CELL LINE CULTURE By Dr.Hesnaa - PowerPoint Presentation

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CELL LINE CULTURE

By

Dr.Hesnaa

Saeed

Al-

Mossawi

Uses

Identification

Further studies

(e.g., Pathogenicity, antiviral sensitivity, research)

Limitations

Absence of detection system for the agent

Inappropriate culture systems

Viruses that cannot be

cultured

A negative viral culture results does not mean that

the agent

is absent

Specimens used to culture viruses

Blood specimens

EDTA

Heparin

Serum

Stool

Throat swabs

Naso-paryngeal aspirates

Stools, rectal swabs

Urine

Saliva

Cerebro

-spinal fluid

Biopsy

Skin (

filoviridae

)

Organs (fixation with formaldehyde 10%)

Virus isolation in

traditional

cell cultures (monolayer

cultures)1 . Primary culture2 . Semi - continuous

cell

culture3 . Continuous cell culture

Primary cultures

Viable

cell suspensions may be obtained by dissociating tissues or organs, e.g. human amnion, with trypsin, collagenase or other enzymes.

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. .

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 ).

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.

Type of 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

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. for the growth of eukaryotic cells.Osmolarity . The growth of cells in culture depends on an optimum range of osmotic pressures, usually between 280 and 320 mmol/kgSerum. 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.Antibiotics. antibiotics providing broad spectrum protection from bacterial contaminants

Subculture of semi-continuous or continuous cell cultures

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.

Subculture of semi-continuous or continuous cell

cultures….CON

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).