ANIMAL CELL CULTURE FRESHNEY PDF

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Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Sixth Edition. Author(s). R. Ian Freshney. Culture of Animal Cells. SIXTH EDITION. A Manual of Basic Technique and Specialized Applications. R. IAN FRESHNEY. #WILEY-BLACKWELL. WWW. Culture of Animal Cells: A Manual of Basic Technique, Fifth Edition, by R. Ian Freshney. Copyright and transfer it to the tissue culture laboratory in dissection. BSS (DBSS) or choice [Freshney, (colon carcinoma);Speirs etal.


Animal Cell Culture Freshney Pdf

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Since the publication of the sixth edition of this benchmark text, numerous advances in the field have been made - particularly in stem cells, 3D culture, scale-up. Culture of human stem cells / editors, R. Ian Freshney, Glyn N. Stacey, . technique is to be found in Freshney (), Culture of Animal Cells. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (6th ed.) by R. Ian Freshney. Read online.

Grander, D. Oncol, 15, Culture of immortalized cells. Courtenay, V. Cancer, 38, Freshney, M. In Culture of hematopoietic cells ed.

Basic Principles of Cell Culture

Freshney, I. Pragnell, and M. Wiley-Liss, New York p.

Vlodavsky, I. Cell, 19, Nicosia, R. In Vitro, 26, Ham, R.

In Vitro, 14, Jacobs, J. Green, H. Cell, 1, Macpherson, I.

Culture of Animal Cells (6th ed.)

Virology, 16, Gaush, C. L, and Smith, T. Graham, F. L, Smiley, J. Virol, 36, Knowles, B. Gey, G. Puck, T. USA, 41, Gluzman, Y. Cell, 23, Fogh, J. Cancer Inst, 58, Soule, H. Giard, D. Cancer Inst. Greene, L A. USA, 73, Gallagher, R. Blood, 54, Gillis, S.

Andersson, L.

Cancer, 23, Moore, G. Dulbecco, R. Virology, 8, USA, 53, Barnes, D. Leibovitz, A. Peehl, D. In Vitro, 16, Stacey, G. Cell contamination leads to inaccurate data: we must take action now. Nature, However, there are many applications in which large numbers of cells are required, for example extraction of a cellular constituent cells can provide 7 mg DNA ; to produce viruses for vaccine production typically 5 x cells per batch or other cell products interferon, plasminogen activator, interleukins, hormones, enzymes, erythropoietin, and antibodies ; and to produce inocula for even larger cultures.

Animal cell culture is a widely used production process in biotechnology, with systems in operation at scales over litres. This has been achieved by graduating from multiples of small cultures, -an approach which is tedious, labour-intensive, and expensive, to the use of large 'unit process' systems.

Although unit processes are more cost-effective and efficient, achieving the necessary scale-up has required a series of modifications to overcome limiting factors such as oxygen limitation, shear damage, and metabolite toxicity. One of the aims of this chapter is to describe these limitations, indicate at what scale they are likely to occur, and suggest possible solutions. This theme has to be applied to cells that grow in suspension and also to those that will only grow when attached to a substrate anchorage-dependent cells.

Another aspect of scale-up that will be discussed is increasing unit cell density to fold by the use of cell immobilization and perfusion techniques. Free suspension culture offers the easiest means of scale-up because a 1 litre vessel is conceptually very similar to a litre vessel. The changes concern the degree of environmental control and the means of maintaining the correct physiological conditions for cell growth, rather than significantly altering vessel design.

Monolayer systems for anchorage-dependent cells are more difficult to scale-up in a single vessel, and consequently a wide range of diverse systems have evolved. In small scale cultures there is leeway for some error. If the culture fails it is a nuisance, but not necessarily a disaster. Large scale culture failure is not only more serious in terms of cost, but also the system demands that conditions are more critically met.

