Enye yezinzuzo eziyinhloko zesiko lamangqamuzana amandla okusebenzisa amakhemikhali angokomzimba okukhiqizwa kwamangqamuzana (okungukuthi izinga lokushisa, i-pH, ingcindezi ye-osmotic, ukungezwani kwe-O2 kanye ne-CO2) kanye nemvelo yokuphila (okungukuthi i-hormone nokugxilwa kwezakhi).Ngaphezu kwezinga lokushisa, indawo yesiko ilawulwa indlela yokukhula.
Nakuba imvelo yemvelo yesiko ingacacile njengemvelo yayo ebonakalayo namakhemikhali, ukuqonda okungcono kwezingxenye ze-serum, ukuhlonza izici zokukhula ezidingekayo ukuze kwande, kanye nokuqonda kangcono imvelo encane yamaseli esikweni.(Okungukuthi ukusebenzisana kwamaseli, ukusabalalisa kwegesi, ukusebenzisana ne-matrix) manje kuvumela imigqa yeseli ethile ukuthi ithuthukiswe kumidiya engenayo i-serum.
1.Indawo yesiko ithinta ukukhula kwamaseli
Sicela uqaphele ukuthi izimo zesiko lamaseli zihlukile kuhlobo lweseli ngalinye.
Imiphumela yokuchezuka ezimeni zesiko elidingekayo ohlotsheni oluthile lweseli isukela ekuboniseni ama-phenotypes angavamile kuya ekuhlulekeni okuphelele kwesiko leseli.Ngakho-ke, sincoma ukuthi ujwayelane nolayini weselula onentshisekelo kuwo futhi ulandele ngokuqinile imiyalelo enikezwe umkhiqizo ngamunye owusebenzisayo ekuhloleni kwakho.
2.Izinyathelo zokuphepha zokudala indawo ethuthukisiwe yesiko lamaseli kumaseli akho:
Imidiya yezamasiko neserum (bona ngezansi ukuze uthole ulwazi olwengeziwe)
Amazinga we-pH kanye ne-CO2 (bona ngezansi ukuze uthole ulwazi olwengeziwe)
Hlakulela ipulasitiki (bheka ngezansi ukuze uthole ulwazi olwengeziwe)
Izinga lokushisa (bona ngezansi ukuze uthole ulwazi olwengeziwe)
2.1 Imidiya Yezamasiko kanye neSerum
I-culture medium iyingxenye ebaluleke kakhulu yendawo yesiko, ngoba inikeza izakhi, izici zokukhula namahomoni adingekayo ekukhuleni kwamangqamuzana, futhi ilawula i-pH nengcindezi ye-osmotic yesiko.
Nakuba ukuhlolwa kokuqala kwesiko lamangqamuzana kwenziwa kusetshenziswa imidiya yemvelo etholwe ekukhishwe kwezicubu noketshezi lomzimba, isidingo sokuma, ikhwalithi yemidiya, kanye nesidingo esikhulayo siholele ekuthuthukisweni kwemidiya eqondile.Izinhlobo ezintathu eziyisisekelo zemidiya imidiya ye-basal, imidiya ye-serum encishisiwe kanye nemidiya engena-serum, futhi inezidingo ezihlukene zokwengezwa kwe-serum.
2.1.1 I-Basic medium
I-Gibco cell Culture medium
Imigqa eminingi yamaseli ikhula kahle emithonjeni yezindaba eyisisekelo equkethe ama-amino acid, amavithamini, usawoti we-inorganic, nemithombo ye-carbon (efana ne-glucose), kodwa lezi zakhi zemidiya eziyisisekelo kufanele zengezwe nge-serum.
2.1.2 I-serum medium encishisiwe
Ibhodlela eline-Gibco Low Serum Medium
Elinye isu lokunciphisa imiphumela emibi ye-serum ekuhlolweni kwesiko lamaseli ukusebenzisa imidiya encishisiwe ye-serum.I-serum medium encishisiwe iyifomula eyisisekelo emaphakathi enomsoco kanye nezici ezitholakala ezilwaneni, ezinganciphisa inani le-serum edingekayo.
2.1.3 I-Serum-free medium
Ibhodlela eline-Gibco-free medium
I-serum-free medium (SFM) ivimbela ukusetshenziswa kwe-serum yezilwane ngokufaka esikhundleni se-serum ngokudla okufanele kanye nokwakhiwa kwamahomoni.Amasiko amaningi ayisisekelo kanye nemigqa yamaseli inokwakheka okuphakathi kwe-serum-free, okubandakanya ulayini wokukhiqiza amaprotheni we-Chinese Hamster Ovary (CHO), imigqa ehlukahlukene yamaseli e-hybridoma, imigqa yezinambuzane i-Sf9 kanye ne-Sf21 (Spodoptera frugiperda), kanye ne-The host ukukhiqizwa kwegciwane. (isibonelo, 293, VERO, MDCK, MDBK), njll. Enye yezinzuzo eziyinhloko zokusebenzisa i-serum-free medium yikhono lokwenza okuphakathi kukhethele izinhlobo ezithile zamaseli ngokukhetha inhlanganisela efanele yezinto ezikhula.Ithebula elilandelayo libonisa izinzuzo kanye nokubi kwemidiya ye-serum-free.
