CryovialsNthawi zambiri amagwiritsidwa ntchito posungirako ma cell a cell ndi zida zina zofunika kwambiri zamoyo, mu ma dewars odzazidwa ndi nayitrogeni wamadzimadzi.

Pali magawo angapo nawo bwino kuteteza maselo mu madzi asafe. Pomwe mfundo yayikulu ndikuundana pang'onopang'ono, njira yeniyeni yomwe imagwiritsidwa ntchito imadalira mtundu wa cell ndi cryoprotectant yomwe imagwiritsidwa ntchito. Pali zinthu zingapo zoyendetsera chitetezo ndi njira zabwino zomwe muyenera kuziganizira posunga ma cell pamalo otsika kwambiri.

Nkhaniyi ikufuna kufotokoza mwachidule momwe ma cryovials amasungidwa mu nayitrogeni wamadzimadzi.

Kodi Cryovials ndi chiyani

Ma Cryovials ndi ang'onoang'ono, otsekeredwa Mbale zomwe zimapangidwira kusunga zitsanzo zamadzimadzi pamtunda wotsika kwambiri. Amawonetsetsa kuti ma cell omwe amasungidwa mu cryoprotectant samalumikizana mwachindunji ndi nayitrogeni wamadzimadzi, kuchepetsa chiopsezo cha ma fractures a ma cell pomwe amapindulabe ndi kuzizira kwambiri kwa nayitrogeni wamadzimadzi.

Mabotolo nthawi zambiri amapezeka m'mabuku osiyanasiyana ndi mapangidwe - amatha kukhala mkati kapena kunja kwa ulusi ndi zotsika kapena zozungulira. Mawonekedwe osabala komanso osabereka amapezekanso.

Ndani Amagwiritsa NtchitoCyrovalsKusunga Ma cell mu Liquid Nitrogen

Mitundu yambiri ya NHS ndi ma laboratories achinsinsi, komanso mabungwe ofufuza omwe ali ndi zingwe zosungira magazi, epithelial cell biology, immunology ndi stem cell biology amagwiritsa ntchito ma cryovials ku cryopreserve cell.

Maselo osungidwa motere akuphatikizapo B ndi T Maselo, CHO Maselo, Hematopoietic Stem ndi Progenitor Cells, Hybridomas, Intestinal Cells, Macrophages, Mesenchymal Stem and Progenitor Cells, Monocytes, Myeloma, NK Cells ndi Pluripotent Stem Cells.

Chidule cha Momwe Mungasungire Cryovials mu Liquid Nitrogen

Cryopreservation ndi njira yomwe imateteza maselo ndi zinthu zina zamoyo zomwe zimapangidwira pozizizira kuzizira kwambiri. Maselo amatha kusungidwa mu nayitrogeni wamadzimadzi kwa zaka zambiri popanda kuwonongeka kwa maselo. Ichi ndi chidule cha ndondomeko zogwiritsidwa ntchito.

Kukonzekera Maselo

Njira yeniyeni yokonzekera zitsanzo idzasiyana malinga ndi mtundu wa selo, koma nthawi zambiri, maselo amasonkhanitsidwa ndi centrifuged kuti apange pellet yolemera kwambiri. Pellet iyi imayikidwanso mu supernatant yosakanikirana ndi cryoprotectant kapena cryopreservation medium.

Cryopreservation Medium

Sing'anga iyi imagwiritsidwa ntchito kusunga ma cell m'malo otentha kwambiri omwe angakumane nawo poletsa mapangidwe a makristasi a intra and extracellular ndipo ma cell amafa. Ntchito yawo ndi kupereka malo otetezeka, oteteza maselo ndi minofu panthawi yachisanu, kusungirako, ndi kusungunuka.

Sing'anga monga fresh frozen plasma (FFP), heparinised plasmalyte solution kapena seramu-free, njira zopanda zigawo za nyama zimasakanizidwa ndi cryoprotectants monga dimethyl sulphoxide (DMSO) kapena glycerol.

