animal cell culture

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Animal Cell Technology- Challenges for 21st Century:

Animal Cell Technology- Challenges for 21 st Century BY -- PALLAV SINGH ( pallav.s1987@gmail.com )

What is Animal Cell Technology ? :

What is Animal Cell Technology ? discipline of cell biology- aims to understand structures, functions and behaviors of differentiated animal cells. also to ascertain their abilities to be used in industrial and medical purposes. goal- accomplishments of clonal expansion of differentiated cells with useful ability, optimization of their culture conditions, modulation of their ability to produce medically and pharmaceutically important proteins & the application of animal cells to gene therapy and artificial organs.

History:

History backs almost 100 years ago. Ross Harrison (1907)- frog embryo nerve fiber outgrowth in vitro. Carrel(1912)- explants of chick connective tissue, heart muscle contractile for 2-3 months. Rous & Jones(1916)- trypsinization and subculture of explants. Keilova (1948)- use of antibiotics in tissue culture. Gey et al. (1952)- First Human cell line HeLa established. Eagle(1955)- development of defined media.

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Sorieul & Ephrussi (1961)- Cell fusion; somatic cell hybridization. Kleinsmith & Pierce (1964)- Pluripotency of embryonal stem cells. Wiktor (1964)- Rabies, Rubella vaccines in WI-38 human lung fibroblasts. Raham & Van der Eb (1973)- DNA transfer- calcium phosphate. Kohler & Milstein (1975)- Hydridomas -monoclonal antibodies. Rheinwald & Green (1975)- Skin culture. Ham & McKeehan (1978) -Serum free media. Butler(1991)- Industrial scale culture of transfected cells for biopharmaceuticals. Freshney (2004)- Exploitation of tissue engineering.

Types of Tissue Culture:

Types of Tissue Culture Organ Culture Cell Culture Histotypic Culture Organotypic Culture

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TYPE OF TISSUE CULTURE

Primary Culture :

Primary Culture cells that are placed in culture directly from the tissue of origin. these are called primary cultures until the first subculture. Isolation of tissues-Mechanical & Enzymatic Mechanical methods- sieving, syringing, vigorous pipetting Enzymatic methods- warm trypsin, cold trypsin & collagenase treatment

Epithelial Cell -Primary Culture:

Epithelial Cell -Primary Culture

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TYPES OF PRIMARY CELL CULTURE Mouse embryos Chick embryos Human biopsy materials Transplantable animal tumour Chick embryo organ rudiments (brain, heart, lungs, liver, gizzard, kidney, spinal cord, skin, muscle) ISOLATION OF TISSUES Must comply with local legislation and medical ethical rules. Sterilize the site with 70% alcohol. Remove tissue aseptically. Transfer to the laboratory in transport medium If delay in transporting to lab, keep at 4C for up to 72hour.

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PRIMARY CULTURE Is a stage of the culture after first isolation of the cells but before the first subculture. 4 stages: 1) acquisition of samples, 2) isolation of tissues, 3) disaggregation, 4) culture seeding into culture vessel. After isolation, a primary cell culture is obtained by allowing cells to migrate out from the fragment of tissue adhering to a suitable substrate by disaggregation. PRIMARY EXPLANT Suitable for small amount of tissues example skin biopsy. Attachment on the substrate by using plasma clots, or fibrinogen and trombin. Disaggregation by mechanical and enzymatic.

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Mouse, mammals, Embryo Eggs (best: for TC : embryo, young) because stage of differentiation) organ explant Grow in media Explants Explants with outgrowth Primary explant Finely cut Finely cut tissue or explant

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Mouse, mammals, Embryo Embryonated Eggs (best: for TC : embryo, young) because stage of differentiation) organ explant Grow in media -monolayer -suspension cells Cell culture Finely cut Finely cut tissue or explant Enzymic digestion

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Primary Explant Culture

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Enzymatic disaggregation Warm trypsin, 37˚C for 30 mins, cell damaged if too long exposure. Cold preexposure, soak at 4C overnight and 37C for less 30 mins. Advantage: higher yield of viable cells, preserve more cell types Other enzyme -collagenase benefit for connective tissues and muscle (fibrous tissue) - pronase, dipase, DNase, hyaluronidase Mechanical disaggregation (prevent proteolytic damage) Scrapping or spillage Sieving Syringes Trituration by pipette

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Warm trypsin disaggregation

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Add to stirrer flask Collect the supernate containing cells, centrifuge, resuspend in medium, and store on ice Cold trypsin disaggregation

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Mechanical disaggregation

Established Cell Lines :

Established Cell Lines After the first subculture, primary culture may be called secondary cultures, and thereafter, if continued passage is possible, a cell line. An established or immortalised cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene. These cell lines have been the workhorses of cell culture, from their use in studying the control of the cell cycle to vaccine production and large-scale industrial production of recombinant proteins.

