Cell culture and its applications in biomedical sciences
Learning goals
- Understand fundamentals of prokaryotic and eukaryotic cell culture
- Describe genetic manipulation methods
- Recognize biomedical applications and assays
- Recognize ethical considerations
Introduction to cell culture
Principles of cell culture
Cell culture involves growing cells under controlled in vitro conditions that mimic their natural
environment. Key principles include aseptic technique, regular monitoring of cell morphology and
growth, and subculturing (cells need to have room again to grow). Proper maintenance ensures
healthy, reproducible cells for biological assays and experimental studies.
What is cell culture?
- Maintenance and growth of cells in controlled
laboratory environments
- Allows in vitro study of biological mechanisms
- Cells are isolated, not in a tissue -> allows you to
look at biological mechanisms
- Ex vivo cells are in their environment, but of of the animal
Why cell culture matters
- Support drug discovery and toxicity testing
- Enables cancer and disease modeling
- Powers regenerative medicine and tissue engineering
- Alternative to the use of animals -> animal free models
- Allow the use of human material -> metabolic pathways differ between species
Fundamentals of cell culture
Culture environment
- Temperature (37°C typical for mammalian cells)
- CO2 levels regulate pH
- Humidity and oxygen influence metabolism
- There are even incubators that can induce hypoxia
Aseptic techniques and biosafety
- Biosafety cabinets, sterilization, PPE use -> everything under the hood is clean, clean
everything that you bring from outside to put under the hood with ethanol. These hoods
have HEPA filters to be sure that the air stays clean and that nothing comes out.
- Contamination control (bacteria, fungi, mycoplasma) -> need to check regularly for
unwanted cells, not only in your culture but also in the hood and incubator
The lab user needs to have good knowledge of the SOP, and respect the work, also from others. You
always have to wear a lab coat, not only to protect the cells but also to protect yourself. The ritual
you need to follow when you enter the lab is: 1) lab coat, 2) washing hands, 3) ethanol
decontamination, 4) gloves. And as any other lab you are not allowed to eat or drink but you are also
not allows to bring electronical devices.
1
,Prokaryotic vs eukaryotic systems
The prokaryotic cells are easier to
control, it doesn’t need that much
attention. The prokaryotic cells
also grow in suspension, so there
is no adhesion. The eukaryotic
cells are much more difficult to
control the environment, and
they grow a lot slower than
bacteria.
Bacterial culture & genetic manipulation
Bacterial growth basics
There are 4 phases in bacterial/cellular growth: lag, log,
stationary, and death phase.
- Lag phase: there is no increase in the number of cells.
- Log phase: there is enough medium for the cells to
grow so they will grow exponentially until the nutrients
run out.
- Stationary phase: the cells reach a plateau, the division
of the cells is equal to the amount of dead cells.
- Death phase: there are not enough nutrients to keep
the cells alive, so they will start dying very fast .
When you are culturing bacteria, you want them to be in the
log phase.
Genetic modification methods
- Transformation – uptake of plasmid DNA
- Conjugation – cell-to-cell plasmid exchange
- Transduction – bacteriophage-mediated DNA transfer
Transformation
- Bacteria take up foreign DNA from their
environment
- Occurs naturally or can be induced
artificially
- Leads to new genetic makeup/altered
trait
- Key technique in molecular biology (e.g.,
cloning)
2
,Conjugation
- Horizontal gene transfer
- Donor bacteria transfer genetic material (plasmid) to recipient
bacteria
- Donor has a fertility factor, forms a pilus and connects to
recipient
- Relaxosome to nick the DNA, transferosome to induce transfer
Transduction
It is the process of
horizontal gene transfer in
bacteria where genetic
material is moved from one
bacterium to another by a bacteriophage. The
bacteriophage acts as a vehicle to transfer a piece of
bacterial DNA to a new host cell.
- Through the infection, machinery is controlled by the bacteriophage
- Host DNA broke into fragments
- Bacteriophage mistakenly packs bacterial DNA
- Injects the bacterial DNA into another bacterium
Applications in biomedical sciences from the transduction, transfection and conjugation can be
recombinant insulin production in E.coli, plasmid vectors for mammalian transfection, or CRISPR
plasmid cloning and propagation
Eukaryotic cell culture techniques
Primary vs immortalized cells
Primary cells are derived
directly from tissue and have a
limited lifespan.
Immortalized cell lines have
continuous growth, and are
convenient for experiments.