The re-liquated chitsanzo pellet ali aliquoted mu polypropylene cryovials mongaKampani ya Suzhou Ace Biomedical Cryogenic Storage Mbale.

Ndikofunika kuti musadzaze ma cryovials chifukwa izi zidzakulitsa chiwopsezo chosweka komanso kutulutsa zomwe zili mkati (1).

Kuzizira Kwambiri Kwambiri

Nthawi zambiri, kuzizira koyendetsedwa pang'onopang'ono kumagwiritsidwa ntchito poteteza bwino ma cell.

Zitsanzo zikayikidwa mu mbale za cryogenic, zimayikidwa pa ayezi wonyowa kapena mufiriji ya 4 ℃ ndipo kuzizira kumayambika mkati mwa mphindi zisanu. Monga kalozera wamba, maselo amazizidwa pamlingo wa -1 mpaka -3 pamphindi (2). Izi zimatheka pogwiritsa ntchito choziziritsa kukhosi kapena poyika vial mu bokosi lotsekeredwa lomwe limayikidwa mufiriji yoyendetsedwa ndi -70 ° C mpaka -90 ° C.

Kusamutsa ku Liquid Nayitrojeni

Mbale zozizira za cryogenic zimasamutsidwa ku tanki yamadzimadzi ya nayitrogeni kwa nthawi yosadziwika ngati kutentha kosakwana -135 ℃ kumasungidwa.

Izi kopitilira muyeso-otsika kutentha angapezeke mwa kumizidwa mu madzi kapena nthunzi gawo asafe.

Liquid kapena Vapor Phase?

Kusungirako mu gawo lamadzimadzi nayitrogeni amadziwika kuti amasunga kutentha kozizira kokhazikika, koma nthawi zambiri sikuvomerezeka pazifukwa izi:

• Kufunika kwa ma voliyumu akulu (kuzama) kwa nayitrogeni wamadzimadzi komwe ndi kowopsa. Kuwotcha kapena kupuma chifukwa cha izi ndi chiopsezo chenicheni.
• Milandu yolembedwa yopatsirana ndi tizilombo toyambitsa matenda monga aspergillus, hep B ndi kufalikira kwa ma virus kudzera mu sing'anga yamadzi ya nayitrogeni (2,3)
• Kuthekera kwa nayitrogeni wamadzimadzi kutayikira mu mbale zomizidwa. Akachotsedwa kusungirako ndikutenthedwa mpaka kutentha kwa chipinda, nayitrogeniyo imakula mofulumira. Chifukwa chake, vial imatha kusweka ikachotsedwa mumadzi osungiramo nayitrogeni, kupangitsa ngozi ku zinyalala zonse zowuluka komanso kukhudzana ndi zomwe zili mkatimo (1, 4).

Pazifukwa izi, kusungirako kutentha kwambiri kumakhala kokhazikika mu gawo la nayitrogeni wa nthunzi. Pamene zitsanzo ziyenera kusungidwa mu gawo lamadzimadzi, machubu apadera a cryoflex ayenera kugwiritsidwa ntchito.

Choyipa cha gawo la nthunzi ndikuti kutentha koyima kumatha kuchitika zomwe zimapangitsa kusinthasintha kwa kutentha pakati pa -135 ℃ ndi -190 ℃. Izi zimafunika kuwunika mosamala komanso mwakhama kuchuluka kwa nayitrogeni wamadzimadzi komanso kusiyanasiyana kwa kutentha (5).

Opanga ambiri amalimbikitsa kuti ma cryovials ndi oyenera kusungidwa mpaka -135 ℃ kapena kugwiritsidwa ntchito mu gawo la nthunzi lokha.