Inzuzo
Khulisa ukucaca
Ukusebenza okungaguquki okwengeziwe
Ukuhlanza okulula nokucubungula phansi komfula
Hlola ngokunembile ukusebenza kweseli
Khulisa ukukhiqiza
Ukulawulwa okungcono kokusabela komzimba
Ukutholwa kwemidiya yeselula okuthuthukisiwe
Ububi
Izidingo zefomula emaphakathi yohlobo lweseli
Kudingeka ukuhlanzeka okuphezulu kwe-reagent
Ukwehla kokukhula
2.2.1 pH izinga
Imigqa eminingi yamaseli ezincelisayo evamile ikhula kahle ku-pH 7.4, futhi umehluko phakathi kwemigqa yamaseli ehlukene mincane.Kodwa-ke, eminye imigqa yamaseli eguquliwe iboniswe ukuthi ikhula kangcono endaweni ene-acidic kancane (pH 7.0 - 7.4), kuyilapho imigqa yeseli ye-fibroblast evamile ikhetha indawo ene-alkaline kancane (pH 7.4 - 7.7).Imigqa yeseli yezinambuzane efana ne-Sf9 ne-Sf21 ikhula kahle kakhulu ku-pH 6.2.
2.2.2 Izinga le-CO2
Indawo yokukhula ilawula i-pH yesiko futhi ivimbela amaseli kusiko ukuze amelane nezinguquko ku-pH.Ngokuvamile, lokhu kugcina kumthamo kufinyelelwa ngokuqukatha okuphilayo (isibonelo, i-HEPES) noma amabhafa asuselwa ku-CO2-bicarbonate.Ngenxa yokuthi i-pH yendawo ephakathi incike ebhalansini ethambile ye-carbon dioxide (CO2) ne-bicarbonate (HCO3-) encibilikisiwe, izinguquko ku-CO2 yasemkhathini zizoshintsha i-pH yendawo.Ngakho-ke, uma usebenzisa i-medium buffered nge-CO2-bicarbonate-based buffer, kuyadingeka ukusebenzisa i-CO2 yangaphandle, ikakhulukazi uma uhlakulela amaseli ezitsheni zamasiko avulekile noma uhlakulela imigqa yeseli eguquliwe ezindaweni eziphezulu.Nakuba abacwaningi abaningi bevame ukusebenzisa i-5-7% CO2 emoyeni, ukuhlolwa okuningi kwesiko lamaseli kuvame ukusebenzisa i-4-10% CO2.Kodwa-ke, i-medium ngayinye ine-tension ye-CO2 enconywayo kanye nokugxila kwe-bicarbonate ukuze kuzuzwe i-pH efanele kanye nengcindezi ye-osmotic;ukuze uthole ulwazi olwengeziwe, sicela ubheke imiyalo yomkhiqizi ophakathi.
2.3 Ukutshala amapulasitiki
Amapulasitiki empucuko yamaseli atholakala ngezindlela ezihlukahlukene, osayizi kanye nezindawo ukuze avumelane nezicelo ezihlukahlukene zesiko lamaseli.Sebenzisa inkomba yepulasitiki yesiko le-cell culture kanye negayidi yesiqukathi seseli yesiko ngezansi ukuze ikusize ukhethe ipulasitiki efanele yesicelo sakho sesiko leseli.
Buka wonke amapulasitiki e-Thermo Scientific Nunc cell culture (isixhumanisi sokukhangisa)
2.4 Izinga lokushisa
Izinga lokushisa elilungile lesiko lamaseli lincike kakhulu ekushiseni komzimba komsingathi lapho amangqamuzana ehlukanisiwe, futhi ngokwezinga elincane ekushintsheni kwe-anatomical kwezinga lokushisa (isibonelo, izinga lokushisa lesikhumba lingase libe phansi kunalelo lemisipha yohlaka lwamathambo. ).Ngokwesiko lamaseli, Ukushisa ngokweqile kuyinkinga enkulu kunokushisa ngokweqile.Ngakho-ke, izinga lokushisa ku-incubator ngokuvamile libekwe ngaphansi kancane kwezinga lokushisa elilungile.
2.4.1 Izinga lokushisa eliphezulu lemigqa yamaseli ehlukene
Iningi lamaseli womuntu kanye nezilwane ezincelisayo zigcinwa ku-36°C kuya ku-37°C ukuze zikhule kahle.
Amaseli ezinambuzane atshalwa ku-27°C ukuze akhule kahle;zikhula kancane kancane emazingeni okushisa aphansi kanye namazinga okushisa aphakathi kuka-27°C no-30°C.Ngaphezulu kuka-30°C, amandla amangqamuzana ezinambuzane ayancipha, ngisho noma ebuyela ku-27°C, amaseli ngeke alulame.
Imigqa yamaseli ezinyoni idinga u-38.5°C ukuze ifinyelele ekukhuleni okuphezulu.Nakuba la maseli angagcinwa ku-37°C, azokhula kancane kancane.
Imigqa yamaseli etholakala ezilwaneni ezinegazi elibandayo (njengezilwane ezihlala emanzini, izinhlanzi zasemanzini abandayo) zingabekezelela izinga lokushisa elibanzi lika-15°C kuya ku-26°C.
Isikhathi sokuthumela: Feb-01-2023