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Mammalian Cell Culture
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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. 2
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Basic equipments used in cell culture
Clean bench Incubator Microscope Culture ware Liquid nitrogen (LN2) tank 3
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Classification of cell cultures
• Primary culture (directly from animal) • Extended culture (multipassage culture) – cell strain • Established (transformed) cell lines
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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
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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 Initial loss of growth potential Immortal Variant (cell line) Human fibroblasts Mouse embryonic fibroblasts
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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
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Cell-Cell & Cell-Matrix Adhesive Interactions
Basal lamina Extracellular Matrix (ECM) Basal surface Connexon Tight junction Gap junction Apical surface
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Enzymatic Disaggregation
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
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Mechanical Disaggregation
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
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Isolation of cells Incubation and growth 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
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Types of cell culture Primary cell culture vs. Cell line culture
Definition Advantage Disadvantage Cells : directly from a donor organism Cell line : subcultured indefinitely Best experimental in vitro models Retain characteristics of normal cells 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 Difficult to obtain Susceptible to contamination
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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 :
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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
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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
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Optimal Cell Environment
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
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Manipulation of cultured cells
Cell split Protease Treatment Trypsin EDTA Protease chelator Cells detach suspension Adherent cell Suspension Suspended cell - No need protease!! Reference :
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Contamination Chemical contaminants
- Endotoxins, plasticizers or metal ions Biological contaminants - Mycoplasma, bacteria, fungus or yeast Bacteria contamination Fungi contamination Fungi Cells Yeast contamination Reference :
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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
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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
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Resources Reputable Cell Repositories ATCC : www.atcc.org ECACC
: DSMZ : 한국세포주은행 :
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TRANSFECTION of DNA to Mammalian Cells
Ca2+Phosphate Liposome Electrotransfection Viral Transduction
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Induced Pluripotent Stem Cell
Shinya Yamanaka, Pioneer Of Induced Pluripotent Stem Cell (iPS) Technology Induced Pluripotent Stem Cell (4 selected out of 24 genes expressed in ES) Shinya Yamanaka Kyoto Univ
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Principles of Confocal Microscopy
Non-Confocal Image Confocal Image 9/18/2018
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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 9/18/2018
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Introduction_Pinhole
Beam path in a confocal LSM. A microscope objective is used to focus a laser beam onto the specimen, where it excites fluorescence, for example. The fluorescent radiation is collected by the objective and efficiently directed onto the detector via a dichroic beamsplitter. The interesting wavelength range of the fluorescence spectrum is selected by an emission filter, which also acts as a barrier blocking the excitation laser line. The pinhole is arranged in front of the detector, on a plane conjugate to the focal plane of the objective. Light coming from planes above or below the focal plane is out of focus when it hits the pinhole, so most of it cannot pass the pinhole and therefore does not contribute to forming the image.
