Transcript 슬라이드 1

Mammalian Cell Culture
Introduction
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.
Equipments
Basic equipments used in cell culture
• Clean bench
• Incubator
• Microscope
• Culture ware
• Liquid nitrogen (LN2) tank
Classification of cell cultures
• Primary culture (directly from animal)
• Extended culture (multipassage culture) – cell strain
• Established (transformed) cell lines
Primary Culture
 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
Stages in the establishment of a cell culture
Phase
I
II
III
Cell Strain
Growth
Rate of
Culture
Cell
senescence
Cell generations
Human fibroblasts
Initial loss
of growth
potential
Growth
Rate of
Culture
Mouse embryonic fibroblasts
Immortal
Variant
(cell line)
senescence
Days after initiation of culture
Physical connections between cells
• 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
Cell-Cell & Cell-Matrix Adhesive Interactions
Tight junction
Apical surface
Gap junction
Basal surface
Connexon
Basal lamina
Extracellular
Matrix (ECM)
Isolation of Cells
Enzymatic Disaggregation
• 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
Isolation of Cells
Mechanical Disaggregation




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
Isolation of cells
Direct purification
Releasing of mononuclear cells
Explant culture
Incubation and growth
Appropriate medium
Special adhesion surfaces
Removal of dead cells
Enrichment of viable cells
Separation of cell types
Types of cell culture
Primary cell culture vs. Cell line culture
Primary cell culture
Cell line culture
Definition
Cells : directly from a donor
organism
Advantage
•Best experimental in vitro
models
•Easy to maintain in culture
•Retain characteristics of
normal cells
•Typically easy to manipulate
Disadvantage
Cell line : subcultured
indefinitely
•Easy to obtain large quantities
•Difficult to obtain
•Cell line may change overtime
•Susceptible to
contamination
•Unclear how well they
represent function of original
cell type
Concepts in mammalian cell line culture
 Maintaining cells in culture
 Manipulation of cultured cells
Types of cell
 Attached cell
 Suspended cell
Attached cell
Suspended cell
Reference : www.atcc.org
Maintaining cells in culture
Temperature
Gas mixture : 37 C, 5% CO2
Growth medium : pH, glucose concentration, growth factor, etc
Culture condition : suspension, adherent cultures, organotypic 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
Soluble Factors
Culture Medium
Serum
Hormones
Growth Factors
Attachment Factors
Cells & Cell Products
Cell-Cell interaction :
Metabolites & Products
Rate of Metabolism
Optimal Cell Environment
Matrix Interaction
•Culture Surface :
Plastics, Glass,
Membranes, Microsheres
•Coating :
Gelatin, Collagen,
Matrigel, Feederlayer,
Biomatrix
Physiological Parameters
pH
Temperature
Oxygen
Osmolarity
Static or Dynamic Culture
Manipulation of cultured cells
Cell split
Protease Treatment
Trypsin
Protease
EDTA
+
chelator
Adherent cell
Cells detach
Suspension
suspension
Suspended cell - No need protease!!
Reference : www.atcc.org
Contamination
 Chemical contaminants
- Endotoxins, plasticizers or metal ions
 Biological contaminants
- Mycoplasma, bacteria, fungus or yeast
Cells
Fungi
Bacteria contamination
Fungi contamination
Yeast contamination
Reference : http://www.unc.edu
Cell line cross-contamination
 can be a major problem
 Regular authentication required
 There are many methods :
isoenzyme analysis
human lymphocyte antigen (HLA) typing
Short tandem repeat (STR) analysis
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
Resources
Reputable Cell Repositories
 ATCC
: www.atcc.org
 ECACC
: www.hpacultures.org.uk
 DSMZ
: www.dsmz.de
 한국세포주은행
: http://cellbank.snu.ac.kr
TRANSFECTION of DNA to Mammalian Cells
Ca2+Phosphate
Electrotransfection
Liposome
Viral Transduction
Induced Pluripotent Stem Cell
(4 selected
out of 24 genes
expressed in ES)
Shinya Yamanaka
Kyoto Univ
Principles of Confocal Microscopy
Non-Confocal Image
7/8/2015
Confocal Image
Wide-field vs. confocal microscopy
a – light source
b – dichroic filter
c – objective lens
d – focal plane
e – specimen
f – light detector
g – confocal aperture
7/8/2015
Introduction_Pinhole
Introduction_LSM700
Introduction_LSM700
Z-stack
X/Y/Z Stack
Z-Drive
Spectral Unmixing - General Concept
32 Channel
Detector
Collect Lambda St
ack
FITC
Raw Image
Sytox-green
Courtesy: Duncan McMillan, Carl Zeiss Microimaging
