ANIMAL CELL CULTURE

A T I

nimal cell culture (ACC) is the process of culture of animal cells outside the tissue (in vitro) from which they were obtained.

he process of ACC is carried out under strict laboratory conditions of asepsis, sterility and controlled environment involving temperature, gases and pressure.

t should mimic the in vivo environment successfully such that the cells are capable of survival and proliferation

Some of the important products which are produced from animal cell cultures are : (i) enzymes (asperagenase, collagenase, urokinase, pepsin, hyaluronidase, ren nin, trypsm, tyrosin hydroxylase) (ii) hormones (leutinizing hormone, follicle stimulating hormone, chorionic hormone and erythropoietin) (iii) vaccines (foot and mouth disease vaccine, vaccines for influenza, measles and mumps, rubella and rabies) (iv) monoclonal antibodies (v) interferons

ANIMAL CELL CULTURE

M A A I

any cell lines, especially those derived from normal tissues, are considered to be :

nchorage-Dependent Cell Lines: they can only grow when attached to a suitable substrate.

nchorage-Independent Cel Lines: Some cell lines are no longer considered normal (frequently designated as Transformed Cells) are frequently able to grow either attached to a substrate or floating free in suspension n addition, some normal cells, such as those found in the blood, do not normally attach to substrates and always grow in suspension.

Types of Cells
Cultured cells are usually described based on their morphology (shape and appearance) or their functional characteristics. There are three basic morphologies: 1. Epithelial-like: cells that are attached to a substrate and appear flattened and polygonal in shape. 2. Lymphoblast-like: cells that do not attach normally to a substrate but remain in suspension with a spherical shape. 3. Fibroblast-like: cells that are attached to a substrate and appear elongated and bipolar, frequently forming swirls in heavy cultures. Culture conditions play an important role in determining shape and that many cell cultures are capable of exhibiting multiple morphologies.

When cells are surgically removed from an organism and placed into a suitable culture environment, they will attach, divide and grow. This is called a Primary Culture. There are two basic methods for doing this. 1) Explant Cultures: small pieces of tissue are attached to a glass or treated plastic culture vessel and bathed in culture medium. After a few days, individual cells will move from the tissue explant out onto the culture vessel surface or substrate where they will begin to divide and grow. 2) Enzymatic Dissociation: adding digesting (proteolytic) enzymes, such as trypsin or collagenase, to the tissue fragments to dissolve the cement holding the cells together. This creates a suspension of single cells that are then placed into culture vessels containing culture medium and allowed to grow and divide.

Subculturing : When the cells in the primary culture vessel have grown and filled up all of the available culture substrate, they must be Subcultured to give them room for continued growth.

Cell Culture Systems: Two basic culture systems are used for growing cells. These are based primarily upon the ability of the cells To either grow attached to a glass or treated plastic substrate (Monolayer Culture Sytems). Monolayer cultures are usually grown in tissue culture treated dishes, T- flasks, roller bottles, or multiple well plates. Or floating free in the culture medium (Suspension Culture Systems). Suspension cultures are usually grown either: 1. In magnetically rotated spinner flasks or shaken Erlenmeyer flasks where the cells are kept actively suspended in the medium; 2. In stationary culture vessels such as T-flasks and bottles where, although the cells are not kept agitated, they are unable to attach firmly to the substrate.

Basic environmental requirements for satisfactory Animal Cell Culture: Controlled temperature Good substrate for cell attachment Appropriate medium and incubator that main tains the correct pH and osmolality Avoiding Contamination

Contamination: Cell culture contamination is of two main types: chemical and biological Chemical - caused by agents, such as endotoxins, plasticizers, metal ions or traces of chemical disinfectants Biological - Biological contaminants in the form of fast growing yeast, bacteria and fungi usually have visible effects on the culture (changes in medium turbidity or pH). Also, mycoplasmas and viruses, are not easy to detect visually and usually require special detection methods.

Major requirements to avoiding contamination: Proper training in and use of good aseptic technique on the part of the cell culturist. Properly designed, maintained and sterilized equipment, plasticware, glassware, and media. The careful and selective (limited) use of antibiotics designed for use in tissue culture can also help avoid culture loss due to biological contami nation.

Cell Culture Uses: Some of the important areas where cell culture is currently playing a major role are: Model Systems Toxicity Testing Cancer Research Virology Cell-Based Manufacturing Genetic Engineering Gene Therapy Drug Screening and Development Mammalian Cell Products

Requirements for Animal and Tissue Culture:

1. 2. 3. 4. 5. 6. 7. 8.

Air conditioned room Hot room with temperature recorder Aseptic area for microscopic work Dark room Service room Sterilization room Preparation room Storage area

Substrates for Cell Growth

A large number of substrates which may be adhesive (e.g. plastic, glass, palladium, metallic surfaces, etc.) or non-adhesive (e.g. agar, agarose, etc) types may be used for anchorage dependent cells : (i) Plastic as a substrate. Polystyrene (Before use they are treated with gamma radiation or electric arc) teflon or polytetrafluoroethylene (PTFE), thermamox (TPX), polyvinylchloride (PVC), polycarbonate, etc. plastic beads of polystyrene, sephadex and polyacrylamide

(ii) Glass as a substrate. Test tubes, slides, coverslips, pipettes, flasks, rods, bottles, Petti dishes, several apparatus, etc. These are sterilized by using chemicals, radiations, dry beat (in oven) and moist heat (in autoclave). (iii) Palladium as a substrate. palladium deposited on agarose used as a substrate for growth of fibroblast and glia (non-neuronal cells that maintain homeostasis).

