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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.

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Presentation Transcript
slide3
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

Introduction

slide4
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

slide5
Areas where cell culture technology is currently playing a major role.

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

Toxicity testing

Study the effects of new drugs

Cancer research

Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells

Why is cell culture used for?

contd
Virology

Cultivation of virus for vaccine production, 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-functional gene

Contd….
slide7
Choice of media depends 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 filtered and incubated at 4 C

Culture media

slide8
Laminar cabinet-Vertical are preferable

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

slide9
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

slide10
Vial from liquid nitrogen is placed into 37 C water bath, agitate vial continuously until 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 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

slide11
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 nitrogen

Freezing cells for storage

slide12
Cells are cultured as anchorage dependent or independent

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

slide13
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 remained to be adherent

Add complete medium depends on the subculture

requirement either to 75 cm or 175 cm flask

Adherent cells

slide14
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

slide15
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 insect cells

Common cell lines

slide16
Cell culture contaminants of two types

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

slide17
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

slide18
Cytotoxicity causes inhibition of cell growth

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

Cell toxicity

slide19
Calcium phosphate precipitation

DEAE-dextran (dimethylaminoethyl-dextran)

Lipid mediated lipofection

Electroporation

Retroviral Infection

Microinjection

Transfection methods

transfection
Transfection
  • What 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…)

MethodApplication

CaPO4, DEAE DNA Transfection

Liposome Based DNA Transfection

Polyamine Based DNA Transfection

early years

1928: Frederick Griffith transforms Streptococcus pneumoniae

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 technique

Early Years
  • Transfection = “transformation” + “infection”
  • The infection of cells by the isolated nucleic acid from a virus, resulting in the production of a complete virus
transfection vs transformation
Transfection vs. Transformation
  • Transformation: genetically altering cells by transporting in foreign genetic material.
  • Transfection: the process of transporting genetic material and/or macromolecules into eukaryotic cells through typically non-viral methods.
purpose uses of transfection
Purpose/uses of Transfection
  • Study gene function
  • Study protein expression
  • Transfer DNA into embryonic stem cells
how it works
How it works
  • Utilize Chemical, lipid or physical methods (direct microinjection, electroporation, biolistic particle delivery) for transportation of genetic materials or macromolecules.
transfection method 1 deae dextran

Add chloroquine to transfection medium

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
  • Facilitates DNA binding to cell membranes and entry via endocytosis
  • DEAE-D associates with DNAcomplex binds to cell surface
transfection method 1 deae dextran1
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 liposome mediated
Transfection method 3: liposome-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

how it works chemical and lipid methods
How it works – Chemical and lipid methods
  • Neutralize negative charges on DNA
  • Chemical 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 endocytosis.
transfection method 2 electroporation
Transfection method 2: electroporation
  • 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

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

electroporate

Selection process for transfectant

transfection method 2 electroporation1
Transfection method 2: electroporation
  • Variants: amplitude and length of electric pulse
  • Less affected by DNA concentration-linear correlation between amount of DNA present and amount taken up
  • Can be used for stable transfections
how it works physical methods
How it works – Physical methods
  • Electroporation: use of high voltage to deliver nucleic acids; pores are formed on cell membrane.
  • 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 delivery of nucleic acids and penetration of cell wall.
advantages disadvantages
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 requiring incorporation of genetic material into the chromosome.

Disadvantages:

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
slide37

Exploring protein function

1) Where is it localized in the cell?

Approaches:

a) Make antibodies - immunofluorescence

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

 Fuse to GFP

2) What is it doing in the cell?

Approaches:

a) Reduce protein levels - RNA interference

b) Increase protein levels “over-express”

c) “Express” mutant versions

Transfection!!!!

slide38

Transfection =

Introduction of DNA into mammalian cells

Gene is transcribed and translated into protein

= “expressed”

slide39

Direct introduction of the DNA

Electroporation - electric field temporarily disrupts plasma membrane

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

Microinjection

slide40

Infection:

Use recombinant viruses to deliver DNA

Retroviruses

Adenoviruses

Virally-mediated introduction of the DNA

slide41

Carrier-mediated introduction of the DNA

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

Calcium Phosphate

DEAE Dextran

liposomes

micelles

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

slide42

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

slide43

DNA “expression” vector transfected:

