Cell Culture
Method that enable growth of eukaryotic or prokaryotic cells in physiological conditions.
Pros Cons
Reduced cell complexity Not representative for in vivo
Analysis of individual identified cell types Introduces expression of carcinogenic genes
Control cellular environment Can acquire mutations during subculturing
Manipulate gene expression in individual cell Loss of anatomic relations
Loss of synaptic pathways
Changes in gene expression
Bacterial cell culture
- Epidemiological studies
- Diagnose infections
- Genetic manipulation → pigments, antibiotics/drugs, biotensids, aroma additives
- Food and beverage production
- Detecting food contaminants
- Develop vaccines and therapeutics
Culture medium: formulation based on amino acids, vitamins, carbon sources, inorganic salts.
- Liquid
o bulk up culture
- Agar
o Select individual colonies from mixed culture
o Enumerate number of CFUs
o Transporting strains from lab to lab without danger
Removal of cells: Confluency:
- Chemically → trypsin → Adhesive cells
- Mechanically → Phosphate-Buffered Saline (PBS) → non-adhesive cells
E.coli → gram-negative, non-sporulating, rod-shaped, facultative anaerobic, and coliform.
Optimal growth at 37 degrees Celsius.
Bacterial transformation
Bacteria absorb DNA from environment resulting in new characteristics in bacteria.
1. Identify gene for a protein
2. Put gene into bacteria
3. Grow lots of bacteria
4. Bacteria transcribe and translate the protein
5. Purify the plasmid DNA or protein
1
,Transformation solution: Covers negative DNA
plasmid with positive Ca2+ to make DNA less polar.
Making cells competent:
Heat-Shock: treat with cations → heat-shock
Electroporation: wash with demi water → electric
shock
Plating:
- Antibiotic selection
- Culture overnight and determine
transformation efficiency
Transformed bacteria (with the plasmid) will
make beta-lactamase, which breaks down
ampicillin. This enables them to grow on
ampicillin plates.
Satellite colonies: non-transformed cells that surround a transformed colony after local
ampicillin centration drops due to the b-lactamase being secreted.
Store plasmids in Cryopreserve glycerol stock
- Restriction analysis/sequencing to confirm plasmid integrity
o Confirm size of plasmid DNA (correct plasmid?)
(A) → turbid, high metabolic
rates modify pH, changing color
of phenol red to yellow.
(B) → multicellular ovoid string-
like structures.
(C) → multicellular, highly
connected, thin filaments
(hyphae).
Virus → difficult to detect, may morphological changes in cells.
Mammalian cell culture
- Gene therapy Culture conditions:
- Model systems Gases, pH, pressure, and temperature
- Toxicity testing Suitable media
- Cancer research Effective growth factors
- Drug screening and Proper cell attachment substrates
development Temperature – 37 degrees Celsius for mammalian cells
- Virology/vaccine production
- Genetic engineering
- Genetic counselling
2
, Cell types
Primary cells → cells directly isolated from human tissue
- Stringent biosafety restriction associated
- Characterized as finite. Rely on continuous supply of stock since proliferation ceases
after limited amount of cell divisions.
Transformed cells → generated naturally or genetically.
- Manipulated genotype may result in karyotypic abnormalities and non-physiological
phenotypes.
Self-renewing cells → embryonic stem cells, IPSCs, neural and intestinal stem cells.
- Can differentiate into diversity of other cells, while self-renewing property allows for
long-term maintenance.
Immortalized cells → modified to bypass senescence and divide indefinitely in culture.
- They are not necessarily transformed (cancer-like) unless they exhibit uncontrolled
growth.
Culture types
Primary culture → directly isolated from parental tissue
- Limited lifespan
- Connot be passaged further
- Significant characteristics of parent tissue are available
Cell line → cells that have been modified or selected to grow indefinitely
- From primary culture
- Lifespan is better
- Can be moved to indefinite cultures
- Significant characteristics of parent tissues are lost
o Finite cell line → proliferate only define number, slow growth rate.
o Continuous cell line → proliferate indefinitely/immortalized, fast growth rate.
Transfection
Insert foreign nucleic acids into eukaryotic cells
1. Trypsin → detach cells
2. Collect & centrifuge
3. Resuspend & count
4. Plate
Stable transfection → integrates foreign DNA into host nuclear genome or maintains episomal
vector in host nucleus as extra-chromosomal element.
- Long-term genetic and pharmacological studies.
Transient transfection → does not require integrating nucleic acids into host cell genome.
- Short-term studies investigating effects of knock-in/-down of a gene.
