6/03/2020

Animal Cell Culture

RAVI SHUKLA
E-mail : ravi.shukla@rmit.edu.au

School of Science, RMIT University

Let’s culture our Cells !!!

In this module …
LECTURE 1
Introduction to the Art and Science of Animal Cell and Tissue
Culture (basics)…

LECTURE 2
More details on the technique aspects…

LECTURE 3 - 5
More details and Applications of Tissue Culture technology

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In this Lecture…

• What is tissue culture…

• How old is this science?... Some historical account..
• Why do we need cell cultures?…
• What are the basic requirements of tissue culture ?...
• Some of the properties of cells grown in culture….

https://sciencing.com
Plant leaf https://www.pinterest.com.au/pin/98516310583
421653/

Distinctive feature:
Cells in plant tissue are much
more organised and easily
identifiable
Courtesy : Mohd Abdel Gawad

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Quick Revision
Characteristics of animal cells

•Size: between 6-30 µm.

•Shape: (spherical or ellipsoidal or irregular).
•Cell structure:
a) No cell wall.
b) Only thin and fragile plasma membrane (Proteins
and lipids)
c) Negatively charged surface (anchorage)
d) Receptors
e) Some animal cells are non-anchorage dependent.
f) cytoskeleton (actin filaments, intermediate
filaments and microtubules) to provide cell
mechanical strength, control shape and guide cell
movement.

Cell/Tissue Culture

Cell culture is the process by which cells and tissue

are aseptically grown under artificially controlled in
vitro conditions.

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Let’s Discuss

How to culture your own cells ?

Think about uniqueness of animal cells
What will be 3 most important things for keeping cells alive out side the body ?

Cell membrane should be intact: aqueous conditions, osmotic balance of

salts in intracellular and extracellular environment, pH

What other things will matter ?

How long will you be able to keep cells alive ?
Depends on: source of cells, nature and stage of cells, in vitro culture conditions

Will cells divide and reproduce in the culture ?

Growth factors, colonial behaviour, specialised requirements of cells

Animal Cell/Tissue Culture

Aseptic artificially controlled in vitro conditions

• Defined temperature , humidity and CO2(incubator )
• Culture medium (cell nutrients & growth factors)

Different types of cells grown in culture

• connective tissue elements such as fibroblasts, skeletal tissue, cardiac,
epithelial tissue (liver, breast, skin, kidney)
• Lung, liver, embryo
• Blood (RBCs, lymphocytes, monocytes & macrophages, Dendritic cells)
• Nerve cells
• Stem cells
• Many different types of tumor cells.

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What you learned from

Plant Tissue Culture ?
Aseptic handling techniques
Unique features of plant cells e.g Totipotency
Tissue culture media
Types of plant tissue cultures
(Explant, Suspension, Callus, embryo etc)
Time line and growth pattern
Contamination of the cultures
Observation, data recording , interpretation and analysis

Why do we need in vitro Cultures..

• Mechanisms of cell cycle control
• Production of cells for biochemical analysis
• Cancer cell signalling studies
• Study of differentiation processes
• Stem cells and cell based therapies
• Detection, production and function of growth factors and hormones
• Detection and production of viruses and vaccines
• Production of monoclonal antibodies (hybridoma technology)
• Production of artificial tissues/ organs and regenerative medicine
• Study of specialised cell function
• The study of cell-cell and cell-matrix interactions
• Study of drug screening and toxicity evaluations
• Study of material-cell interactions and biocompatibility evaluations

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History of Animal Tissue Culture

1878: Claude Bernard proposed that physiological systems of an organism can be maintained in a
living system after the death of an organism.
1885: Roux maintained embryonic chick cells in a saline culture.
1897: Loeb demonstrated the survival of cells isolated from blood and connective tissue in serum
and plasma.
1907: Harrison cultivated frog nerve cells in a lymph clot held by the 'hanging drop' method and
observed the growth of nerve fibers in vitro for several weeks.
1911: Lewis and Lewis made the first liquid media consisted of sea water, serum, embryo extract,
salts and peptones. They observed limited monolayer growth.
1913: Carrel introduced strict aseptic techniques so that cells could be cultured for long periods
1916: Rous and Jones introduced proteolytic enzyme trypsin for the subculture of adherent cells
1940s: The use of the antibiotics penicillin and streptomycin in culture medium decreased the
problem of contamination in cell culture.