7/8/2015
Derive Emission Fing
erprints
Unmixed Image
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
# of Events
Measurement of DNA
G0-G1
S
G2-M
Fluorescence Intensity
• 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
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
Viable Cell
Damaged Cell
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
104
Fluorescence
Transmission
102
oocysts
10
1
ghosts
debris
10
0
Side Scatter
103
Flow Cytometry Dot Plot
Flow cytometric scatter plot of
gamma
irradiated C. parvum oocysts. The oocysts region is
clearly distinguished from ghosts and debris.
Images on the right show Sytox green fluorescence
and transmission images of these regions. Note
ghosts do not take up Sytox green dye.
Forward Scatter
100
101
102
103
Green Fluorescence
104
Specific Organelle Probes
Probe
BODIPY
NBD
DPH
TMA-DPH
Rhodamine 123
DiO
diI-Cn-(5)
diO-Cn-(3)
Site
Golgi
Golgi
Lipid
Lipid
Mitochondria
Lipid
Lipid
Lipid
Excitation
505
488
350
350
488
488
550
488
Emission
511
525
420
420
525
500
565
500
BODIPY - borate-dipyrromethene complexes
NBD - nitrobenzoxadiazole
DPH - diphenylhexatriene
TMA - trimethylammonium
Organelle Staining Dyes
•
•
•
•
Mitochondria
Endosomes
Golgi
E.R.
by
by
by
by
Rhodamine 123
Ceramides
BODIPY-Ceramide
DiOC6(3) Carbocyanine
Calcium Related Applications
• Probe Ratioing
– Calcium Flux (Indo-1)
– pH indicators (BCECF, SNARF)
Molecule-probe
Calcium - Indo-1
Calcium- Fluo-3
Calcium - Fura-2
Calcium - Calcium Green
Magnesium - Mag-Indo-1
Phospholipase A- Acyl Pyrene
Excitation
351
488
363
488
351
351
nm
nm
nm
nm
nm
nm
Emission
405, >460 nm
525 nm
>500 nm
515 nm
405, >460 nm
405, >460 nm
Probes for Ca2+Ions
•
•
•
•
INDO-1
QUIN-2
Fluo-3
Fura -2
Ex350
Ex350
Ex488
Ex330/360
Em405/480
Em490
Em525
Em510
Calcium ratios with Indo-1
1
1
2
2
3
3
460 nm
405/35
nm
Export data from measured regions
to Microsoft Excel
Changes in the fluorescence were
measured using the Bio-Rad calcium
ratioing software. The same region in
each wave length was measured and
the relative change in each region was
recorded and exported to a spread
sheet for analysis..
Ratio: intensity1 (460nm) / intensity2 (405/35nm)
0.8
Export data from Excel data
base to Delta Graph
cell 1
cell 2
cell 3
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
50
100
150
200
FRAP
Intense laser Beam
Bleaches Fluorescence
%F
Recovery of fluorescence
Zero time
10 seconds
Time
30 seconds
Fluorescence-Activated
Cell Sorting (FACS)
Flow Cytometry
4 colors - simultaneous
collection
FITC
PE
PETR
PE-CY5
530
580 630
680 730
Emission wavelength (nm)
780
Page 45
© 1988-2002 J.Paul Robinson, Purdue University Cytometry Laboratories BMS 602 LECTURE 9.PPT
Purity / Doublet discrimination
Voltage pulse
46
Purity / Doublet discrimination
Area vs. Width vs. Height
47
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
Flow cytometry measurements
SCATTER
FLUORESCENCE
G
M
L
IMAGE
CD56 – NK Cells
CD3 – T cells
CD4 – T cells – Helper
CD8 – T cells - Cytotoxic
This is a subset of cells
It is CD3+ CD4+
FOUR COLOR PATTERN
1
10
2
10
3
10
4
4
10
10
2
CD4
CD4 -->
1
10
1
10
10
10
10
2
CD8
10
CD8 -->
2
10
CD56 -->
1
10
CD56 - NK
10
3
3
3
10
10
4
4
This is a subset of cells
It is CD3+ CD56+
10
1
CD3 -->
10
2
10
3
10
4
10
1
10
10
3
10
4
CD3
CD3
1
10
2
CD56 -->
CD56
10
3
10
4
2
CD4
CD4 -->
1
10
1
10
10
10
3
2
10
CD4 -->
CD4
10
10
10
10
CD8
2
1
CD8 -->
10
3
3
10
10
4
10
4
4
CD3
2
CD3 -->
CD3 -->
10
1
10
2
CD56 -->
CD56
10
3
10
4
10
1
10
2
10
3
CD8 -->
CD8
Data from Dr. Carleton Stewart
10
4
Purity / Doublet discrimination
FSC Area vs. FSC Height
53
Purity
ideal
real
AND
!
AND
!
54
Purity / Doublet discrimination
FSC parameter
55
Droplet sorting
Flowcell
Cuvette
Nozzle
Deflection
Plates
Collection
Tubes
56
Droplet sorting
Nozzle
Sort pulse
(Drop Charge)
Charged droplet
57
+
++
--
Droplet sorting
-
Deflection Plates
++
58
+
0
+
-
--
Sort speed
Sorting speed is dependent on
fluid pressure and nozzle size
Example:
At 70 psi preessure, using a 70 µm nozzle,
the frequency of drop formation can be up to
~90 kHz.
I.e. 90000 drops per second can be formed.
59
Purity
Purity of the sort is mainly dependent on the sample
The major source of contamination during a sort are
DOUBLETS!
60
Tips for high quality sort
result
Sort twice
1. Yield
2. Purity
Enrich (or deplete) cells
magnetically
1. MACS sort
2. FACS sort
61
Sample preparation
Basic Ab staining

