Cell Culture. Basics of Cell Culture. Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. But in practice it refers to the culturing of cells derived from animal cells.
Cell culture was first successfully undertaken by Ross Harrison in 1907
Roux in 1885 for the first time maintained embryonic chick cells in a cell culture
Second was the use of trypsin to remove adherent cells to subculture further from the culture vessel
Third was the use of chemically defined culture medium.
Major development’s in cell culture technology
Model systems for
Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies
Study the effects of new drugs
Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells
Why is cell culture used for?
Cultivation of virus for vaccine production, also used to study there infectious cycle.
Production of commercial proteins, large scale production of viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measles
Cells having a functional gene can be replaced to cells which are having non-functional geneContd….
Commonly used Medium are GMEM, EMEM,DMEM etc.
Media is supplemented with antibiotics viz. penicillin, streptomycin etc.
Prepared media is filtered and incubated at 4 C
Incubation facilities- Temperature of 25-30 C for insect & 37 C for mammalian cells, co2 2-5% & 95% air at 99% relative humidity. To prevent cell death incubators set to cut out at approx. 38.5 C
Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin) & media components (e.g. glutamine & serum) at -20 C
Microscope- An inverted microscope with 10x to 100x magnification
Tissue culture ware- Culture plastic ware treated by polystyrene
Basic equipments used in cell culture
Swab all bottle tops & necks with 70% ethanol
Flame all bottle necks & pipette by passing very quickly through the hottest part of the flame
Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little finger
Work either left to right or vice versa, so that all material goes to one side, once finished
Clean up spills immediately & always leave the work place neat & tidy
Basic aseptic conditions
Centrifuge the vial for 10 mts at 1000 rpm at RT, wipe top of vial with 70% ethanol and discard the supernatant
Resuspend the cell pellet in 1 ml of complete medium with 20% FBS and transfer to properly labeled culture plate containing the appropriate amount of medium
Check the cultures after 24 hrs to ensure that they are attached to the plate
Change medium as the colour changes, use 20% FBS until the cells are established
Working with cryopreserved cells
Dislodge the cells by trypsin-versene
Dilute the cells with growth medium
Transfer the cell suspension to a 15 ml conical tube, centrifuge at 200g for 5 mts at RT and remove the growth medium by aspiration
Resuspend the cells in 1-2ml of freezing medium
Transfer the cells to cryovials, incubate the cryovials at -80 C overnight
Next day transfer the cryovials to Liquid nitrogen
Freezing cells for storage
Cell lines derived from normal tissues are considered as anchorage-dependent grows only on a suitable substrate e.g. tissue cells
Suspension cells are anchorage-independent e.g. blood cells
Transformed cell lines either grows as monolayer or as suspension
Culturing of cells
Cells are washed with PBS (free of ca & mg ) solution.
Add enough trypsin/EDTA to cover the monolayer
Incubate the plate at 37 C for 1-2 mts
Tap the vessel from the sides to dislodge the cells
Add complete medium to dissociate and dislodge the cells
with the help of pipette which are remained to be adherent
Add complete medium depends on the subculture
requirement either to 75 cm or 175 cm flask
As the suspension cells reach to confluency
Asceptically remove 1/3rd of medium
Replaced with the same amount of pre-warmed medium
-MCF-7 breast cancer
HL 60 Leukemia
HEK-293 Human embryonic kidney
HeLa Henrietta lacks
Primate cell lines
Vero African green monkey kidney epithelial cells
Cos-7 African green monkey kidney cells
And others such as CHO from hamster, sf9 & sf21 from insect cells
Common cell lines
Chemical-difficult to detect caused by endotoxins, plasticizers, metal ions or traces of disinfectants that are invisible
Biological-cause visible effects on the culture they are mycoplasma, yeast, bacteria or fungus or also from cross-contamination of cells from other cell lines
Contaminant’s of cell culture
As trypan blue dye is permeable to non-viable cells or death cells whereas it is impermeable to this dye
Stain the cells with trypan dye and load to haemocytometer and calculate % of viable cells
- % of viable cells= Nu. of unstained cells x 100
total nu. of cells
Observed effect on the morphological alteration in the cell layer or cell shape
Characteristics of abnormal morphology is the giant cells, multinucleated cells, a granular bumpy appearance, vacuoles in the cytoplasm or nucleus
Cytotoxicity is determined by substituting materials such as medium, serum, supplements flasks etc. at atime
Lipid mediated lipofection
Transfection is a method of transporting DNA, RNA and/or various macromolecules into an eukaryotic cell by using chemical, lipid or physical based methods.
CaPO4, DEAE DNA Transfection
Liposome Based DNA Transfection
Polyamine Based DNA Transfection
1944: Avery, Macleod and McCarty discover DNA is the transforming factor
1964: Foldes and Trautner use the term transfection
1965: Vaheri and Pagano describe the DEAE-Dextran transfection techniqueEarly Years
Add plasmid DNA to DEAE-Dextran medium
Incubate cells with mixture…efficient transfection results in 25-75% death
DMSO “shock”Transfection method 1: DEAE-Dextran
HBS = HEPES-Buffered Saline
Harvest cells and resuspend in electroporation buffer
Add DNA to cell suspension…for stable transfection DNA should be linearized, for transient the DNA may be supercoiled
Selection process for transfectant
Provides the ability to transfer in negatively charged molecules into cells with a negatively charged membrane.
Liposome-mediated transport of DNA has high efficiency. Good for long-term studies requiring incorporation of genetic material into the chromosome.
