TFF Tangential Flow Filtration Ua ke nrog Ultracentrifugation Rau Kev Npaj Me Me Extracellular Vesicle

Me me extracellular vesicles (sEVs) yog nanoscale vesicles secreted los ntawm cov hlwb uas muaj nucleic acids, proteins, me me molecules, thiab lwm yam Cheebtsam, thiab ua lub luag hauj lwm tseem ceeb nyob rau hauv kev sib txuas lus intercellular. Exosome-hom me me extracellular vesicles sib koom siab zoo sib xws nrog lawv niam thiab txiv hlwb. Muab piv nrog cov khoom siv nanoparticles, lawv pom cov kab mob tiv thaiv kab mob qis dua thiab qis dua cytotoxicity, ua rau lawv cov neeg sib tw tau zoo li tshiab.hauv vivo tsheb xa tshuaj. Yuav luag txhua hom hlwb tuaj yeem zais cov vesicles no, thiab kev tshawb fawb ntawm lawv daim ntawv thov raws li cov neeg nqa tshuaj maj mam txav ntawm chav kuaj mus rau kev siv tshuaj kho mob. Txawm li cas los xij,loj -scale ntau lawm tseem yog ib qho tseem ceeb fwj.

 

Cov txheej txheem tam sim no rau Extracellular Vesicle Isolation

Tam sim no extracellular vesicle cais txoj kev suav nrogultracentrifugation, ceev gradient centrifugation, loj-exclusion chromatography (SEC), thiab polyethylene glycol (PEG) nag lossis daus.

PEG nag lossis daus muaj cov txiaj ntsig siab thiab kev ua haujlwm yooj yim, tab sis nws cuam tshuam nrog kev sib kis loj los ntawm cov protein tsis xav tau. Density gradient centrifugation and size-exclusion chromatography can provide high-purity products, but their efficiency is relatively low and the processes are time-consuming. Ultracentrifugation yog dav siv, tab sis nws cov ntim ntim yog txwv los ntawm lub peev xwm centrifuge, ua rau nws nyuaj rau scale.

 

Daim ntawv thov ntawm Tangential Flow Filtration rau sEV Separation

Tangential flow filtration (TFF) twb tau siv rau kev sib cais ntawm nanoparticles xws li liposomes thiab lentiviral vectors. Nyob rau hauv TFF, cov khoom noj ntws tangentially hla lub membrane nto, uas yuav pab txo cov membrane fouling thiab ua rau cov txheej txheem yooj yim rau scale.

Txij li thaum me me extracellular vesicles feem ntau yog los ntawm30-200 nmdiam, a0.45 μm membrane cassettetuaj yeem xaiv nyob rau hauv TFF system kom tshem tawm cov protein ntau thiab cov vesicles, thaum a300 kDa membrane cassettetuaj yeem siv los tshem tawm me me molecular-cov proteins hnyav thiab mloog zoo rau cov hlwv me me uas muaj nyob hauv kab lis kev cai nruab nrab.

 

Tus cwj pwm ntawm cov khoom siv raw tom qab TFF sib cais

Nanoparticle nrhiav kev txheeb xyuas (NTA) tau pom tias cov particle diameters tau muab faib ua ntau yam ntawm0-250 nm.

 

 

Txawm li cas los xij, kis tau tus kab mob electron microscopy (TEM) tau nthuav tawm ntau cov kab mob sib kis, thaum tsis pom cov hlwv ntxiv. Cov txiaj ntsig no tau qhia tias yuav tsum muaj kev lim dej ntxiv hauv qab no txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm.

 

Tus cwj pwm ntawm TFF Concentrate tom qab 100 kDa Ultrafiltration

Lub TFF concentrate tau ua tiav ntxiv siv a100 kDa ultrafiltration tube.

NTA qhia tau hais tias cov particle diameters nyob rau hauv cov concentrates tau tom qab ib thiab ob rounds ntawm ultrafiltration tseem nyob rau hauv ntau yam ntawm0-250 nm. Txawm li cas los xij, kev ua ultrafiltration ob zaug ua rau txo qis ntawm cov khoom tau txais.

TEM tsom xam pom tias tsis muaj vesicle -zoo li cov qauv tau pom nyob rau hauv ib qho - sij hawm los yog ob - lub sij hawm ultrafiltration concentrates, thaum muaj ntau ntawm cov kab mob muaj protein ntau tseem nyob.