Examples of Established cell lines:

Examples of Established cell lines May be derived from Normal or Tumor cells. Cell line Organism Origin Tissue HeLa Human Cervical cancer 293-T Human Kidney (embryonic) A-549 Human Lung carcinoma ALC Murine Bone marrow CHO Hamster Ovary HB54 Hybridoma Hybridoma FM3 Human Metastatic lymph node

HeLa-Established Cell line:

HeLa-Established Cell line

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Mouse kidney cells Secondary Hamster kidney cells Primary cell cultures split several times:

Physical Environment for Culture:

Physical Environment for Culture the aim is to provide an environment that mimics, to the greatest extent possible, the in vivo environment of that specific cell type. the cell culture incubator, the culture dish or apparatus, and the medium together create this environment in vitro. provides an appropriate temperature, pH, oxygen, and CO 2 supply, surface for cell attachment, nutrient and vitamin supply, protection from toxic agents, the hormones and growth factors that control the cell's state of growth and differentiation.

Media:

Media Support survival and growth Natural Media Clots- Plasma clots (male fowl) Biological Fluids- Amniotic, Ascitic fluid, serum Tissue extracts- Chicken and bovine embryo extract Artificial Media Defined & Complex media Immediate Survival Prolonged Survival Indefinite growth Specialized function Classes Serum containing media Serum free media Chemically defined Media Protein free media

Serum Containing Media:

Serum Containing Media MEM, DMEM, M199, F12, DMEM/F12,etc EMEM with 5-20% serum Provide plasma protein, peptides, lipids carbohydrates, minerals and enzyme Hormones (cortisone, insulin and testosterone and prostaglandin) Growth factors (PDGF, TGF-p, epidermal growth factors etc) Supply protein ( fibronectin , spreading factor Binding factors (albumin, transferrin ) Increase viscosity of medium Protease inhibitor Buffer Minerals (Na, K, Fe, Zn, and Cu etc)

Disadvantages of using serum:

Disadvantages of using serum Serum may inhibit growth of some cell types, e.g., epidermal keratinocytes . 2. Serum may contain some cytotoxic or potentially cytotoxicconstituents . For example, foetal calf serum contains the enzyme polyamine oxidase which converts polyamines like spermidine and sperrnine (secreted by fast growing cells) into cytotoxic polyaminoaldehydes . 3. There is a large variation in serum quality from one batch to another ; this requires costly and time consuming testing every time a new batch has to be used . 4. Some growth factors may be inadequate for specific cell types and may need supplementation . 5. It interferes with downstream processing when cell cultures are used for production of biochemicals . 6. The supply of serum is always lower than its demand.

Serum Free Media:

Serum Free Media Analytical approach Synthetic approach Limiting factor approach Defined Media EMEM, DME, Ham,s F12, CMRL 1066, RPMI 1640, Iscove,s modified Dulbecco,s medium

Serum-Free Media -advantages:

Serum-Free Media -advantages 1 . Improved reproducibility of results from different laboratories and over time since variation due to batch change of serum is avoided. 2. Easier downstream processing of products from cultured cells. 3. Toxic effects of serum are avoided. 4. Biassays are free from interference due to serum proteins. 5. There is no danger of degradation of sensitive protein by serum proteases. 6. They permit selective culture of differentiated and producing cell types from the heterogenous cultures.

Serum-Free Media - disadvantages:

Serum-Free Media - disadvantages 1. Most serum-free media are specific to one cell type . Therefore, different media may be required for different cell lines. 2. Reliable serum-free preparations, for most of the media formulations are not available commercially. This necessitates time consuming task of preparing the desired formulations in the laboratory. 3. A greater control of pH , temperature etc. is necessary as compared to that with serum containing media. 4. Growth rate and the maximum cell density attained are lower than those with serum containing media. 5. Cells tend to become fragile during prolonged agitated cultures unless biopolymers or synthetic polymers are added.

