basics of animal cell culture

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Deals with definitions, requirements, types of culture, merits, demerits, limitations and applications

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Slide 1: 

Basics of Animal Cell Culture Mrs. Rani ashok Asst. Prof. of Zoology Lady Doak College, madurai – 2 Email: eaarani@rediffmail.com

Animal Cell Culture (ACC) : 

Animal Cell Culture (ACC) Process of culture of animal cells outside the tissue (in vitro) from which they were obtained Carried out under strict laboratory conditions of asepsis, sterility and controlled environment involving temperature, gases and pressure 1/28/2011 2 Rani Ashok, LDC, Madurai

Cell Culture : 

Cell Culture Merits Minimises animal use Optimizes growth pattern Enables control of the extracellular environment Allows monitoring of various elements and secretions without interference from other biological molecules 1/28/2011 3 Rani Ashok, LDC, Madurai

Cell Culture : 

Demerits Cells are devoid of in vivo interactive environment. Recreation of the in vivo environment is unattainable. Productions of unwanted proteins due to de-differentiation of cells in artificial condition Cell Culture 1/28/2011 4 Rani Ashok, LDC, Madurai

LIMITATIONS (From Freshney, 2005) : 

LIMITATIONS (From Freshney, 2005) Category Examples Necessary Expertise Environmental control Quantity and cost Sterile handling Chemical contamination Microbial Contamination Cross-contamination Workspace Incubation, pH control Containment and disposal of biohazards Capital equipment for scale-up Medium, serum Disposable plastics 1/28/2011 5 Rani Ashok, LDC, Madurai

LIMITATIONS (From Freshney, 2005) : 

LIMITATIONS (From Freshney, 2005) Category Examples Genetic instability Phenotypic instability Identification of cell type Heterogeneity, variability Dedifferentiation Adaptation Selective overgrowth Markers not always expressed Histology difficult to recreate and atypical Geometry and microenvironment change cytology 1/28/2011 6 Rani Ashok, LDC, Madurai

History, Scope & Terms associated with ACC : 

History, Scope & Terms associated with ACC 1/28/2011 7 Rani Ashok, LDC, Madurai

History : 

History 1880 - Arnold showed that leucocytes can divide outside the body 1885: Roux maintained embryonic chick cells in saline 1903 - Jolly studied the behavior of animal tissue explants immersed in serum, lymph, or ascites fluid. 1907 - Ross Granville Harrison cultured frog tadpole spinal chord in a lymph drop hanging from a cover slip of a cavity slide. 1913 - Carrel developed a complicated methodology for maintaining cultures free of contamination 1965: Harris & Watkins successfully fused human and mouse cells by virus 1975: Kohler & Milstein produced the first Hybridomas capable of secreting monoclonal antibodies. 1/28/2011 8 Rani Ashok, LDC, Madurai

Cell Culture - Scope : 

Cell Culture - Scope Indispensible technology study regulation of cell proliferation, differentiation and product formation under controlled conditions define entire human genome dissect the pathways of intracellular and intercellular signaling which ultimately regulate gene expression 1/28/2011 9 Rani Ashok, LDC, Madurai

Terms associated with cell culture : 

Terms associated with cell culture Split ratio- divisor of the dilution ratio of a cell culture at subculture Passage number- number of times that the culture has been cultured Generation number- number of doublings that a cell population has undergone 1/28/2011 10 Rani Ashok, LDC, Madurai

Culture … : 

Culture … Organ culture- culture of native tissue that retains most of the in vivo histological features Histotypic culture- culturing of the cells for their re-aggregation to form a tissue-like structure Organotypic culture- recombination of different cell types to form a more defined tissue or an organ 1/28/2011 11 Rani Ashok, LDC, Madurai

Confluency : 

Confluency How “covered” the growing surface appears Optimal confluency for moving cells to a new dish is 70-80% too low, cells will be in lag phase and won’t proliferate Too high and cells may undergo unfavorable changes and will be difficult to remove from plate. 1/28/2011 12 Rani Ashok, LDC, Madurai

