Chimodzi mwazabwino zazikulu za chikhalidwe cha cell ndikutha kuwongolera kapangidwe kake ka kubalana kwa maselo (ie kutentha, pH, kuthamanga kwa osmotic, kupsinjika kwa O2 ndi CO2) komanso chilengedwe (mwachitsanzo, mahomoni ndi michere).Kuphatikiza pa kutentha, chikhalidwe cha chikhalidwe chimayendetsedwa ndi kukula kwapakati.
Ngakhale kuti chilengedwe cha chikhalidwe cha chikhalidwe sichidziwika bwino monga momwe chilengedwe chimakhalira ndi mankhwala, kumvetsetsa bwino zigawo za seramu, kuzindikira zinthu za kukula zomwe zimafunikira kuti zichuluke, komanso kumvetsetsa bwino za microenvironment ya maselo mu chikhalidwe.(Ndiye kuyanjana kwa ma cell, kufalikira kwa gasi, kulumikizana ndi matrix) tsopano kulola kuti mizere ina ya cell ipangidwe mu media yopanda seramu.
1.Culture chilengedwe chimakhudza kukula kwa maselo
Chonde dziwani kuti zikhalidwe zama cell ndizosiyana pamtundu uliwonse wa selo.
Zotsatira zapatuka pazikhalidwe zomwe zimafunikira pamtundu wina wa selo zimachokera ku mawonekedwe achilendo a phenotype mpaka kulephera kwathunthu kwa chikhalidwe cha maselo.Chifukwa chake, tikukulimbikitsani kuti mudziwe bwino za mzere wa cell womwe mukufuna ndikutsatira mosamalitsa malangizo omwe amaperekedwa pachinthu chilichonse chomwe mumagwiritsa ntchito poyesera.
2.Kusamala popanga malo okhathamiritsa a chikhalidwe cha ma cell anu:
Culture media ndi seramu (onani pansipa kuti mudziwe zambiri)
pH ndi CO2 milingo (onani pansipa kuti mudziwe zambiri)
Lima pulasitiki (onani m'munsimu kuti mudziwe zambiri)
Kutentha (onani pansipa kuti mudziwe zambiri)
2.1 Cultural Media ndi Serum
Sing'anga ya chikhalidwe ndi gawo lofunika kwambiri la chikhalidwe cha chikhalidwe, chifukwa limapereka zakudya, kukula ndi mahomoni ofunikira pakukula kwa maselo, ndikuwongolera pH ndi osmotic pressure ya chikhalidwe.
Ngakhale kuyesa koyambirira kwa chikhalidwe cha ma cell kunkachitika pogwiritsa ntchito zinthu zachilengedwe zomwe zidatengedwa kuchokera kuzinthu zamtundu ndi zamadzi am'thupi, kufunikira kokhazikika, mtundu wa media, komanso kufunikira kowonjezereka kudapangitsa kuti pakhale zofalitsa zotsimikizika.Mitundu itatu yoyambira ya media ndi basal media, media media yocheperako komanso media yopanda seramu, ndipo ali ndi zofunika zosiyanasiyana pakuwonjezera seramu.
2.1.1 Basic medium
Gibco cell Culture medium
Mizere yambiri ya maselo imakula bwino muzinthu zoyambira zomwe zimakhala ndi ma amino acid, mavitamini, mchere wa inorganic, ndi magwero a kaboni (monga shuga), koma zopangira izi ziyenera kuwonjezeredwa ndi seramu.
2.1.2 Kuchepetsa kwa seramu
Botolo lokhala ndi Gibco Low Serum Medium
Njira ina yochepetsera zotsatira zoyipa za seramu mu kuyesa kwa chikhalidwe cha ma cell ndikugwiritsa ntchito media yochepetsera seramu.Kuchepetsedwa kwa seramu ndi njira yoyambira yomwe ili ndi michere yambiri komanso zinthu zochokera ku nyama, zomwe zimatha kuchepetsa kuchuluka kwa seramu yofunikira.
2.1.3 Sing'anga yopanda seramu
Botolo la Gibco lopanda seramu
Serum-free medium (SFM) imalepheretsa kugwiritsa ntchito seramu ya nyama posintha seramu ndi zakudya zoyenera komanso kupanga mahomoni.Zikhalidwe zambiri zoyambira ndi ma cell amakhala ndi ma serum opanda ma seramu apakati, kuphatikiza mzere wopangira mapuloteni aku China Hamster Ovary (CHO), mizere yosiyanasiyana ya ma cell a hybridoma, mizere ya tizilombo Sf9 ndi Sf21 (Spodoptera frugiperda), komanso kwa Wopanga ma virus. (mwachitsanzo, 293, VERO, MDCK, MDBK), etc. Ubwino umodzi waukulu wogwiritsa ntchito sing'anga yopanda seramu ndikutha kupanga sing'anga kusankha mitundu yeniyeni ya maselo posankha kuphatikiza koyenera kwa zinthu zakukula.Gome ili m'munsili likutchula ubwino ndi kuipa kwa zofalitsa zopanda seramu.