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Introduction_LSM700
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Introduction_LSM700 Z-stack X/Y/Z Stack Z-Drive
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Spectral Unmixing - General Concept
32 Channel Detector Collect Lambda Stack Raw Image Derive Emission Fingerprints FITC Sytox-green Unmixed Image Courtesy: Duncan McMillan, Carl Zeiss Microimaging 9/18/2018
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Introduction_LSM700 3D projection and rotation
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Scanning an image
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Applications Organelle Structure & Function
Mitochondria (Rhodamine 123) Golgi (C6-NBD-Ceramide) Actin (NBD-Phaloidin) Lipid (DPH)
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Fluorescence Microscope image of Hoechst stained cells (plus DIC)
Image collected with a 470T Optronics cooled camera
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Measurement of DNA Use for DNA content and cell viability
G0-G1 S G2-M Fluorescence Intensity # of Events 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 Ratio of fluorescence (green) vs (violet) different for different subclasses of chicken thymocytes shift of emission toward green as [dye] increased fixed rat thymocytes low dye/Protein ratio, Hi affinity binding with hi QE yield blue fluorescence as increased [dye], lower affinity binding to secondary sites, emission shifts toward green, and DNA precipitates
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PI - Cell Viability How the assay works: Viable Cell Damaged Cell
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
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Crypto Green Fluorescence Side Scatter Fluorescence Transmission
4 Fluorescence Transmission 10 Flow Cytometry Dot Plot 3 10 Side Scatter 2 10 oocysts 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. Crypto isolated from cow (purchased) gamma irradiated 900 Krads (kills alm ost all) Stained with Sytox Green (membrane impermeant) i.e. gamma irradiation damages oocyst wall to all dye to penetrate and stain DNA Ghosts - don’t stain because trophozoites have left and there is no DNA to stain Dead cells stain because of the damaged membrane from the gamma irradiation Live cells will not stain either ghosts 1 10 debris Forward Scatter 10 10 10 1 10 2 10 3 10 4 Green Fluorescence
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Specific Organelle Probes
Probe Site Excitation Emission BODIPY Golgi NBD Golgi DPH Lipid TMA-DPH Lipid Rhodamine 123 Mitochondria DiO Lipid diI-Cn-(5) Lipid diO-Cn-(3) Lipid BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH - diphenylhexatriene TMA - trimethylammonium
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Organelle Staining Dyes
Mitochondria by Rhodamine 123 Endosomes by Ceramides Golgi by BODIPY-Ceramide E.R. by DiOC6(3) Carbocyanine
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Calcium Related Applications
Probe Ratioing Calcium Flux (Indo-1) pH indicators (BCECF, SNARF) Molecule-probe Excitation Emission Calcium - Indo nm 405, >460 nm Calcium- Fluo nm 525 nm Calcium - Fura nm >500 nm Calcium - Calcium Green 488 nm 515 nm Magnesium - Mag-Indo nm 405, >460 nm Phospholipase A- Acyl Pyrene 351 nm 405, >460 nm
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Probes for Ca2+Ions INDO-1 Ex350 Em405/480 QUIN-2 Ex350 Em490
Fluo-3 Ex488 Em525 Fura -2 Ex330/360 Em510
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Calcium ratios with Indo-1
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.. 1 1 2 2 3 3 460 nm 405/35 nm Export data from measured regions to Microsoft Excel 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 cell 1 cell 2 cell 3 Ratio: intensity1 (460nm) / intensity2 (405/35nm) 50 100 150 200 base to Delta Graph Export data from Excel data
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FRAP Intense laser Beam Bleaches Fluorescence Zero time 10 seconds
Recovery of fluorescence Zero time 10 seconds 30 seconds
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Fluorescence-Activated Cell Sorting (FACS) Flow Cytometry
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4 colors - simultaneous collection
FITC PE PE- TR PE-CY5 Emission wavelength (nm) Page 45 © J.Paul Robinson, Purdue University Cytometry Laboratories BMS 602 LECTURE 9.PPT
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Purity / Doublet discrimination
Voltage pulse
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Purity / Doublet discrimination
Area vs. Width vs. Height
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Immunofluorescence staining
specific binding nonspecific binding Slide from Dr. Carleton Stewart
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Direct staining Fluorescent probe attached to antibody
Specific signal: weak, 3dyes/site Nonspecific binding: low Slide from Dr. Carleton Stewart
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Avidin-Biotin method I
biotinylated primary Ab biotin avidin biotinylated dye
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Flow cytometry measurements
SCATTER FLUORESCENCE IMAGE G M L
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FOUR COLOR PATTERN This is a subset of cells It is CD3+ CD56+ CD8 CD4
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+ CD8 CD4 CD56 - NK CD3 CD3 CD3 As additional antibodies are combined, further subsetting can be achieved and heretofore unknown populations of cells discovered (CD3+CD4+CD8-CD56+). The increased dimensionality of data may make its visualization difficult. CD8 CD4 CD4 CD56 CD56 CD8 Data from Dr. Carleton Stewart
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Purity / Doublet discrimination
FSC Area vs. FSC Height
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Purity ideal real AND !
