History of animal cell culture, cell final

History of animal cell culture, cell

culture media and reagents, culture of mammalian cells, tissues

and organs

Submitted by:Dr. Vijayata

DEPARTMENT OF ANIMAL BIOTECHNOLOGY

History of animal cell culture

EVENT

1907 Harrison Frog embryo nerve fiber outgrowth in vitro. (cultivated frog nerve cells in a lymph clot held by the 'hanging drop' method andobserved 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/30s

Carrel & Ebeling, 1923

Subculture of fibroblastic cell lines

1925–1926

Strangeways & Fell

Differentiation in vitro in organ culture

1940s Keilova, 1948; Cruikshank & Lowbury, 1952

Introduction of the use of antibiotics in tissue culture (penicillin and streptomycin )

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 subculturesVirus 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 & Ephrussi

Cell fusion–somatic cell hybridization

1962 Macpherson & Stoker

Establishment and transformation of BHK21

1964 Klein smith & Pierce

Pluripotency of embryonal stem cells

1965 Ham Serum-free cloning of Chinese hamster cells

1967 Hoober & Cohen

Epidermal growth factor

1968 Stoker et al. Anchorage-independent cell proliferation

1969 Metcalf Colony formation in hematopoietic cells

1975 Kohler & Milstein

Hybridomas—monoclonal antibodies

1976 Illmensee & Mintz

Totipotency of embryonal stem cells

1976 Hayashi & Sato Growth factor-supplemented serum-free media

1977 Nelson-Rees & Flandermeyer

Confirmation of HeLa cell cross-contamination of many cell lines

1978 Ham & McKeehan MCDB-selective, serum-free media

1980–1987

Peehl & Ham, 1980; Hammond et al., 1984; Knedler & Ham, 1987

Development of many specialized cell lines

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; Collins & Kennedy, 1999

Development of laminar-flow cabinets

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

Cell culture media and reagents

• 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. )

Basal media

Serum-free

media

Reduced-serum media

The choice of culture media is dependent on the requirements of cells being cultured.

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.

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.

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

REAGENTS

The components or reagents of suitable culture media include:

Balanced salt solution

Buffers and chemicals

Cell dissociation

reagents

Supplements

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)

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

Supplements :

• L-glutamine• Glutamax™• Non-essential amino acids (NEAA)• Growth Factors and Hormones (e.g.:

insulin)• Vitamines (MEM Vitamin Solution)

• 2-Mercaptoethanol• Lipid Supplement• CHO Supplement• Antibiotics and Antimycotics

(Penicillin, streptomycin, gentamicin, amphotericin B)

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.

Culture of mammalian cells, tissues and organs

Cell culture

Explant culture

Organ culture

• Tissue culture is used as a generic term to include the in vitro cultivation of organs, tissues and cells.

• The ability to survive and grow tissues outside the body in an artificial environment.

• Tissue culture can be subdivided into three major categories

Tissue culture

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.

Organ culture techniques

1. Plasma clot method or Watch glass method2. Organ Culture on Agar (Agar gel method)i. Raft methodii. Grid method3. Organ Culture in Liquid Media

1. Plasma clot method or Watch glass method

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.

Dame Honor Fell and Robinson introduced plasma clot substrate originally to study development of avian limb bone rudiment.

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.

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.

• 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.

Organ Culture in Liquid Media

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

1. Slide or cover slip cultures

a) Single cover slip with plasma clotb) Double cover slip with plasma clotc) Single cover slip with liquid medium

2. Carrel flask cultures

3. Roller test tube cultures

EXPLANTATION TECHNIQUES

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.

Single cover slip with plasma clot

• 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.

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

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.

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.

(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).

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.

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.

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

Thank you