Tissue culture was first devised at the beginning of the twentieth century [as a method for studying the behavior of animal cells free of systemic variations that might arise in vivo both during normal homeostasis and under the stress of an experiment
including recombinant protein producing lines of Chinese Hamster Ovary (CHO), various hybridoma cell lines, the insect lines Sf9 and Sf21 (Spodopterafrugiperda), and for cell lines that act as hosts for viral production (e.g., 293, VERO, MDCK, MDBK), and others.
Solutions most commonly include sodium, potassium, calcium, magnesium, and chloride. Used for washing tissues and cells and are usually combined with other agents to treat the tissues and cells.
Bicarbonate buffered media requires CO2 atmosphere HEPES Strong chemical buffer range pH 7.2 – 7.6 (does not require CO2)Trypsin 0.05% - EDTA 0.53 mM (1X), liquidTrypsin 0.5% - EDTA 5.3 mM (10X), liquidTrypsin 0.25% - EDTA 1 mM (1X), liquid
Originally, the term is not limited to animal cells, but includes the in vitro cultivation of plant cells.
Microscopic anatomy of cells and tissue, There are a variety of platforms used to facilitate the growth of 3 dimensional cellular structures such as nanoparticle facilitated magnetic levitation, gel matrices scaffolds, and hanging drop platesTissue at gas-liquid interfaceOrgans cannot be propagated so each piece of tissue can only be used once, which makes it difficult to assess the reproducibility of a response.
Raft or lens can be usedThe raft makes easy to transfer the tissue, excess fluid can also be removed.
In this approach the When agar is used in medium, no extra mechanical support is required. Agar does not allow to liquefy the support. The tumours obtained from adults fail to survive on agar media, whereas embryonic organs grow well.
A modification of the original grid method is widely used to study the growth and differentiation of adult and embryonic tissues.
Incubate with growth medium•Cells migrate, multiply and form primary monolayer, Used for isolation of virus in the ‘latent’ state. Eg. Adenoid tissue explant culture for adenovirusTissue at solid-Liquid interface,Cell migrate to form outgrowth the cells migrating out are known as the outgrowthAdvantage- for isolation of virus in the ‘latent’ state
Tissue at solid-Liquid interface,Cell migrate to form outgrowthAdvantage- for isolation of virus in the ‘latent’ state
place upto six D3.5 Carrel Flasks in a rack with their necks flamed andpointing to the right; even though polystyrene culture flasks can also be used provided they have a wide neck for handling the explants.
Disaggregated tissue;Cells form monolayer at solid-liquid interfaceCell culture refers to cultures derived from dissociated cells taken from the original tissue ('primary cell culture'). Cells are dispersed (mechanically and/or enzymatically) into a cell suspension
History of animal cell culture, cell final
DEPARTMENT OF ANIMAL BIOTECHNOLOGY History of animal cellculture, cell culture media and reagents, culture of mammalian cells, tissues and organs Submitted by: Dr. Vijayata
EVENT1907 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 months1916 Rous & Jones Trypsinization and subculture of explants1920s Carrel & Subculture of fibroblastic cell lines/30s Ebeling, 19231925– Strangeways & Differentiation in vitro in organ culture1926 Fell1940s Keilova, 1948; Introduction of the use of antibiotics in tissue Cruikshank & culture (penicillin and streptomycin ) Lowbury, 1952
1943 Earle Establishment of the L-cell mouse fibroblast cell line; first continuous cell line1949 Enders Growth of virus in cell culture1952 Dulbecco Use of trypsin for generation of replicate subcultures Virus plaque assay1952 Gey Establishment the first human cell line, HeLa, from a cervical carcinoma,1955 Eagle Development of defined media1959 Puck & Marcus Cloning of HeLa on a homologous feeder layer1961 Sorieul & Cell fusion–somatic cell hybridization Ephrussi
1962 Macpherson & Establishment and transformation of BHK21 Stoker1964 Klein smith & Pluripotency of embryonal stem cells Pierce1965 Ham Serum-free cloning of Chinese hamster cells1967 Hoober & Epidermal growth factor Cohen1968 Stoker et al. Anchorage-independent cell proliferation1969 Metcalf Colony formation in hematopoietic cells1975 Kohler & Hybridomas—monoclonal antibodies Milstein1976 Illmensee & Totipotency of embryonal stem cells Mintz1976 Hayashi & Sato Growth factor-supplemented serum-free media1977 Nelson-Rees & Confirmation of HeLa cell cross- Flandermeyer contamination of many cell lines
1978 Ham & McKeehan MCDB-selective, serum-free media1980– Peehl & Ham, 1980; Development of many specialized cell lines1987 Hammond et al., 1984; Knedler & Ham, 19871982 Darnell, Regulation of gene expression1983 Evans Regulation of cell cycle1984 Collen Production of recombinant tissue-type plasminogen activator in mammalian cells1989 Weinberg Oncogenes , malignancy, and transformation1990s Kruse et al., 1991; Development of laminar-flow cabinets Collins & Kennedy, 19991991 Griffiths Salk polio vaccine grown in monkey kidney cells1998 Thomson et al., Culture of human embryonic stem cells2000+ Dennis et al., 2001 Human Genome Project: genomics, proteomics, genetic deficiencies and expression errors
• 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 Basalmedia is dependent on the mediarequirements of cellsbeing cultured. Reduced- Serum- serum free media media
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 enrichedwith nutrients and animal-derived factors, which reduce the amountof 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.
REAGENTSThe components or reagents of suitable culture media include: Cell Balanced Buffers and dissociation Supplements salt solution chemicals reagents
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 (Dulbeccos Phosphate-Buffered Salines) HBSS (Hanks Balanced Salt Solutions) PBS (Phosphate-Buffered Salines) EBSS (Earles Balanced Salt Solutions)
Buffers and chemicals :• HEPES (4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid )• Sodium Bicarbonate solutionCell 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 • 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.
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
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 techniques1. 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 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 extractwith 15 drops of plasma in a watch glass placed on a cottonwool 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.
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 supportRaft 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.
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.
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
EXPLANTATION TECHNIQUES1. Slide or cover slip culturesa) Single cover slip with plasma clotb) Double cover slip with plasma clotc) Single cover slip with liquid medium2. Carrel flask cultures3. Roller test tube cultures
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.
• 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 Maximovs 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; forthick 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 generationsDisadvantages – 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