Preparation and Sterilization of Medium:

Preparation and Sterilization of Medium The various media constituents and other reagents used in cell cultures must be carefully sterilized either by autoclaving or by filtration. Heat stable constituents tike water, salts, supplements like peptone or tryptose etc. are autoclaved at 121°C for 20 min. But heat labile constituents like serum, trypsin, proteins, growth factors etc. must be sterilized by filtration through a 0.2 mm porosity membrane filter. Each filtrate should be tested for sterility to avoid failure due to contamination. In case of soda glass, caps should be left slack to avoid breaking during autoclaving. Autoclaving is preferred to filtration since it is cheaper, needs less labour and is uniformly effective. Therefore wherever possible, autoclaving should be resorted to and autoclavable versions of the media should be used. Most of the media, however, now available commercially are usually presterilized. .

Selection of Media:

Selection of Media Cell type Medium Supplemented with Permanent cell line MEM. DME, McCDoy,5a, RPMI1640 Serum or protein Permanent cell line F10, F12,DME Purified proteins and hormones Permanent cell line in monolayer CMRL 1066, MCDB 411, DME, IMDM Clonal Growth of permanent cell lines F12, MCDB 301 etc

Subculturing :

Subculturing Subculturing or "splitting cells," is required to periodically provide fresh nutrients and growing space for continuously growing cell lines. The frequency of subculture and the split ratio, or density of cells plated depend on the characteristics of each cell line being carried. Subculturing - Adherent Cells Suspension culture.

Growth Curve:

Growth Curve

MEASURING PARAMETERS OF GROWTH:

MEASURING PARAMETERS OF GROWTH increasing the number of cells increasing the size of the cells increasing the amount of intercellular substance. Cell counting Hemacytometer Electronic Particle Counter Cell viability assay Measurement of DNA amount RNA amount Protein amount

Cloning :

Cloning The purpose of cloning is to assure that all cells in the culture are descended from a single cell, that is, genetically identical. This prevents the rapid and unpredictable changes in culture phenotype that may occur in mixed cell populations when conditions change to favor one cell type over another. Recloning established cell lines will frequently give a line with more consistent characteristics. If one wishes to change the cell's properties through mutation or transfection , cloning is necessary to assure that all cells in the study population are similar.

Freezing and Thawing Cells :

Freezing and Thawing Cells provides a backup in case cells are lost due to contamination, carelessness, equipment failure, or a natural disaster. Some types of primary cultures can be prepared in large batches, a large number of vials frozen, and the cells thawed sequentially and studied as secondary cultures so that a large number of experiments can be performed on early passage cells from the same preparation. Alternatively, as one tries to establish a cell line, a few vials of the cells should be frozen every three to five passages to have a permanent record of any changes that may occur with passage number. For normal human cells, which have a limited life span in vitro, expansion and freezing of an early passage bank is the only method that allows similar cells to be used in many different laboratories. Whenever a new property of a cell line is thoroughly characterized or a cell line recloned, a number of vials should be frozen at that point in order to be able to return to these cells if the lines being carried change.

Cryopreservation:

Cryopreservation Freeze preservation of animal cells is now routine in all cell line banks. A cryoprotective agent like DMSO or glycerol is generally added to minimize injury to cells during freezing and thawing. Frozen ampoules are generally stored in liquid nitrogen refrigerators which are rather convenient and quite safe.

Contamination:

Contamination Contamination with other cell lines- cross contamination Yeast Fungi Viruses: especially bovine Pestiviruses BVDV – Virus of Bovine Virus diarrhea CSFV – Virus of the classical swine but also BDV ( Borna Disease Virus) • Bacteria • Mycoplasma

Cross contamination:

Cross contamination

Contamination by Yeast:

Contamination by Yeast

Contamination by Fungi:

Contamination by Fungi

Contamination by viruses:

Contamination by viruses

Contamination by Bacteria:

Contamination by Bacteria

Contamination by Mycoplasma:

Contamination by Mycoplasma

Mycoplasma in Electron Microscope:

Mycoplasma in Electron Microscope

Sources of Mycoplasma in Cell culture:

Sources of Mycoplasma in Cell culture

Recommendation for improvement of contamination :

Recommendation for improvement of contamination

Scale Up :

Scale Up Large scale production of cells or cell products May range from laboratory small scale to industrial scale production Use of fermentors specially designed to fulfil the requirements Use of techniques like Roller Bottles Microcarrier Beads Cells in Hollow Fibers Collagen Gels Feeder Layers

Cell Culture: Applications :