Insert gene

in here

For expression in cells

Polyadenylation

site

GFP

CMV

Promoter

SV40

Promoter

To generate stable cell line

pCMV/GFP

Ampicillin

resistance

Neomycin

resistance

For amplification of the plasmid in bacteria

Polyadenylation

site

pUC

Bacterial origin of replication

slide44

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

PROTEIN X

GFP

GFP

PROTEIN Y

PROTEIN

GFP

Z

slide45

EXPERIMENT:

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”

slide47

Nuclei

Mitochondria

Secretory Pathway:

Endoplasmic Reticulum

Golgi Complex

Endocytotic Pathway:

Endosomes

  • Compartments/organelles examined
  • Protein sequences sufficient for localization
  • Vital stains
slide48

Nucleus

Transport through nuclear pore

signal = basic amino acid stretches

example: P-P-K-K-K-R-K-V

slide50

Nuclear Stain:

Hoechst 33258 binds DNA

slide51

Mitochondria

Transmembrane transport signal

Example: 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

slide53

Mitochondrial dye = MitoTracker Red

Diffuses through membranes

Non-fluorescent until oxidized

Accumulates in mitochondria and oxidized

Mitotracker

DNA

slide54

lysosome

plasma

membrane

late

endosome

nuclear envelope

endoplasmic reticulum

early

endosome

CYTOSOL

cis

Golgi

network

Golgi

stack

trans

Golgi

network

Golgi apparatus

Cellular components of the secretory and endocytic pathways

slide55

Endoplasmic Reticulum

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

at amino terminus

Retention in E.R. lumen:

Signal = K-D-E-L-COOH

at carboxy terminus

slide56

Endoplasmic Reticulum marker

ER-Tracker Blue-White

Live bovine pulmonary artery endothelial cells

slide58

Golgi

nucleus

From the ER, secreted and membrane proteins move to the Golgi, a

series of membrane-bound compartments found near the nucleus

slide59

Golgi marker

BODIPY-TR ceramide

Ceramide = lipid

When metabolized, concentrates in the Golgi

Red fluorophore

slide60

Cultured Epithelial Cells

DNA (Hoechst)

Golgi (ceramide)

slide61

MDCK Cells

Madin-Darby Canine Kidney

Polarized Epithelial Cells

DNA (Hoechst)

Golgi (ceramide)

Lysosomes (LysoTracker)

Molecular Probes, Inc.

slide62

Rhodamine transferrin

Does the fluorescent green protein co-localize?

slide64

Immunofluorescence / confocal microscopy 실험방법

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.

slide66

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

slide68

Model systems

E. coli

- 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

slide69

Yeast

  • Eukaryote와 prokaryote 사이의 차이점
  • 연구에 이용
  • - Genetic study easy
  • - haploid, single-celled
  • Grow very rapidly, inexpensive
  • 2가지 종류 사용
  • → Saccaromyces cerevisiae; budding
  • → Schizosaccaromyces pombe; fission
slide70

Cloning the yeast origin of replication

- 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

slide71

Nematoda worm

- Small and easy to keep

- Three days to develop

Multicellular organism

Caenorhabditis elegans

Fruit fly

Identification of mutant

→ mutation 특징 확인이 쉬움

- 짧은 시간에 많은 자손 생산

- 알에서 성체가 되기까지 12일 소요

- Drosophila melanogaster

slide72

Zebrafish

- Reproduction rapidly and high number

- vertebrate

- 발생 과정이 사람과 유사

- 알이 투명하여 발달 확인 가능

Danio rerio

Amphibians

- 알의 크기가 큼 →발생학 연구에 이용

- 1920년 슈페만 형성체 발견

- Xenopus leavis

slide73

Chicken

배 발생 연구에서 알 사용됨

; 진화상 인간과 유사

- 알이 크고, 분화 관찰에 좋음

- Gallus gallus

Mouse

- vertebrate, mammals

Human과 더욱 가까움

; 생리학, 발생학적으로 유사

- Expensive to maintain, Reproduce slowly

Knock out mouse 이용

→ 특정 gene의 기능 확인

slide74

Human cell culture

Human blood나 tissue에서 분리

Primary 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