Enye yeenzuzo eziphambili zenkcubeko yeeseli kukukwazi ukulawula ikhemistri yenyama yokuveliswa kweeseli (okt ubushushu, i-pH, uxinzelelo lwe-osmotic, uxinzelelo lwe-O2 kunye ne-CO2) kunye nokusingqongileyo (okt ihomoni kunye noxinzelelo lwezondlo).Ukongeza kwiqondo lokushisa, indawo yenkcubeko ilawulwa yindlela yokukhula.
Nangona indawo ye-physiological yenkcubeko ayicacanga njengendawo yayo yomzimba kunye neekhemikhali, ukuqonda okungcono kwamacandelo e-serum, ukuchongwa kwezinto zokukhula ezifunekayo ekwandeni, kunye nokuqonda okungcono kwe-microenvironment yeeseli kwinkcubeko.(Oko kukuthi ukusebenzisana kweeseli, ukusasazwa kwerhasi, ukusebenzisana ne-matrix) ngoku ivumela imigca ethile yeeseli ukuba ikhuliswe kwimidiya engenaserum.
1.Imeko yenkcubeko ichaphazela ukukhula kweeseli
Nceda uqaphele ukuba iimeko zenkcubeko yeeseli zahlukile kuhlobo ngalunye lweseli.
Iziphumo zokutenxa kwiimeko zenkcubeko ezifunekayo kuhlobo oluthile lweeseli ukusuka ekubonakalisweni kweephenotypes ezingaqhelekanga ukuya kukusilela ngokupheleleyo kwenkcubeko yeseli.Ke ngoko, sicebisa ukuba uqhelane nomgca weseli onomdla kuwo kwaye ulandele ngokungqongqo imiyalelo enikelwe kwimveliso nganye oyisebenzisayo kuvavanyo lwakho.
2.Izilumkiso zokudala imeko ephuculweyo yenkcubeko yeeseli kwiiseli zakho:
Imidiya yenkcubeko kunye neserum (jonga ngezantsi ngolwazi oluthe kratya)
amanqanaba e-pH kunye ne-CO2 (jonga ngezantsi ngolwazi oluthe kratya)
Lima iplastiki (jonga ngezantsi ngolwazi olungakumbi)
Ubushushu (jonga ngezantsi ngolwazi oluthe kratya)
2.1 Imidiya yeNkcubeko kunye neSerum
Indawo yenkcubeko yeyona ndawo ibalulekileyo yendawo yenkcubeko, kuba ibonelela ngezondlo, izinto zokukhula kunye namahomoni afunekayo ekukhuleni kweeseli, kwaye ilawula i-pH kunye noxinzelelo lwe-osmotic yenkcubeko.
Nangona uvavanyo lokuqala lwenkcubeko yeeseli lwenziwa kusetyenziswa imithombo yeendaba yendalo efunyenwe kwiincindi zethishu kunye nolwelo lomzimba, imfuno yokumisela umgangatho, umgangatho wemidiya, kunye nokwanda kwemfuno kukhokelele kuphuhliso lwemithombo yeendaba eqinisekileyo.Iindidi ezintathu ezisisiseko zeendaba ziyi-basal media, ukunciphisa i-serum media kunye ne-serum-free media, kwaye zineemfuno ezahlukeneyo zokuncedisa i-serum.
2.1.1 Indlela esisiseko
Gibco cell inkcubeko medium
Uninzi lwemigca yeeseli ikhula kakuhle kwimithombo yeendaba esisiseko equlethe i-amino acids, iivithamini, iisalti ze-inorganic, kunye nemithombo yekhabhoni (efana ne-glucose), kodwa ezi zinto zisisiseko zemithombo yeendaba kufuneka zongezwe ngeserum.
2.1.2 Ukunciphisa i-serum medium
Ibhotile ene-Gibco Low Serum Medium
Esinye isicwangciso sokunciphisa imiphumo emibi ye-serum kwiimvavanyo zenkcubeko yeseli kukusebenzisa i-serum-reduced media.Ukunciphisa i-serum medium yifomula ephakathi etyebileyo kwizondlo kunye nezinto eziphuma kwizilwanyana, ezinokunciphisa umthamo we-serum efunekayo.
2.1.3 Isixhobo esisimahla seSerum
Ibhotile ene-Gibco serum-free medium
I-serum-free medium (SFM) ithintela ukusetyenziswa kwe-serum yezilwanyana ngokutshintsha i-serum kunye nesondlo esifanelekileyo kunye nokuqulunqwa kwehomoni.Iinkcubeko ezininzi eziphambili kunye nemigca yeeseli zineserum-free ye-medium formulation, kubandakanya i-Chinese Hamster Ovary (CHO) iphinda ivelise iprotein yokuvelisa, iiseli ezahlukeneyo ze-hybridoma, imigca yezinambuzane Sf9 kunye ne-Sf21 (Spodoptera frugiperda), kunye ne-host host yemveliso yentsholongwane. (umzekelo, i-293, i-VERO, i-MDCK, i-MDBK), njl. Enye yeenzuzo eziphambili zokusebenzisa i-serum-free medium kukukwazi ukwenza i-medium ekhethiweyo kwiintlobo ezithile zeeseli ngokukhetha indibaniselwano efanelekileyo yezinto ezikhulayo.Le theyibhile ilandelayo idwelisa izinto ezilungileyo kunye nezingeloncedo kwimidiya engenaserum.