This section describes the factors that need to be considered as the culture size gets larger. It is far more difficult to get a reliable measure of cell viability because the methods employed either stress the cells or use a specific, and not necessarily typical, parameter of cell physiology. An additional difficulty is that in many culture systems the cells cannot be sampled most anchoragedependent cultures , or visually examined, and an indirect measurement has to be made.

Trypan blue 0. In the presence of serum, therefore, erythrocin B 0. Cells are counted in the standard manner using a haemocytometer.

Some caution should be used when interpreting results as the uptake of the dye is pHand concentration-dependent, and there are situations in which misleading results can be obtained. Two relevant examples are membrane leakiness caused by recent trypsinization and freezing and thawing in the presence of dimethyl sulfoxide. A colorimetric method using the MTT assay see Chapter 7 can be used both to measure viability after release of cytoplasmic contents into the medium from artificially lysed cells, and for microscopic visualization within the attached cell 1.

The most commonly used parameter is glucose utilization, but oxygen utilization, lactic or pyruvic acid production, or carbon dioxide production can also be used, as can the expression of a product, such as an enzyme. When cells are growing logarithmically, there is a very close correlation between nutrient utilization and cell numbers.

A method which is not so influenced by growth rate fluctuations is the lactate dehydrogenase LDH assay. The reaction is initiated by the addition of pyruvate 2. To measure viable cells a reverse assay can be performed by controlled lysis of the cells and measuring the increase in LDH. It is preferable to use borosilicate glass e. Pyrex because it is less alkaline than soda glass and withstands handling and autoclaving better.

Cells usually attach readily to glass but, if necessary, attachment may be augmented by various surface treatments see Section 3. In suspension culture, cell attachment has to be discouraged, and this is achieved by treatment of the culture vessel with a proprietary silicone preparation siliconization. Examples are Dow Corning which has to be baked on or dimethyldichlorosilane Repelcote, Hopkins and Williams which requires thorough washing of the vessel in distilled water to remove the trichloroethane solvent.

Complex systems use a combination of stainless steel and silicone tubing to connect various components of the system.

Silicone tubing is very permeable to gases, and loss of dissolved carbon dioxide can be a problem. It is also liable to rapid wear when used in a peristaltic pump. Thick-walled tubing with additional strengthening sleeve should be used. Custom-made connectors should be used to ensure good aseptic connection during process operation. These are available from all fermenter supply companies; see Appendix. Safe removal of samples of the culture at frequent intervals is essential.

An entry with a vaccine stopper through which a hypodermic syringe can be inserted provides a simple solution, but is only suitable for small cultures. The use of specialized sampling devices, also available from fermenter supply companies, is recommended.

These automatically enable the line to be cleared of static medium containing dead cells, thus avoiding the necessity of taking small initial samples which are then discarded, and increasing the chances of retaining sterility. Air filters are required for the entry and exit of gases. Even if continuous gassing is not used, one filter entry is usually needed to equilibrate pressure and for forced input or withdrawal of medium. The filters should have a 0.

It is also helpful in reducing cell attachment to glass by suppressing the action of serum in the attachment process. However, its most beneficial action is to protect cells from shear stress and bubble damage in stirred and sparged cultures, and it is especially effective in low serum or serum-free media. Shifts in pH during the initial stages of a culture create many problems, including a long lag phase and reduced yield.

Avoid using stationary phase cells as an inoculum since this will mean a long lag phase, or no growth at all.

Ideally, cells in the late logarithmic phase should be used. Always inoculate at a high enough cell density. Fibroblastic cells Fibroblastic or fibroblast-like cells are bipolar or multi-polar, have elongated shapes and grow attached to a substrate. Epithelial-like cells Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches.

Lymphoblast-like cells Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface. The levels of glucose and L-glutamine can influence cell growth, and correct levels for each cell line should be checked before attempting to put it into culture typical levels for glucose and L-glutamine are mM and 2 mM, respectively.

