Chap. 6 Culture of Animal Cells1
Introduction

Tissue culture (組織培養): started in early 20th century, indicates the culture of 3D tissue
or tissue fragments, so this name has remained since.

Cell culture: culture of dispersed cells.
I. Biology of cultured cells
The culture environment

The cultured cells do not possess exactly identical properties characteristic of the same
cell type in vivo because the cellular environment has changed. Cell-cell and cell-matrix
interactions are reduced, thus favoring the spreading, migration and proliferation of the
cultured cells.

Four routes of influences:

Nature of substrate on which the cells are grown.

The composition and physico-chemical properties (pH, osmotic pressure) of the
medium.

The composition of gas

The incubation temperature
Cell adhesion

Most cells from solid tissues need to attach before they can proliferate, and grow as
adherent monolayers (unless they are transformed2 and become anchorage independent).

Cell adhesion is mediated by specific cell surface molecules for interactions with the
ECM molecules.

Cell adhesion molecules:
1
Freshney, RI. (2005) Culture of animal cells: a manual of basic technique. Wiley-Liss, New York
2
The term, transformation, is used very loosely, here transformation refers to an alteration in characteristics (anchorage
independence, loss of contact inhibition).
1

Cell-cell adhesion molecules (CAM, Ca2+-independent), cadherins (Ca2+-dependent):
these proteins interact with each other and connect neighboring cells.

Integrins: responsible for the interaction with ECM molecules (e.g. fibronectin,
entactin, laminin and collagen). Many ECM proteins contain RGD (Arg-Gly-Asp)
motifs, which promotes the binding with integrin and hence cell adhesion.

Proteoglycans: interact with matrix constituents such as other proteoglycans or
collagen, but not via the RGD motif.
Cell proliferation
Cell cycle

Gap 1 (G1) phase: the cell
either progresses towards DNA
synthesis and another cell
division cycle, or exits the cell
cycle reversibly (G0), or
commits to differentiation (分
化) irreversibly. During this
phase, cell cycle is controlled
to determine whether the cells
re-enter the cycle, withdraw, or
2
differentiate.

S phase (DNA synthesis): DNA replicates. The checkpoint between the S and G2 phases
checks the accuracy of DNA synthesis. If errors occur in DNA synthesis, the cell will halt
the cycle to allow DNA repair or entry into apoptosis (凋亡, if repair is unsuccessful).

G2 phase: cells prepare for re-entry into M phase.

Mitosis (M) phase: the chromatins condense into chromosomes, and the two sets of sister
chromatids segregate to each daughter cell.
Control of cell proliferation

Entry into the cell cycle is regulated by extracellular mitogenic growth factors (GF) such
as epidermal GF, FGF or platelet-derived GF (PDGF). The GF binding to the cell
membrane receptors initiates signal transduction pathways, often involving protein
phosphorylation and secondary messengers such as cyclic adenosine monophosphate
(cAMP), or Ca2+.

Intracellular control is mediated by positive-acting factors, such as cyclins (upregulated
by signal transduction cascades) or negative-acting factors such as p53 (blocks cell cycle
progression at checkpoints).

High cell density (cells/cm2) inhibits the proliferation of normal cells (contact inhibition).
Differentiation and dedifferentiation

Differentiation: the development of special properties that a cell would have expressed in
vivo (e.g. neural stem cells can differentiate into neurons).

Dedifferentiation: the loss of the characteristic properties of the cells (e.g.
dedifferentiated hepatocytes could lose their characteristic enzyme arginase and could not
store glycogen or secrete serum proteins). Usually occurs during 2D culture.

During cell culture, the differentiated properties are often limited by the promotion of
cell proliferation, which is necessary for cell propagation. So if cells are isolated from
tissues and differentiation is required, generally two sets of conditions are used in
series—one to optimize cell proliferation and one to optimize cell differentiation.

Serial passage at relatively low seeding cell density (promote cell proliferation and
constrain differentiation).
3

After sufficient cells are obtained, culture the cells at high seeding cell density using
medium containing serum and appropriate hormones (to promote differentiation).

Maintenance of differentiation can be aided by artificial matrices (e.g. cellulose) or other
natural tissue matrix glycoproteins (e.g. fibronectin).

Commercial products (e.g. Matrigel), reproduce the characteristics of ECM. Matrigel contains laminin,
entacin proteoglycan, growth factors and other undefined components.
It is a liquid at 4C, but
polymerizes into gel at 37C. It can enhance the cell proliferation and differentiation in vitro.
Evolution of cell lines

After the first passage (or subculture), the
primary culture becomes known as a cell
line and may be propagated a number of
times.

