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Summary Summar Human Cells in Culture (XM_0115)
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This document consists of a summary of the course Human Cells in Culture, corresponding to the Master Drug Discovery Sciences.
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Lecture 1. Cell culture equipment and mediumLearning goals:
o To get to know the beginnings of (human) cell culture and understand their role in research
today
o To get familiar with the standard equipment used in cell culture
o To understand the function of each cell culture medium component
o To recognize the limitation of FBS and to learn more about serum free media
Origin of cell and tissue culture
In 1885, zoologist wilhelm roux maintained embryonic chicken cells in warm saline solution. In the early
1900s the origin of the long term culture started with the frog embryo nerve fiber in 1907 by Harrison
and the chick embryo fragments in 1910 (Burrows). There was a huge improvement by immortalization
of the first continues cell line, the mouse fibroblast cell line L-929, cultured by Wilton Earle in 1941 in a
mixture of horse serum, embryonic chick extract and Earle’s balanced salt solution (EBSS). George Grey
developed the first human immortalized cell line HeLa in 1952. HeLa cells come from the donator
Henrietta Lacks. They are the first immortal human cells obtained from a cervical carcinoma. They have
been stored for more than 70 years.
Applications of cell culture
Cell culture was first used to develop vaccines for the polio virus in the 1930s. Later WI-31 cells and Vero
cells were used to cultivate many viruses, including the H5N1 influenza virus. The AstraZeneca Covid19
vaccine was produced using HEK293 cells. Cell culture is also used for the production of hormones (e.g.
insulin) antibodies and other biologicals.
Cell cultures are very useful for the replacement of in vivo models, for example the use of ovarian cancer
cells in the development of cisplatin. These cell cultures make it possible to work with human material.
This is also very useful for ethical reasons (reduction, replacement, refinement of animals (3R)). As
Russell and Burch described it in 1959: “Mammalian tissue cultures have become one of the most
important replacement techniques, and indeed one of the most important developments in biology”. At
the moment animal models cannot be replaced fully, but a lot of tests can already be replaced and this
will be more in the future.
Potential limitations of cell culture
An in vitro model is just as good as the cells that are used and the most used cells are cancer cell lines.
In cell culture there is often a lack of training of users and a lack of following the Good Cell Culture
Practice (GCCP). In addition, cell culture is not always cheap.
Some in vitro challenges are (1) the limited understanding of cell and environment, because of the de-
differentiation of cells outside the body, (2) the lack of cell interactions in mono-cultures, and (3) the lack
of immune response, due to the fact that there are often no immune cells in in vitro systems.
Origin and evolution of cells
Many cell lines are derived from cancer biopsies, just like the first human cells from cancer biopsies
(HeLa). Besides cancer cell lines there are also primary cells with a normal karyotype.
Cell culture is a selective process that favours expansion of some cell over others. Trypsinisation and
subculturing may induce changes over time. Usually by the third passage the culture becomes more
stable. HeLa cells and MCF-7 cells change over time in culture and multiple sub-cones exist containing
differences in copy number variations (CNVs) and also major chromosomal aberrations.
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,Primary cells stop dividing after a while. Finite cell lines have a defined number of population doublings
due to senescence/hayflick limit, which is the limit on cell replication imposed by shortening of
telomeres with each division (senescence is the end stage).
Laboratory needs for cell culture
To perform cell culture, there as a need to work asepsic to prevent contaminations. The cells need to be
contained to prevent exposure of workers or environment from hazard. This containment is done by
using laminal flow cabinets and biological safety cabinets (BSC). These cabinets are used to reduce traffic
to avoid dust, spores etc.
There are two different laminar flow cabinets, a horizontal
laminal flow or a vertical laminar flow. The principle of
both is the same, room air is collected and filtered by a
primary filter. Fans are used to circulate the air and before
the air flows out, it is filtered by the HEPA filter. The
difference between the two laminar flows is that in the
horizontal laminar flow, the room air is collected at the
top and the filtered air flows out horizontally, and in the
vertical laminar flow the room air is collected at the
bottom and the filtered air flows out from the top
(vertical). In addition, the vertical laminar flow cabinet contains a class screen.
There are different classes of BSC. The
first class is not suitable for cell culture.
The most common BSC is the class II A2.
The class II B2 BSC is used for hazardous
chemicals. In BSC class II A2 air flows in and passes the
primary filter. When air flows out back into the cabinet it
passes the HEPA recirculating filter and when air flows
out to the outside it passes the HEPA exhaust filter and
the optional pathogen trap. In BSC class II B2 air flows in
and passes the primary filter. When air flows out back
into the cabinet it passes the HEPA recirculating filter and
when air flows out to the outside it passes the HEPA
exhaust filter and the charcoal filter and/or the pathogen
trap.
Other standard equipment for cell cultures are incubators to create a humid environment and regulate
gas levels, sterile plastic ware (dishes, pipettes), vacuum pump system, centrifuge, inverted
microscope, water bath, and an autoclave to sterilize things).
Incubators for cell culture
Incubators are used to mimic the human body which is between 36.5 and 37 degrees Celsius. There are
two basic types of incubators, dry incubators and humic 𝐶𝑂2 incubators. Dry incubators are more
economical, but require the cell cultures to be incubated in sealed flasks to prevent evaporation. Placing
a water dish in a dry incubator can provide some humidity, but they do not allow precise control of
atmospheric conditions in the incubator. Humid 𝐶𝑂2 incubators are more expensive, but allow superior
control of culture conditions. They can be used to incubate cells cultured in Petri dishes or multiwell
plates, which require a controlled atmosphere of high humidity and increased 𝐶𝑂2 tension. This
incubator contains a sensor which detects gas levels and injects automatically more 𝐶𝑂2 when needed.
