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Animal tissue culture, Examples cell lines:
L929 [mouse L-cells] - first…
Animal tissue culture
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Tissue culture medias
Complete media
- contains all constituents & supplements
- sufficient for use specified
- comprises:
- Amino acids - essential & non-essential
- Vitamins - A, D, E, K (depends)
- Salts
- Glucose / Sugar molecules
- Organic supplements - peptides, nucleotides
- Serum / Hormones & Growth factors
- Antibiotics - broad & narrow
Chemically defined media
- Based on: body fluids & nutritional biochemistry analyses
- Eagle's Basal Medium
- Eagle's Minimal Medium
- contain:
- calf / human / horse serum
- protein hydrolysates
- embryo extract
- Well defined
- Complex combination of: organic salts, amino acids, vitamins, glutamine, glucose, protein supplements
- prolonged growth / high output of cell culture goals
- cell cultures required gas phase: O2 & CO2
- made up of acid solution
- may contain buffer
- Medium recommended bicarbonate conc. & CO2 tension - achieve correct pH & osmolality
Natural media
- Based on: tissue extracts & body fluids
- Chick embryo extract
- serum
- lymph
Modified media
- Based on specific needs of cell lines
- without serum
- Contian NaHCO3, CO2
- Dulbecco's Modified Eagle Medium (DMEM)
- Roswell Park Memorial Institute (RPMI) 1640 medium
Types
- cultured at liquid-gas interface - gel, grid, raft
- favors retention of spherical or 3D shape
- retain differentiated properties of source tissue // retain specific histological interactions
- which do not grow rapidly
- cannot be propagated
- requires grater effort & provide poorer reproducibility of sample
- culture at plastic /glass - liquid interface
- migration is promoted, form outgrowth
- outgrowth from primary explants is disperse into a cell suspension
- propagation of cell lines becomes feasible - disaggregated tissue
- Cells form monolayer - monolayer suspension
- at solid-liquid interface
- cell suspension may disperse by enzymatic treatment & reseeded into fresh vessels
- 2 types:
- Anchorage dependent - monolayer culture
- Anchorage independent - suspension culture
Laboratory facilities'
Facilities
- Minimum
- Sterile area, clean, quiet, no through traffic
- Separate from animal house & microbiology labs
- Preparation area
- Wash up area
- Space for incubators
- Storage areas:
- Liquids & Chemicals - ambient & 4-20°C;
- chemicals in sealed container
- Glassware - shelving
- Plastics - shelving
- Small items - drawers
- Specialized equipment/slow turnover - cupboards
- CO2 cylinder
- Space fr liquid N2 freezer
- Sink
- Desirable
- Filtered air/air-conditioning
- Service bench adjacent to culture area
- Separate prep room
- Hot room with temperature recorder
- Separate sterilizing room
- Separate cylinder store
- Useful
- Piped CO2 & compressed air
- Storeroom for bulk plastics
- Quarantine room
- Containment room
- Liquid N2 storage tank & separate storeroom for N2 freezers
- Microscope room
- Darkroom
- Vacuum line
Equipments
- Basic
- Laminar-flow hood - biohazard if for human cells
- Incubator - humid CO2 incubator
- 5% CO2 cylinder - for gassing cultures
- Liquid CO2 cylinder, without siphon - CO2 incubator
- Balance
- Sterilizer - autoclave, pressure cooker
- Refrigerator
- Freezer - 20°C
- Inverted microscope
- Deep washing sink
- Soaking bath or sink
- Pipette cylinder
- Pipette washer
- Still or water purifier
- Bench centrifuge
- Liquid N2 freezer
- Liquid N2 storage dewer
- Slow-cooling device for cell freezing
- Magnetic stirrer racks for suspension culture
- Hemocytometer
- Beneficial
- Cell counter
- Peristaltic pump
- Pipettor
- pH meter
- Sterilizing oven
- Hot room
- Temperature recorders on sterilizing oven & autoclave in hot room
- Phase-contrast, fluorescence microscope
- Automatic dispenser
- Trolley or carts
- Drying oven - high & low temperature
- Roller racks - roller bottle culture
- Piped CO2 supply from cylinder store
- Automatic changeover dewvice on CO2 cylinders
- Additional
- Glassware washing machine
- Low temperature freezer - <70°C
- Conductivity meter
- Osmometer
- Polyethylene bag sealer - packaging sterile items for long-term storage
- Computer for freezer records and cell line database
- Colony counter
- High-capacity centrifuge
- Digital camera and monitor for inverted miscroscope