Contact Inhibition : 

Contact Inhibition When cells contact each other, they cease their growth. Cells arrest in G0 phase of the cell cycle Transformed cells will continue to proliferate and pile upon each other 1/28/2011 13 Rani Ashok, LDC, Madurai

Anchorage Dependence : 

Anchorage Dependence Cells that attach to surfaces in vivo require a surface to attach to in vitro. Other cells or specially treated plastic or other biologically active coatings Blood cells are primary exception. Transformed cells may not require attachment 1/28/2011 14 Rani Ashok, LDC, Madurai

Substrates for Cell Growth : 

Substrates for Cell Growth Glass Plastic Metals Palladium Modification of Substrate Surface 1/28/2011 Rani Ashok, LDC, Madurai 15

Glass : 

Glass most commonly used substrate easily washed and readily sterilized optically clear to allow microscopic observation of cultures Glass made of alum-borosilicate (e.g. Pyrex) is preferred to soda-lime glass as soda-lime glass releases alkali into the medium. Soda-lime glass can be detoxified by boiling in weak acid, before use Slides, test tubes and flasks, used as substrates must be washed with non­toxic detergent 1/28/2011 Rani Ashok, LDC, Madurai 16

Plastic : 

Plastic nonautoclavable. Sterile substrates available are meant for single use. most commonly used plastic polystyrene.; Polyethylene,; polycarbonate,; perspex; polyvinyl chloride; teflon; Cellophane; cellulose acetate, etc. Thin teflon films are available as petri dishes or as membranes to be used as rafts. These films are permeable to O2 and CO2 and can be sectioned for microscopy. 1/28/2011 Rani Ashok, LDC, Madurai 17

Metals : 

Metals Stainless steel and titanium, being relatively chemically inert and negatively charged, are suitable for use as substrates. If the stainless steel is not of proper grade, it will release toxic ions. Stainless steel should be acid washed, to remove surface impurities. Palladium deposited on agarose is used as a substrate for growth of fibroblasts and glia. 1/28/2011 Rani Ashok, LDC, Madurai 18

Modification of Substrate Surface : 

Modification of Substrate Surface The efficiency of the cultured cells through increased cell attachment can be enhanced by treating the surface of the substrate with purified fibronectin or collagen. The chemical is poured onto the surface of the dish, excess chemical drained, and then dried and sterilized using UV light. The surface of the substrate is also treated with a monolayer of special types of cells. This layer is called feeder layer, since it feeds the growing cells. The cells used as feeder layer are glial cells, normal foetal intestine, mouse embryo, fibroblasts, etc. The feeder layer is used for the growth of neurons, epithelium of breast and coelom, and production of transgenic animals. 1/28/2011 Rani Ashok, LDC, Madurai 19

CULTURE MEDIA : 

CULTURE MEDIA Classification criteria Natural media Artificial media Serum medium Serum-free media 1/28/2011 20 Rani Ashok, LDC, Madurai

Culture Media … : 

Culture Media … Classified based on Source – Natural/ Artificial Composition – Defined/ Undefined Consistency – Solid, Semi Solid, Liquid Serum content – Serum and Serum-free media Special Need 1/28/2011 Rani Ashok, LDC, Madurai 21

Media : 

Media Artificial environment Appropriate source of energy for the cells which they can easily utilize and compounds which regulate the cell cycle May or May not contain serum 1/28/2011 22 Rani Ashok, LDC, Madurai

Serum : 

Serum Advantages of serum in culture medium Disadvantages of serum in culture medium Serum binds and neutralizes toxins Serum contains a complete set of essential growth factors, hormones, attachment and spreading factors, binding and transport proteins It contains the protease inhibitors It increases the buffering capacity It provides trace elements. It is not chemically defined and therefore it’s composition varies a lot It is sometimes source of contamination by viruses, mycoplasma, prions etc. It increases the difficulties and cost of down stream processing It is the most expensive component of the culture medium. 1/28/2011 23 Rani Ashok, LDC, Madurai