Yog li ntawd,100 kDa ultrafiltration tsis tuaj yeem siv tau zoo los ntxuav cov kab lis kev cai nruab nrab tom qab TFF concentration.

 

 

Tus cwj pwm ntawm TFF Concentrate Purified los ntawm Ultracentrifugation

NTA qhia tau hais tias cov particle diameters ntawm cov khoom purified yog feem ntau faib nyob rau hauv ntau yam ntawm0-250 nm.

Qhov tseem ceeb, TEM qhia meej meej vesicle-zoo li cov qauv thoob plaws ntau qhov kev pom. Cov txiaj ntsig no tau pom tias tom qab tsom mus rau kab lis kev cai nruab nrab siv TFF,Ultracentrifugation tuaj yeem ua haujlwm tom qab rau kev sib cais ntxiv thiab ua kom huv ntawm cov hlwv extracellular.

 

 

Tus cwj pwm ntawm sEVs cais los ntawm Mesenchymal Stem Cell Conditioned Medium Siv TFF Ua ke nrog Ultracentrifugation

NTA qhia tau hais tias cov particle diameters ntawm cov khoom purified yog feem ntau faib nyob rau hauv ntau yam ntawm0-250 nm.

Vesicle-zoo li cov qauv raug soj ntsuam los ntawm TEM. Western blot tsom xam pom tau hais tias zoo ntawm cov hlab ntsha extracellular-cov proteins uas cuam tshuamCD9, CD63, thiab ALIXnyob rau hauv lub purified extracellular vesicles.

Cov txiaj ntsig no tau lees paub tias cov hlwv me me ntawm cov hlwv me me muaj cov exosomes, qhia tias txoj kev no kuj tuaj yeem siv rau.exosome cais thiab rho tawm.

 

 

Xaus: TFF Ua ke nrog Ultracentrifugation rau siab -Quality sEV Preparation

Txoj kev tshawb no qhia tau tiasTFF concentration ib leegua rau ib yam khoom uas muaj ntau ntau ntawm cov kab mob sib kis thiab yog li tsis txaus kom tau txais kev tsim nyog cov vesicles extracellular.

Ib yam li ntawd, siv a100 kDa ultrafiltration raj rau kev lim dej theem nrabtsis tau txais txiaj ntsig zoo ntawm cov txheej txheem vesicle.

Los ntawm kev sib txuas TFF concentration nrog kev ua kom huv ntawm ultracentrifugation tom ntej, qhov zoo ntawm ob txoj kev tuaj yeem ua ke kom tau txaissiab -cov khoom me me extracellular vesicle zoo.

Piv nrog cov pa ultracentrifugation ib leeg, cov txheej txheem ua ke tuaj yeem ua tau zootxo lub sijhawm ua haujlwm tag nrho thiab txhim kho kev sib cais.

Hauv kev ua haujlwm no,TFF ua kom muaj txiaj ntsig sai ntawm - cov qauv ntim loj, hais txog qhov txwv ntawm cov pa ultracentrifugation hauv kev ua cov qauv loj. Ultracentrifugation tom qab ntawd ua kom huv si sib sib zog nqus kom tshem tawm cov co-cov protein ntau cov impurities thaum pab tswj cov purity thiab kev ncaj ncees ntawm cov vesicles.

Tsis tas li ntawd, cov txheej txheem tsis xav tau qhov sib ntxiv ntawmexogenous tshuaj nag lossis daus reagents, pab khaws cia cov kev ua ub no ntawm cov hlab ntsha mus rau qhov loj tshaj plaws.

Los ntawm kev xav ntawm kev lag luam txhais lus, cov txheej txheem no tseem ceeb heevscale - nce peev xwmthiab tej zaum yuav pab daws cov teeb meem cuam tshuam nrog rau qhov loj -scale zus tau tej cov extracellular vesicles raukev xa tshuaj, tshuaj kho dua tshiab, thiab kev tsim khoom lag luam. Nws kuj tseem muab cov txheej txheem sim ua kom zoo rau cov txheej txheem tom ntej thiab kev sim ua haujlwm - nplai tsim.

 

Kev siv

Wang Ling, Li Mengshi, Xu Sha, et al.Tsim ib txoj hauv kev rau kev sib cais ntawm me me Extracellular Vesicles los ntawm Tangential Flow Filtration Ua ke nrog Ultracentrifugation. Suav Journal of Biologicals, 2022, 35(9): 1096–1101. DOI: 10.13200/j.cnki.cjb.003713.