Kusungunula Maselo Anu A Cryopreserved

Njira yosungunula imakhala yovutitsa kwa chikhalidwe chozizira, ndipo kagwiridwe koyenera ndi kachitidwe kake kakufunika kuti zitsimikizidwe kuti ma cell azitha kugwira bwino ntchito, kuchira, komanso kugwira ntchito kwake. Ma protocol omwe amasungunuka amatengera mitundu ya ma cell. Komabe, kusungunuka mwachangu kumawonedwa ngati koyenera:

• Chepetsani kukhudza kulikonse pakuchira kwa ma cell
• Thandizani kuchepetsa nthawi yowonekera ku ma solutes omwe amapezeka muzojambula zozizira
• Chepetsani kuwonongeka kulikonse ndi ayezi recrystallization

Masamba osambira m'madzi, mikanda, kapena zida zapadera zomwe zimagwiritsidwa ntchito nthawi zambiri kusungunula zitsanzo.

Nthawi zambiri 1 cell line imasungunuka nthawi imodzi kwa mphindi 1-2, pozungulira pang'onopang'ono mumadzi osambira a 37 ℃ mpaka pangotsala madzi oundana pang'ono mu vial asanatsukidwe m'malo okulirapo otenthedwa.

Kwa maselo ena monga mazira a mammalian, kutentha pang'onopang'ono ndikofunikira kuti apulumuke.

Maselo tsopano ali okonzeka kutengera chikhalidwe cha ma cell, kudzipatula kwa ma cell, kapena ngati pali ma cell a hematopoietic stem cell - maphunziro otheka kuti atsimikizire kukhulupirika kwa ma cell tsinde opereka chithandizo chamyeloablative chisanachitike.

Ndichizoloŵezi chachilendo kutenga ma aliquots ang'onoang'ono a chitsanzo chotsukidwa kale chomwe chimagwiritsidwa ntchito powerengera maselo kuti adziwe kuchuluka kwa maselo kuti apangidwe mu chikhalidwe. Kenako mutha kuwunika zotsatira za njira zodzipatula zama cell ndikuzindikira momwe ma cell akhalira.

Njira Zabwino Kwambiri Zosungira Ma Cryovials

Kusungidwa bwino kwa zitsanzo zosungidwa mu cryovials kumadalira zinthu zambiri mu protocol kuphatikizapo kusungidwa koyenera ndi kusunga zolemba.

• Gawani ma cell pakati pa malo osungira- Ngati mabuku amalola, gawani maselo pakati pa Mbale ndikuzisunga m'malo osiyanasiyana kuti muchepetse chiopsezo cha kutayika kwa chitsanzo chifukwa cha kulephera kwa zida.
• Pewani kuipitsidwa- Sankhani kugwiritsa ntchito kamodzi kokha mbale za cryogenic kapena autoclave musanagwiritse ntchito
• Gwiritsani ntchito mbale zazikuluzikulu zama cell anu- Mbale zimabwera mosiyanasiyana pakati pa 1 ndi 5mls. Pewani kudzaza Mbale kuti muchepetse chiopsezo chosweka.
• Sankhani mkati kapena kunja ulusi cryogenic Mbale- Mbale za ulusi wamkati amalimbikitsidwa ndi mayunivesite ena kuti atetezeke - amathanso kupewa kuipitsidwa pakudzazidwa kapena kusungidwa mu nayitrogeni wamadzimadzi.
• Pewani Kutuluka- Gwiritsani ntchito zisindikizo zojambulidwa ndi ma jekeseni awiri opangidwa mu screw-cap kapena O-rings kuti zisatayike ndi kuipitsidwa.
• Gwiritsani ntchito ma barcode a 2D ndikuyika mbale- kuti zitsimikizidwe kuti ziwoneka bwino, Mbale zokhala ndi malo akuluakulu olembera zimathandiza kuti vial iliyonse ikhale ndi zilembo zokwanira. Ma barcode a 2D amatha kuthandizira pakusungirako ndikusunga zolemba. Makapu amitundu ndi othandiza kuti muzindikire mosavuta.
• Kusamalira kokwanira kosungirako- Kuonetsetsa kuti maselo sakutayika, zombo zosungiramo ziyenera kuyang'anitsitsa kutentha ndi kuchuluka kwa nayitrogeni wamadzimadzi. Ma alamu ayenera kuikidwa kuti adziwitse ogwiritsa ntchito zolakwika.

