfilmsindiumtinoxid eller ITO-interdigiterede elektroder på samme objektglas med elektroderne opad. Placer derefter forsigtigt glasgliden i en plasmarenser.Når du har lukket gasventilen og tændt pumpen, skal du vente i to minutter for at opnå en sensoraflæsning på 600 til 800 millitorr. Tænd derefter afbryderen og vent 30 sekunder, før du drejer RF-strømknappen fra lav til høj og venter i et minut. Følg derefter den omvendte rækkefølge for at slukke for sættet.Umiddelbart efter åbning af plasmarenserens kammer løftes og drejes PDMS 180 grader, så kanalsiden vender nedad. Placer kanalen på toppen af ITO-substratet for at starte bindingsprocessen. Brug pincet til forsigtigt at trykke ned på hjørnerne af PDMS i ca. tre sekunder.Læg primingmediet i en milliliter sprøjte med en 23-gauge nål. Fugt langsomt og forsigtigt kanalen ved at stikke nålen direkte ind i indløbsbrønden, før mediet slippes uden at indføre luftbobler. Efter inkubation i mindst tre minutter fjernes primemediet ved hjælp af en pipettespids på 10 mikroliter.Til sidst vaskes kanalen med dielektroforesemedium tre gange ved at indsætte mediet i kanalen.

Research

JoVE Journal

Bioengineering

Preparation of the Microfluidic Device for Electrodeformation of RBCs

Amplitude-Modulated Electrodeformation to Evaluate Mechanical Fatigue of Biological Cells

Red blood cells (RBCs) are known for their remarkable deformability. They repeatedly undergo considerable deformation when passing through the ...

Preparation of Dielectrophoresis Medium and Electrodeformation Prime Medium

Fremstilling af dielektroforesemedium og elektrodeformationsprimmedium

Preparation of Human RBC Suspension for Electrodeformation Experiment

Fremstilling af human RBC-suspension til elektrodeformationseksperiment

Preparing the Electrodeformation Setup and Performing Fatigue Testing on Human RBC

Forberedelse af elektrodeformationsopsætningen og udførelse af træthedstest på human RBC