Augstas kapacitātes ārpusšūnu vezikulu izdalīšana ar plūsmas lauka frakcionēšanas metodi mikrofluīdikā

Abstract

Visas šūnas, tostarp vēža šūnas, izdala nanoizmēra ārpusšūnu vezikulas (EVs), kas satur svarīgas biomolekulas – miRNS, proteīnus un daudzas citas. EVs nodrošina šūnu savstarpējo komunikāciju, regulējot gan normālos gan patoloģiskos apstākļus. Šobrīd tās ir intensīvi pētītas kā potenciālie biomarķieri dažādu slimību diagnostikā un terapijā. Neskatoties uz EV priekšrocībām to izolēšanas metodes kavē EV pielietojumu klīniskajos pētījumos. Uz doto brīdi ir izstrādāti dažādi risinājumi pielietojot mikrofluīdikas ierīces, kas neietekmē EV integritāti un tām ir laba atkārtojamība, taču šīs ierīces ir domātas maza tilpuma paraugiem - serumam un asinīm. Šī darba mērķis ir izveidot mikroiekārtu, kas izdala totālo EV daudzumu no liela tilpuma paraugiem (>10 mL) un ar augstu efektivitātes līmeni (>90%). Tika izveidots kontroles paraugs, ar kuru tika testēts PDMS (polidimetilsiloksāns) un dažādu citu mikrofluīdikas materiālu EV un mazmolekulāro hidrofobisko molekulu absorbcijai. OSTE (Off-stoichiometry thiol-ene polymer) no visiem testētajiem materiāliem tika atrasts kā vispiemērotākais mikrofluīdikas ierīču izveidē, kas būtu domāts EV izolēšanai. Tika izgatavots PDMS materiāla mikroiekārtas prototips uz kura tika optimizēti parauga ievadīšanas spiedieni izmantojot kontroles paraugu. Tika noskaidrots, ka mazāki spiedieni sniedz labākus rezultātus, taču ir nepieciešams veikt turpmāko šo iekārtu optimizāciju un iekārtu izgatavošanas protokolu optimizēšanu no OSTE.

All cells, including cancer cells, secrete nano-sized extracellular vesicles (EVs), which contain important biomolecules - miRNAs, proteins and others. EVs provide cell-to-cell communication by regulating both normal and pathological conditions. They are currently being intensively studied as potential biomarkers in the diagnosis and treatment of various diseases. Despite the benefits of EVs, their isolation still cause the major problems for their application for their use in clinical trials. There are some solutions developed as microfluidic devices that do not affect the integrity of EV and have good repeatability, but these devices are designed for small volume samples - serum and blood. The aim of this work is to create a microfluidic device that isolates total amount of EVs in large volume samples (> 10 mL) and with a high level of efficiency (> 90%). A control sample was developed to test the effect on absorption of PDMS (polydimethylsiloxane) and various other microfluidic materials by using EVs and low molecular weight hydrophobic molecule. OSTE (Off-stoichiometry thiol-ene polymer) of all tested materials was found to be the most suitable for microfluidic device production that could be used for EV isolation. A prototype PDMS material microdevice was made on which the sample injection pressure was optimized using a control sample. It was found that lower pressure gives better results, but OSTE does not need to optimize this equipment and optimize equipment manufacturing protocols, however, further optimization of this equipment and optimization of equipment manufacturing protocols from OSTE is required.