1. Cell rov qab
Tom qab cov cellopreserved raug tshem tawm los ntawm cov kua nitrogen, lawv tau thawed nrog tas li co nyob rau hauv ib da dej 37 degree. Hloov cov hlwb thawed mus rau hauv ib lub raj centrifuge, ntxiv pre-warmed DMEM ua tiav nruab nrab ntawm 37 degree (ntawm cov fetal bovine serum yog li 10 feem pua), maj mam tshuab sib npaug, centrifuge ntawm 500 g rau 2 min, thiab muab pov tseg lub supernatant.
Ntxiv DMEM ua tiav nruab nrab los ntxuav thiab pov tseg cov supernatant. Ntxiv DMEM ua tiav qhov nruab nrab, sib tov maj mam los ntawm pipetting, ua lub cell ncua kev kawm ntawv, inoculate nws nyob rau hauv ib tug petri tais / hwj, thiab kab lis kev cai nyob rau hauv lub cell incubator muaj 5 feem pua CO2.
2. Cell hla
Thaum lub cell ceev nce mus txog 80 feem pua ~ 90 feem pua (cov qoob loo ntxov ntxov tsis txaus, cov hlwb lig dhau los tsis zoo, subculture ntawm qhov sib piv ntawm 1: 2 txog 1: 10 lossis ntau dua, feem ntau 1: 3 txog 1: 5 cell tiam, uas yog, los ntawm cell inoculation Thaum lub sij hawm ntawm kev sib cais thiab rov cog qoob loo, qhov nruab nrab tag nrho raug tshem tawm thiab ntxuav ob zaug nrog 1X PBS.
Ntxiv trypsin (ceeb toom: tus nqi ntawm kev zom zaub mov yog qhov zoo tshaj plaws los npog cov hlwb, thiab qhov zoo tshaj plaws digestion kub yog 37 degree . Saib cov hlwb nyob rau hauv lub microscope: soj ntsuam cov digested hlwb nyob rau hauv lub inverted microscope, yog hais tias lub cytoplasm yog retracted, lub hlwb. tsis txuas ua ke lawm. Cov hlais, qhia tias cov hlwb tau zom tau zoo nyob rau lub sijhawm no), zom lawv, thiab muab tso rau hauv lub cell incubator li ntawm 2-3 feeb.
Ntxiv qhov tsim nyog ntawm DMEM ua tiav qhov nruab nrab kom tsis txhob trypsinization, hloov mus rau ib lub raj centrifuge thiab centrifuge ntawm 500 g rau 2 min, pov tseg cov supernatant, ntxiv DMEM ua tiav nruab nrab los ntxuav, thiab pov tseg cov supernatant.
Ntxiv DMEM ua tiav qhov nruab nrab, sib tov los ntawm maj mam muab tso, pipette 10 microliters rau suav, thiab tom qab ntawd txuas ntxiv mus rau kab lis kev cai hauv lub cell incubator uas muaj 5 feem pua CO2 raws li qhov xav tau ntawm tes ntim.
3. Cell cryopreservation
Thaum lub cell ceev nce mus txog 80 feem pua ~ 90 feem pua, tshem tawm qhov nruab nrab thiab ntxuav 2 zaug nrog 1X PBS. Ntxiv trypsin rau kev zom thiab tso rau hauv lub cell incubator li ntawm 2-3 min. Ntxiv DMEM ua tiav nruab nrab kom tsis txhob trypsin plab zom mov, hloov mus rau ib lub raj centrifuge thiab centrifuge ntawm 500 g rau 2 min, muab pov tseg lub supernatant, ntxiv DMEM ua tiav nruab nrab los ntxuav, thiab pov tseg cov supernatant. Ntxiv 1ml ntawm cov tshuaj khov nab kuab (90 feem pua fetal bovine serum, 10 feem pua DMSO. Feem ntau hais lus, cov ntsiab lus hauv cov ntshav tuaj yeem hloov kho ntawm 10 feem pua thiab 90 feem pua . Ntxiv cov kua qaub rau hauv cov kua dej khov tuaj yeem muab cov khoom noj rau cov hlwb ntawm ib sab, thiab tuaj yeem Muab cov tshuaj tiv thaiv uas tsis yog-permeable thaum lub sij hawm cell cryopreservation, xws li sucrose, albumin, thiab lwm yam los tiv thaiv cov hlwb zoo dua), muab tso rau hauv lub raj cryopreservation (muaj isopropyl cawv hauv lub raj kom ntseeg tau tias qhov kub thiab txias yuav txo tau), muab tso rau tam sim ntawd. Txias hauv lub tub yees 4 degree rau 30 feeb, tom qab ntawd muab tso rau hauv lub tub yees -20 degree rau 30 feeb, thiab tom qab ntawd muab tso rau hauv lub tub yees -80 degree thaum hmo ntuj.