Serum media : 

Serum media Fetal Bovine Serum/ Fetal calf serum Equine Serum 1/28/2011 24 Rani Ashok, LDC, Madurai

Fetal Bovine Serum : 

Fetal Bovine Serum By-product of meat industry Produced from blood collected bovine fetus at the abattoir (Slaughter houses). Collected after the pregnant uterus has been removed from the slaughtered cow. 1/28/2011 25 Rani Ashok, LDC, Madurai

Fetal Bovine Serum Contd… : 

Fetal Bovine Serum Contd… Serum is collected as early as 5 minutes after slaughter or as long as 25–40 minutes after, at which point the fetuses may still be alive. Invasive procedures, involving insertion of a 12–16 gauge needle between the 4th and 5th rib of the fetal heart are often employed. Most widely used serum for cell culture 1/28/2011 26 Rani Ashok, LDC, Madurai

Fetal Bovine Serum Contd… : 

Fetal Bovine Serum Contd… Low in antibodies Contains more growth factors The globular protein, bovine serum albumin (BSA) is a major component of fetal bovine serum. The rich variety of proteins in fetal bovine serum maintains cultured cells in a medium in which they can survive, grow, and divide. 1/28/2011 27 Rani Ashok, LDC, Madurai

Fetal Bovine Serum Contd… : 

Fetal Bovine Serum Contd… Contains more growth factors The globular protein, bovine serum albumin (BSA) is a major component of fetal bovine serum. The rich variety of proteins in fetal bovine serum maintains cultured cells in a medium in which they can survive, grow, and divide. 1/28/2011 28 Rani Ashok, LDC, Madurai

Serum – Free Media : 

Serum – Free Media Serum-free media are media designed to grow a specific cell type or perform a specific application in the absence of serum. The use of serum-free media (SFM) represents an important tool, that allows cell culture to be done with a defined set of conditions as free as possible of confounding variables. 1/28/2011 29 Rani Ashok, LDC, Madurai

Serum-free media … : 

Serum-free media … Serum-free cell culture media allow users to standardize their cell culture conditions by avoiding the use of undefined and highly variable serum products derived from humans or animals, e.g. human AB serum or fetal calf serum (FCS). Contains less then 30µg protein/ml such as albumin, fibronectin and fetuin serve a variety of functions that include adsorbing toxic compounds, providing protection against shear forces in bioreactors, creating a matrix for cellular attachment to surfaces, and acting as a carrier for lipids and other growth factors. 1/28/2011 30 Rani Ashok, LDC, Madurai

Advantages of using serum-free media : 

Advantages of using serum-free media Increased definition. More consistent performance. Easier purification and downstream processing. Precise evaluations of cellular function. Increased growth and/or productivity. Better control(s) over physiological responsiveness. Enhanced detection of cellular mediators. 1/28/2011 31 Rani Ashok, LDC, Madurai

BIOLOGY OF CULTURED CELLS : 

BIOLOGY OF CULTURED CELLS CELL GROWTH KINETICS PRIMARY CULTURE SUBCULTURE DEVELOPMENT OF CELL LINES 1/28/2011 32 Rani Ashok, LDC, Madurai

Growth Kinetics : 

Growth Kinetics Growth patterns and kinetics in batch culture - growth phases In batch culture: - lag phase - logrithmic or exponential growth phase - deceleration phase - stationary phase - death phase 1/28/2011 33 Rani Ashok, LDC, Madurai

Slide 34: 

Typical growth curve for a bacterial population 1/28/2011 34 Rani Ashok, LDC, Madurai

Classification of Cell Cultures : 

Classification of Cell Cultures Primary Culture Cells taken directly from a tissue to a dish Secondary Culture Cells taken from a primary culture and passed or divided in vitro. These cells have a limited number of divisions or passages. After the limit, they will undergo apoptosis. Apoptosis is programmed cell death 1/28/2011 35 Rani Ashok, LDC, Madurai