1940s: First chemically defined Cell culture media were developed

1952: George Otto Gey established first human cell line from a cervical carcinoma patent (Henrietta
Lacks) and named it HeLa cells.

1975: Kohler and Milstein produced the first hybridoma capable of secreting a monoclonal
antibody.

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Ross Granville Harrison, “America’s most famous unknown

scientist.”
Ross Granville Harrison in 1907 discovered a way to grow cells
outside the body. He successfully cultured frog neuroblasts in
a lymph medium and thereby took the first step toward current
research on precursor and stem cells. At the time, “tissue culture”
was a curiosity, but later in 1998, historians of science Meyer
Friedman and Gerald W. Friedland named it one of “medicine’s
ten greatest discoveries.
Twice Harrison was seriously considered for the Nobel Prize. In 1917 the Nobel
committee recommended him for science’s greatest honour, but due to the World War, a
prize was not awarded in his field. In 1933 he was one of two finalists, but because the
full value of tissue culture was not yet appreciated, the Nobel went to geneticist
Thomas Hunt Morgan.

He helped bring together American chemists and the British discoverers of penicillin to
dramatically speed up production of the antibiotics.

One of the most important uses of Harrison’s methodology was initiated by John F.
Enders, who found a way to grow poliomyelitis virus in tissue culture. In 1954, when
Enders and his assistants received the Nobel for discoveries leading to the polio
vaccine, Harrison rejoiced that his 1907 observation had led to this success.

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Cell Culture in Lab: Requirements

Infrastructure Engineering Controls

laminar air flow hood (verticle) Dedicated lab space

Water bath Incubator Sink
Phase contrast microscope HEPA filters
Autoclave nitrogen/ CO2 Supply
Refrigerator
Hot air oven
Ultrapure tissue culture grade water Consumables
Deep Freezer
Media and sera
Centrifuge
Buffers & balanced salt solutions
Cryo Container/ Liquid nitrogen Biochemicals
Hemocytometer/ Digital cell counter Disinfectant
Glassware
Plasticware

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Cell Culture in Lab: Requirements

Consumables
Washing buffers (PBS, HBSS)
Culture medium (DMEM, RPMI 1640)
Serum (Defined vs Semi defined medium) (10%)
Dissociation media (Trypsin EDTA/ Collagenase)
Antibiotics
Growth factors (EGF, VEGF, FGF, insulin, TGF β etc…
Glass ware and plastic ware (pipettes culture dishes etc….)

Operator Requirements

Aseptic conditions
personal protective equipments
proper waste disposal

Work Ethics

Tissue Source
Genetic manipulations
Viral work
Cross contamination

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A Quick look at
an advanced cell culture research laboratory
fibroblasts
Finite
autoclave
primary epithelial
CO2
disinfectant

ethics PPE
Stem cells
Sigma
continuous
Explant

scrapper
ATCC microscope

macrophages
plasticware

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Terminology…

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General Characteristics of cells in culture

Primary Cultures and finite cell lines Established Cell Lines

• Anchorage dependence • Transformed cells show altered phenotypes

– cells need a solid or firm surface to stick – no anchorage dependence
to
– no dependence on serum (growth factors)
• Serum (growth factor) dependence
– no density-dependent inhibition of growth
– growth factors in serum are essential for
– altered cytoskeleton
growth of cells in culture
• May form tumors
• Density-dependent inhibition
– cells grow only to a certain density, then
stop
– may involve cell - cell contacts
• Cytoskeletal organization
– cells are flat & extended
– elongated network of actin filaments
• Cells grow as a monolayer
• Do not form tumor after in vivo
administration (except embryonic stem cells
and some cancer cells)

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Cell Types: Morphology

Lymphoblast like Fibroblast Like Epithelial Like

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Culture Types: Attachment

Suspension Culture
Peripheral blood cells (RBCs, WBCs)
Leukemic cell lines

Adherent Cultures
Anchorage dependent (Mostly Primary and finite)
Fibroblast
Epithelial
Special (Stem, Endothelial, Nerve, Adipocyte)

