Post on 16-Feb-2021
Mammalian Cell Culture
What is cell culture ?
• Cultivation or growth of cells under controlled conditions
outside of the host organism
• In practice the term "cell culture" has come to refer to the
culturing of cells derived from multicellular eukaryotes,
especially animal cells.
Introduction
Basic equipments used in cell culture
• Clean bench
• Incubator
• Microscope
• Culture ware
• Liquid nitrogen (LN2) tank
Equipments
• Primary culture (directly from animal)
• Extended culture (multipassage culture) – cell strain
• Established (transformed) cell lines
Classification of cell cultures
A culture derived directly from a tissue
A stage from cell isolation to first subculturing
Best resembling natural tissue
Limited growth potential
Limited life span
May give rise to a cell strain or be immortalized
Strain : a lineage of cells originating from one
primary culture
Primary Culture
Human fibroblasts
Mouse embryonic fibroblasts
Stages in the establishment of a cell culture
Cell Strain
Cell senescence
Growth Rate of Culture
Phase I II III
Cell generations
Days after initiation of culture
senescence
Growth Rate of Culture
Initial loss of growth potential
Immortal Variant (cell line)
• Cells in multicellular organisms are in contact with each
other or extracellular matrix
• Cell connections involve multiple ligands and cell adhesion
receptors
• The interaction between cell adhesion receptors and their
ligands are relatively weak
• A lot of weak interactions make a strong bond
Physical connections between cells
Cell-Cell & Cell-Matrix Adhesive Interactions
Basal lamina
Extracellular Matrix (ECM)
Basal surface
Connexon
Tight junction
Gap junction
Apical surface
• Faster than explant • Break the connections : cell - cell cell - extracellular matrix • Enzymes Trypsin Collagenase II (from Pseudomonas perfringens) Elastase Hyaluronidase or heparinase - proteoglycan DNase Pronase (bacterial protease) – fibronectin / laminin Calcium chelators : EDTA or EGTA
Enzymatic Disaggregation
Isolation of Cells
Mincing Collecting cells when tissue is sliced Pressing the tissue through a series of sieves Repeated pipetting Quicker than enzymatic methods Causes more mechanical damage
Mechanical Disaggregation
Isolation of Cells
Isolation of cells
Direct purification Releasing of mononuclear cells Explant culture
Appropriate medium Special adhesion surfaces Removal of dead cells Enrichment of viable cells Separation of cell types
Incubation and growth
Types of cell culture
Primary cell culture vs. Cell line culture
Primary cell culture Cell line culture
Definition
Advantage
Disadvantage
•Best experimental in vitro models
•Retain characteristics of normal cells
•Difficult to obtain
•Susceptible to contamination
Cells : directly from a donor organism
Cell line : subcultured indefinitely
•Easy to maintain in culture
•Easy to obtain large quantities
•Typically easy to manipulate
•Cell line may change overtime
•Unclear how well they
represent function of original
cell type
Types of cell
Concepts in mammalian cell line culture
Maintaining cells in culture Manipulation of cultured cells
Reference : www.atcc.org
Attached cell Suspended cell
Attached cell Suspended cell
Temperature
Gas mixture : 37 C, 5% CO2
Growth medium : pH, glucose concentration, growth factor, etc
Culture condition : suspension, adherent cultures, organotypic culture
Maintaining cells in culture
Manipulation of cultured cells
Nutrient depletion in the growth media
Accumulation of dead cells.
Cell-to-cell contact : Contact inhibition (senescence)
Cellular differentiation
Media Change & Passaging cells
Transfecting
Sterile techniques
Antibiotics & antifungals
pH indicator
Cells & Cell Products Cell-Cell interaction : Metabolites & Products Rate of Metabolism
Soluble Factors Culture Medium Serum Hormones Growth Factors Attachment Factors
Physiological Parameters pH
Temperature
Oxygen
Osmolarity
Static or Dynamic Culture
Matrix Interaction •Culture Surface :
Plastics, Glass,
Membranes, Microsheres
•Coating :
Gelatin, Collagen,
Matrigel, Feederlayer,
Biomatrix
Optimal Cell Environment
Suspended cell - No need protease!!
Manipulation of cultured cells Cell split
Adherent cell Suspension
Reference : www.atcc.org
Protease Treatment
Trypsin EDTA
Protease + chelator
Cells detach suspension
Contamination
Chemical contaminants
- Endotoxins, plasticizers or metal ions
Biological contaminants
- Mycoplasma, bacteria, fungus or yeast
Bacteria contamination Fungi contamination
Fungi
Cells
Yeast contamination
Reference : http://www.unc.edu
can be a major problem
Regular authentication required
There are many methods :
isoenzyme analysis
human lymphocyte antigen (HLA) typing
Short tandem repeat (STR) analysis
Cell line cross-contamination
Applications of cell culture : What can we do with cells?