Cell Lines : 

Cell Line Cells that have undergone a mutation and becomes immortal (won’t undergo apoptosis after a limited number of passages) i.e. Attain indefinite growth. Transformed cell line A cell line that has been transformed by a tumor inducing virus or chemical that has the ability to cause tumors if injected into animal. Hybrid cell line (hybridoma) Fused cells with two different characteristics Cell Lines 1/28/2011 36 Rani Ashok, LDC, Madurai

Slide 37: 

1/28/2011 37 Rani Ashok, LDC, Madurai Primary cell culture

Disaggregation of cells : 

Disaggregation of cells Cells can be allowed to migrate out from an explant Mechanical dissociation (mincing) Enzymatic dissociation Exception – hematopoietic cells do not need to be disaggregated, they already are 1/28/2011 38 Rani Ashok, LDC, Madurai

Enzymatic Disaggregation : 

Enzymatic Disaggregation The two important enzymes used in tissue disaggregation are collagenase and trypsin. CollagenaseIt is used for disaggregation of embryonic, normal and malignant tissues, by digesting the collagen of intracellular matrix TrypsinUse of trypsin for disaggregation of tissue is called trypsinization. It may be of two types:1. Warm trypsinization and 2. Cold trypsinization. 1/28/2011 Rani Ashok, LDC, Madurai 39

Disadvantages of Cell Culture : 

Disadvantages of Cell Culture Contamination can be Chemical (culture medium) Or Biological (adding antibiotics). Finding a Happy Environment. De-differentiation Origin of Cells Major differences from in-vivo(loss of ECM) 1/28/2011 40 Rani Ashok, LDC, Madurai

TYPES OF CULTURE METHODS : 

TYPES OF CULTURE METHODS 1/28/2011 41 Rani Ashok, LDC, Madurai

TYPES OF CULTURE : 

TYPES OF CULTURE From Freshney, 2005 1/28/2011 42 Rani Ashok, LDC, Madurai

Slide 43: 

1/28/2011 Rani Ashok, LDC, Madurai 43

APPLICATIONS OF CELL & TISSUE CULTURE : 

APPLICATIONS OF CELL & TISSUE CULTURE 1/28/2011 44 Rani Ashok, LDC, Madurai

Cell culture is used for … : 

Cell culture is used for … Investigation - the normal physiology and biochemistry of cells. Study - the effect of various chemicals or drugs on specific cell types (cytotoxicity tests). Large scale production - of valuable biologicals "vaccines and antibodies". Use of tissue cultures to generate - artificial tissues “biotechnology or tissue engineering". 1/28/2011 45 Rani Ashok, LDC, Madurai

Cell culture tests can be designed to evaluate various effects : 

Cell culture tests can be designed to evaluate various effects Reduced growth rate. Breakdown of membrane permeability Tissue specificity of response. Ability to metabolize toxic compounds. Genetic effects/mutagenicity. 1/28/2011 46 Rani Ashok, LDC, Madurai

Slide 47: 

Cell-cell interaction Embryonic induction, Cell proliferation, Contact inhibition Cell products Product formation, Exocytosis, Secretion Genetics Genetic analysis, genetic manipulation, Transformation, Immobilization Intracellular activity Transcription, Protein synthesis, Energy metabolism, Drug metabolism Intracellular flux RNA, Hormones, Metabolites, Signal transduction, Membrane trafficking Environmental Interaction Infection, Drug action, Ligand receptor interactions, Mutagenesis, Carcinogenesis AREAS OF MAJOR INTEREST IN TISSUE CULTURE Immunology Cell surface Receptors, Hybridoma, cytokines 1/28/2011 47 Rani Ashok, LDC, Madurai

Slide 48: 

Thank You 1/28/2011 48 Rani Ashok, LDC, Madurai