ISOLATION OF LIVE CELLS FROM DIFFERENT MICE TISSUES UP TO NINE DAYS AFTER DEATH

Abstract

Some limited reports suggest that cells can survive in the cadavers for much longer than it was previously thought.  In our study we explored how time after death, tissue type (muscle, brain and adipose tissue), storage temperature of cadavers (4 °C or at room temperature) and form of tissue storage (stored as cadavers or tissue pieces in phosphate buffered saline) affect the success of harvesting live cells from mice after death. Cells were isolated from dead tissues and grown in standard conditions. Some cells were used for RNA extraction and RT² Profiler™ PCR Array for cell lineage identification was performed to establish which lineages the cells obtained from post mortem tissues belong to. Results of our study showed that viable cells can be regularly isolated from muscle and brain tissue 3 days post mortem and with difficulty up to 6 days post mortem. Viable cells from brain tissue can be isolated up to 9 days post mortem. No cells were isolated from adipose tissue except immediately after death. In all instances viable cells were isolated only when tissues were stored at 4 °C. Tissue storage did not affect cell isolation. Isolated cells were progenitors from different germ layers. Our results show that live cells could be obtained from mouse cadavers several days after death.

IZOLACIJA ŽIVIH CELIC IZ RAZLIČNIH TKIV MIŠI DO DEVET DNI PO SMRTI

Izvleček: Nekatere raziskave kažejo, da je preživetje celic v truplih precej daljše, kot je bilo znano do sedaj. V naši raziskavi smo proučevali, kako na uspešnost izolacije živih celic po smrti miši vplivajo različen čas izolacije po smrti, vrsta tkiva (mišično, možgansko in maščobno), temperatura shranjevanja trupel ter oblika shranjenega tkiva (kot koščki tkiv ali kot celi kadavri). Izolacija in gojenje celic iz tkiv mrtvih miši sta potekali pod standardnimi pogoji. Da bi ugotovili, katerim celičnim linijam pripadajo izolirane celice, je bil del celic uporabljen za izolacijo RNK in nadaljno uporabo v sistemu identifikacije izvornih celičnih linij z verižno reakcijo s polimerazo v realnem času. Rezultati naše raziskave so pokazali, da je žive celice mogoče izolirati iz mišičnega in možganskega tkiva 3 dni po smrti, pogojno tudi do 6 dni po smrti. Iz možganskega tkiva je bilo žive celice mogoče izolirati tudi do 9 dni po smrti. Iz maščobnega tkiva je bilo celice mogoče izolirati zgolj takoj po smrti, ne pa tudi v kasnejših časovnih intervalih. V vseh primerih so bile celice izolirane samo v primeru shranjevanja tkiv pri 4°C. Oblika shranjenega tkiva na izolacijo celic ni vplivala. Izolirane celice so pripadale različnim zarodnim plastem. Rezultati raziskave so pokazali, da je žive celice iz mišjih trupel mogoče izolirati tudi več dni po smrti.

Ključne besede: miš; truplo; matične celice; možgansko tkivo; mišično tkivo; maščobno tkivo