Cell Culture

Laboratory of Molecular Genetics, KNU

Cell Culture

Laboratory of Molecular Genetics, KNUBasics of Cell Cul-

ture


• 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 under-taken by Ross Harrison in 1907

• Roux in 1885 for the first time maintained embryonic chick cells in a cell culture

Introduction

Laboratory of Molecular Genetics, KNU• First development was the use of antibiotics which inhibits the growth of contaminants.• 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


Areas where cell culture technology is currently playing a major role.

• Model systems for Studying basic cell biology, interactions be-

tween disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies

• Toxicity testing Study the effects of new drugs• Cancer research Study the function of various chemicals, virus

& radiation to convert normal cultured cells to cancer-ous cells

Why is cell culture used for?


Contd….• Virology Cultivation of virus for vaccine produc-

tion, also used to study there infectious cycle.• Genetic Engineering Production of commercial proteins,

large scale production of viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measles

• Gene therapy Cells having a functional gene can be

replaced to cells which are having non-func-tional gene


• Choice of media de-pends on the type of cell being cultured

• Commonly used Medium are GMEM, EMEM,DMEM etc.

• Media is supplemented with antibiotics viz. penicillin, streptomycin etc.

• Prepared media is fil-tered and incubated at 4 C

Culture media


• Laminar cabinet-Vertical are preferable

• Incubation facilities- Tem-perature of 25-30 C for in-sect & 37 C for mam-malian 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 me-dia kept at 4 C, enzymes (e.g. trypsin) & media components (e.g. glu-tamine & serum) at -20 C

• Microscope- An inverted microscope with 10x to 100x magnification

• Tissue culture ware- Cul-ture plastic ware treated by polystyrene

Basic equipments used in cell culture

Laboratory of Molecular Genetics, KNU• If working on the bench use a Bunsen flame to heat the air surrounding the Bunsen• 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


• Vial from liquid nitrogen is placed into 37 C water bath, agitate vial continuously un-til medium is thawed

• 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 appro-priate 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 estab-lished

Working with cryopreserved cells


• Remove the growth medium, wash the cells by PBS and remove the PBS by aspiration

• 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 nitro-gen

Freezing cells for storage


• Cells are cultured as anchor-age dependent or indepen-dent

• Cell lines derived from normal tissues are considered as an-chorage-dependent grows only on a suitable substrate e.g. tissue cells

• Suspension cells are anchor-age-independent e.g. blood cells

• Transformed cell lines either grows as monolayer or as suspension

Culturing of cells


• Cells which are anchorage dependent • 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 re-

mained to be adherent• Add complete medium depends on the

subculture• requirement either to 75 cm or 175 cm

flask

Adherent cells


• Easier to passage as no need to detach them• As the suspension cells reach to confluency• Asceptically remove 1/3rd of medium• Replaced with the same amount of pre-warmed

medium

Suspension cells


• Human cell lines• -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 in-

sect cells

Common cell lines


Cell culture contaminants of two types

• Chemical-difficult to de-tect caused by endotox-ins, plasticizers, metal ions or traces of disinfec-tants that are invisible

• Biological-cause visible ef -fects 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


• Cell viability is determined by staining the cells with trypan blue

• 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

Cell viability


• Cytotoxicity causes inhibition of cell growth• Observed effect on the morphological alter-

ation 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 ma-terials such as medium, serum, supplements flasks etc. at atime

Cell toxicity


• Calcium phosphate precipitation• DEAE-dextran (dimethylaminoethyl-dex-

tran)• Lipid mediated lipofection• Electroporation• Retroviral Infection• Microinjection

Transfection methods

Transfection of eukaryotic cells

TransfectionWhat is Transfection?

Transfection is a method of transporting DNA, RNA and/or various macromolecules into an eukaryotic cell by using chemical, lipid or physical based methods.

Methods: (just a few examples…) Method Application

CaPO4, DEAE DNA Transfection Liposome Based DNA Transfection Polyamine Based DNA Transfection

Early Years• Transfection = “trans-

formation” + “infection”

• The infection of cells by the isolated nucleic acid from a virus, resulting in the production of a complete virus

1928: Frederick Griffith transforms Streptococcus pneumoniae

1944: Avery, Macleod and McCarty discover DNA is the transforming factor

1965: Vaheri and Pagano describe the DEAE-Dextran transfection technique

1964: Foldes and Trautner use the term transfection

Transfection vs. Transforma-tion

Transformation: genetically altering cells by transporting in foreign ge-netic material.

Transfection: the process of trans-porting genetic material and/or macromolecules into eukaryotic cells through typically non-viral methods.

Purpose/uses of Transfec-tion

Study gene functionStudy protein expressionTransfer DNA into embryonic stem cells

How it worksUtilize Chemical, lipid or physical methods (direct microinjection, electroporation, biolistic particle delivery) for transportation of ge-netic materials or macromolecules.