Anchorage independent
Epithelial
Special (Stem)

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Adherent Cell Culture Vs Suspension Cell Culture

• Appropriate for most cell types,
including primary cultures
• Requires periodic passaging, but
allows easy visual inspection
under inverted microscope
• Cells are dissociated
enzymatically (e.g. trypsin) or
mechanically from substrate
• Growth is limited by surface area,
which may limit product yields
• Requires tissue-culture treated
vessel
• Used for cytology, harvesting
products continuously, and many
research applications
• Appropriate for cells adapted to suspension
culture and a few other cell lines that are
nonadhesive (e.g., hematopoietic)
• Easier to passage, but requires daily cell counts
and viability determination to follow growth
patterns; culture can be diluted to stimulate
growth
• Does not require enzymatic or mechanical
dissociation
• Growth is limited by concentration of cells in the
medium, which allows easy scale-up
• Can be maintained in culture vessels that are not
tissue-culture treated, but requires agitation (i.e.,
shaking or stirring) for adequate gas exchange
• Used for bulk protein production, batch
harvesting, and many research applications

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Cell Culture : Methods

Suspension Culture (Primary and Established)

Feeding and Passaging (sub culturing)

Adherent Cultures

Primary (Single Cell Suspension and Explant cultures)

Established (Feeding and Passaging)

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Establishment and characterization of Neuroglial

cell lines from the brain tumors

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Establishment of Neuroglial cell lines from the

brain tumors

a.) Explant (5 days)

b.) Primary (Fibroblast kind) during
senescence (HNGC-1)
c.) Established (HNGC-2)

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Explant Tissue Culture

Primary Cell Culture HNGC-1

Repeat Feeding and Passaging (Sub Culturing- finite cells)

Cellular senescence and Telomere Shortening

Spontaneous transformation
Chromosomal Aberrations and
Gene mutations

Immortal cell line

HNGC-2

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Chromosomal Abnormalities

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HNGC-1 and -2 are Neuroglial cell lines

HNGC-1 HNGC-2

Neuroglial cell lines from the brain tumors

S-100 Glial Marker

Tuj 1 Neuronal Marker

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HNGC-2 leads to in vivo tumour development

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Learning today…..
• Animal cells can be grown in the laboratory
• They need TLC due to highly fragile cell membrane
• Aqueous environment is necessary to maintain cells
• Cell culture is more of an art and needs specialised
labs and infrastructure
• Lab grown cells may or may not attached to the
culture vessels (substrates)
• They may be identified by their morphology
• New terminology/ vocabulary needed to learned to
work in this area
• Lab grown cells could be used for many applications
and advanced research

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References
Parameswaran V, Shukla R, Bhonde R, Sahul Hameed AS. Development of a Pluripotent ES-like Cell Line from Asian
Sea Bass (Latescalcarifer)—An Oviparous Stem Cell Line Mimicking Viviparous ES Cells. Mar Biotechnol. 2007, 9,
766-775

SahulHameed AS, Parameswaran V, Shukla R, Bright Singh IS, Thirunavukkarasu AR, Bhonde RR. Establishment and
characterization of India’s first marine fish cell line (SISK) from the kidney of sea bass (Latescalarifer). Aquaculture.
2006, 257, 92.

Shiras A, Bhosale A, Shepal V, Shukla R, BaburaoVS, Prabhakara K, Shastry P. A Unique model system for tumour
progression in GBM comprising two developed human neuro-epithelial cell lines with differential transforming potential
and coexpressing neuronal and glial markers. Neoplasia. 2003, 5, 520-532.

Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition R. Ian Freshney ISBN:
978-0-470-52812-9 796 pages

Web resources
http://www.atcc.org/en/Products/Cells_and_Microorganisms/Cell_Lines.aspx

http://www.cellbankaustralia.com/Cell-Lines

http://www.qiagen.com/knowledge-and-support/spotlight/protocols-and-applications-
guide/animal-cell-culture/

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