This document provides a history and overview of animal cell culture techniques. It discusses the development of cell culture media and reagents used to support cell growth in vitro. It also describes different techniques for culturing mammalian cells, tissues, and organs, including organ culture, explant culture, and cell culture. The goal of animal cell culture is to maintain cells, tissues, or organs outside of their natural environment for research purposes.

1. DEPARTMENT OF ANIMAL BIOTECHNOLOGY
History of animal cell
culture, cell culture media and
reagents, culture of
mammalian cells, tissues and
organs
Submitted by:
Dr. Vijayata

2. History of animal cell culture

3. EVENT
1907 Harrison Frog embryo nerve fiber outgrowth in vitro.
(cultivated frog nerve cells in a lymph clot held by
the 'hanging drop' method and
observed the growth of nerve fibers in vitro )
1912 Alexis carrel Explants of chick connective tissue; heart muscle
contractile for 2–3 months
1916 Rous & Jones Trypsinization and subculture of explants
1920s Carrel & Subculture of fibroblastic cell lines
/30s Ebeling, 1923
1925– Strangeways & Differentiation in vitro in organ culture
1926 Fell
1940s Keilova, 1948; Introduction of the use of antibiotics in tissue
Cruikshank & culture (penicillin and streptomycin )
Lowbury, 1952

4. 1943 Earle Establishment of the L-cell mouse fibroblast cell line;
first continuous cell line
1949 Enders Growth of virus in cell culture
1952 Dulbecco Use of trypsin for generation of replicate
subcultures
Virus plaque assay
1952 Gey Establishment the first human cell line, HeLa, from
a cervical carcinoma,
1955 Eagle Development of defined media
1959 Puck & Marcus Cloning of HeLa on a homologous feeder layer
1961 Sorieul & Cell fusion–somatic cell hybridization
Ephrussi

5. 1962 Macpherson & Establishment and transformation of BHK21
Stoker
1964 Klein smith & Pluripotency of embryonal stem cells
Pierce
1965 Ham Serum-free cloning of Chinese hamster cells
1967 Hoober & Epidermal growth factor
Cohen
1968 Stoker et al. Anchorage-independent cell proliferation
1969 Metcalf Colony formation in hematopoietic cells
1975 Kohler & Hybridomas—monoclonal antibodies
Milstein
1976 Illmensee & Totipotency of embryonal stem cells
Mintz
1976 Hayashi & Sato Growth factor-supplemented serum-free
media
1977 Nelson-Rees & Confirmation of HeLa cell cross-
Flandermeyer contamination of many cell lines

6. 1978 Ham & McKeehan MCDB-selective, serum-free media
1980– Peehl & Ham, 1980; Development of many specialized cell lines
1987 Hammond et al.,
1984; Knedler & Ham,
1987
1982 Darnell, Regulation of gene expression
1983 Evans Regulation of cell cycle
1984 Collen Production of recombinant tissue-type
plasminogen activator in mammalian cells
1989 Weinberg Oncogenes , malignancy, and transformation
1990s Kruse et al., 1991; Development of laminar-flow cabinets
Collins & Kennedy,
1999
1991 Griffiths Salk polio vaccine grown in monkey kidney cells
1998 Thomson et al., Culture of human embryonic stem cells
2000+ Dennis et al., 2001 Human Genome Project: genomics, proteomics,
genetic deficiencies and expression errors

7. Cell culture media and reagents

8. • The culture medium is the Combination of ingredients will
support the cell growth by providing all the essential nutrients ,
growth factors, and hormones for cell growth, as well as
regulating the pH and the osmolarity of culture.
• The three basic classes of media are: (differ in their requirement
for supplementation with serum. )
The choice of culture
Basal
media is dependent on the media
requirements of cells
being cultured.
Reduced- Serum-
serum free
media media

9. Basal (Basic) Media :
• Basal Medium is a defined medium that contains essential and
nonessential amino acids, vitamins, inorganic salts, organic
compounds, and trace elements, but does not contain the Growth
Supplements necessary for cell growth.
• Balanced salt solutions (BSS) e.g. phosphate-buffered saline
(PBS)
• DMEM and RPMI 1640 (with or without glutamine)
Reduced-Serum Media :
• Reduced-serum media are basal media formulations enriched
with nutrients and animal-derived factors, which reduce the amount
of serum that is needed.

10. Serum-Free Media :
• Serum-free media (SFM) circumvents issues with using
animal sera by replacing the serum with appropriate
nutritional and hormonal formulations.
• Serum-free media formulations exist for many primary
cultures and cell lines, including Chinese Hamster Ovary
(CHO), hybridoma cell lines, VERO, MDCK, MDBK cell
lines etc.
• One of the major advantages of using serum-free media is the
ability to make the medium selective for specific cell types by
choosing the appropriate combination of growth factors.