Transfection method 1: DEAE-Dextran

• Facilitates DNA binding to cell membranes and entry via endocytosis

• DEAE-D associates with DNAcomplex binds to cell surface

Add chloroquine to transfection medium

DMSO “shock”

Add plasmid DNA to DEAE-Dextran medium

Incubate cells with mixture…efficient transfection results in 25-75% death

Transfection method 1: DEAE-Dextran

• Major variants: number of cells, concentration of DNA and concentration of DEAE-Dextran

• Only method that cannot be used for stable transfections

Transfection method 3: lipo-some-mediated

• Use of cationic lipids– chemical and physical similarities to biological

lipids– spontaneous formation of complexes with

DNA, called lipoplexes• High efficiency for in vitro transfections• Can carry larger DNA than viruses• Safer than viruses• Low in vivo efficiency

http://homepage.usask.ca/~sdw132/images/lipid_nanoparticle.gif

http://homepage.usask.ca/~sdw132/images/lipid_nanoparticle.gif

How it works – Chemical and lipid methods

Neutralize negative charges on DNAChemical method: Calcium phosphate; creates precipitates that settle on cells and are taken in.Lipid or Polymer methods: interact with DNA, promotes binding to cells and uptake via endocyto-sis.

Experimental method/process

(chemical methods)

HBS = HEPES-Buffered Saline

Transfection method 2: elec-troporation

• Use of high-voltage electric shocks to introduce DNA into cells• Cell membranes: electrical capacitors unable to pass current• Voltage results in temporary breakdown and formation of

pores

Harvest cells and resuspend in electroporation buffer

Selection process for transfectant

Add DNA to cell suspension…for stable transfection DNA should be linearized, for transient the DNA may be supercoiled

electroporate

Transfection method 2: elec-troporation

• Variants: amplitude and length of electric pulse • Less affected by DNA concentration-linear corre-

lation between amount of DNA present and amount taken up

• Can be used for stable transfections

How it works – Physical methods

Electroporation: use of high voltage to deliver nucleic acids; pores are formed on cell mem-brane.Direct Microinjection: Use of a fine needle. Has been used for transfer of DNA into embryonic stem cells.Biolistic Particle delivery: Uses high-velocity for de-livery of nucleic acids and penetration of cell wall.

Advantages/Disadvan-tages

Advantages: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 requir-ing incorporation of ge-netic material into the chromosome.

Disadvan-tages:Chemical Reagents: not useful for long-term stud-ies.Transfection efficiency is dependent on cell health, DNA quality, DNA quan-tity, confluency (40-80%)Direct Microinjection and Biolistic Particle delivery is an expensive and at times a difficult method.


Exploring protein function1) Where is it localized in the cell?

2) What is it doing in the cell?

Approaches:a) Make antibodies - immunofluorescence

Approaches:a) Reduce protein levels - RNA

interference

b) “Express” the protein in cells with a tag Fuse to GFP

b) Increase protein levels “over-express”

c) “Express” mutant versions

Transfection!!!!


Transfection = Introduction of DNA into mammalian cells

Gene is transcribed and translated into protein= “expressed”


Direct introduction of the DNA

Electroporation - electric field temporarily disrupts plasma membrane

Biolistics (gene gun)- fire DNA coated particles into cell

Microinjection


Infection:Use recombinant viruses to deliver DNA

RetrovirusesAdenoviruses

Virally-mediated introduction of the DNA


Positively charged carrier molecules are mixed with the DNA and added to cell culture media:

Calcium PhosphateDEAE Dextranliposomesmicelles

Carrier-DNA complexes bind to plasma membrane and are taken up

Carrier-mediated introduction of the DNA


Types of Transfection

Transient:Expression assayed 24-48

hours post transfection

Stable:Integration of the transfected

DNA into the cell genome - selectable marker like neomycin resistance required

“stably transfected” cell line

Laboratory of Molecular Genetics, KNUDNA “expression” vector transfected:

pCMV/GFP

CMV

Promoter

Insert genein here

Polyadenylationsite

SV40

Promoter

Neom

ycinresistance

PolyadenylationsitepUC

Bacterial origin of replicationAm

picillin

resistance

For expression in cells

To generate stable cell line

For amplification of the plasmid in bacteria

GFP


PROTEIN X

PROTEIN Y

GFP

GFP

GFP ZPROTEIN

Three ways to make Green fluorescent protein “GFP” fusion constructs:


EXPERIMENT:

Transfect unknown GFP fusion proteinProtein X, Y or Z

Visualize GFP protein fluorescence by fluorescence microscopy in living cells

Counter-stain with known marker to compare localization patterns in living cells

= “vital stain”


Some Cellular Organelles


•Compartments/organelles examined•Protein sequences sufficient for localization•Vital stains