Most Abs bind well at RT or 4℃, for 10-15min

In some cases, 37 ℃ more preferable
 Chemokines and their receptors – exceptionally sensitive to
variation and staining techniques

62
Do not ignore the specified/complicated staining
protocols for some special cases
Sample preparation
Additional considerations for sample preparation
63

Dead cell discrimination necessary
• DNA-intercalating dyes(EMA, PI, 7-AAD, DAPI)
• Phospatidylserine-binding reagents(AnnexinV)
• Amine reactive dyes(UViD, ViViD, GrViD, OrViD)

Qdot: it can relatively easily form aggregates!
Other things influencing
sorting
64

Protein concentration and Refractive Index

Autofluorescence and High Background

Sheath and Cell buffers

Buffering capacity and sorting

Sheath pressure and formulation

Tissue Disaggregation

Cell filtration
Other things influencing
sorting
Protein concentration and Refractive Index
Use the least amount of protein(BSA or FBS)
to minimize FSC and SSC signal interference
Autofluorescence and High Background
NADH and Flavins increase cellular auto FL
Unbounded Ab and phenol red increase background
FL
65
Other things influencing
sorting
Sheath and Cell buffers
Avoid high conc. of protein to minimize
foaming
Buffering capacity and sorting
Final conc. of 25mM HEPES in PBS, HBSS or
Phenol-free culture media with neutral pH
66
Other things influencing
sorting
Sheath pressure and formulation
Sort at appropriate psi. for state of cells
Sort into tubes that have protein-rich media(50%FBS in
PBS)
Use temp. control collection module
Tissue Disaggregation and Cell filtration
Use DNAse in presence of magnesium chloride to help
reduce cellular aggregation
Use 35um nylon mesh(suitable for leukocytes)
※Choose an appropriate mesh size for each cell types
67
Stem Cell Sorting
Cell Sorting
• Physically separating cells based on
some measurable characteristic
• Placing these cells into containers
Fluorescence Activated
Cell Sorting
488 nm laser
FALS Sensor
Fluorescence detector
-
+
Charged Plates
Single cells sorted
into test tubes
Purdue University Cytometry Laboratories
Sample in
Sheath
Stream
Charge
Piezoelectric
crystal oscillator
Sheath in
Sensors
Sensor
Laser beam
SORT DECISIONS
SMALL BEAD LARGE BEAD
Last attached
droplet
SORT LEFT
-2KV
+2KV
RIGHT
LEFT
Waste
SMALL BEAD
SORT RIGHT
LARGE BEAD
Frequency Histogram
Stem Cells
Stem cells →
•
•
•
•
72
progenitor cells
→ mature cells
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.
Cancer Stem Cells
Minority of cancer cells with tumorigenic potential
NORMAL
TUMORAL
• SELF-RENEWAL
• DIFFERENTIATION
• PROLIFERATIVE ABILITY
ABERRANT REGULATION
73
Modified from Bjerkvig R et al. Nat Rev Cancer. 2005;5:899-904
Stem Cells:
identifying properties
• SELF-RENEWAL
• DIFFERENTIATION
• EXTENSIVE PROLIFERATION POTENTIAL
•
•
•
•
•
74
Are the minority subpopulation in a given tissue
Mainly appear to be in a quiescent cell-cycle state
long-lived cells giving rise to short-lived, differentiated cells
Highly influenced by signals form their microenvironment
Characterized by specific surface markers
Therapeutic implications
• Resistance to treatment
→ absence of the targeted biological property (imatinib mesylate
in CML)
→ quiescent state
→ expression of efflux proteins protecting vs xenobiotic toxins
• Relapse
• Metastasis
Strategies to target cancer stem cells:
• Immunotherapy against stem-cell-specific markers
• Combination of
treatment vs tumor burden and treatment
vs cancer stem cells
• Therapies promoting differentiation of cancer stem cells
75
Assays in stem cell research
Surrogate in vitro and in vivo studies
• Clonogenic assays
• Repopulation experiments in immunodeficient mice strains
STEM CELLS
• 1960s: transplantation experiments in immunodeficient mice
→very small population of cells responsible for reconstitution
→surface marker phenotype negative for lineage-specific antigen
CANCER STEM CELLS