11. COMMON CELL CULTURE MEDIA
• Minimum Essential Medium (MEM)
• GMEMm (Glasgow Minimum Essential Medium)
• EMEM (Eagle’s MEM)
• DMEM (Dulbecco’s Modified Eagle Medium)
• Medium 199
• BME (Basal Medium Eagle)
• Ham's F-10 Medium
• Ham's F-12 Medium
• RPMI 1640 medium
• Leibovitz L-15 medium
• CMRL 1066
• Dulbecco's Modified Eagle's Medium (DMEM-001)
• MCDB 131
• McCoy's 5A

12. REAGENTS
The components or reagents of suitable culture media include:
Cell
Balanced Buffers and
dissociation Supplements
salt solution chemicals
reagents

13. Balanced salt solution :
• Balanced salt solutions can provide an environment that
maintains the structural and physiological integrity, pH and
osmotic pressure of cells in vitro.
• Maintain osmolarity
• Regulate membrane potential (Na+, K+, Ca2+)
• Ions for cell attachment and enzyme cofactors
DPBS (Dulbecco's Phosphate-Buffered Salines)
HBSS (Hanks' Balanced Salt Solutions)
PBS (Phosphate-Buffered Salines)
EBSS (Earle's Balanced Salt Solutions)

14. Buffers and chemicals :
• HEPES (4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid )
• Sodium Bicarbonate solution
Cell dissociation reagents :
Are the cell detachment solutions of proteolytic and collagenolytic enzymes.
• Papain, powder
• Trypsin 0.25% (1X), liquid
• Trypsin 2.5% (10X), liquid
• Dispase (Neutral Protease), powder
• Elastase, powder
• Hyaluronidase
• Pepsin A, powder
• Collagenase type 1,2,3,4
• Trypsin Inhibitors

15. Supplements :
• L-glutamine • 2-Mercaptoethanol
• Glutamax™ • Lipid Supplement
• Non-essential amino acids (NEAA) • CHO Supplement
• Growth Factors and Hormones • Antibiotics and Antimycotics
(e.g.: insulin) (Penicillin, streptomycin,
gentamicin, amphotericin B)
• Vitamines (MEM Vitamin Solution)
Serum :
• Foetal Calf/Bovine Serum (FCS & FBS) provides the best growing
conditions.
• Source of growth and adhesion factors, hormones, lipids and minerals
for the culture of cells.
• Regulates cell membrane permeability and serves as a carrier for
lipids, enzymes, micronutrients, and trace elements into the cell.

16. Culture of mammalian cells,
tissues and organs

17. Tissue culture
• Tissue culture is used as a
generic term to include the
in vitro cultivation of Organ
culture
organs, tissues and cells.
• The ability to survive and
grow tissues outside the Explant
culture
body in an artificial
environment. Cell culture
• Tissue culture can be
subdivided into three
major categories

18. Organ culture
• The entire embryos or organs are excised from the body and
culture.
• Organ culture refers to a three-dimensional culture of tissue retaining
some or all of the histological features of the tissue in vivo.
(Histological structure maintained)
• The tissue is cultured at the liquid–gas interface (on a grid or gel),
which favors the retention of a spherical or three-dimensional shape.
Advantages
• Normal physiological functions are maintained.
• Cells remain fully differentiated.
Disadvantages :
• The particular cells of interest may be very small in number in a piece
of tissue so the response produced may be difficult to detect and
quantify.

19. Organ culture techniques
1. Plasma clot method or Watch glass method
2. Organ Culture on Agar (Agar gel method)
i. Raft method
ii. Grid method
3. Organ Culture in Liquid Media

20. 1. Plasma clot method or
Watch glass method
Dame Honor Fell and Robinson introduced plasma
clot substrate originally to study development of
avian limb bone rudiment.
A plasma clot is prepared by mixing 5 drops of embryo extract
with 15 drops of plasma in a watch glass placed on a cotton
wool pad.
The cotton wool pad is put in a Petri dish. Time to time cotton
is moistened so that excessive evaporation should not occur.
A small piece of organ tissue is placed on the top of plasma
clot present in the watch glass.

21. 2.Organ Culture on Agar
• Media solidified with agar is used
• Media consist of : 7 parts 1% agar in Basal Salt Solution + 3 Parts
chick embryo extracts + 3 parts horse serum(7:3:3)
• Embryonic cultures grow well
• No need of mechanical support as media with agar serves good support
Raft Method :
• Tissue is placed onto a raft of lens paper or rayon acetate, which is
floated on serum in a watch glass.
• Rayon acetate rafts are made to float on the serum by treating their 4
corners with silicone.
• In a combination of raft and clot techniques, the explants are first
placed on a suitable raft, which is then kept on a plasma clot.
• This modification makes media changes easy, and prevents the sinking
of tissue into liquefied plasma.

22. Grid Method
• Initially devised by Trowell in 1954, the grid method utilizes 25 mm
x 25 mm pieces of a suitable wire mesh or perforated stainless steel
sheet.
• Skeletal tissues are generally placed directly on the grid but softer
tissues like glands or skin are first placed on rafts, which are then
kept on the grids.
• The grids themselves are placed in a culture chamber filled with fluid
medium up to the grid.
• The chamber is supplied with a mixture of O2 and CO2 to meet the
high O2 requirements of adult mammalian organs.

23. Organ Culture in Liquid Media
• The liquid media consist of all the ingredients except agar.
• When liquid media are used for organ culture, generally perforated
metal gauze or cellulose acetate or a raft of lens paper is used.
• These possibility provides support.

