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بسم اهلل الرحمن الرحيم ” صدق اهلل العظيم (أل عمار :ايه )8 Virus isolation in traditional cell cultures and newer cell culture formats By Wafaa Kamel Mowafy Prof. of Microbiology and Immunology , Mansoura Faculty of Medicine • The discovery in the early 1900s that human cells could be propagated in vitro provided virologists with an alternative to embryonated eggs and laboratory animals for in vitro isolation of viruses. By the early 1970s, diagnostic virology expanded dramatically, largely because of the availability of highly of purified reagents and commercially prepared cell lines. Virus isolation in traditional cell cultures (monolayer cultures) 1. Primary culture 2. Semi -continuous cell culture 3. Continuous cell culture Cell monolayers are most commonly used for culture of viruses. The are three categories, namely (1) primary, (2) semi continuous, and (3) continuous cell cultures. • Primary cultures. Viable cell suspensions may be obtained by dissociating tissues or organs, e.g. human amnion, with trypsin, collagenase or other enzymes. Advantages . Disadvantages. • Semi continuous cell cultures (cell strains ) – Semi continuous cell cultures are established with the successful subculture of primary cell monolayers. These cultures consist mostly of spindle shaped fibroblast cells. Established from human embryonic tissue, or neonatal foreskin. . – Advantages. – Disadvantages. • Continuous cell cultures ( cell lines ). Continuous cultures are produced either by transformation ( spontaneous or engineered ) of cell strains in vitro, or by culture of cells taken from tumors e.g Hela ( human cervical carcinoma ) and a human rhabdomyosarcoma cell line (RD cells ). – Advantages. – Disadvantages. The following continuous cell lines are commonly used: Hela and HEp2 are used for cultivation of HSV, adenovirus, poliovirus and some coxsackie viruses. Vero cells will also support growth of these viruses and are used with BHK21 cells for growth of arboviruses. RK13 cells and BHK21 cells for isolation and propagation of rubella virus . RD cells for the isolation of coxsackie A virus . • Newer cell culture formats 1. Centrifugation-enhanced inoculation and pre-CPE detection of viruses in cell culture. 2. Virus isolation in transgenic cell lines 3. Virus isolation in cocultured cells. – Centrifugation-enhanced inoculation and pre- CPE detection of viruses in cell culture. Cytospinning ( shell vials ). In this assay cells are grown on a coverslip in a vial , infected with the specimen and submitted to low- speed centrifugation followed by over night incubation. Labeled antibody to early antigen is used to stain cells after 24- 48 hours. Figure 5.17a: Tissue culture cells are grown on coverslips on the bottom of shell vials. Reproduced from Athmanathan, S., S. R. Bandlapally, and G. N. Rao, BMC Clin. Pathol. 2 (2002): 1-5. Figure 5.17b: Detection of Herpes Virus Simplex 1 using the shell vial technique and immunofluorescence. Modified from J. H. Shelhamer, et. al., Ann. Intern. Med. 124 (1996): 585-599. Shell vial technique • Centrifugation Culture (Shell Vial Technique) – Used a lot in clinical labs • Engineered cell lines (Enzyme-linked virus- inducible system , Transgenic cell lines) • The application of transgenic cells in cell cultures involves the stable introduction of genetic elements into a cell such that when a virus, and only a particular virus, enter this cell ,a virus specific event is trigged that results in the production of an easily measurable enzyme. ( baby hamster kidney – inducible B -galactosidase gene with HSV – inducible promoter ) was designed for sensitive and specific detection of both HSV-1 and HSV-2. ً When these cells are infected with either HSV-1 or HSV-2 , the B - galactosidase is induced and then an X- gal colorimetric substrate is added, infected cells turn blue, while other viruses do not induce the enzyme and the cells remain colorless. • This method has been incorporated into a commercial kit that has been marketed as the enzyme – linked virusinducible system{ ELVIS}. • There are advantages to this method including its rapidity, use of relatively inexpensive substrate as compared to antibodies, and the ability to use cells to look for both the color changes and CPE. The method is adaptable for other viruses, both DNA and RNA viruses. • Virus isolation in cocultered cells • Techniques involving combination of different cell types grown together as a single monolayer in a vial and the application of various MAbs, each labeled with a different fluorochrome for the detection of several viruses in the same vial. • . – The choice of cells has been focused on the ability to isolate and identify viruses that have a common pathological presentation, such as enteroviruses or respiratory viruses. – . An example of This, R- mix uses a combination of A549 cells and mink lung cells and is useful for the detection of adenoviruses, parainfluenza viruses, andٌ RSV. Three