With each passage, the cells with higher
proliferating ability gradually predominate,
while slowly proliferating cells will be
diluted out. By the 3rd passage, the culture
becomes more stable.

Normal cells can divide only a number of times and then will die out a phenomenon
known as senescence. Senescence is attributed to the lack of telomerase (responsible for
replicating the telomere sequence), which results in
shortened telomeres and stop of cell proliferation.

Exceptions to this are germ cells, some stem cells and
transformed cells which can express telomerase, hence
these cells can proliferate indefinitely and evolve to
become continuous cell lines.
The development of continuous cell lines

Some cell lines may give rise to continuous cell lines due
to genetic variation (in vitro transformation).
Transformation may occur spontaneously or be induced
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chemically or virally. This often involves the deletion or mutation of the p53 gene (which
would normally arrest cell cycle progression).

For continuous cell lines, there is usually considerable variation in the chromosome
number among cells in the population (heteroploidy, 異倍性).
II. Equipment

Laminar flow hood is essential for aseptic operations and should be: (1) Large enough (at
least 120 cm (W)60 cm (Depth)); (2) Quiet (noisy hoods are more fatiguing); (3) easily
cleaned and comfortable to sit.

Two types of laminar flow hoods

Horizontal flow: give the most stable airflow and
best sterile protection.

Vertical flow: gives more protection to the operator
(particularly for handling biohazardous materials).
The air passes through the HEPA filter to ensure the
sterility.
5

CO2 Incubator:

Should be large enough and have forced air circulation, temperature control to
within 0.2C and a safety thermostat that cuts off if the incubator overheats.

Many incubators have a water jacket to distribute heat evenly around the cabinet.
III.Culture vessels

Polystyrene flasks have been commonly used in the labs. Polystyrene is hydrophobic and
is not suitable for cell growth. These vessels are treated by -irradiation, or with an
electric ion discharge to produce a wettable, charged surface.
6

For monolayer cultures, the cell yield is proportional to the surface area. If the cell yield
required is too large, multilayer flasks may increase the surface areas. Roller bottles are
an alternative option.
7
Left: Roller bottles. Right: Multichamber flasks (Cell Factory, http://www.nuncbrand.com/en/frame.aspx?ID=10781)
A Cell Factory is a stack of chambers sealed together into a single unit, sharing common vent and fill ports.
Each chamber has a flat growth surface. Cell Factory provides a large amount of growth surface in a small area
with easy handling and low risk of contamination. A 40 chamber unit with a growth area of 25,280 cm2
corresponds to 14 large roller bottles (1750 cm2 each).

Anchorage-independent cells can be grown in suspension in any type of flask or plate.

Spinner flasks are common to culture suspended cells whose rotation is driven by a
magnetic stirrer.

The rotational speed must be kept low (<100 rpm) to avoid damage from shear
stress.

Since CO2 and O2 are needed for the cells, the caps are loosened one full turn for
gas exchange. Some flasks are equipped with gas-permeable cap.

Cell attachment and growth can be improved by:

Treatment with denatured ECM molecules (chondronectin enhances chondrocyte adherence and laminin
promotes the adherence of epithelial cells).

Commercial matrices include Matrigel (that contains laminin, fibronectin and
proteoglycans), Pronectin F (Protein polymer Technologies), and Cell-tak (BD
Biosciences).

Feeder Layers

Some cells (e.g. embryonic stem cells), especially at low cell densities, require
support from living cells (e.g. fibroblasts). These cells, grown as a monolayer, serve
as the feeder layer cells. This is due to the supplementation of the medium by the
metabolites or growth factors secreted from the fibroblasts.
8

Feeder layers may make the surface suitable, or even selective, for attachment for
other cells. The interaction of a cell with the feeder cells is different from the
interaction of the cell with a synthetic substrate, which causes a change in
morphology and reduces the cells’ ability to proliferate.

Three dimensional matrices

Many functional and morphological characteristics are lost during serial subculture,
so 3-D culture is attempted.

3-D matrices include collagen gel, cellulose sponge, microcarriers, etc..
Collagen sponge
IV.

Polyester carrier
Aseptic technique
Contamination by microorganisms remains a major problem in tissue culture. Bacteria,
mycoplasma, yeast and fungal spores may be introduced via the operator, the atmosphere,
work surfaces, solutions and many other sources.