Culture Media
There are two types of media used for culture of animal cell and tissue, the natural media and the synthesized media.
Natural media These are of three types : (i) coagulans or plasma clots (ii) biological fluid The most commonly used fluids are human placental, cord serum and foetal calf serum. (iii) tissue extract extract from some tissues such as embryo, liver, spleen, leukocytes, tumour, bone marrow, etc. Synthetic media Synthetic media are prepared artificially by adding several nutrients (organic and inorganic), vitamins, salts, O2 and CO2 gas phases, serum proteins, carbohydrates, cofactors, etc. Synthetic media are of two types, (i) Serum-containing media (i.e. the media containing serum) and (ii) Serum-free media (i.e. media devoid of serum). Example of some of the media are: minimal essential medium (MEM) (Eagle, 1955), 199 (Morgan et al. 1950), CMRL 1066 (Parker et al, 1957), RPMI 1640 (Moore et al, 1967) and F12 (Ham, 1965).

Disaggregation of tissue Some of tissues consist of cells which are tightly aggregated. Tissue like epithelium is impregnated with Ca2+ and Mg2++ ions that provide integrity to it. Therefore, for getting primary culture it is necessary that tissue must be disaggregated either mechanically or using enzymes or chemicals so that cell suspension could be obtained.

Culture conditions affecting cell types and origin of cell lines.

Some products of medical use derived from animal cell cultures.

Erythropoietins Human growth hormones Products Erythropoietin-a Erythropoietin-p hGH Somatotropin Anti-lipopolysacharide Murine anti-idiotype/human B-cell lymphoma Anti-fibrin 99 99 Tcm-FAb (breast) PR-356CYT-356-in-lll Urokinase type plasminogen activator Tissue type plasminogen activator Recombinant plasminogen activator HIV vaccines (gpl20) Malaria vaccine Polio vaccines Application Anaemia resulting from cancer and chemothcraoy Anaemia secondary to kindney disease Human growth deficiency in children, renal cell carcinoma Chronic renal insufficiency, Turners' syndrom Treatment of sepsis B-cell lymphoma Blood clot Blood cancer Prostate adinocarcinoma Acute myocardial infarction, acute stroke, pulmonary embolism, deep vein thrombosis

Monoclonal antibodies (therapeutic)

Monoclonal antibodies (diagnostics)

Plasminogen activator

Vaccines

AIDS prophylaxis and treatment Malaria prophylaxis Poliomyelitis prophylaxis

Cultivation of Animal Cells En Masse in Bioreactor

The cells are cultivated in two different phases as Suspension Culture. In suspension culture the cells are dispersed in liquid medium and grow freely but not attached with any solid. The medium is agitated so that they should not form sediments. Immobilized cells on solid phase. Adherent cells are permitted to get attached on solid support and they grow as immobilized culture. Therefore, it depends on choice of cells whether they are capable of growing freely in suspension or as immobilized culture.

A compact loop bioreactor

Somatic Cell Fusion In animals fusion of two different cells and production of a hybrid cell have been successfully achieved. These hybrid cells have significant biotechnological applications in many more areas such as: (i) study of control of gene expression and differentiation, (ii) gene mapping, (iii) malignancy, (iv) viral replication, and (v) antibody production through hybridoma technology

Organ Culture

Not whole but pieces of organs can be cultured on artificial medium. The culture media on which organ is cultured are the same as described for cell and tissue culture. However, it is more easy to culture embryonic organs than the adult animals. Methods of culturing embryonic organ and adult organs differ.

The embryonic organs can be cultured by applying any of the following three methods:
Organ Culture on Plasma Clots Organ Culture on Agar Organ Culture in Liquid Media Whole Embryo Culture

MONOCLONAL ANTIBODY PRODUCTION

Hybridoma technology HAT medium is often used for preparation of monoclonal antibodies. Laboratory animals (e.g., mice) are first exposed to an antigen against which we are interested in isolating an antibody. Once splenocytes are isolated from the mammal, the B cells are fused with HGPRT negative, immortalized myeloma cells using polyethylene glycol or the Sendai virus. Fused cells are incubated in the HAT medium. Aminopterin in the medium blocks the de novo pathway. Hence, unfused myeloma cells die, as they cannot produce nucleotides by de novo or salvage pathway. Unfused B cells die as they have a short lifespan. In this way, only the B cell-myeloma hybrids survive. These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells). The incubated medium is then diluted into multiwell plates to such an extent that each well contains only 1 cell. Then the supernatant in each well can be checked for desired antibody. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, and are known as monoclonal antibodies.

Antibodies can be purified by anyone of the following techniques

(i) ion-exchange chromatography. (ii) antigen affinity chromatography. Hybridoma cells are grown in dialysis based mini-fermentors. This technology produces high density cultures that can be 20 to 30 times that achieved in static cell culture systems. Depending on the particular hybridoma these high density cultures on average produce antibody concentrations of 1 mg/ml.

IMPORTANT APPLCATIONS OF ANIMAL CELL CULTURE

Tissue plasminogen activator (abbreviated TPA or PLAT) is a protein involved in the breakdown of blood clots. As an enzyme, it catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for clot breakdown.

PRODUCTION PROCESS

Blood Factor VIII: Current therapy of Haemophilia is the transfusion of Blood Factor VIII. Factor VIII gene introduced into mammalian cells (Hamster Kidney Cells) ----- Factor VIII secreted into medium

Erythropoeitin: Erythropoietin is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. It is a cytokine for erythrocyte (red blood cell) precursors in the bone marrow. Recombinant human EPO (r-HuEPO) has been produced in mammalian cell system (e.g. Chinese hamster ovary cell lines) and commercialized. It is an effective treatment in patients suffering from AIDS, cancer and renal failure.