ANIMAL CELL TECHNOLOGY

Lecturer: Nguyen Thi Hong Minh

Email: nguyen-thi-hong.minh@usth.edu.vn
Tel. 094 393 6511
 Overview

 L1: Introduction to animal cell technology

 L2: Basic method in animal cell culture

 L3: Animal cell as tool for drug discovery

 L4: Animal cell as tool for biologist

 L5: 2D/3D culture, antibody production

 L6: Good laboratory practice (GLP) and good cell culture practice

 L7: Student presentation

Basic methods in animal cell culture

 Type of culture
 Culture media and its environment
 Maintaining cells in culture
 Cryopreservation and thawing frozen cells
 Type of culture

 Organ Culture
 Primary Culture
 Continuous Cultures
 Type of cell culture
Tissue of interest

Organ or tissue culture in vitro

Primary culture Senescence
 Subculture is the process of
sub-culture (passage) dispersion and re-culture the
Cell line
cells
 Two types of continuous
cultures: finite cell line and
Finite cell line Immortalization
infinite cell line

Immortalised cell line Transformed cell line

(cancerous cells)
 Organ culture
Culture of whole organs, intact organ fragments or
entire embryos
Studying their continued function or development
Prolong organ donor

hours or days
 Organ culture

Advantages
oMost accurate reflection of organism’s physiology
oCells remain fully differentiated.
Disadvantages
oScale-up is not recommended.
oGrowth is slow.
oFresh tissues are required for every experiments.

U of Minnesota 2007 – heart perfusion w/stem cells created a “new heart”.

 Type of culture

 Organ Culture
 Primary Culture
 Continuous Cultures
 Primary culture

 Primary cell culture is the primary step of cell culturing in which the
cell is first isolated from tissue and then proliferated under the
appropriate conditions until they consume all available contents for
their growth.

 Primary cells are cells isolated directly from human or animal tissue by
using enzymatic or mechanical methods
 Primary culture

From the outgrowth cells from a piece of tissue that is

disaggregated by enzymatic, chemical, or mechanical
methods.
Primary culture
 Classification of primary cells

 According to the genus (by species or tissues) from

which they are isolated, they can be categorized in:
 Epithelial cells
 Fibroblasts
 Keratinocytes
 Melanocytes
 Endothelial cells
 Muscle cells
 Hematopoietic and mesenchymal stem cells
Human keratinocytes Endothelial cells

Fibroblast cells
Epithelial cells
 Primary cell culture
 Primary cells are morphologically similar to the parent tissue and
considered by many researchers to be more physiologically similar to in vivo
cells.

 These cultures are capable of only a limited number of cell divisions, after
which they enter a non-proliferative state called senescence and eventually
die out.

 Retain differentiated phenotype

 Primary cell culture is generally more difficult than culture of continuous cell
lines.
 Primary culture of cardiomyocytes

E16 after 1
day culture

15
Primary culture of cardiomyocytes

16
E18 after 2 days culture
Primary cell culture: Advantage &
Disadvantage
Advantages Disadvantages
They are thought to represent the best Preparation of primary cultures is labor
experimental models for in vivo intensive.
situations.

Usually retain many of the differentiated Very susceptible to contamination.

characteristics of the cell in vivo

May not fully act like tissue due to

Not “dedifferentiated”
(lose specialized characteristics) complexity of media.

Considerable variation in population

and between preparations.
Can be maintained in vitro only for a
limited period of time.
 Type of culture

 Organ Culture
 Primary Culture
 Continuous Cultures
 Continuous culture

 Derived from subculture (passage) of

primary culture
Subculture is the process of
dispersion and re-culture the
cells.

 Comprised of a single cell type

 Two types of continuous cultures

 Finite cell line

 Infinite cell line

Continuous cultures
 Finite Cell Lines

Formed after the first sub-culturing

(passaging) of a primary cell
culture.

Proliferate for a limited number

of cell divisions, after which they
will senesce (after
approximately thirty cycles of
division).
 Finite Cell Line:
Advantage & Disadvantage of

Advantages Disadvantages
Can obtain a large population of Cells have a tendency to
similar cells. differentiate over time in culture.
Most cellular characteristics are Over time the culture tends to
maintained. select for aberrant cells.
It is essential to establish a system of master and working banks in
order to maintain such lines for long periods.
 Continuous cell lines (Infinite cell
line)

Derived from a primary culture

Immortalized:
Spontaneously (e.g.: spontaneous genetic mutation)
By transformation vectors (e.g.: virus or plasmids)
Loss of anchorage dependency (anchorage independent) and
contact inhibition
Infinite life span in vitro
Immortalized cell lines are also known as transformed cells and
frequently correlates with tumorigenicity (cancer cells).
Contact inhibition (density-
dependent inhibition)
 Continuous cell lines (Infinite
cell line)

Advantage
Continuous cell lines are generally easier to work with
than primary or finite cell cultures.
Disadvantage
These cells have undergone genetic alterations and
their behavior in vitro may not represent the in vivo
situation.
 Cell morphology

Most mammalian cells in culture can be divided into

three basic categories based on their morphology:

Fibroblastic (or fibroblast-like) cells are

bipolar or multipolar, have elongated
shapes and grow attached to the surface
of growth container.
Epithelial-like cells are polygonal in shape with
more regular dimensions and grow attached to the
surface of growth container in discrete patches.
Lymphoblast-like cells are spherical in shape
and usually grown in suspension without attaching
to a surface.
 Common Infinite cell line