• 1990s: AML cells transplanted in immunodeficient mice
→cells able to sustain tumor growth are a minority subpopulation
→reconstitution of the phenotypic heterogeneity of donor tumor
76
Brain tumor: Neurosphere
assay
• Cell culture system for normal neural stem cells
→ long-term self-renewing
→ multi-lineage-differentiating
• Galli R et al. Cancer Res. 2004;64:7011-7021:
isolation and serial propagation of „cancer neurospheres“
→ long-term self-renewing
→ multi-lineage-differentiating
→ in vivo tumorigenicity
• Singh SK et al. Nature. 2004;432:396-401:
Cell surface marker CD133 identifies glioma stem cells
77
Cancer stem cells models
78
•
Acute myelogenous leukemia: [CD34+,CD38-]
•
Breast Cancer: [CD44+, CD24-/low]
•
Brain tumor: [CD133+]
•
Prostate cancer: [CD44+]
•
Colon cancer: [CD133+]
Stem cell markers
Markers Commonly Used to Identify Stem Cells and to Characterize Differentiated Cell Types
79
Stem cell markers_continued
80
Stem cell sorting procedure
81
Stem cell sorting procedure
Side population sorting and
analysis
82
FACS Aria Ⅲ cell sorter – Multi-color
83
The Cell Cycle
G2
S
M
G1
G0
Quiescent cells
Definitions & Terms
• Ploidy
– related to the number of chromosomes in a cell
• Haploid: Number of chromosomes in a gamete (germ cell) is
called the HAPLOID number for that particular species
• Diploid: The number of cells in a somatic cell for a particular
species
• Hyperdiploid: greater than the normal 2n number of
chromosomes
• Hypodiploid: Less than the normal 2n number of chromosomes
• DNA Tetraploidy: Containing double the number of
chromosomes
• DNA Index: The ratio between the mode of the relative DNA
content of the test cells (in G0/G1phase) to the mode of the
relative DNA content in normal G0/G1 diploid cells
• Coefficient of Variation - CV: The ratio between the SD of the
mode of the G0/G1 cell populations expressed as a percentage.
DNA Probes
• Key feature of DNA probes is that
they are STOICHIOMETRIC - thus
number of molecules of probe
bound is equivalent to number of
molecules of DNA
DNA PROBES
Ethidium
Propidium
NH2
NH2
NH2
NH2
N+
N+
C2H5
C2H2)3 N+
CH3
C2H5
C2H5
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
Normal Cell Cycle
300
G0 - G1G0
G2
Cell Count
225
G0
M
G1
s
150
75
0
G2 M
s
0
200
2N
400
600
DNA Content
4N
800
1000
A typical DNA Histogram
4n
2n
G0-G1
G2-M
# of Events
S
Fluorescence Intensity
Action of DNA Probes
• 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
• Antibiotics
– Mithramycin, Chromamycin A3
– Bind the G-C rich regions of DNA
Action of DNA Probes
• Acridine Orange - AO
– Intercalates into the DS DNA
– Binds to single stranded nucleic acids by
stacking on the charged phosphates
– Blue excitation and green emission for DS DNA
– Red fluorescence emission for SS DNA
• Pyronyn Y is a related dye to AO
– Used for RNA measurement after blocking
DNA with non-fluoresceinated DNA probe
Measurement of Apoptosis
• Apoptosis is programmed cell death where
the cell goes through a highly regulated
process of “dying”.
• Characteristics are condensation of the
chromatin material
• Blebbing of nuclear material
• Often accompanied by internucleosomal
degradation of DNA giving rise to
distinctive 'ladder' pattern on DNA gel
electrophoresis.
Detection of Apoptotic
Cells
• DNA Fragments:After fixing or
permeabilising cells, low molecular
weight fragments of DNA are
extracted and an extra peak ('subG1') of apoptotic cells can be seen
in the DNA histogram.
• Internucleosomal degradation of
DNA can be visualised by labelling
enzymatically the ends of the
broken strands of DNA.
Detection Methods for
Apoptotis
• Phosphatidyl serine, can be
detetected by incubating the cells
with fluorescein-labelled Annexin V
• By staining with the dye, Hoechst
33342 (UV)
• By staining with the dye PI (visible)
• By staining with the dye YOPRO-1
(visible)
Flow Cytometry of Apoptotic
Cells
Apoptotic cells
# Events
Normal G0/G1 cells
PI - Fluorescence