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세포배양의 원리 생명과학 실험기법 II 박태식 맹효진 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 Successive sub-culture Immortalization Loss of control of cell growth Clonal cell line Transformed cell line Immortalised cell line Senescence in vitro에서 배양되는 세포의 종류 Primary cultures • 동물조직으로부터 직접 세포를 분리 배아 또는 성체? Normal or neoplastic? • tissue explants 또는 single cells • 초반에는 여러 종류의 세포가 존재 – 나중에는 fibroblast 가 잘자람 • 제한된 배양능력 in vitro • 분화된 표현형을 보유 • 부착이 잘 안됨- collagen-coated 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% CO2, temperature 37oC, 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 pH and osmolarity (260-320 mOsm/L). • Provide nutrients and energy source. Basal Media의 구성성분 무기염 • 삼투압 유지 (osmolarity) • 세포막전위 유지 (Na+, K+, Ca2+) • 세포부착과 조효소 공급 pH Indicator – Phenol Red • 세포최적성장 pH 7.4 Buffers (Bicarbonate and HEPES) • Bicarbonate buffered media requires CO2 atmosphere • HEPES Strong chemical buffer range pH 7.2 – 7.6 (does not require CO2) 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 (56oC 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 ‘Mr Frosty’ Used to freeze cells Cryopreservation 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 -80oC 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 -80oC – rapid yet ‘slow’ freezing • Liquid nitrogen -196oC 세포수 측정 (Hemocytometer) Diagram represent cell count using hemocytometer. Automated cell count Cellometer lets you: • View cell morphology, for visual confirmation after cell counting • Take advantage of 300+ cell types and easy, wizard-based parameter set-up • Save sample images with results securely on your computer, plus autosave results on the network for added convenience and data protection 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?