Cell Culture: Applications Cell culture has become one of the major tools used in cell and molecular biology. Some of the important areas where cell culture is currently playing a major role are briefly described below: Model Systems : Cell cultures provide a good model system for studying 1) basic cell biology and biochemistry, 2) the interactions between disease-causing agents and cells, 3) the effects of drugs on cells , 4) the process and triggers for aging , and 5) nutritional studies. Toxicity Testing: Cultured cells are widely used alone or in conjunction with animal tests to study the effects of new drugs , cosmetics and chemicals on survival and growth in a wide variety of cell types. Especially important are liver- and kidney-derived cell cultures. Cancer Research: Since both normal cells and cancer cells can be grown in culture, the basic differences between them can be closely studied. By the use of chemicals, viruses and radiation, to convert normal cultured cells to cancer causing cells. Thus, the mechanisms that cause the change can be studied. Cultured cancer cells also serve as a test system to determine suitable drugs and methods for selectively destroying types of cancer. Virology : One of the earliest and major uses of cell culture is the replication of viruses in cell cultures (in place of animals) for use in vaccine production . Cell cultures are also widely used in the clinical detection and isolation of viruses, as well as basic research into how they grow and infect organisms.

Cell Culture: Applications :

Cell-Based Manufacturing : While cultured cells can be used to produce many important products, three areas are generating the most interest. The first is the large-scale production of viruses for use in vaccine production. These include vaccines for polio, rabies, chicken pox, hepatitis B and measles. Second, is the large-scale production of cells that have been genetically engineered to produce proteins that have medicinal or commercial value. These include monoclonal antibodies, insulin, hormones, etc. Third, is the use of cells as replacement tissues and organs. Artificial skin for use in treating burns and ulcers is the first commercially available product. However, testing is underway on artificial organs such as pancreas, liver and kidney. A potential supply of replacement cells and tissues may come out of work currently being done with both embryonic and adult stem cells. These are cells that have the potential to differentiate into a variety of different cell types. It is hoped that learning how to control the development of these cells may offer new treatment approaches for a wide variety of medical conditions. Genetic Counseling: Amniocentesis, a diagnostic technique that enables doctors to remove and culture fetal cells from pregnant women, has given doctors an important tool for the early diagnosis of fetal disorders. These cells can then be examined for abnormalities in their chromosomes and genes using karyotyping, chromosome painting and other molecular techniques. Cell Culture: Applications

Cell Culture: Applications :

Genetic Engineering: The ability to transfect or reprogram cultured cells with new genetic material (DNA and genes) has provided a major tool to molecular biologists wishing to study the cellular effects of the expression of theses genes (new proteins). These techniques can also be used to produce these new proteins in large quantity in cultured cells for further study. Insect cells are widely used as miniature cells factories to express substantial quantities of proteins that they manufacture after being infected with genetically engineered baculoviruses. Gene Therapy: The ability to genetically engineer cells has also led to their use for gene therapy. Cells can be removed from a patient lacking a functional gene and the missing or damaged gene can then be replaced. The cells can be grown for a while in culture and then replaced into the patient. An alternative approach is to place the missing gene into a viral vector and then “infect’’ the patient with the virus in the hope that the missing gene will then be expressed in the patient’s cells. Drug Screening and Development: Cell-based assays have become increasingly important for the pharmaceutical industry, not just for cytotoxicity testing but also for high throughput screening of compounds that may have potential use as drugs. Originally, these cell culture tests were done in 96 well plates, but increasing use is now being made of 384 and 1536 well plates. Cell Culture: Applications

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ADVANTAGES OF TISSUE CULTURE Ability to control the environment for maximising cell growth Characterization and homogeneity of samples Economy, Scale and mechanization Invivo modelling LIMITATIONS Necessary Expertise Quantity and Cost Dedifferentiation and Selection Origin of cells Instability

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ADVANTAGES 1. Control of the environment a)Physiochemical parameters i.e. pH, temperature, osmotic pressure, oxygen, carbon dioxide b)Physiological conditions supplementation of medium with defined constituents i.e serum, hormones and other regulatory substances Nutrient concentrations need to be regulated c)Microenvironment Regulation of matrix (cell attachment and growth improved by pretreating the subtrate : fibronectin, denatured collagen, cell-cell interaction, gaseous diffusion)

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Control of environment..continue Treatment with specific biological compounds, can induce specific alterations in the attachment and behavious of specific cell types. e.g. chodronectin enhances chondrocytes adherence , laminin promotes epithelial cell. Preservation of cell lines indefinitely - stored in liquid nitrogen (-196 o C).