Secretory Pathway:Endoplasmic ReticulumGolgi Complex

Endocytotic Pathway:Endosomes

Mitochondria

Nuclei


Transport through nuclear poresignal = basic amino acid stretches example: P-P-K-K-K-R-K-V

Nucleus

Laboratory of Molecular Genetics, KNUImport of proteins into nucleus through nuclear pore


Nuclear Stain:

Hoechst 33258 binds DNA


Transmembrane transport signalExample: H2N-M-L-S-L-R-Q-S-I-R-F-F-K-P-A-A-T-R-T-L-C-S-S-R-Y-L-L

Mitochondria


Protein being transported across mitochondrial membranes

Laboratory of Molecular Genetics, KNUMitochondrial dye = MitoTracker Red

Non-fluorescent until oxidized

Accumulates in mitochondria and oxidized

Diffuses through membranes

Mitotracker

DNA


nuclear envelope

endoplasmic reticulum

lysosome

earlyendosome

lateendosome

Golgi apparatus

cisGolgi

network

transGolgi

network

Golgistack

CYTOSOL

plasmamembrane

Cellular components of the secretory and endocytic pathways


Entry into E.R.:Transmembrane transport signal= hydrophobic amino acid stretches

Example: H2N-M-M-S-F-V-S-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q

Retention in E.R. lumen:

Signal = K-D-E-L-COOH

Endoplasmic Reticulum

at amino terminus

at carboxy terminus


ER-Tracker Blue-White

Live bovine pulmonary artery endothelial cells

Endoplasmic Reticulum marker


Mitotracker Red and ER-blue/white


Golginucleus

From the ER, secreted and membrane proteins move to the Golgi, a series of membrane-bound compartments found near the nucleus


BODIPY-TR ceramide

Golgi marker

Ceramide = lipidWhen metabolized, concentrates in the Golgi

Red fluorophore


Golgi (ceramide)

DNA (Hoechst)

Cultured Epithelial Cells


Golgi (ceramide)

Lysosomes (LysoTracker)

DNA (Hoechst)

MDCK CellsMadin-Darby Canine KidneyPolarized Epithelial Cells

Molecular Probes, Inc.


Rhodamine transferrin

Does the fluorescent green protein co-localize?


Immunofluorescence / confocal microscopy 원리


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.

Immunofluorescence / confocal microscopy 실험방법

High-throughput loss-of-function screens using RNAi cell microarrays.

An example of a high-density, high-content RNAi cell-microarray screen in cultured D. melanogaster Kc167 cells.


Experimental Animals


Model systems

E. coli- easy and inexpensive to maintain- 사람 장내 서식 , 배설물의 주성분- 해로운 박테리아의 생장 저해Pathogenic 은 sickness, death 유발- 돌연변이 잘 유발 → Metabolism 연구에 이용Eukaryote 에서의 과정 일어나지 않음


• Yeast- Eukaryote 와 prokaryote 사이의 차이점

연구에 이용

- Genetic study easy

- haploid, single-celled- Grow very rapidly, inexpensive - 2 가지 종류 사용

→ Saccaromyces cerevisiae; budding

→ Schizosaccaromyces pombe; fission


Cloning the yeast origin of replication

- yeast genome cut restriction enzyme- fragments clone into plasmid vector- recombinant plasmids growth and se-lect- yeast survive in media lacking leucine- selected yeast cells contain leu2+ gene


Nematoda worm- Small and easy to keep- Three days to developMulticellular organismCaenorhabditis elegans

Fruit flyIdentification of mutant → mutation 특징 확인이 쉬움- 짧은 시간에 많은 자손 생산- 알에서 성체가 되기까지 12 일 소요- Drosophila melanogaster


Zebrafish- Reproduction rapidly and high number- vertebrate- 발생 과정이 사람과 유사- 알이 투명하여 발달 확인 가능Danio rerio

Amphibians- 알의 크기가 큼 →발생학 연구에 이용- 1920 년 슈페만 형성체 발견- Xenopus leavis


Chicken배 발생 연구에서 알 사용됨 ; 진화상 인간과 유사- 알이 크고 , 분화 관찰에 좋음- Gallus gallus

Mouse- vertebrate, mammalsHuman 과 더욱 가까움 ; 생리학 , 발생학적으로 유사- Expensive to maintain, Reproduce slowlyKnock out mouse 이용 → 특정 gene 의 기능 확인


Human cell cultureHuman blood 나 tissue 에서 분리Primary culture ; derived directly from living tissue - Grow for a short period time and stop- 세포 분열 횟수 제한

Plants- Grow slowly, long generation time- 세포벽 때문에 형질전환이 어려움- Large genome- Transposon 연구에 이용 ; corn- Arabidopsis thaliana

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Cell Culture