A range of inorganic ions, amino acids and vitamins are essential for cell survival and will usually be included in basal growth media from proprietary sources. Both oxygen and carbon dioxide are essential and are provided either as a mixture of CO2 and air supplied to the culture vessel or by sealing the vessel tightly to retain the CO2 produced by cell metabolism.

Aseptic technique Skill in aseptic technique is important to maintain sterility during media preparation and cell cultivation procedures. Furthermore, it is a vital component in ensuring operator protection from infectious agents that may be present in culture materials. Dedicate separate medium for each cell line. Because of the risks of contamination and cross-infection, cell culture in the virus diagnostic laboratory is best carried out in closed vessels, usually screw-capped tubes and flat-sided bottles.

WHO does not recommend the use of well plates for the isolation of polioviruses from stool specimens as this method is inappropriate to conditions encountered in many laboratories of the Global Polio Laboratory Network. Preparation of glassware Due to the difficulty of cleaning and recycling glassware to culture quality, many laboratories have resorted to using disposable cell culture plastic ware.

If a laboratory chooses to use glassware, however, it must ensure that all glassware is meticulously cleaned and sterilized so that cell cultures will not be affected by traces of proteinaceous material, detergent, pyrogens, water deposits and other residual materials which may get deposited on the glassware.

These detergents are easily rinsed from glassware without leaving residues. DO NOT use domestic dishwashing liquid detergent under any circumstances.

Periodically inspect brushes for wear to avoid scratching glass. Even the smallest residual amounts of cleansers, disinfectants or acids can affect the growth of cell cultures. If glassware is hazy, has a film or blotches are evident, then additional cleaning is required before use.

Chromic acid, however, is a hazardous substance, with safety and environmental concerns. If chromic acid must be used, follow all normal safety precautions for using concentrated acids and acid solutions. As with any other cleaning process, all cleaning solutions must be completely rinsed from the glassware through copious changes of tap water followed by several changes of distilled water. Selection of cell culture systems Many cell culture systems support the growth of polioviruses and other enteroviruses.

Regional reference laboratories RRL are advised to obtain cell cultures from the official collections. National poliomyelitis laboratories can in turn apply to their designated RRL for supplies of these cell lines. Preparation of cell culture systems Cells should be received with documented evidence for the key characteristics relating to the quality of cell cultures as described above. In handling cell cultures, laboratory personnel must be concerned not only with preventing microbial contamination of the cultures, but also with avoiding contamination of the working environment with cell culture materials.

All cultures must be considered potentially hazardous, whether inoculated or un-inoculated. After use all cultures and their fluids should be decontaminated by autoclaving.

Cross-contamination between different cell types, especially continuous cell lines, is an ever-present hazard. To avoid this, different cell lines should never be processed at the same time. All working areas should be thoroughly cleaned between the preparations of different cell types.

Cell culture media employed in virology can be divided into two main categories, growth media and maintenance media. After a monolayer has formed and prior to inoculation with virus, the growth medium is removed and replaced with maintenance medium. Fetal calf serum is the serum of choice: it is good for promoting cell growth and it lacks viral inhibitors.

If serum from other sources is used, it must be pre-tested for the presence of inhibitors to the viruses being studied. Isolation of cells Cells can be isolated from tissues for ex vivoculture in several ways. Cells can be easily purified from blood; however only the white cells are capable of growth in culture. Mononuclear cells can be released from soft tissues by enzymatic digestionwith enzymes such as collagenase, trypsin or protease, which break down the extracellular matrix.

Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are available for culture.

This method is known as explantculture. Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived from tumours, most primary cell cultures have limited lifespan. After a certain number of population doublings cells undergo the process of senescence and stop dividing, while generally retaining viability. An established or immortalised cell linehas acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene.

There are numerous well established cell lines representative of particular cell types. Culture Conditions Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured invariably consists of a suitable vessel containing a substrate or medium that supplies the essential nutrients amino acids, carbohydrates, vitamins, minerals , growth factors, hormones, and gases O2, CO2 , and regulates the physicochemical environment pH, osmotic pressure, temperature.