Chemical Reagents: not useful for long-term studies.
Transfection efficiency is dependent on cell health, DNA quality, DNA quantity, confluency (40-80%)
Direct Microinjection and Biolistic Particle delivery is an expensive and at times a difficult method.Advantages/Disadvantages
1) Where is it localized in the cell?
a) Make antibodies - immunofluorescence
b) “Express” the protein in cells with a tag
Fuse to GFP
2) What is it doing in the cell?
a) Reduce protein levels - RNA interference
b) Increase protein levels “over-express”
c) “Express” mutant versions
Introduction of DNA into mammalian cells
Gene is transcribed and translated into protein
Electroporation - electric field temporarily disrupts plasma membrane
Biolistics (gene gun)- fire DNA coated particles into cell
Use recombinant viruses to deliver DNA
Virally-mediated introduction of the DNA
Positively charged carrier molecules are mixed with the DNA and added to cell culture media:
Carrier-DNA complexes bind to plasma membrane and are taken up
Expression assayed 24-48 hours post transfection
Integration of the transfected DNA into the cell genome - selectable marker like neomycin resistance required
“stably transfected” cell line
For expression in cells
To generate stable cell line
For amplification of the plasmid in bacteria
Bacterial origin of replication
Three ways to make Green fluorescent protein “GFP” fusion constructs:
Transfect unknown GFP fusion protein
Protein X, Y or Z
Visualize GFP protein fluorescence by fluorescence microscopy in living cells
Counter-stain with known marker to compare localization patterns inliving cells
= “vital stain”
Transport through nuclear pore
signal = basic amino acid stretches
Hoechst 33258 binds DNA
Transmembrane transport signal
Diffuses through membranes
Non-fluorescent until oxidized
Accumulates in mitochondria and oxidized
Cellular components of the secretory and endocytic pathways
Entry into E.R.:
Transmembrane transport signal
= hydrophobic amino acid stretches
at amino terminus
Retention in E.R. lumen:
Signal = K-D-E-L-COOH
at carboxy terminus
Live bovine pulmonary artery endothelial cells
From the ER, secreted and membrane proteins move to the Golgi, a
series of membrane-bound compartments found near the nucleus
Ceramide = lipid
When metabolized, concentrates in the Golgi
Madin-Darby Canine Kidney
Polarized Epithelial Cells
Molecular Probes, Inc.
Does the fluorescent green protein co-localize?
Immunofluorescence / confocal microscopy
B or T cells in suspension, adherent cells on chambered coverglass or chamberslides, cryostat sections of unfixed, OCT embedded tissue:
1. wash cells 1x cold RPMI (no wash for cryostat sections).
2. Fix 20 min 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4, 0.03M sucrose on ice. **
3. wash 2x PBS/1%BSA. From now on everything can be at room temp. or on ice.
4. Permeabilize 5 min RT in 0.2% saponin, PBS, 0.03M sucrose, 1% BSA.
5. wash 1x PBS/BSA.
6. Block 15 min 5% normal goat serum (NGS) in PBS/BSA.
7. wash 1x PBS/BSA.
8. 1° diluted in PBS/BSA 60 min RT; 100 l per tube or section.
9. wash 3x PBS/BSA.
10. Block 15 min 5% NGS in PBS/BSA.
11. wash 1x PBS/BSA.
12. 2° diluted in PBS/BSA 30 min RT; 100 l per tube or section.
13. wash 3x PBS/BSA; (wash 1x in PBS/BSA then 2 x 10 min in Molecular Probes SlowFade Light buffer if using Slow Fade Light S-7461 to coversip); pellet cells and put up in two drops of Molecular Probes Slowfade Light antifade medium. Pipet about 15 m l on slide and coverslip. For chambered coverglass just put two-three drops in each chamber after wash.
An example of a high-density, high-content RNAi cell-microarray screen in cultured D. melanogaster Kc167 cells.
- easy and inexpensive to maintain
- 사람 장내 서식, 배설물의 주성분
- 해로운 박테리아의 생장 저해
Pathogenic은 sickness, death 유발
- 돌연변이 잘 유발 → Metabolism 연구에 이용
Eukaryote에서의 과정 일어나지 않음
- yeast genome cut restriction enzyme
- fragments clone into plasmid vector
- recombinant plasmids growth and select
- yeast survive in media lacking leucine
- selected yeast cells contain leu2+ gene
- Small and easy to keep
- Three days to develop
Identification of mutant
→ mutation 특징 확인이 쉬움
- 짧은 시간에 많은 자손 생산
- 알에서 성체가 되기까지 12일 소요
- Drosophila melanogaster
- Reproduction rapidly and high number
- 발생 과정이 사람과 유사
- 알이 투명하여 발달 확인 가능
- 알의 크기가 큼 →발생학 연구에 이용
- 1920년 슈페만 형성체 발견
- Xenopus leavis
배 발생 연구에서 알 사용됨
; 진화상 인간과 유사
- 알이 크고, 분화 관찰에 좋음
- Gallus gallus
- vertebrate, mammals
Human과 더욱 가까움
; 생리학, 발생학적으로 유사
- Expensive to maintain, Reproduce slowly
Knock out mouse 이용
→ 특정 gene의 기능 확인
Human blood나 tissue에서 분리
; derived directly from living tissue
- Grow for a short period time and stop
- 세포 분열 횟수 제한
- Grow slowly, long generation time
- 세포벽 때문에 형질전환이 어려움
- Large genome
- Transposon 연구에 이용 ; corn
- Arabidopsis thaliana