Immortalized cell lines
Cell lines that have acquired the ability to proliferate indefinitely under in vitro conditions. Bypass
normal senescence and apoptosis mechanisms.
- Spontaneous: occurs naturally
(e.g. tumor-derived lines like HeLa)
- Induced: achieved by genetic
manipulation, e.g.
o Viral oncogenes (SV40 T-
antigen, HPV E6/E7)
o Telomerase activation
(hTERT expression)
o Inactivation of tumor
suppressors (p53, Rb)
3
, Primary cells
Cells obtained directly from tissues or
organs and cultured for the first time in
vitro. Retain morphological and functional
characteristics of their tissue of origin.
If your study passes in the immortalized cell
line, you can move on to primary cells to see how it will look like in the actual biological environment
-> gives you a good idea of how your study will look in vivo.
Isolation methods:
- Enzymatic digestion: trypsin, collagenase, or dispase: break down ECM and release individual
cells
- Mechanical disaggregation: homogenization of tissue to separate cells without enzymes (can
induce a lot of death, so be careful)
- Explant culture: small tissue fragments placed on a culture surface; cells migrate out and
proliferate
Stem cells
- Self-renewing, pluripotent cells
- Can differentiate into multiple lineages
- Enable modeling and regenerative medicine
When you receive stem cells you have to validate them -> force
them to differentiate in the 3 cells they should differentiate into
(bone, fat, cartilage) -> if 1 cell line is not differentiating in one of
them we will discard them, they are not doing what they are
supposed to and we cannot use them.
Induced-pluripotent stem cells (iPSC)
- Self-renewing, pluripotent cells
- Can differentiate into multiple lineages
- Enable modeling and regenerative medicine
The capacity to differentiate is way higher -> they are not
normal stem cells. We can take skin cells (fibroblasts) and
force them to be stem cells and than force them to express
biomarkers for stem cells -> these cells are GMO, we are
programming them by entering a plasmid. These cells have
the potential to become everything you want -> just give
them the right information to differentiate -> often subjected
to growing teratoma, not easy to keep under control.
3D cultures and organoids
- 3D systems mimic tissue architecture
- Organoids: miniature functional models (brain, kidney, gut)
4
Learning goals
- Understand fundamentals of prokaryotic and eukaryotic cell culture
- Describe genetic manipulation methods
- Recognize biomedical applications and assays
- Recognize ethical considerations
Introduction to cell culture
Principles of cell culture
Cell culture involves growing cells under controlled in vitro conditions that mimic their natural
environment. Key principles include aseptic technique, regular monitoring of cell morphology and
growth, and subculturing (cells need to have room again to grow). Proper maintenance ensures
healthy, reproducible cells for biological assays and experimental studies.
What is cell culture?
- Maintenance and growth of cells in controlled
laboratory environments
- Allows in vitro study of biological mechanisms
- Cells are isolated, not in a tissue -> allows you to
look at biological mechanisms
- Ex vivo cells are in their environment, but of of the animal
Why cell culture matters
- Support drug discovery and toxicity testing
- Enables cancer and disease modeling
- Powers regenerative medicine and tissue engineering
- Alternative to the use of animals -> animal free models
- Allow the use of human material -> metabolic pathways differ between species
Fundamentals of cell culture
Culture environment
- Temperature (37°C typical for mammalian cells)
- CO2 levels regulate pH
- Humidity and oxygen influence metabolism
- There are even incubators that can induce hypoxia
Aseptic techniques and biosafety
- Biosafety cabinets, sterilization, PPE use -> everything under the hood is clean, clean
everything that you bring from outside to put under the hood with ethanol. These hoods
have HEPA filters to be sure that the air stays clean and that nothing comes out.
- Contamination control (bacteria, fungi, mycoplasma) -> need to check regularly for
unwanted cells, not only in your culture but also in the hood and incubator
The lab user needs to have good knowledge of the SOP, and respect the work, also from others. You
always have to wear a lab coat, not only to protect the cells but also to protect yourself. The ritual
you need to follow when you enter the lab is: 1) lab coat, 2) washing hands, 3) ethanol
decontamination, 4) gloves. And as any other lab you are not allowed to eat or drink but you are also
not allows to bring electronical devices.