Ubwino
Wonjezerani kumveka bwino
Kuchita kosasinthasintha
Kuyeretsa kosavuta ndi kukonza kunsi kwa mtsinje
Onetsetsani bwino ntchito ya maselo
Wonjezerani zokolola
Kuwongolera bwino momwe thupi limayendera
Kuzindikirika kwama cell media
Kuipa
Zofunikira za fomula yapakati pamtundu wa ma cell
Amafuna apamwamba reagent chiyero
Kuchepetsa kukula
2.2.1 pH mlingo
Mizere yodziwika bwino ya ma cell a mammalian imakula bwino pa pH 7.4, ndipo kusiyana pakati pa mizere yosiyana ya maselo kumakhala kochepa.Komabe, mizere ina yosinthidwa ya maselo yasonyezedwa kuti ikukula bwino m'malo a acidic pang'ono (pH 7.0 - 7.4), pamene mizere ya cell ya fibroblast imakonda malo amchere pang'ono (pH 7.4 - 7.7).Mizere yama cell a tizilombo monga Sf9 ndi Sf21 imakula bwino pa pH 6.2.
2.2.2 CO2 mlingo
Sing'anga yakukulira imawongolera pH ya chikhalidwe ndikusunga ma cell mu chikhalidwe kuti asasinthe pH.Nthawi zambiri, kusunga uku kumatheka ndi kukhala ndi organic (mwachitsanzo, HEPES) kapena CO2-bicarbonate-based buffers.Chifukwa pH ya sing'anga imadalira kusungunuka kwa mpweya woipa wa carbon dioxide (CO2) ndi bicarbonate (HCO3-), kusintha kwa CO2 mumlengalenga kudzasintha pH ya sing'anga.Choncho, pamene ntchito sing'anga buffered ndi CO2-bicarbonate ofotokoza bafa, m'pofunika ntchito exogenous CO2, makamaka pamene culturing maselo lotseguka chikhalidwe mbale kapena culturing kusandulika mizere selo pa woipa kwambiri.Ngakhale ofufuza ambiri amagwiritsa ntchito 5-7% CO2 mumlengalenga, kuyesa kwa chikhalidwe cha selo nthawi zambiri kumagwiritsa ntchito 4-10% CO2.Komabe, sing'anga iliyonse imakhala ndi mphamvu ya CO2 ndi ndende ya bicarbonate kuti ikwaniritse pH yoyenera ndi kuthamanga kwa osmotic;kuti mudziwe zambiri, chonde onani malangizo opanga sing'anga.
2.3 Kulima mapulasitiki
Mapulasitiki amtundu wa cell amapezeka m'mitundu yosiyanasiyana, makulidwe ndi mawonekedwe kuti agwirizane ndi machitidwe osiyanasiyana amtundu wa cell.Gwiritsani ntchito kalozera wa cell Culture pulasitiki pamwamba ndi chiwongolero cha cell culture pansipa kuti zikuthandizeni kusankha pulasitiki yoyenera kugwiritsa ntchito chikhalidwe chanu.
Onani mapulasitiki onse a Thermo Scientific Nunc cell culture (ulalo wotsatsa)
2.4 Kutentha
Kutentha koyenera kwa chikhalidwe cha ma cell kumadalira kwambiri kutentha kwa thupi la wolandirayo komwe maselo amasiyanitsidwa, komanso pang'ono ndi kusintha kwa kutentha kwa thupi (mwachitsanzo, kutentha kwa khungu kumatha kukhala kotsika kuposa kwachigoba). ).Kwa chikhalidwe cha ma cell, Kutentha kwambiri ndi vuto lalikulu kuposa kutenthedwa.Choncho, kutentha mu chofungatira zambiri anapereka pang`ono pansi akadakwanitsira kutentha.
2.4.1 Kutentha koyenera kwa ma cell osiyanasiyana
Mizere yambiri ya maselo aumunthu ndi mammalian imasungidwa pa 36 ° C mpaka 37 ° C kuti ikule bwino.
Maselo a tizilombo amalimidwa pa 27 ° C kuti akule bwino;amakula pang’onopang’ono pa kutentha kotsika ndi kutentha kwapakati pa 27°C ndi 30°C.Pamwamba pa 30 ° C, mphamvu ya maselo a tizilombo imachepa, ngakhale itabwerera ku 27 ° C, maselo sadzakhalanso.
Mizere ya Avian cell imafunika 38.5 ° C kuti ifike kukula kwakukulu.Ngakhale kuti maselowa amatha kusungidwa pa 37 ° C, amakula pang'onopang'ono.
Mizere ya ma cell yochokera ku nyama zamagazi ozizira (monga amphibians, nsomba za m'madzi ozizira) zimatha kupirira kutentha kwapakati pa 15 ° C mpaka 26 ° C.
Nthawi yotumiza: Feb-01-2023