R- mix vials are inoculated for each specimen. Preliminary results using these cell mixtures indicate that they perform similarly to or better than cell culture or single – cell shell vial cultures and are more cost effective . • Selection for culture media A range of media have been formulated for growth of vertebrate cells in culture. These incorporate various conc. Of amino acids, vitamins, enzymes, growth factors, and inorganic salts. Glucose, fructose, or galactose are also added along with glutamine to provide a carbon source for cell metabolism. • There is a variety of formulations for cell culture media: – Dulbecco,s Minimal Essential Medium (DMEM) is in common use for continuous cell lines. – CMRL medium is particularly suited for the propagation of semi -continuous cell lines. – RPMI 1640 is recommended for growth of Lymphoblastoid cells in suspension. • Conditions for growth of cell cultures • Optimum pH. A pH range of 7.1-7.5 is required for the growth of eukaryotic cells. Most culture media use bicarbonate buffer systems (co2 / Hco 3) to maintain Ph. These media are formulated with NaHco 3and Co2 is either provided by the cultured cells as a metabolic product or by enrichment of the atmosphere using a co 2 incubator. • It is common to supplement the bicarbonate buffer system with HEPES buffer , it overrides all other buffers present and obviates the need for co2-enriched atmosphere. • Osmolarity. The growth of cells in culture depends on an optimum range of osmotic pressures, usually between 280 and 320 mmol/kg. • Serum. Balanced salt solutions will support cell proliferation only when supplemented with serum, lactalbumin hydrolysate, or other supplements. The serum has several functions : It provides essential amino acids, nucleic acid precursors, and fatty acids. It provides hormones and inhibits the protease used for routine dissociation of cells for culture. • Fetal or new – born calf serum are used at conc. 5-15 % to promote cell growth and at reduced conc. Of 0-2 % for maintenance of confluent monolayer cultures. • Serum should be stored at –70 C , repeated freezing and thawing should be avoided. • Antibiotics. antibiotics providing broad spectrum protection from bacterial contaminants are : benzylpencillin, 20-100 units/ml, gentamicin, 16-50 ug / ml, and tetracycline, 10 ug/ ml.,Amphotericin B, 0.5 ug/ ml, or nystatin, 50 units/ ml. are recommended for control of fungal infections. • Stock solutions should be stored at –20 C . • Subculture of semi-continuous or continuous cell cultures ( method -1 ) 1. Pour off culture medium and wash the cell sheet twice with phosphat buffered saline(PBS) 2. Add sufficient amount of trypsin-EDTA solution to cover cells. 3. Incubate at room temperature until cell sheet appears opaque. At this stage the cells will be rounded but not detached when observed with an inverted microscope. This process usually takes 1-3 min. • 4 .Remove excess trypsin solution. Cells will be detached from the culture vessel after approximately 2-3 min • 5. Add a small amount of chilled growth medium and aspirate several times with a 10 ml pipette to suspend and separate cells. • 6. Dilute a small sample of the cell suspension with additional growth medium for cell counting or dispense directly into new growth vessels. Semi -continuous fibroblast are generally passed, one-to-two split. A one – to-six up to a one-to-ten split is common for continuous cultures. • 7. When the monolayer reaches confluence, the growth medium should replaced with maintenance medium ( 2% serum). • Method -2 • 1-2 as method-1 • 3. Incubate the cells until cell detachment occurs. • 4. Add about 10 ml of growth medium , mix and transfer to centrifuge tube. Centrifuge for 10 min. at 1000rpm, remove supernatant and resuspend pellet in growth medium. • 5,6 and 7 steps as method 1. • Determination of cell numbers and viability by haemocytometer: • Disperse cells and dilute o.1ml of cell suspension with o.9 ml trypan blue solution in a separate container. • Moisten the supporting ridges of the haemocytometer chamber and apply a cover –slip. • After 4-5 min in trypan blue solution , resuspend cells and fill both sides of the counting chamer without overflowing. • Count the number of cells in the four corner squares and in the center of each side (10 squares). • Calculate the number of cells in the original suspension. Each squar represents an area of.1mm2 and has a depth of o.1 mm therefore the volume held by each squar is o.1 mm3 and the sample volume is 1o x o.1 or 1 mm3. To determine the cell conc.of the original flask the following formula is used: • Number of cells counted x 10 ( dilution factor) x 1000(no. of mm3 in cm3 = number of cells /ml. for example when 250 cells counted in 10 squars , the cell conc. in the original solution 250x10x1000 =2.5x106 cells/ ml. • To determine viability , make separate counts of both stained ( dead) and non stained (live) cells in a given area .A total of at least 100 cells should be counted.