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