Isolation of cell lines for in vitro culture

Isolation of cell lines for in vitro culture Resected Tissue Cell or tissue culture in vitro Primary culture Sub-culture Secondary culture Sub-culture Cell Line Single cell isolation Immortalization Successive sub-culture Loss of control of cell growth Clonal cell line Transformed cell line Immortalised cell line Senescence

in vitro에서 배양되는 세포의 종류 Secondary cultures • • • primary cell culture로부터 유도됨 Isolated by selection or cloning 여러 종류의 세포가 존재 제한된 배양능력 in vitro 분화된 표현형을 보유 부착이 잘 안됨- collagen-coated

in vitro에서 배양되는 세포의 종류 Continuous cultures • primary 또는 secondary culture • Immortalized: • Spontaneously (e. g. : spontaneous genetic mutation) • By transformation vectors (e. g. : viruses &/or plasmids) • • • 성장속도가 증가되어 배양액에서 계속적으로 배양 가능 균일한 세포 종류 배양기에 부착 잘됨 in vitro에서 계속적으로 배양가능 분화된 표현형 (Differentiated phenotype): • 암에서 유도된 세포의 성질을 가짐 • 유전적으로 불안정

세포 종류에 따른 세포 형태 Fibroblastic Epithelial Endothelial Neuronal

세포 배양 환경 (in vitro) 세포성장에 필요한 성분 • Substrate 또는 liquid (cell culture flask or scaffold material) chemically modified plastic or coated with ECM proteins suspension culture • 영양분 (Nutrients): culture media • 환경 (5% CO 2, temperature 37 o. C, humidity) Oxygen tension maintained at atmospheric but can be varied • 위생적 환경 (aseptic technique, 항생제 and 항진균제) Mycoplasma tested

세포 배양 환경 (in vitro) Basal Media (배지) • Maintain p. H and osmolarity (260 -320 m. Osm/L). • Provide nutrients and energy source. Basal Media의 구성성분 무기염 • 삼투압 유지 (osmolarity) • 세포막전위 유지 (Na+, K+, Ca 2+) • 세포부착과 조효소 공급 p. H Indicator – Phenol Red • 세포최적성장 p. H 7. 4 Buffers (Bicarbonate and HEPES) • Bicarbonate buffered media requires CO 2 atmosphere • HEPES Strong chemical buffer range p. H 7. 2 – 7. 6 (does not require CO 2) Glucose • 에너지 공급

세포 배양 환경 (in vitro) Components of Basal Media Keto acids (oxalacetate and pyruvate) • Intermediate in Glycolysis/Krebs cycle • Keto acids added to the media as additional energy source • Maintain maximum cell metabolism Carbohydrates • Energy source • Glucose and galactose • Low (1 g/L) and high (4. 5 g/L) concentrations of sugars in basal media Vitamins • Precursors for numerous co-factors • B group vitamins necessary for cell growth and proliferation • Common vitamins found in basal media is riboflavin, thiamine and biotin Trace Elements • Zinc, copper, selenium and tricarboxylic acid intermediates

세포 배양 환경 (in vitro) Supplements L-glutamine • Essential amino acid (not synthesised by the cell) • Energy source (citric acid cycle), used in protein synthesis • Unstable in liquid media - added as a supplement Non-essential amino acids (NEAA) • Usually added to basic media compositions • Energy source, used in protein synthesis • May reduce metabolic burden on cells Growth Factors and Hormones (e. g. : insulin) • Stimulate glucose transport and utilisation • Uptake of amino acids • Maintenance of differentiation Antibiotics and Antimycotics • Penicillin, streptomycin, gentamicin, amphotericin B • Reduce the risk of bacterial and fungal contamination • Cells can become antibiotic resistant – changing phenotype • Preferably avoided in long term culture

세포 배양 환경 (in vitro) Fetal Calf/Bovine Serum (FCS & FBS) • • Growth factors and hormones Aids cell attachment Binds and neutralise toxins Long history of use • • Infectious agents (prions) Variable composition Expensive Regulatory issues (to minimise risk) Heat Inactivation (56 o. C for 30 mins) – why? • Destruction of complement and immunoglobulins • Destruction of some viruses (also gamma irradiated serum) Care! Overdoing it can damage growth factors, hormones & vitamins and affect cell growth

실험실에서 세포 배양하는 법 Revive frozen cell population Isolate from tissue Containment level 2 cell culture laboratory Maintain in culture (aseptic technique) Typical cell culture flask Sub-culture (passaging) Count cells Cryopreservation ‘Mr Frosty’ Used to freeze cells

세포의 지속적 배양 (Passaging Cells) Check confluency of cells Remove spent medium Wash with PBS Incubate with trypsin/EDTA Why passage cells? • To maintain cells in culture (i. e. don’t overgrow) • To increase cell number for experiments/storage How? • 70 -80% confluency • Wash in PBS to remove dead cells and serum • Trypsin digests protein-surface interaction to release cells (collagenase also useful) • EDTA enhances trypsin activity • Resuspend in serum (inactivates trypsin) • Transfer dilute cell suspension to new flask (fresh media) • Most cell lines will adhere in approx. 3 -4 hours Resuspend in serum containing media Transfer to culture flask 70 -80% confluence 100% confluence

세포의 보존 Passage cells Resuspend cells in serum containing media Centrifuge & Aspirate supernatant Resuspend cells in 10% DMSO in FCS Transfer to cryovial Freeze at -80 o. C Transfer to liquid nitrogen storage tank Why cryopreserve cells? • Reduced risk of microbial contamination. • Reduced risk of cross contamination with other cell lines. • Reduced risk of genetic drift and morphological changes. • Research conducted using cells at consistent low passage. How? • Log phase of growth and >90% viability • Passage cells & pellet for media exchange • Cryopreservant (DMSO) – precise mechanism unknown but prevents ice crystal formation • Freeze at -80 o. C – rapid yet ‘slow’ freezing • Liquid nitrogen -196 o. C

세포수 측정 (Hemocytometer) Diagram represent cell count using hemocytometer.

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The ideal growth curve for cells in culture

오염 (contamination) A cell culture contaminant can be defined as some element in the culture system that is undesirable because of its possible adverse effects on either the system or its use. 1 -Chemical Contamination Media Incubator Serum water 2 -Biological Contamination Bacteria and yeast Viruses Mycoplasmas Cross-contamination by other cell culture How Can Cell Culture Contamination Be Controlled?