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,Biosafety levels
Different biosafety levels
Risk group 1 No or low individual and
community risk
Risk group 2 Moderate individual and
low community risk
Risk group 3 High individual and low
community risk, e.g.
working with HIV
Risk group 4 High individual and high
community risk, e.g.
working with Ebola
In different countries there are different laws on what counts as GMO and how waste needs to be
disposed.
Cell culture medium
Minimum requirements for medium were developed by Albert Fischer, Harry Eagle. Traditionally the
medium is not very well defined. Medium contains serum (calf, horse, human), amino acids, vitamins,
inorganic salts and glucose. General medium is not optimized for all cell lines and adaptions for more
differentiated cell lines are required.
pH
Most cells grow at a pH of 7.4 and diminished cell growth occurs above a pH of 7.7 and below a pH of
6.5. In the human body, sodium bicarbonate (𝑁𝑎𝐻𝐶𝑂3 ) and 𝐶𝑂2 make up the pH and this pH is very
specific. The kidney (regulates bicarbonate) and the lung (regulates 𝐶𝑂2 ) regulate the acid-base in the
human body. In cell culture there is no lung and kidney and this is why the incubator contains 𝐶𝑂2 . When
transformed cells grow beyond the confluence, lactate is released from glucose which lowers the pH.
This causes exhausting of the nutrient and growth factor and the accumulation of toxic metabolites.
Phenol red is used in cell culture as an indicator of the pH. When phenol red changes from pink to yellow
it means that the culture is more acidic, probably due to an increase in lactate secretion. Typically
cultures are fed and passaged before this happens.
pH buffering
instead of sodium bicarbonate, HEPES can be used. In cell culture, normally 5% of 𝐶𝑂2 us dissolved in
culture medium which establishes an equilibrium with 𝑁𝑎𝐻𝐶𝑂− . The higher the 𝐶𝑂2 the more
bicarbonate is required.
Oxygen
In the atmosphere there is 21% oxygen, but this is never that high in
tissues in vivo (2-9% depending on the distance to oxygenated blood.
Cells are never too far away from the capillaries, because the 𝑂2
gradient decreases the further the cells are away from the capillaries,
which results in hypoxic or even necrotic cells. Carriers are important to
balance the correct oxygen tension. In the absence of carrier (e.g.
hemoglobin) reactive oxygen species may arise. Oxygen toxicity may be
reduced by adding betamercaptoethanol or glutathione (selenium is a
cofactor in glutathione synthesis). Many transformed cells become more glycolytic meaning that they
do not require high amounts of oxygen. Serum will improve the oxygen tolerance. Sometimes, cells are
on purpose grown in hypoxic conditions, in which the oxygen is below 2%.
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, Temperature
On average the optimal temperature in humans is 36.7 °C but this varies depending on the animal
species. Cells can stay alive several days at lower temperatures (4°C) but cannot tolerate more than 2°C
higher temperature for more than few hours and will die rapidly above 40°C. Under certain conditions,
cells can be frozen.
Osmolarity
Osmolarity is the amount of particles per kg of solved,
expressed as mOsm/kg. The human plasma has on osmolarity
of 290 mOsm/kg. In practice an osmolarity between 260 and
320 mOsm/kg is acceptable for most cell lines. Changes in this
osmolarity are usually obtained by changing the NaCl
concentration. It is important to understand that the addition of
drugs dissolved in strong acids and bases may affect the
osmolarity. When the environment of the cell is hypotonic, it
means that the osmolarity is too low, resulting in a swelling of
the cell. When the environment of the cell is hypertonic, it
means that the osmolarity is too high, resulting in the shrinking
of the cell.
Medium
Medium is a base with a balanced salt solution to imitate the natural cellular environment. There are
different mediums present, (1) medium 199, made by Morgan in 1950, (2) Eagle’s Minimal Essential
Medium (MEM) made in 1959, (3) DMEM, made by Dulbecco and Freeman in 1959, containing the
double amount of amino acids and four times the vitamins concentration compared to MEM, (4) Ham’s
F12, made by Ham in 1965 and was developed for low or serum free cultures, and (5) RPMI, made by
Moore in 1976 which was a modified suspension culture medium, but is nowadays also used for
adherent cultures.
The medium contains (1) amino acids, both essential like arginine, cysteine, glutamine, and tyrosine, but
also non-essential (NEAA) if for example cell types cannot synthesize them. The essential amino acid
glutamine, required by most cell cultures for energy and as carbon source, is unstable at 37 °C. GlutaMAX
(alanyl-glutamine dipeptide) is more stable and bioavailable due to the dipeptidases. (2) Vitamins are
also present in the medium. (3) Lastly inorganic salts, like 𝑁𝑎+ , 𝐾 + , 𝑀𝑔2+ , 𝐶𝑎2+ , 𝐶𝑙 − , 𝑆𝑂42−, 𝑃𝑂43−,
and 𝐻𝐶𝑂3−, are present in the medium, which contribute to the osmolarity.
Glucose
In cells there is between 4 and 20 mM glucose present depending on the cell
type. 5mM glucose is needed for normal cells, and this is higher for cancer or
iPSC cells. In physiological conditions, when the amount of glucose is above 10
mM, the SGLT2 is exhausted, resulting in diabetic conditions. Glucose is used
as energy source and converted to pyruvate by glycolysis, that may be
converted to lactate in hypoxic conditions.
Antibiotics
The most common antibiotics used is penicillin/streptomycin (pen/strep). The use of antibiotics is
optimal but not always necessary, however, it is recommended for primary cells. Antibiotics often result
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