Contamination may be minimized if
1.
Cultures are checked carefully by eye and on a microscope.
2.
Antibiotics are added to important cultures. However, do not add antibiotics
during the routine cell culture so that cryptic (隱密的) contamination is
revelaed.
3.
Reagents are checked for sterility before use.
4.
Bottles of media are not shared with other people or used for different cell lines.
Elements of aseptic environment

Quiet area

Free from the draft (a current of air, may be from doors or windows, etc).
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
No through traffic

No equipments that generate drafts (e.g. air conditioners, centrifuges, etc.) around

The hood and the area should be used exclusively for tissue culture. Nonsterile
activities should be carried out elsewhere.

Work Surface

Swab the surface with 70% alcohol initially and in between procedures, and wop up
any spillage immediately.

Arrange the work area so that you have (a) easy access to all items; (b) a wide space
in the center of the hood. Do not leave too many items too close to you otherwise
you will inevitably brush the tip against a nonsterile surface. Furthermore, the
laminar flow will fail in a hood that is crowded with equipment.

Dry skin and loosely adherent microorganisms on the hands are the greatest risks wash
hands.

The caps and necks of the bottle should be flamed before they are open and after they are
closed. Do not leave bottles open and do not keep bottles vertical when open. Also
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don’t let hands or any items come between an open vessel or sterile pipette and the
HEPA filter.

Laminar flow hood efficiency relies on the pressure drop across the filter. When
resistance builds up, the pressure drop increases and the flow rate falls. Below 0.4 m/s,
the stability of the laminar airflow is lost, and sterility can no longer be maintained. The
HEPA filter should be monitored every 6 months for airflow and holes. The primary
filter should be replaced more often.

One major source of contamination in the Petri dishes is the capillary space between the
lid and the base. Trapped medium forms a bridge with the non-sterile outside air and
may cross-contaminate wells in a multiwell plate. Avoid the medium entering the space.

Humidified incubators should be cleaned regularly by removing all racks or trays, and
washing the interior and the racks with detergents. Traces of detergents should then be
removed with 70% alcohol. Roccall (2%, a fungicide) or copper sulfate (1%) may be
placed in the incubator to retard fungal growth.
V. Media

Early media formulations include Eagle’s Basal Medium (1955) and Eagle’s Minimal
Essential Medium (1959). These media are suitable for many cell lines and still widely
adopted now. Subsequent modifications are aimed at replacing serum and optimizing
media for different cell types (e.g. RPMI 1640 for lymphoid), etc.
Physicochemical properties

pH

Most cell lines grow at pH 7.4. The optimum could vary though. Some fibroblasts
grow better at pH 7.4-7.7, yet insect cells grow better at 6.2.

Phenol red is commonly used as an indicator. It is red at 7.4, orange at 7.0, yellow
at 6.5, lemon yellow below 6.5, more pink at 7.6 and purple at 7.8.

CO2 and bicarbonate

CO2 in the air can lower the pH by
H2O+CO2H2CO3H+ + HCO311
(1)

This pH shift can be neutralized by bicarbonate
NaHCO3Na+ + HCO3-

(2)
The increased HCO3- pushes equation (1) to left until equilibrium is reached at pH
7.4.

Buffering


Culture media must be buffered because

Overproduction of CO2 and lactate can lower the pH.

Open dishes cause the pH to rise (due to the escape of CO2)
In spite of its poor buffering capacity, bicarbonate is still widely used due to its low
cost, and low cytotoxicity.

HEPES is a much stronger buffer in the pH range of 7.2-7.6; it is frequently used at
10-20 mM.

Oxygen

Providing correct O2 concentration is important because:

Most cells require oxygen for respiration in vivo. Many cells (e.g. transformed
cells) may change their metabolism to anaerobic metabolism under low oxygen
tension.


High oxygen concentration may be toxic due to elevated levels of free radicals.
Oxygen requirements in organ and cell cultures are distinct:

Cell culture: atmospheric or lower oxygen tensions are preferred.

Organ culture: high oxygen tension is required (may be up to 95% O2 in the gas
phase for late-stage embryos).

The high requirement for oxygen of organ culture may be due to the diffusion
limitation or the difference between the differentiated and rapidly proliferating cells.

The requirement for selenium (Se) in the medium is related to oxygen toxicity, as Se
is a cofactor3 for glutathione synthesis, and glutathione (2-ME and DTT) is a freeradical scavenger, so Se is important in removing free radicals.
3
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's
biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules"
that assist in biochemical transformations.
12

Oxygen tolerance and Se may be provided by serum, so DO control is more critical
in SFM.

To avoid oxygen diffusion limitation, keep the depth of the medium within the
range of 2-5 mm (0.2-0.5 ml/cm2) in the static culture.

Osmolality4 (a measure of osmotic pressure)

In practice, osmolalities of 260-320 mOsm/kg are acceptable for most cells.