• Human cell lines

• MCF-7 breast cancer cells

• HL 60 Leukemia cells
• HEK-293 Human embryonic kidney cells
• HeLa cervical cancer cells
• Primate cell lines
• Vero African green monkey kidney epithelial cells
• Cos-7 African green monkey kidney cells

ATCC (American Type Culture Collection)

 Summary the advantages cell
culture
Category
Physico-chemical Summary the
Advantages
Control of pH, temperature, osmolarity, dissolved gases
environment
Physiological conditions advantages
Control of hormone and nutrient concentrations
Microenvironment Regulation of matrix, cell-cell interaction
Cell line homogeneity
Characterization
cell
Availableculture
of selective media
Easy to perform
Preservation Can be stored in liquid nitrogen
Validation & accreditation Origin, history, purity can be recorded
Replicates Quantitation is easy
Reagent saving Lower cost
Control of treatment Ability to define dose, concentration, time
Mechanization Available with microtitration and robotics
Reduction of animal use Cytotoxicity and screening of pharmaceutics, cosmetics, etc.
 Limitations of cell culture

✔ Requires expertise
✔ Phenotypic and genetic instability
Dedifferentiation: loss of the phenotypic characteristics typical of the
tissue from which the cells had been isolated.
Adaptation
Chromosomal abnormality: heterogeneity in growth rate and the
capacity to differentiate within the population

✔ Lacks the several systemic components involved in homeostatic

regulation as in vivo
 Why do we need cell culture?

To overcome problems in studying cellular behavior:

Confounding effects of surrounding tissues
Variations that might arise in animals under experimental stress

Economy:
Cell-based assays (high throughput screening)
Production of cell material (e.g., vaccine, antibody, hormone)

To reduce the number of animals used

The list of major differences between primary cells
and cell lines
Summary
Basic methods in animal cell culture

 Type of culture
 Culture media and its environment
 Maintaining cells in culture
 Cryopreservation and thawing frozen cells
 Culture Media

•Culture medium is available in 3 forms from commercial suppliers:

I. Powdered form: it needs to be prepared and sterilized by the
investigator.
II.Concentrated form: to be diluted by the investigator.
III.Working solution: to be used directly without further manipulation.

Powdered form Concentrated form Working solution

 Chemical composition in media

Balanced Salt Solution (BSS)

a solution made to a physiological pH and salt concentration
for buffering system
inorganic salts : Na+, K+, Mg2+, Ca2+, Cl-, SO 2-, PO 3-, HCO
L-Amino acids
most cultured cells require glutamine as a source of energy and carbon
Glucose
Vitamins
Organic supplements
proteins, nucleosides, citric acid cycle intermediates, pyruvate, lipids
pH indicator = phenol red
 Advantages and disadvantages
Advantages
• Contains various growth factors and
hormones which stimulates cell
growth and functions
• Helps in attachment of the cells
• Acts as a spreading factor
• Acts as a buffering agent which helps
in maintaining the pH of the culture
media
• Functions as a binding protein
• Minimizes mechanical damages or
damages caused by viscosity
Disadvantages
• Testing needs to be done to maintain
the quality of each batch before using
• Lack of uniformity in the
composition of serum
• May contain some of the growth
inhibiting factors
• Presence of serum in media may
interfere with the purification and
isolation of cell culture products
• Increase the risk of contamination
 Supplements
L-glutamine
• Essential amino acid (not synthesised by the cell)
• Energy source (citric acid cycle), used in protein synthesis
• Unstable in liquid media - added as a supplement

Non-essential amino acids (NEAA)

• Usually added to basic media compositions
• Energy source, used in protein synthesis
• May reduce metabolic burden on cells

Growth Factors and Hormones (e.g.: insulin)

• Stimulate glucose transport and utilisation
• Uptake of amino acids
• Maintenance of differentiation

Antibiotics and Antimycotics

• Penicillin, streptomycin, gentamicin, amphotericin B
• Reduce the risk of bacterial and fungal contamination
• Cells can become antibiotic resistant – changing phenotype
• Preferably avoided in long term culture
Antibiotics
Culture environment
Basic Requirement for Growing Animal Cells
 Bicarbonate buffer

The increased
- HCO3 concentration pushes the
equation to the left until the equilibrium is reached at pH 7.4.

Compound Eagle’s MEM DMEM

(EMEM)
NaHCO3 26 mM 44 mM
CO2 5% 10%
Basic methods in animal cell culture

 Type of culture
 Culture media and its environment
 Maintaining cells in culture
 Cryopreservation and thawing frozen cells
Basic cell culture techniques

 Subculture
 Cell counting
 Cryopreservation and thawing frozen
cells
 What is subculture?

Subculture or passaging
 Transfer of cells from a previous culture into fresh growth medium
 Propagation of cell line

Why and When to do subculture?

Type of cell culture and their
growth characteristics
 Cell characteristics
Normal cells
Diploid chromosome
Finite lifespan
Longer doubling time
Normal growth characteristic
Contact inhibition
Density dependent
Anchorage dependent
Inability to form tumor in
animal
Transformed cells
Aneuploid chromosome
Indefinite lifespan
Shorter doubling time
Altered growth control
Loss of contact inhibition
Loss of density dependent
Loss of anchorage dependent
Ability to form tumor in animal
Why to do subculture
When to do subculture?
Basic methods in animal cell culture

 Type of culture
 Culture media and its environment
 Maintaining cells in culture
 Cryopreservation and thawing frozen cells