Most cells are anchorage dependent and must be cultured while attached to a solid or semi-solid substrate adherent or monolayer culture , while others can be grown floating in the culture medium suspension culture. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed.

Vlodavsky, I.

Cell, 19, Nicosia, R. In Vitro, 26, Ham, R. In Vitro, 14, Jacobs, J. Green, H. Cell, 1, Macpherson, I. Virology, 16, Gaush, C. L, and Smith, T.

Copyright information

Graham, F. L, Smiley, J. Virol, 36, Knowles, B. Gey, G. Puck, T. USA, 41, Gluzman, Y. Cell, 23, Fogh, J. Cancer Inst, 58, Soule, H. Giard, D. Cancer Inst. Greene, L A. USA, 73, Gallagher, R.

10 editions of this work

Blood, 54, Gillis, S. Andersson, L. Cancer, 23, Moore, G. Dulbecco, R. Virology, 8, USA, 53, Barnes, D. Leibovitz, A.

Peehl, D. In Vitro, 16, Stacey, G. Cell contamination leads to inaccurate data: we must take action now.

Nature, However, there are many applications in which large numbers of cells are required, for example extraction of a cellular constituent cells can provide 7 mg DNA ; to produce viruses for vaccine production typically 5 x cells per batch or other cell products interferon, plasminogen activator, interleukins, hormones, enzymes, erythropoietin, and antibodies ; and to produce inocula for even larger cultures.

Animal cell culture is a widely used production process in biotechnology, with systems in operation at scales over litres. This has been achieved by graduating from multiples of small cultures, -an approach which is tedious, labour-intensive, and expensive, to the use of large 'unit process' systems.

Although unit processes are more cost-effective and efficient, achieving the necessary scale-up has required a series of modifications to overcome limiting factors such as oxygen limitation, shear damage, and metabolite toxicity. One of the aims of this chapter is to describe these limitations, indicate at what scale they are likely to occur, and suggest possible solutions.

This theme has to be applied to cells that grow in suspension and also to those that will only grow when attached to a substrate anchorage-dependent cells. Another aspect of scale-up that will be discussed is increasing unit cell density to fold by the use of cell immobilization and perfusion techniques. Free suspension culture offers the easiest means of scale-up because a 1 litre vessel is conceptually very similar to a litre vessel.

The changes concern the degree of environmental control and the means of maintaining the correct physiological conditions for cell growth, rather than significantly altering vessel design. Monolayer systems for anchorage-dependent cells are more difficult to scale-up in a single vessel, and consequently a wide range of diverse systems have evolved. In small scale cultures there is leeway for some error. If the culture fails it is a nuisance, but not necessarily a disaster.

Large scale culture failure is not only more serious in terms of cost, but also the system demands that conditions are more critically met. This section describes the factors that need to be considered as the culture size gets larger.

It is far more difficult to get a reliable measure of cell viability because the methods employed either stress the cells or use a specific, and not necessarily typical, parameter of cell physiology. An additional difficulty is that in many culture systems the cells cannot be sampled most anchoragedependent cultures , or visually examined, and an indirect measurement has to be made.

Trypan blue 0. In the presence of serum, therefore, erythrocin B 0. Cells are counted in the standard manner using a haemocytometer. Some caution should be used when interpreting results as the uptake of the dye is pHand concentration-dependent, and there are situations in which misleading results can be obtained.

Two relevant examples are membrane leakiness caused by recent trypsinization and freezing and thawing in the presence of dimethyl sulfoxide. A colorimetric method using the MTT assay see Chapter 7 can be used both to measure viability after release of cytoplasmic contents into the medium from artificially lysed cells, and for microscopic visualization within the attached cell 1.

The most commonly used parameter is glucose utilization, but oxygen utilization, lactic or pyruvic acid production, or carbon dioxide production can also be used, as can the expression of a product, such as an enzyme.