Muab tso rau hauv cov kua nitrogen rau hnub tom qab, nws tuaj yeem khaws cia tsawg kawg yog ob xyoos, thiab yog tias koj tsis muab tso rau hauv cov kua nitrogen, nws tuaj yeem khaws cia rau peb lub hlis.
Lub hauv paus ntsiab lus ntawm cell cryopreservation thiab rov qab yog: qeeb khov thiab thawing, uas yog qhov zoo dua rau kev tswj cov cell viability. Cryopreservation ntawm cov hlwb uas tsis muaj tus neeg saib xyuas kev tiv thaiv yuav ua rau tsim cov dej khov hauv lub cev, uas yuav ua rau cov neeg kho tshuab puas tsuaj rau cov hlwb, ua rau muaj kev hloov pauv hauv intracellular ib puag ncig osmotic siab, pH, electrolytes, thiab lwm yam, thiab tom qab ntawd txhawb kev tuag ntawm tes.
4. Cov teeb meem xav tau kev saib xyuas
(1) Preheating kab lis kev cai tov: muab lub raj mis npaj uas muaj cov kab lis kev cai tov, PBS thiab trypsin rau hauv 37 degree dej da dej rau preheat;
(2) Siv 75 feem pua piv txwv li los so lub ultra-ntxuav workbench thiab tes uas tau irradiated nrog ultraviolet rays;
(3) Kev tso kawm kom raug ntawm cov cuab yeej siv: xyuas kom muaj chaw ua haujlwm txaus, uas tsis yog yooj yim rau kev khiav lag luam tab sis kuj txo cov pa phem;
(4) Teeb lub teeb cawv: nco ntsoov tias cov nplaim taws yuav tsum tsis txhob me dhau;
(5) Kev ua haujlwm aseptic nruj;
(6) Kev zom zaub mov nruab nrab ntawm cov hlwb ua ke: Lub sijhawm zom zaub mov raug cuam tshuam los ntawm ntau yam xws li hom kev zom zaub mov, lub sijhawm npaj, thiab cov nyiaj ntxiv rau cov kab lis kev cai. Thaum lub sij hawm txoj kev zom zaub mov, yuav tsum tau them nyiaj mloog mus rau cov kev hloov nyob rau hauv cov duab ntawm cov kab mob hlwb. Thaum lub cytoplasm shrinks, Yog hais tias qhov kev twb kev txuas yog xoob, los yog muaj cov cim ntawm floating nyob rau hauv tej daim, lub plab zom mov yuav tsum tau txiav tam sim ntawd;
(7) Tag nrho cov kev khiav hauj lwm ntawm cov hlwb dhau los yuav tsum nyob ze rau ntawm cov nplaim taws ntawm lub teeb cawv li sai tau. Nws yog qhov zoo tshaj los ua haujlwm nrog tsuas yog ib lub xovtooj ntawm ib lub sijhawm, siv ib txheej khoom siv rau txhua lub xovtooj. zam kev kis kab mob;
(8) Lub raj mis lub qhov ncauj ntawm lub xov tooj ntawm tes yuav tsum tau ua kom tsis muaj menyuam ntawm lub teeb cawv txhua zaus nws qhib lossis kaw.