- Time-lapse video equipment
- Cell sizer - Scharfe, Coulter
- Portable temperature recorder for checking hot room or incubators
- Plastic shredder / sterilizer
- Controlled-rate cooler - cell freezing
- Fluorescence-activated cell sorter
- Confocal microscope
- Microtitration plate scintillation counter
- Centrifugal elutriator centrifuge and rotor
Layout
- Small scale
- Mid-scale
- Mid-scale with prep room
- Large scale
Applications
Basic
- Intracellular activity - DNA transcription, Protein synthesis, Energy metabolism, Drug metabolism, Cell cycle
- Intracellular flux - RNA processing, Hormone receptor, Metabolic flux, Calcium mobiliazation
- Genomic - Genetic analysis, Transfection, Infection, Transformation
- Proteomics - Gene products, Cell phenotype, Metabolic pathways
- Cell-cell interactions - Morphogenesis, Paracrine control, Cell proliferation, Cell adhesion & motility
Applied
- Cell products - Biotechnology, Bioreactor design, Product harvesting, Downstream processing
- Immunology - Cell surface epitope, Hybridomas, Cytokines & signalling, Informations
- Pharmacology - Drug action, Ligand receptor interactions, Drug metabolism, Drug resistant
- Tissue engineering - Tissue constructs, Matrices and scaffolds, Stem cell sources, Propagation, Differentiation
- Toxicology - Infection, Cytotoxicity, Mutagenesis, Carcinogenesis, Irritation, Inflammation
Advantages & Limitations
Advantages
- Physio-chemical environment: Control pH, temperature, osmolality, dissolved gas
- Physiological conditions: Control of hormone and nutrient conc.
- Microenvironment: Regulation of matrix, cell-cell interaction, gaseous diffusion
- Cell line homogeneity: Availability of selective media, cloning
- Characterization: Cytology and immunostanding are easily performed
- Preservation: Can be stored in liquid nitrogen
- Validation & Accreditation: Origin, history, purity can be authenticated and recorded
- Replicates & Variability: Quantitation is easy
- Reagent saving: Reduced volumes, direct access to cells, lower cost
- Control of conc. & time: Able to define dose, conc., time
- Mechanization: Available with microtitration and robotics
- Reduction of animal use: Cytotoxicity and screening of pharmaceutics, cosmetics
Limitation
- Necessity of expertise - Sterile handling, avoidance of chemical contamination, detection of microbial contamination, awareness and detection of mis-identification
- Environmental control - Isolation and cleanliness of workplace, incubation, pH control, containment and disposal of biohazards
- Quality & cost - Capital equipment of scale-up, medium, serum, disposable plastics
- Genetix instability - Heterogeneity, variability
- Phenotypic instability - Dedifferentiation, adaptation, selective overgrowth
- Identification of cell type - Markers not always expressed, histology is difficult to recreate and atypical, geometry and microenvironment changes cytology
Fundamental
Primary culture
- stage of culture where cells complete occupy the substrate
- cells are required to be subcultured (passage)
Cell line /sub-clone
- after the first subcultured
- derived from primary culture
- limited life span
- cells with highest growth capacity predominate
- resulting a genotypic & phenotypic uniform in the cell population
Cell strain
- positively selected specific subpopulation of cell line by cloning
- acquired additional genetic changes to the initiation of parent line
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Subculture // Passage
Washing :arrow_right: Trypsinization :arrow_right: Incubation :arrow_right: Re-suspension :arrow_right: Reseeding :arrow_right: Incubation
Examples cell lines:
- L929 [mouse L-cells] - first cloned cell strain isolated by capillary cloning - 1948
- HeLa [Henrietta Lacks] - first continuous human cell line - 1952
- human cervix's epithelial cells
- 3T3-A31 - mouse
- A549 - human lungs
- SP2/0 - mouse spleen, hybridoma B-lymphocytes
- Confluent monolayer growing in flask after ~1 week
- Medium removed and monolayer washed in D-PBSA (with or without EDTA)
- Trypsin added
- Trypsin removed leaving residual film
- Incubate at 37'C for 10mins
- Cells rounding up after incubation
- Cells resuspended in medium ready for counting and reseeding
- Cells reseeded in a flesh flask
- Incubate at 37'C
- Cells spreading after a few hours
- Incubate at 37'C for 7days