BIO 150
E1 | Cell Culture Basics: Plate, split, and freeze human cells
LABSTER
Cell culture is when scientists remove cells from an organism and grow
them in artificial environment, often a petri dish or other container in a
lab. This allows the scientist to perform experiments on the cells in a
controlled environment.
Q: What is one of the advantages of using cell cultures?
A: It’s an easy and highly cost-effective tool to study life sciences
- This is an excellent tool for researchers in their study of
different diseases in order to manufacture vaccines, select
drugs, improve gene therapy or understand disease
mechanisms. But it has many other applications, such as
producing recombinant proteins.
Q: Why is cell culture referred to as in vitro?
A: Because it’s a process performed outside a living organism
-In vitro refers to the study of cells or biomolecules outside
their normal biological context.
Q: For adherent cells, what does adhesion provide?
A: All options (A signal to grow and divide; A signal to enhance
survival; A signal to transduce differentiation signals)
- Adhesion initiates important signaling cascades that are
important for adherent cells. This is the main reason we use
special coated flasks for culturing cells.
First mission is to prepare the reagents.
When culturing cells, we have to provide them different essential
nutrients. Like we eat everyday, cells need to get enough energy to
keep growing.
These nutrients usually come from the basal culture medium and
serum. They are stored in the fridge-freezer to preserve the integrity of
their components.
The basal culture medium is a liquid that generally contains amino
acids, carbohydrates, (water-soluble) vitamins, and minerals. (+ trace
elements)
RPMI-1640 is widely used for suspension cells like lymphocytes. It can
look pink or colorless, depending on if it contains phenol red or not.
Conditioned medium (a special type of medium) is made from any
used medium that still has nutritional value, usually after being in
contact with cells. It has many applications, such as cell differentiation
induction, or delivery of engineered viruses and even therapeutical
agents.
DMEM (Dulbecco’s Modified Eagle Medium) — one of the most
used culture mediums to grow adherent cells. Besides its basic
components, it also has phenol red which acts as pH indicator and also
protects media from damage by light. (Medium to be used)
___ — This medium is formulated specifically for the growth of
pluripotent stem cells and provides essential nutrients that keep their
pluripotency intact. It also contains phenol red.
Q: Why is L-glutamine an important amino acid in cell culture?
A: Because cells get much of their energy from its catabolism
- L-glutamine is catabolized into a compound directly involved
in the Krebs cycle. Therefore, it helps cells produce most of
the energy they need to keep growing.
Q: What is the function of phenol red?
A: Indicate ph and protect from light
- Cells gro well in a pH around 7.4 and can tolerate little
variations but not drastic changes.
- Phenol red indicated pH by changing the medium color: it
turns yellow when pH decreases below 6.8 and it turns pink
when pH increases above 8.0
- This is a good way to keep track of the health of the cells.
Q: What is conditioned media useful for?
A: Eliciting cellular responses
- Conditioned media can help cells grow a bit faster.
- Besides providing the basic nutrients to cells, it also has
many other growth factors that were secreted by the cells
previously cultivated in this medium. THerefore, it can
promote the growth of cells that grow slower than needed.
Q: What are the optimal conditions to store cell culture media?
A: At 4 °C in the dark
- It’s important to store the culture media inside the fridge to
preserve their components as more stable as possible.
Otherwise, some of them could be degraded affecting the
cell growth.
Sometimes we need to add serum to the culture medium.
Serum, such as fetal bovine serum (FBS) usually comes from animal
fetuses because it has a high concentration of growth factors and
hormones that will stimulate the cells to grow and divide.
Q: What is one of the important components of the serum?
A: Growth factors
Q: What is the best way to store the serum?
A: In aliquots, frozen at -20 °C in a non-frost free freezer
- To avoid many freeze-thaw cycles and therefore ensure the
preservation of its components.
Q: Why do we have to pre-warm all the reagents we need to use at
37 °C?
A: To avoid thermal shock to cells
- We should leave the reagents in the water bath for up to 30
mins to warm up.
The airflow hood protects the working environment from dust and other
airborne contaminants by maintaining a constant, uni-directionsl flow of
filtered air over the work area.
Remember that every time you put something inside the hood it must
first be rinsed with 70% ethanol. This is to kill any contaminants that
might be sitting on the outside of the medium bottle from the fridge, the
water bath or the lab bench. Using aseptic technique is careful work.
Q: What is the atmosphere inside an air flow hood like?
A: Very clean but not absolutely sterile
- High Efficiency Particulate Air (HEPA) filters are responsible
for the clean atmosphere inside the hood, but it’s not 100%
sterile.
Q: How must the sash, the adjustable transparent cover, of the air
flow good be set?
A: At an indicated level when in use
Q: Which rule must you follow when working with animal cells in
an air flow hood?
A: Never open any item outside of the hood
- Work using aseptic technique to avoid any type of
contamination in your culture. This means that the opening
of any item must always happen inside the hood.
Q: What is the main characteristic of a vessel specifically
designed to culture adherent cells, such as fibroblasts?
A: The surface is specifically coated
- We can culture adherent cells in these flasks because one of
the polystyrene surfaces of the flask is chemically treated to
promote cell adhesion. Otherwise, these types of cells would
not survive.
STEP:
1. Remove 50 mL of medium from the DMEM bottle using the pipette
controller and serological pipettes.
2. Discard the medium into the disposal bottle and discard the
serological pipette in the trash bin.
3. Add 50 mL of serum to the DMEM culture medium bottle using a
new serological pipette.