Biological products :
enzymes, synthetic hormones,
immunobiologicals , vaccines
Toxicity testing
Test pharmaceutical drugs
Watch disease mechanisms
Observe the regenerative process
Observe the developmental process
Cancer research
Gene therapy
ATCC : www.atcc.org ECACC : www.hpacultures.org.uk DSMZ : www.dsmz.de 한국세포주은행 : http://cellbank.snu.ac.kr
Resources
Reputable Cell Repositories
http://www.dsmz.de/http://www.dsmz.de/
TRANSFECTION of DNA to Mammalian Cells Ca2+Phosphate Liposome
Electrotransfection Viral Transduction
Schematic diagram of a generic viral vector and Helper (packaging) cell system
Generic strategy for engineering a virus into a vector
Retrovirus Packaging Cell Lines
Retrovirus system
Lentivirus system with a Pseudotyped Envelope
VSVG(Vesicular stomatitis virus G protein) A pseudotype envelope protein Extremely broad tropism
Induced Pluripotent Stem Cell
Shinya Yamanaka Kyoto Univ
(4 selected out of 24 genes expressed in ES)
5/13/2013
Principles of Confocal Microscopy Non-Confocal Image Confocal Image
Resolution =~ 0.61λ/N. Aperture of Lens
사용하는 빛의 파장이 짧을 수록 Resolution이 좋다 해상도; 가시광선
FLUORESCENCE (형광)과 형광물질
형광 형광물질
Absorbs Blue Light --> Emits Green Light Absorbs Green Light --> Emits Red Light
Optics of a Fluorescence Microscope
separate the excitation and emission light paths
Confocal scanning laser microscopy (CSLM)
Introduction_Pinhole
Confocal microscopy uses the objective lens as both the objective and the condenser. This allows the illuminating light to be focused on a relatively thin plane. In addition, a ‘pin-hole’ is used to further minimize the light coming from other planes
Introduction_LSM700
Introduction_LSM700
X/Y/Z Stack
Z-Drive
Z-stack
5/13/2013
Spectral Unmixing - General Concept
32 Channel
Detector
Collect Lambda St
ack
Derive Emission Fing
erprints
FITC Sytox-green
Raw Image
Unmixed Image Courtesy: Duncan McMillan, Carl Zeiss Microimaging
Introduction_LSM700
3D projection and rotation
Scanning an image
Applications
• Organelle Structure & Function
– Mitochondria (Rhodamine 123)
– Golgi (C6-NBD-Ceramide)
– Actin (NBD-Phaloidin)
– Lipid (DPH)
Fluorescence Microscope image of Hoechst stained cells (plus DIC) Image collected with a 470T Optronics cooled camera
• Use for DNA content and cell viability
– 33342 for viability
• Less needed to stain for DNA content than for viability
– decrease nonspecific fluorescence
• Low laser power decreases CVs
Measurement of DNA
G0-G1 S
G2-M
Fluorescence Intensity
# o
f Eve
nts
Specific Organelle Probes
BODIPY Golgi 505 511
NBD Golgi 488 525
DPH Lipid 350 420
TMA-DPH Lipid 350 420
Rhodamine 123 Mitochondria 488 525
DiO Lipid 488 500
diI-Cn-(5) Lipid 550 565
diO-Cn-(3) Lipid 488 500
Probe Site Excitation Emission
BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH - diphenylhexatriene TMA - trimethylammonium
Fluorescence-Activated Cell Sorting (FACS) Flow Cytometry
FLOW CYTOMETRY
The scatter (i.e., direction change): the size and shape of the cell Cells labeled with fluorochromes which are excited by the laser
46
Purity / Doublet discrimination Voltage pulse
47
Purity / Doublet discrimination Area vs. Width vs. Height
Flow cytometry measurements
L
M
G
SCATTER FLUORESCENCE IMAGE
49
Purity / Doublet discrimination FSC Area vs. FSC Height
50
AND
AND
!
!
ideal real
Purity
Page 52 © 1988-2002 J.Paul Robinson, Purdue University Cytometry Laboratories BMS 602 LECTURE 9.PPT
4 colors - simultaneous collection
Emission wavelength (nm) 530 580 630 680 730 780
FITC PE PE- TR
PE-CY5
Immunofluorescence staining
specific binding
nonspecific binding
Slide from Dr. Carleton Stewart
Direct staining
• Fluorescent probe attached to antibody
• Specific signal: weak, 3dyes/site
• Nonspecific binding: low
Slide from Dr. Carleton Stewart
Avidin-Biotin method I
biotinylated primary Ab
biotin
avidin
biotinylated dye
10 1 10 2 10 3 10 4
CD56 -->
10
11
02
10
31
04
CD
4 -
->
10 1 10 2 10 3 10 4
CD3 -->
10
11
02
10
31
04
CD
4 -
->
CD3 CD3 CD3
10 1 10 2 10 3 10 4
CD3 -->
10
11
02
10
31
04
CD
56 -
->
10 1 10 2 10 3 10 4
CD3 -->
10
11
02
10
31
04
CD
8 -
->
CD56
10 1 10 2 10 3 10 4
CD56 -->
10
11
02
10
31
04
CD
8 -
->
CD56 CD8
10 1 10 2 10 3 10 4
CD8 -->
10
11
02
10
31
04
CD
4 -
->
FOUR COLOR PATTERN
CD
4
CD
8
CD
56 -
NK
C
D8
CD
4
CD
4
Data from Dr. Carleton Stewart
CD56 – NK Cells CD3 – T cells CD4 – T cells – Helper CD8 – T cells - Cytotoxic
This is a subset of cells It is CD3+ CD56+
This is a subset of cells It is CD3+ CD4+
Cell Sorting
• Physically separating cells based on some measurable characteristic
• Placing these cells into containers
488 nm laser
+ -
Fluorescence Activated
Cell Sorting
Charged Plates
Single cells sorted
into test tubes
FALS Sensor
Fluorescence detector
Purdue University Cytometry Laboratories
59
• SELF-RENEWAL
i.e. replenish the repertoire of identical stem cell
• DIFFERENTIATION
i.e. create a heterogeneous progeny differentiating to mature cells
• EXTRAORDINARY PROLIFERATION POTENTIAL
• HOMEOSTATIC CONTROL according to the influence of microenvironment.