The addition of HEPES and drugs to the medium and their subsequent
neutralization can markedly affect the osmolality.

Temperature

The optimal temperature depends on the body temperature of the animal from which
the cells are obtained.

37C is optimal for most human and warm-blooded animal cell lines. Avian cells
can be maintained at 38.5C while insect cells are grown at 27-28C.

Cultured cells can tolerate drops in temperature, can survive several days at 4C and
can be frozen at -196C. But cells can not tolerate more than about 2C above
normal (39.5C) for more than a few hours and will die quickly at 40C.

A large number of flasks should not be stacked together in the incubator otherwise
air circulation would be affected and “cold-spot” (and uneven growth) could occur.

Cells from cold-blooded animals (e.g. cold-water fish) tolerate a wider range
between 15C and 26C, it may require an incubator for cooling as well as heating.

Viscosity

Viscosity is mainly influenced by the serum content.

Viscosity becomes important in suspension culture because of the damage from
shear stress. The damage from shear stress is a concern at low serum concentration
and can be reduced by increasing the viscosity with carboxylmethylcellulose (CMC).
4
1 M (molar) glucose = 1 mole glucose in 1 liter “solution”
1 m (molal) glucose = 1 mole (180 g) glucose dissolved in 1 kg of solvent.
1 m NaCl has 2 osmole (Osm, a non-SI unit that defines the number of moles of a chemical compound that contributes to a
solution's osmotic pressure) because Na+ and Cl- concentrations are 1 m each.
13

Surface tension and foaming

For the suspension culture, the agitation can cause the foaming problem. Foaming
can increase the rate of protein denaturation and the risk of contamination if the
foam reaches the neck of the culture vessel.

Limit gaseous diffusion.

The addition of a silicon antifoam or Pluronic F-68 (0.01-0.1%) helps prevent
foaming by reducing surface tension, and may also protect the cells against shear
stress from bubbles.
Complete media
Salts

Are mainly provided by the balanced salt solution (BSS) which may include sodium
bicarbonate and glucose5.
5
BSS can be used to dilute a.a. and vitamins or other chemicals. Cells can be incubated with BSS for up to about 4 h (usually
with glucose) to investigate the effects of chemicals on the cells.
14

Mainly contain Na+, K+, Mg2+, Ca2+, Cl-, SO42-, PO43- and HCO3-, and are the major
components contributing to the osmolality.

Divalent cations, particularly Ca2+, are required for cell adhesion molecules. [Ca2+] is
reduced in suspension cultures so as to minimize cell aggregation and attachment. Ca2+
also acts as an intermediate in signal transduction so [Ca2+] can influence whether cells
will proliferate or differentiate.

Na+, K+ and Cl- regulate the membrane potential. SO42-, PO43- and HCO3- are regulators
of intracellular pH and they are required for the synthesis of many ECM and nutritional
molecules.
Amino acids

Essential a.a. (Arg, Gly, Ile, Leu, Lys, Met, Phe, Thr, Trp, His, Tyr, Val) are not
synthesized in the body and are thus required for the cultured cells.

Other a.a. can be produced by animal cells and are not essential, but these nonessential
a.a. are often added to the medium to compensate for a particular cell type’s incapacity to
make them.

Gln is often required by most cells as a source of energy and carbon. But Gln is unstable
upon long-term storage, thus the shelf life of Gln-containing medium is not too long.

The oxidation of Gln produces Glu which enters TCA cycle by transamination to 2oxoglutarate. This tends to produce ammonia, which is toxic and can limit cell growth.
Vitamins

Vitamins are required as the cofactors of many enzymes.

Examples of vitamin include water soluble vitamins (e.g. the B group, colin, folic acid,
inositol and nicotinamide), biotin, etc.

Vitamin C (ascorbic acid) is important for some cells, particularly for collagen secreting
cells. But vitamin C tends to oxidize and is unstable, thus it is usually not included in the
medium formulation. Vitamin C is added when needed.
Glucose

Glucose is an important source of carbon and energy.

The accumulation of lactate implies that TCA cycle may not function entirely as it does
in vivo. In this case, much of the carbon is derived from glutamine rather than from
15
glucose. This explains the exceptionally high requirement of some cultured cells for
glutamine or glutamate.
Other supplements

Other compounds, including proteins, peptides, nucleotides, TCA cycle intermediates,
pyruvate and lipids, may appear in complex media. These may act as

Antioxidants: e.g. glutathione

Precursors: adenosine, ATP, AMP, guanine, D-ribose, uracil, etc.