When cells are growing logarithmically, there is a very close correlation between nutrient utilization and cell numbers. A method which is not so influenced by growth rate fluctuations is the lactate dehydrogenase LDH assay.

The reaction is initiated by the addition of pyruvate 2. To measure viable cells a reverse assay can be performed by controlled lysis of the cells and measuring the increase in LDH. It is preferable to use borosilicate glass e. Pyrex because it is less alkaline than soda glass and withstands handling and autoclaving better. Cells usually attach readily to glass but, if necessary, attachment may be augmented by various surface treatments see Section 3. In suspension culture, cell attachment has to be discouraged, and this is achieved by treatment of the culture vessel with a proprietary silicone preparation siliconization.

Examples are Dow Corning which has to be baked on or dimethyldichlorosilane Repelcote, Hopkins and Williams which requires thorough washing of the vessel in distilled water to remove the trichloroethane solvent. Complex systems use a combination of stainless steel and silicone tubing to connect various components of the system. Silicone tubing is very permeable to gases, and loss of dissolved carbon dioxide can be a problem. It is also liable to rapid wear when used in a peristaltic pump.

Thick-walled tubing with additional strengthening sleeve should be used. Custom-made connectors should be used to ensure good aseptic connection during process operation. These are available from all fermenter supply companies; see Appendix. Safe removal of samples of the culture at frequent intervals is essential. An entry with a vaccine stopper through which a hypodermic syringe can be inserted provides a simple solution, but is only suitable for small cultures.

The use of specialized sampling devices, also available from fermenter supply companies, is recommended. These automatically enable the line to be cleared of static medium containing dead cells, thus avoiding the necessity of taking small initial samples which are then discarded, and increasing the chances of retaining sterility.

Air filters are required for the entry and exit of gases. Even if continuous gassing is not used, one filter entry is usually needed to equilibrate pressure and for forced input or withdrawal of medium. The filters should have a 0.

It is also helpful in reducing cell attachment to glass by suppressing the action of serum in the attachment process. However, its most beneficial action is to protect cells from shear stress and bubble damage in stirred and sparged cultures, and it is especially effective in low serum or serum-free media. Shifts in pH during the initial stages of a culture create many problems, including a long lag phase and reduced yield. Avoid using stationary phase cells as an inoculum since this will mean a long lag phase, or no growth at all.

Ideally, cells in the late logarithmic phase should be used. Always inoculate at a high enough cell density. There is no set rule as to the minimum inoculum level below which cells will not grow, as this varies between cell lines and depends on the complexity of the medium being used. Find empirically the optimum stirring rate for a given culture vessel and cell line.

This could vary between r. A unit volume of medium is only capable of giving a finite yield of cells. Factors which affect the yield are: pH, oxygen limitation, accumulation of toxic products e. NH4 , nutrient limitation e. As soon as one of these factors comes into effect, the culture is finished and the remaining resources of the system are wasted.

The aim is, therefore, to delay the onset of any one factor until the accumulated effect causes cessation of growth, at which point the system has been maximally utilized. Simple ways of achieving this are: a better buffering system e. Hepes instead of bicarbonate , continuous gassing, generous headspace volume, enriched rather than basal media, with nutrient-sparing supplements such as non-essential amino acids or lactalbumin hydrolysate, perfusion loops through ultrafiltration membranes or dialysis tubing for detoxification 3 and oxygenation, and attention to culture and process design.Human diploid cells are almost unique in utilizing cystine heavily.

Page 1 of 1 Start over Page 1 of 1. Prepare medium to about pH 7. The medium in the chamber can be vigorously sparged to ensure oxygen saturation and other additions, such as sodium hydroxide for pH control, which would damage the cells if put directly into the culture, can be made.

A Manual of Basic Technique by R. Cultured animal cells are also used to determine the maximum permissible dosage of new drugs. Air filters are required for the entry and exit of gases. It is also used in drug screening and development and large scale manufacturing of biological compounds e.

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