Inzuzo
Yandisa ukucaca
Ukusebenza okuhambelanayo ngakumbi
Ukucocwa lula kunye nokusetyenzwa phantsi komsinga
Vavanya ngokuchanekileyo umsebenzi weeseli
Yandisa imveliso
Ulawulo olungcono lweentshukumo zomzimba
Ukufunyanwa kwemidiya yeseli ephuculweyo
Ukusilela
Uhlobo lweseli iimfuno zefomula ephakathi
Ifuna ukucoceka okuphezulu kwe-reagent
Ukucotha ekukhuleni
2.2.1 inqanaba le-pH
Uninzi lwemigca yeseli ye-mammalian eqhelekileyo ikhula kakuhle kwi-pH 7.4, kwaye umahluko phakathi kwemigca yeeseli ezahlukeneyo mincinci.Nangona kunjalo, ezinye iilayini zeeseli eziguquliweyo ziye zaboniswa ukuba zikhule ngcono kwindawo ene-acidic kancinane (pH 7.0 - 7.4), ngelixa ezinye iiseli zeseli ze-fibroblast zikhetha indawo encinci ye-alkaline (pH 7.4 - 7.7).Imigca yeeseli zezinambuzane ezifana ne-Sf9 kunye ne-Sf21 zikhula kakuhle kwi-pH 6.2.
2.2.2 inqanaba le-CO2
Indlela yokukhula ilawula i-pH yenkcubeko kwaye ikhusela iiseli kwinkcubeko ukuxhathisa utshintsho kwi-pH.Ngesiqhelo, le buffering iphunyezwa ngokuqulatha eziphilayo (umzekelo, HEPES) okanye CO2-bicarbonate-based buffers.Ngenxa yokuba i-pH ye-medium ixhomekeke kwibhalansi ethambileyo ye-carbon dioxide (CO2) kunye ne-bicarbonate (HCO3-), utshintsho kwi-CO2 ye-atmospheric iya kutshintsha i-pH ye-medium.Ngoko ke, xa usebenzisa i-medium buffered nge-CO2-bicarbonate-based buffer, kuyafuneka ukuba usebenzise i-CO2 yangaphandle, ngakumbi xa uhlakula iiseli kwizitya zenkcubeko ezivulekileyo okanye ukulima imigca yeseli eguqulweyo kwiindawo eziphezulu.Nangona uninzi lwabaphandi ngokuqhelekileyo basebenzisa i-5-7% ye-CO2 emoyeni, ezininzi iimvavanyo zenkcubeko yeeseli zihlala zisebenzisa i-4-10% CO2.Nangona kunjalo, i-medium nganye ine-tension ye-CO2 ekhuthazwayo kunye nogxininiso lwe-bicarbonate ukufezekisa i-pH echanekileyo kunye noxinzelelo lwe-osmotic;ngolwazi oluthe kratya, nceda ubhekisele kwimiyalelo yomenzi ophakathi.
2.3 Ukulima iiplastiki
Iiplastiki zenkcubeko yeseli zifumaneka ngeendlela ezahlukeneyo, ubungakanani kunye nemigangatho ukuze zilungele izicelo ezahlukeneyo zenkcubeko yeseli.Sebenzisa isikhokelo seplastiki yenkcubeko yeseli kunye nesikhokelo sesikhongozeli senkcubeko yeseli ngezantsi ukukunceda ukhethe iplastiki elungileyo kwisicelo sakho senkcubeko yeseli.
Jonga zonke iiplastiki zenkcubeko ye-Thermo Scientific Nunc (ikhonkco lentengiso)
2.4 Ubushushu
Elona qondo lobushushu lifanelekileyo kwinkcubeko yeeseli lixhomekeke kumlinganiselo omkhulu kubushushu bomzimba wenginginya apho iiseli zibekwe zodwa, kwaye kancinci kutshintsho lwe-anatomical kwiqondo lobushushu (umzekelo, ubushushu bolusu bunokuba sezantsi kunesihlunu samathambo). ).Kwinkcubeko yeseli, Ukushisa kakhulu yingxaki enkulu kunokushisa.Ke ngoko, iqondo lobushushu kwi-incubator lidla ngokusetwa ngaphantsi kweqondo lobushushu elilelona liphezulu.
2.4.1 Olona bushushu buphezulu kwiiseli ezahlukeneyo
Uninzi lwemigca yeeseli zabantu kunye nezanyisayo zigcinwa kuma-36°C ukuya kuma-37°C ukuze zikhule ngokugqibeleleyo.
Iiseli zezinambuzane zilinywa kwi-27 ° C ukuze zikhule kakuhle;zikhula ngokucothayo kumaqondo asezantsi kunye namaqondo obushushu aphakathi kwe 27°C kunye ne 30°C.Ngaphezulu kwe-30 ° C, amandla eeseli zezinambuzane ayancipha, nokuba abuyele ku-27 ° C, iiseli aziyi kuphinda zibuyele.
Imigca yeeseli ze-Avian idinga i-38.5°C ukuze ifikelele ekukhuleni okuphezulu.Nangona ezi seli zinokugcinwa kuma-37°C, ziya kukhula ngokucothayo.
Imigca yeseli ephuma kwizilwanyana ezinegazi elibandayo (ezifana nezilwanyana ezihlala emanzini nasemanzini, iintlanzi zasemanzini abandayo) ziyakwazi ukunyamezela ubushushu obuphakathi kwe-15°C ukuya kuma-26°C.
Ixesha lokuposa: Feb-01-2023