Lipids: cholesterol, linoleic acid…

Hormones and growth factors: are supplied in the serum in complex media (i.e.
usually there’s no need for extra addition), but they are usually added to SFM.
Antibiotics:

Introduced to avoid contamination. However, they are not encouraged to use during
routine culture because:

They hide the presence of low-level, cryptic contaminants (e.g. mycoplasma
infections).


They encourage the development of antibiotic-resistant organisms.

They have anti-metabolic effects that can cross-react with mammalian cells.

Antibiotics should only be used in primary culture or large-scale experiments.
Fungal and yeast contaminations are particularly hard to control with antibiotics. They may be held, but are seldom eliminated.
Serum
16

Commonly added to the medium at a concentration of 5-20% (v/v). Functions of many
serum proteins remain obscure, but some are known:

Albumin: carrier of lipids, minerals and globulins (these molecules may bind to albumin).

Fibronectin: promotes cell attachment

2-macroglobulin: inhibits trypsin

transferrin: binds iron, making it less toxic but functional.

Growth factors: many growth factors stimulate the cell proliferation (e.g. PDGF,
FGF, vascular endothelial growth factor) or differentiation (e.g. TGF-, insulin-like
growth factor).

Serum proteins also increase the medium viscosity, reduce the shear stress during
pipetting, and enhance the medium’s buffering capacity.

The sera are usually derived from the calf (小牛), fetal bovine (胎牛) or horse. FBS
(fetal bovine serum) is suitable for more demanding cells, but more expensive (may try to
reduce the serum concentration or mix FBS and FCS).

Serum is usually heat inactivated (incubation at 56 C for 30 min) to inactivate
complement proteins (a family of proteins that can cause cell lysis). After that, serum
should be dispensed into aliquots and stored at -20C. One batch of serum will last about
6 months to a year at -20C.
Other supplements (optional):

Additional hormones, nutrients, lipids and minerals (e.g. trace elements that are present
at low concentrations (<10-4 M) such as iron, copper, selenium and zinc) can be added.

Tissue extracts and digests have been traditionally used as supplements. For example,
bactopeptone, tryptose and lactalbumin hydrolysate are proteolytic digests of beef hearts
or lactalbumin and contain mainly amino acids and small peptides.
Serum-free media (SFM)

Disadvantages of serum

Physiological variability: lot-to-lot variation occurs

Shelf life and consistency: one batch can last one year at most, thus consistency is
difficult to maintain.
17

Availability

Downstream processing

Contamination (with prion that causes mad cow disease in cattle and CreutzfeldtJakob disease (CJD) in humans)

Growth inhibitors: serum contains growth stimulators (e.g. PDGF) and inhibitors.
Hydrocortisone, present at 10-8 M in FBS, is cytostatic to many cell types at high
cell densities.

VI.
SFM is desired if the products derived from the cell is aimed for uses in humans.
Preparation and Sterilization
Glassware

The requirements of tissue culture washing are higher than for general glassware.

Glassware must be cleaned very thoroughly to avoid traces of toxic minerals
contaminating the inner surface. Do not let soiled glassware dry out.

For cell propagation, the surface must also carry the correct charge. Alkaline detergents
render the surface unsuitable, and neutralization with dilute HCl is necessary, but many
modern detergents do not alter glass surface and can be removed completely.

Detergents with enzymes (e.g. Tergzyme) may be used to remove proteins on the
glassware. Disinfectant (e.g. Clorox) of 300 ppm should be used.

Plastic flasks are meant for single use, but cells may be reseeded back for a number of
times.
Sterilization
18
19

During autoclave (20 min at 121 C, 100 kPa), the bottles should be loosely capped to
allow the steam to enter.

Sterile filtration

Filtration through 0.1-0.2 m filters is used for heat-labile solutions. Different materials
may be used (e.g. cellulose acetate, polyethersulphone (PES) etc.)

Alternative methods:

Immerse in 70%alcohol for 30 min and dry under UV light in a laminar flow hood.

-irradiation at a level of 2000-3000 Gy is best for plastics.
20

Water purification can be divided into four stages:

RO (or distillation): removes viruses, microorganisms, pyrogen and virtually all
inorganic impurities.

Carbon filtration: remove both organic and inorganic colloids. Total organic carbon
(TOC) should be <10 parts per billion (ppb).

Ion exchange: to remove ionized inorganic material (conductivity should be
monitored and 20 Mcm at 25C)

Microfiltration: remove any microorganism

Note: For water for injection (WFI), RO or distillation at the last stage is required
References:
Freshney, RI. (2005) Culture of animal cells: a manual of basic technique. Wiley-Liss, New
York
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