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Purity / Doublet discrimination
FSC parameter
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Droplet sorting Flowcell Cuvette Nozzle Deflection Plates
Collection Tubes
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Droplet sorting + ++ - - - Charged droplet Nozzle Sort pulse
(Drop Charge) Charged droplet + ++ - - -
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Droplet sorting Deflection Plates - +
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Sorting speed is dependent on fluid pressure and nozzle size
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 drops per second can be formed.
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Purity Purity of the sort is mainly dependent on the sample
The major source of contamination during a sort are DOUBLETS!
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Tips for high quality sort result
Sort twice 1. Yield 2. Purity Enrich (or deplete) cells magnetically 1. MACS sort 2. FACS sort
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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 Do not ignore the specified/complicated staining protocols for some special cases
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Sample preparation Additional considerations for sample preparation
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!
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Other things influencing sorting
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
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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
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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
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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
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Stem Cell Sorting
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Cell Sorting Physically separating cells based on some measurable characteristic Placing these cells into containers
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Fluorescence detector
Fluorescence Activated Cell Sorting 488 nm laser FALS Sensor Fluorescence detector - + Charged Plates Single cells sorted into test tubes Purdue University Cytometry Laboratories
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SORT DECISIONS -2KV +2KV LEFT RIGHT Frequency Histogram Waste
SMALL BEAD LARGE BEAD Frequency Histogram Sample in Sheath Sheath in Laser beam Stream Charge +2KV -2KV Waste SORT RIGHT SORT LEFT SORT DECISIONS Piezoelectric crystal oscillator Last attached droplet LEFT RIGHT Sensors Sensor
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Stem Cells Stem cells → 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.
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Cancer Stem Cells Minority of cancer cells with tumorigenic potential
NORMAL SELF-RENEWAL DIFFERENTIATION PROLIFERATIVE ABILITY ABERRANT REGULATION TUMORAL Modified from Bjerkvig R et al. Nat Rev Cancer. 2005;5:
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Stem Cells: identifying properties
SELF-RENEWAL DIFFERENTIATION EXTENSIVE PROLIFERATION POTENTIAL 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
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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
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Surrogate in vitro and in vivo studies
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
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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: : isolation and serial propagation of „cancer neurospheres“ → in vivo tumorigenicity Singh SK et al. Nature. 2004;432: : Cell surface marker CD133 identifies glioma stem cells
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Cancer stem cells models
Acute myelogenous leukemia: [CD34+,CD38-] Breast Cancer: [CD44+, CD24-/low] Brain tumor: [CD133+] Prostate cancer: [CD44+] Colon cancer: [CD133+]
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Stem cell markers Markers Commonly Used to Identify Stem Cells and to Characterize Differentiated Cell Types
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Stem cell markers_continued
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Stem cell sorting procedure
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Stem cell sorting procedure
Side population sorting and analysis
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FACS Aria Ⅲ cell sorter – Multi-color
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The Cell Cycle M G2 G1 G0 Quiescent cells S
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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.
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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
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DNA PROBES N+ C2H2)3 NH2 C2H5 CH3 Propidium N+ C2H5 NH2 Ethidium
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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
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Normal Cell Cycle s G2 M G0 G0 - G1 400 1000 75 150 225 300 Cell Count
200 400 600 800 1000 75 150 225 300 G0 G0 - G1 M G0 G2 G1 s Cell Count G2 M s 2N 4N DNA Content
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A typical DNA Histogram
G0-G1 G2-M S # of Events Fluorescence Intensity 17
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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
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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
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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.
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Detection of Apoptotic Cells
DNA Fragments:After fixing or permeabilising cells, low molecular weight fragments of DNA are extracted and an extra peak ('sub-G1') 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.
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Detection Methods for Apoptotis
Phosphatidyl serine, can be detetected by incubating the cells with fluorescein-labelled Annexin V By staining with the dye, Hoechst (UV) By staining with the dye PI (visible) By staining with the dye YOPRO-1 (visible)
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Flow Cytometry of Apoptotic Cells
PI - Fluorescence # Events Apoptotic cells Normal G0/G1 cells
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