Chitetezo

Nayitrogeni wamadzimadzi wafala kwambiri mu kafukufuku wamakono koma amakhala ndi chiopsezo chovulala kwambiri ngati atagwiritsidwa ntchito molakwika.

Zida zodzitetezera zoyenerera (PPE) ziyenera kuvalidwa pofuna kuchepetsa chiwopsezo cha chisanu, kupsa ndi zochitika zina zoyipa mukagwira nayitrogeni wamadzimadzi. Valani

• Magolovesi a cryogenic
• Chovala cha Laboratory
• Chishango chonse cha nkhope chomwe chimakwiriranso khosi
• Nsapato zotsekedwa
• Splashproof pulasitiki apuloni

Mafiriji a nayitrogeni amadzimadzi amayenera kuyikidwa m'malo olowera mpweya wabwino kuti achepetse kuopsa kwa mpweya - nayitrogeni yomwe imatuluka imaphwa ndikuchotsa mpweya wa mumlengalenga. Malo osungiramo zinthu zazikulu ayenera kukhala ndi ma alarm ochepa a okosijeni.

Kugwira ntchito awiriawiri pogwira nayitrogeni wamadzimadzi ndikoyenera ndipo kugwiritsa ntchito kwake kunja kwa maola ogwirira ntchito kuyenera kuletsedwa.

Cryovials Kuthandizira Kuyenda Kwanu kwa Ntchito

Kampani ya Suzhou Ace Biomedical imapereka mitundu ingapo yazinthu zomwe zimakwaniritsa zosowa zanu zama cell amitundu yosiyanasiyana. Mbiriyi imaphatikizapo machubu angapo komanso ma cryovial osabala.

Ma cryovials athu ndi awa:

• Lab Screw Cap 0.5mL 1.5mL 2.0mL Cryovial Cryogenic Mbale Conical Pansi Cryotube ndi Gasket

  ● 0.5ml, 1.5ml, 2.0ml ndondomeko, ndi siketi kapena popanda siketi
  ● Mapangidwe owoneka bwino kapena odziyimira okha, osabala kapena osabereka onse akupezeka
  ● Machubu a screw cap amapangidwa ndi polypropylene yachipatala
  ● PP Cryotube Mbale akhoza mobwerezabwereza mazira ndi thawed
  ● Mapangidwe a kapu akunja amatha kuchepetsa mwayi woipitsidwa panthawi ya chithandizo chachitsanzo.
  ● Screw cap cryogenic chubu Ulusi wa Universal screw kuti ugwiritse ntchito
  ● Machubu amakwanira ma rotor ambiri
  ● Cryogenic chubu o-ring machubu oyenera muyezo 1 inchi ndi 2 inchi, 48well, 81well, 96well ndi 100well mabokosi mufiriji
  ● Zimatha kuzimitsa zokha kufika 121°C ndipo zimazizira mpaka -86°C

  GAWO NO	ZOCHITIKA	VOLUME	KAPACOLOR	PCS/BAG	MIZIMBA/NKHANI
  ACT05-BL-N	PP	0.5ML	Black, Yellow, Blue, Red, Purple, White	500	10
  Chithunzi cha ACT15-BL-N	PP	1.5ML	Black, Yellow, Blue, Red, Purple, White	500	10
  ACT15-BL-NW	PP	1.5ML	Black, Yellow, Blue, Red, Purple, White	500	10
  Chithunzi cha ACT20-BL-N	PP	2.0ML	Black, Yellow, Blue, Red, Purple, White	500	10