1
,Prokaryotic vs eukaryotic systems
The prokaryotic cells are easier to
control, it doesn’t need that much
attention. The prokaryotic cells
also grow in suspension, so there
is no adhesion. The eukaryotic
cells are much more difficult to
control the environment, and
they grow a lot slower than
bacteria.
Bacterial culture & genetic manipulation
Bacterial growth basics
There are 4 phases in bacterial/cellular growth: lag, log,
stationary, and death phase.
- Lag phase: there is no increase in the number of cells.
- Log phase: there is enough medium for the cells to
grow so they will grow exponentially until the nutrients
run out.
- Stationary phase: the cells reach a plateau, the division
of the cells is equal to the amount of dead cells.
- Death phase: there are not enough nutrients to keep
the cells alive, so they will start dying very fast .
When you are culturing bacteria, you want them to be in the
log phase.
Genetic modification methods
- Transformation – uptake of plasmid DNA
- Conjugation – cell-to-cell plasmid exchange
- Transduction – bacteriophage-mediated DNA transfer
Transformation
- Bacteria take up foreign DNA from their
environment
- Occurs naturally or can be induced
artificially
- Leads to new genetic makeup/altered
trait
- Key technique in molecular biology (e.g.,
cloning)
2
,Conjugation
- Horizontal gene transfer
- Donor bacteria transfer genetic material (plasmid) to recipient
bacteria
- Donor has a fertility factor, forms a pilus and connects to
recipient
- Relaxosome to nick the DNA, transferosome to induce transfer
Transduction
It is the process of
horizontal gene transfer in
bacteria where genetic
material is moved from one
bacterium to another by a bacteriophage. The
bacteriophage acts as a vehicle to transfer a piece of
bacterial DNA to a new host cell.
- Through the infection, machinery is controlled by the bacteriophage
- Host DNA broke into fragments
- Bacteriophage mistakenly packs bacterial DNA
- Injects the bacterial DNA into another bacterium
Applications in biomedical sciences from the transduction, transfection and conjugation can be
recombinant insulin production in E.coli, plasmid vectors for mammalian transfection, or CRISPR
plasmid cloning and propagation
Eukaryotic cell culture techniques
Primary vs immortalized cells
Primary cells are derived
directly from tissue and have a
limited lifespan.
Immortalized cell lines have
continuous growth, and are
convenient for experiments.
Immortalized cell lines
Cell lines that have acquired the ability to proliferate indefinitely under in vitro conditions. Bypass
normal senescence and apoptosis mechanisms.
- Spontaneous: occurs naturally
(e.g. tumor-derived lines like HeLa)
- Induced: achieved by genetic
manipulation, e.g.
o Viral oncogenes (SV40 T-
antigen, HPV E6/E7)
o Telomerase activation
(hTERT expression)
o Inactivation of tumor
suppressors (p53, Rb)
3
, Primary cells
Cells obtained directly from tissues or
organs and cultured for the first time in
vitro. Retain morphological and functional
characteristics of their tissue of origin.
If your study passes in the immortalized cell
line, you can move on to primary cells to see how it will look like in the actual biological environment
-> gives you a good idea of how your study will look in vivo.
Isolation methods:
- Enzymatic digestion: trypsin, collagenase, or dispase: break down ECM and release individual
cells
- Mechanical disaggregation: homogenization of tissue to separate cells without enzymes (can
induce a lot of death, so be careful)
- Explant culture: small tissue fragments placed on a culture surface; cells migrate out and
proliferate
Stem cells
- Self-renewing, pluripotent cells
- Can differentiate into multiple lineages
- Enable modeling and regenerative medicine
When you receive stem cells you have to validate them -> force
them to differentiate in the 3 cells they should differentiate into
(bone, fat, cartilage) -> if 1 cell line is not differentiating in one of
them we will discard them, they are not doing what they are
supposed to and we cannot use them.
Induced-pluripotent stem cells (iPSC)
- Self-renewing, pluripotent cells
- Can differentiate into multiple lineages
- Enable modeling and regenerative medicine
The capacity to differentiate is way higher -> they are not
normal stem cells. We can take skin cells (fibroblasts) and
force them to be stem cells and than force them to express
biomarkers for stem cells -> these cells are GMO, we are
programming them by entering a plasmid. These cells have
the potential to become everything you want -> just give
them the right information to differentiate -> often subjected
to growing teratoma, not easy to keep under control.
3D cultures and organoids
- 3D systems mimic tissue architecture
- Organoids: miniature functional models (brain, kidney, gut)
4