24. Primary explant culture
• It is developed by Harrison and carrel.
• In primary explant culture, a fragment of tissue is placed at a glass
(or plastic)–liquid interface, are simply allowed to attach to an
appropriate substrate, ( tissue attached to surface by plasma clot or
warming) where, after attachment, cell migration (outgrowth of
cell) is promoted in the plane of the solid substrate.
• Incubated with growth medium

25. EXPLANTATION TECHNIQUES
1. Slide or cover slip cultures
a) Single cover slip with plasma clot
b) Double cover slip with plasma clot
c) Single cover slip with liquid medium
2. Carrel flask cultures
3. Roller test tube cultures

26. 1. Slide or cover slip cultures
• Slides or cover slips are prepared by placing a fragment of
tissue (explantation) onto a cover slip, which is subsequently
inverted over the cavity of a depression slide.
• This is the oldest method of tissue culture and is still quite
widely used.
a) Single cover slip with plasma clot :
• Prepare medium in two parts, one containing 50% plasma in
BSS (balanced salt solution) and the other containing 50%
embryo extract in serum.
• Under aseptic conditions, using a capillary pipette, place 1
drop of plasma containing solution In the centre of cover slip.

27. Single cover slip with plasma clot

28. • Transfer a fragment of tissue (explant) to this drop without crushing
the tissue.
• Add the embryo extract containing solution and mix thoroughly
before clotting starts and then locate the explant.
• Place two small spots of petroleum jelly near the concavity of a
depression slide and invert the slide over the cover slip, apply gentle
pressure so that jelly sticks to cover slip.
• Allow culture medium to clot.
• Turn over the slide and seal the margins of cover slip with paraffin.
• Label and incubate at 37 c.

29. b) Double cover slip with plasma clot :
• Resembles single cover slip method.
• A small drop of BSS is placed on a large coverslip (40mm).
• A square or round cover slip (22mm) is placed over BSS in the centre of
large cover slip.
• Place the explant over the round cover slip.
• The entire preparation is attached to a large depression slide by petrolleum
jelly and wax in such a way that the small cover slip is not in contact with
the slide at any point.
Top View Side View
Maximov's Double Coverslip Method for Preparation of Slide Cultures

30. c) Single cover slip with liquid medium :
• Suitably prepared explants are placed in culture medium in a
watch glass.
• The explants are drawn into the tip of a capillary pipette, and
one explant is deposited in the centre of coverslip.
• The liquid medium can be spread out in a very thin circular
film with the explant protruding above the surface.
• A depression slide with petroleum jelly is applied immediately
and preparation turned over with a quick flip to prevent the
fluid from running out of the crevice between the slide and the
coverslip.
• The coverslip is sealed and the slide incubated at 37 C,upright
or inverted; the tissue grows on the coverglass.

31. 2.Flask cultures
• The main use of flask cultures is in the establishment of a strain from
fresh explants of tissue.
• A good Carrel Flask has excellent optical properties for microscopic
examination.
Advantages:
(i) tissue can be maintained in the same flask for months or even years;
(ii) large number of cultures can be easily prepared and large amount of
tissue can be maintained for a considerable period of time.
There are two types of flask techniques:
(i) Thick clot culture, which allow rapid growth suitable for short-term
cultures and
(ii) Thin clot culture, which can be maintained for a considerable period
of time.

32. (i) Place a drop of plasma on the floor of carrel flask and spread this plasma out
in a circle with the help of spatula,
(ii) transfer the desired number of explants to the plasma and allow clotting to
occur;
(iii) after the plasma clots and explants fixed in position, add extra medium; for
thick clots 1.2ml of dilute plasma and for thin clots 1.2ml of dilute serum ; the
whole thing is left for clotting;
(v) The flasks are gassed with gas phase (5% CO2 in air).

33. 3.Roller test tube cultures
• The feeding ,patching and transfer of culture is done as in
other primary explantation techniques.
• This technique is used for preparing a large number of
cultures, which can be placed in a stationary racks or roller
drums.
• Cultures on plasma clots in test tube are prepared just like
those in flasks, but tissues may be grown on the wall of test
tube without a plasma clot.

34. Cell culture
• Cell culture refers to cultures derived from dissociated cells
taken from the original tissue .
• Cell culture implies that the tissue, or outgrowth from the
primary explant, is dispersed (mechanically or enzymatically)
into a cell suspension, which may then be cultured as a
monolayer on a solid substrate or as a suspension in the culture
medium
• These cultures have lost their histotypic architecture.
Advantages
– Development of a cell line over several generations
Disadvantages
– Cells may lose some differentiated characteristics.

35. Reference:
• Culture of Animal Cells - John Wiley & Sons
• Basic Principles of Cell Culture - R. Ian Freshney
• Principles of Animal cell culture - Basant Kumar Sinha, Rinesh
Kumar
• Handbook for cell culture basics (Gibco)
• Textbook Of Pharmaceutical Biotechnology - Chandrakant
Kokate, Pramod H.J, SS Jalalpure

36. Thank you