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2. Characterization and homogeneity of cells Tissue samples are invariably heterogenous - consists of many types of cells Replicates from one tissue – many cell types After further subculturing (1-2 passgess) – homogeneity attained – uniform type of cells -selective pressure of culture conditions tends to produce a homogenous culture of the most vigorous cell types Further replicates at each subculture – virtually identical to each other -reduced the need for statistical analysis of variance

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Characterization and homogeneity of cells..continue Characterization: chromosomal analysis and DNA content, cytology and immunostaining Free of contamination (extraneous bacteria, viruses, fungi, mycoplasma) Free of contamination from other cell lines Characteristic of line may be perpetuated over several generation Validation and accreditation: Records of origin, history and purity

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3. Economy, scale and mechanization Less reagent or media – cheaper Lower and defined concentration – direct access to the cell Compared to in vivo: 90% loss by excretion and distributon to tissues not under study Screening test: duplicates, triplicates , many variables Reduction of animal use: legal, moral,ethical questions of animal experimentaion is avoided Microtitration, robotics- save time and economics of scale

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4 . Invitro Modeling of invivo condition Development of histotypic (one-cell type) and organotypic (more than cell types) models increased accuaracy of the invitro modeling. delivery of specific experimental compounds to be regulated: C (concentration), T (duration of exposure) and metabolic state

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LIMITATIONS OF TISSUE CULTURE- It’s Challenges Expertise Strict aseptic conditions Understanding the complexity of cells-environment-media requirement Ability to detect microbial (and mycoplasma) contamination and cross contamination with other cell lines To troubleshoot, diagnose and solve TC related- problems Quantity Large expenditure of efforts and materials – production of relatively little tissue Small laboratories 1-10g Larger laboratory 10-100g Industrial pilot plant scale: >100g

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Origin of cells If differentiation are lost – difficult to relate the cultured cells to functional cells in the tissues where they are derived Markers are not always expressed. Media/culture condition may need to be modified, therefore markers are expressed Genetic Instability Major problem with many continuous cell lines Unstable aneuploid chromosomal constitution Heterogeneity in growth rate and capacity to differentiate with the population can produce variability from one passage to the next LIMITATIONS OF TISSUE CULTURE.. continue

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Dedifferentiation Definition: `irreversible loss of the specialised properties that a cell would have expressed in vivo ’ Or `the loss of differentiated properties of tissue when it becomes malignant or growth in culture (A mature cell returning to a less mature state). Loss of the phenotypic characteristics typical of the tissue from which the cells had been isolated (original) Process reversal to differentiation: due to overgrowth of undifferentiated cells of the same or a different lineage Need to provide correct conditions so that many of the differentiated properties of these cells may be restored Serum-free selective media –allowed for the isolation of specific lineages LIMITATIONS OF TISSUE CULTURE..continue

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Major differences between animal cells in vivo and tissue culture in vitro Differences in cell behaviour between cultured cells and their in vivo stem Invivo 3D geometry and in vitro - In 2D monolayers Lost heterotypic cell-cell interaction Specific cell interactions characteristic of the histology of the tissues are lost Cells spreadout, become mobile-Proliferate – increased population When cell line forms, it may represent only one or two cell types-heterotypic

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Major differences between animal cells in vivo and tissue culture in vitro .. The culture environment lacks the several systemic components involved in homeostatic regulation in vivo eg. Hormones Without this control, cellular metabolism maybe more constant in vitro than in vivo but may not be truly representative of the tissue from which the cells were derived

Tissue culture & Social aspects:

Tissue culture & Social aspects the growing expectations of people from this field have itself become a major challenge now. A simple question arises- Do animal cell technology fulfills the expectation of people? The answer is not simple. The large scale implement of animal cell culture to produce various products that are to be cheap and available to every needy, the authenticity of such product, their safety level, etc are to be carefully dealt. Refinement of the methods, the continuous research, public interest and awareness, and the interest of Government towards this field are responsible to answer this question.

References:

References Agusti-Tocco , G., and Sato, G., 1969, Establishment of functional clonal lines of neurons from mouse neuroblastoma , Proc. Natl. Acad. Sci. 64 :311-315. Ambesi-Impiombato , F., Parks, L., and Coon, H., 1980, Culture of hormone dependent functional endothelial cells from rat thyroids, Proc. Natl. Acad. Sci. USA 77 :3455-3459. Arathoon , W. R., and Birch, J. R., 1986, Large-scale cell culture in biotechnology, Science 232 :1390-1395. Barnes, D., and Sato, G., 1980, Methods for growth of cultured cells in serum-free medium, Anal. Biochem . 102 :255-270. Barnes, D., and Sato, G., 1980, Serum-free cell culture: A unifying approach, Cell 22 :649-655. Freshney , R. I., 1992, Culture of Epithelial Cells, Wiley- Liss , New York. ……………………………………………………………………………………. And others

THANK :

THANK YOU