Stem cells → progenitor cells → mature cells
Stem Cells
60
• SELF-RENEWAL
• DIFFERENTIATION
• PROLIFERATIVE ABILITY
ABERRANT REGULATION
Modified from Bjerkvig R et al. Nat Rev Cancer. 2005;5:899-904
Minority of cancer cells with tumorigenic potential
NORMAL
TUMORAL
Cancer Stem Cells
61
Stem cell sorting procedure
62
FACS Aria Ⅲ cell sorter – Multi-color
The Cell Cycle
G1
M G2
S G0 Quiescent cells
Action of DNA Probes
• Phenanthridiniums
– Propidium Iodide
– Ethidium Bromide
– Intercalate between bases in DS nucleic acids
– Excite in the UV or Blue with red emission
– Bind DS RNA - must be treated with RNAse for DNA measurements to be correct
• Benzimides
– Hoechst 33342
– Binds to the A-T rich regions in the small groove of DNA
– UV excitation with blue emission
– Red emission if exceed optimal concentration
DNA PROBES in Cells
N+
C2H2)3
NH2 NH2
N+ C2H5 CH3 C2H5
Hoechst : Live/Fixed Cell DNA 모두 염색
Propidium; Fixed/Dead Cell의 DNA를 염색
Measurement of DNA
G0-G1 S
G2-M
Fluorescence Intensity
# o
f Eve
nts
Propidium; Hoechst33342
PI - Cell Viability How the assay works: • PI cannot normally cross the cell membrane
• If the PI penetrates the cell membrane, it is assumed to be damaged
• Cells that are brightly fluorescent with the PI are damaged or dead
PI
PI
PI
PI
PI
PI
PI
PI PI
PI
PI
PI
PI
PI
Viable Cell Damaged Cell
Normal Cell Cycle
G0 G0 - G1
s G2 M
DNA Content 2N 4N
G2 M G0
G1
s
0 200 400 600 800 1000 0
75
150
225
300
Ce
ll C
ou
nt
A typical DNA Histogram
G0-G1
S
G2-M
Fluorescence Intensity
# o
f Eve
nts
2n 4n
Measurement of Apoptosis
• Condensation of the chromatin material
• DNA Fragments
• Internucleosomal degradation of DNA; distinctive 'ladder' pattern on DNA gel electrophoresis.
• Blebbing of nuclear material
Flow Cytometry of Apoptotic Cells
PI - Fluorescence
# E
vents
Apoptotic cells
Normal G0/G1 cells
Flow Cytometry of Apoptotic Cells
Phosphatidyl serine, can be detetected by incubating the cells with fluorescein-labelled Annexin V
Immune Incompetent Mice
Nude : a thymic a spontaneous deletion in the FOXN1 gene No functinal T cell
Nude mice have a spontaneous deletion in the FOXN1 gene. ( NODSCID: Non Obese Diabatic/Severe Combined Immune Deficiency
Recessive mutated in Prkdc (Protein kinase, DNA activated, catalytic polypeptide) Impaired activity of T, B cell, complement system
NOG : (NOD/Shi-scid/IL-2Rγnull) mouse No activity of T cell, B cell and NK cell Reduced complementary activities Dysfunction of macrophage Dysfunction of dendritic cell No leakiness: no incidence of T, B cells with aging No incidence of lymphoma
http://en.wikipedia.org/wiki/Complement_systemhttp://en.wikipedia.org/wiki/T_cellhttp://en.wikipedia.org/wiki/B_cellhttp://en.wikipedia.org/wiki/NK_cellhttp://en.wikipedia.org/wiki/Macrophagehttp://en.wikipedia.org/wiki/Dendritic_cellhttp://en.wikipedia.org/wiki/Dendritic_cellhttp://en.wikipedia.org/wiki/Dendritic_cellhttp://en.wikipedia.org/wiki/Lymphoma
Tumorigenicity Assays
Cancer Stem Cell 분리와 농축
사람 면역체계를 재건한 마우스 Reconstruction of Human Immune System:
Hu-Mice