3
Method that enable growth of eukaryotic or prokaryotic cells in physiological conditions.
Pros Cons
Reduced cell complexity Not representative for in vivo
Analysis of individual identified cell types Introduces expression of carcinogenic genes
Control cellular environment Can acquire mutations during subculturing
Manipulate gene expression in individual cell Loss of anatomic relations
Loss of synaptic pathways
Changes in gene expression
Bacterial cell culture
- Epidemiological studies
- Diagnose infections
- Genetic manipulation → pigments, antibiotics/drugs, biotensids, aroma additives
- Food and beverage production
- Detecting food contaminants
- Develop vaccines and therapeutics
Culture medium: formulation based on amino acids, vitamins, carbon sources, inorganic salts.
- Liquid
o bulk up culture
- Agar
o Select individual colonies from mixed culture
o Enumerate number of CFUs
o Transporting strains from lab to lab without danger
Removal of cells: Confluency:
- Chemically → trypsin → Adhesive cells
- Mechanically → Phosphate-Buffered Saline (PBS) → non-adhesive cells
E.coli → gram-negative, non-sporulating, rod-shaped, facultative anaerobic, and coliform.
Optimal growth at 37 degrees Celsius.
Bacterial transformation
Bacteria absorb DNA from environment resulting in new characteristics in bacteria.
1. Identify gene for a protein
2. Put gene into bacteria
3. Grow lots of bacteria
4. Bacteria transcribe and translate the protein
5. Purify the plasmid DNA or protein
1
,Transformation solution: Covers negative DNA
plasmid with positive Ca2+ to make DNA less polar.
Making cells competent:
Heat-Shock: treat with cations → heat-shock
Electroporation: wash with demi water → electric
shock
Plating:
- Antibiotic selection
- Culture overnight and determine
transformation efficiency
Transformed bacteria (with the plasmid) will
make beta-lactamase, which breaks down
ampicillin. This enables them to grow on
ampicillin plates.
Satellite colonies: non-transformed cells that surround a transformed colony after local
ampicillin centration drops due to the b-lactamase being secreted.
Store plasmids in Cryopreserve glycerol stock
- Restriction analysis/sequencing to confirm plasmid integrity
o Confirm size of plasmid DNA (correct plasmid?)
(A) → turbid, high metabolic
rates modify pH, changing color
of phenol red to yellow.
(B) → multicellular ovoid string-
like structures.
(C) → multicellular, highly
connected, thin filaments
(hyphae).
Virus → difficult to detect, may morphological changes in cells.
Mammalian cell culture
- Gene therapy Culture conditions:
- Model systems Gases, pH, pressure, and temperature
- Toxicity testing Suitable media
- Cancer research Effective growth factors
- Drug screening and Proper cell attachment substrates
development Temperature – 37 degrees Celsius for mammalian cells
- Virology/vaccine production
- Genetic engineering
- Genetic counselling
2
, Cell types
Primary cells → cells directly isolated from human tissue
- Stringent biosafety restriction associated
- Characterized as finite. Rely on continuous supply of stock since proliferation ceases
after limited amount of cell divisions.
Transformed cells → generated naturally or genetically.
- Manipulated genotype may result in karyotypic abnormalities and non-physiological
phenotypes.
Self-renewing cells → embryonic stem cells, IPSCs, neural and intestinal stem cells.
- Can differentiate into diversity of other cells, while self-renewing property allows for
long-term maintenance.
Immortalized cells → modified to bypass senescence and divide indefinitely in culture.
- They are not necessarily transformed (cancer-like) unless they exhibit uncontrolled
growth.
Culture types
Primary culture → directly isolated from parental tissue
- Limited lifespan
- Connot be passaged further
- Significant characteristics of parent tissue are available
Cell line → cells that have been modified or selected to grow indefinitely
- From primary culture
- Lifespan is better
- Can be moved to indefinite cultures
- Significant characteristics of parent tissues are lost
o Finite cell line → proliferate only define number, slow growth rate.
o Continuous cell line → proliferate indefinitely/immortalized, fast growth rate.
Transfection
Insert foreign nucleic acids into eukaryotic cells
1. Trypsin → detach cells
2. Collect & centrifuge
3. Resuspend & count
4. Plate
Stable transfection → integrates foreign DNA into host nuclear genome or maintains episomal
vector in host nucleus as extra-chromosomal element.
- Long-term genetic and pharmacological studies.
Transient transfection → does not require integrating nucleic acids into host cell genome.
- Short-term studies investigating effects of knock-in/-down of a gene.
3
