> water migrates out of the cells (osmosis) To avoid these genetic changes the cells have to be congelated in liquid nitrogen for their preservation.ĭuring congelation you can cause celular damage: This effect is less pronounced in immortal cancer lines (the majority), primary cell lines are still mortal and they just die. For pharmaceutical investigation only 5 (five!) passages (subculturing) are accepted for a cell line. This means, that the behaviour of the cells changes during subculturing: thereby frequently subcultured cell lines can not be considered as the true (original) cell model.
=> use less concentrated dissociation soln.ĭuring prolonged (sub)cultivation the cell lines can change their phenotypic characteristics and expression pattern (genetic drift). => Centrifuge cells (5 min, 125 g) and/or add an inhibitor (e.g. Dissociation enzymes (trypsin) not inactivated > Dissociating process is too harsh: less pipetting, lower trypsin concentration Medium is viscous (DNA has been released from lysed cells) Caco-2, a higher inoculum should be used. HeLa, a lower inoculum while for slow growing cells, e.g. Subculturing is only done with flasks, not with MTP the numbers given here are an orientation to inoculate experiments. Inoculate monolayer cells with ~1 * 10 4N/cm 2. Place a drop of homogeneous cell suspension onto a improved Neubauer-Kammer - due to capillar force the liquid enters equally to the chamber.Ĭount the cell number (N) of all the 25 small (yellow) squares and multipicate this number with 10,000 to get the N/ml. pipette up and down to wash the cells from bottom and to isolate them Observe the cells under inverted microscope or stereomicroscope: detached cells becomes roundĪdd medium to inactivate trypsin/EDTA: 7.5 ml for T-75 or 2.5 ml for T-25 DO NOT hit or shake flask in order to avoid clumping of the cells Add 0.05 % trypsin/EDTA and incubate about 10 min at RT: 1.5 ml for T-75 or 0.5 ml for T-25 Wash the cells once with DPBS in order to remove traces of FBS which inhibits trypsin: 8 ml for T-75 3 ml for T-25 Label flask or multititerplate (cell line date working lot experiment) Prewarm medium to 37 ☌ (15 ml for T-75, 5 ml for T-25), prewarm trypsin/EDTA and DPBS to RT Place tips, pipettes, (clean) waste flask into the BSC Clean Biological Safety Cabinet (BSC) with 70 % EtOH. => Iniciate subculturing when 80 - 90 % confluent: ~10 5N/cm Cell morphology (dead cells are floating or round) Microbial contamination (medium turbid) Observe the cells under an inverted microscope (phase contrast) or (far less good, but cheaper) under a stereomicroscope: 100x.
In contrast, suspension cells can directly be diluted with fresh growth medium. Therefore, when the cell growth is 80 - 90 % confluent the cells have to be "diluted" = subcultured. When adherent monolayer cells occupate all growth space the cells stop to grow due to contact inhibition. CellBIND from "Corning" or Nunclon from "Nunc") Pretreated plastics accelerates cell attachemnt (e.g. Serum provides attachement factors like fibronectin, vitronectin Seed ~10 4 N/cm 2 and use enough medium: 0.25 ml/cm 2 Centrifuge cells at low revolutions: 5 min 5 g to get a soft pellet and temperature inactivate/remove them afterwards When subculturing, use prewarmed medium Subculture cells at 80 - 90 % confluent Avoid exposition of medium to UV/fluorescent light (peroxide formation) Add 5 - 10 mM HEPES to medium to have a more stable pH Buy and store Gln-free medium and add it as dipeptide Some general recommendations for having happy cells :-)
Lymphoblast-like cells grow suspended and appear spherical.ĭMEM/F12 + 10% FBS + 0.01 mg/ml insulin ? Standard Techniques for Cell Culture Standard Techniques for Cell Cultureīased on hteir morphology and/or functions 3 types of cell lines can be differenciated:Įpithelial-like cells grow attached to a surface and appear flat and polygonal.įibroblast-like cells grow attached to a surface and appear elongated bipolar.
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