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Chapter 10.3 : Cell Culture Techniques: Serial Propagation and Maintenance of Monolayer Cell Cultures

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Abstract:

Monolayer cell cultures are most frequently used in diagnostic virology. They are prepared by treating the tissue or subculture with proteolytic enzymes and/or chelating agents to dissociate the cells and then seeding the cell suspension in a culture vessel. The cells adhere, divide, and form a layer. Adherence to the vessel surface is important for the subsequent survival and growth of the cells.

Citation: Garcia L. 2010. Cell Culture Techniques: Serial Propagation and Maintenance of Monolayer Cell Cultures, p 31-39. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.3
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References

/content/book/10.1128/9781555817435.chap10.3
1. Bird, B. R.,, and F. T. Forrester. 1981. Basic Laboratory Techniques in Cell Culture. U.S. Department of Health and Human Services, Centers for Disease Control, Atlanta, Ga.
2. McGarrity, G. J., 1982. Detection of mycoplasma infection of cell cultures, p. 99131. In K. Maramorosch (ed.), Advances in Cell Culture. Academic Press, Inc., New York, N.Y..
3. Schmidt, N. J., 1989. Cell culture procedures for diagnostic virology, p. 51100. In N. J. Schmidt, and R. W. Emmons (ed.), Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections, 6th ed. American Public Health Association, Washington, D.C.
1. Bird, B. R.,, and F. T. Forrester. 1981. Basic Laboratory Techniques in Cell Culture. U.S. Department of Health and Human Services, Centers for Disease Control, Atlanta, Ga..
2. McGarrity, G. J., 1982. Detection of mycoplasma infection of cell cultures, p. 99131. In K. Maramorosch (ed.), Advances in Cell Culture. Academic Press, Inc., New York, N.Y..
3. Schmidt, N. J., 1989. Cell culture procedures for diagnostic virology, p. 51100. In N. J. Schmidt, and R. W. Emmons (ed.), Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections, 6th ed. American Public Health Association, Washington, D.C..
1. Bird, B. R.,, and F. T. Forrester. 1981. Basic Laboratory Techniques in Cell Culture. U.S. Department of Health and Human Services, Centers for Disease Control, Atlanta, Ga.

Tables

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Table 10.3-1

Characteristicsofmonolayercellcultures

HNK, human neonatal kidney; RMK, rhesus monkey kidney; RK, rabbit kidney; MRC-5 and WI-38, human embryonic lung; HNF, human newborn foreskin; HEp-2, human epidermoid laryngial carcinoma; A-549, human epidermoid lung carcinoma; HeLa, human cervical carcinoma; Vero, African green monkey kidney; McCoy, mouse karyotype; ML, mink lung.

May be subpassaged to yield secondary cultures.

Citation: Garcia L. 2010. Cell Culture Techniques: Serial Propagation and Maintenance of Monolayer Cell Cultures, p 31-39. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.3
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Table 10.3-2

Reagent volumes for trypsinization of monolayer cell cultures

PBS or balanced salt solution.

Trypsin-EDTA (Versene).

Minimum essential medium with 10% FBS. If a cell count is not going to be performed, resuspend the cells in a volume equal to the split ratio of the given cell line.

Citation: Garcia L. 2010. Cell Culture Techniques: Serial Propagation and Maintenance of Monolayer Cell Cultures, p 31-39. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.3
Generic image for table
Table 10.3-3

Recommended split ratios for commonly used celllines

Adapted from Bird and Forrester ( ).

LLC-MK, rhesus monkey kidney; BHK-21, hamster kidney; RK-13, rabbit kidney. For other cell lines, see Table 10.3-1 , footnote

Citation: Garcia L. 2010. Cell Culture Techniques: Serial Propagation and Maintenance of Monolayer Cell Cultures, p 31-39. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.3
Generic image for table
Table 10.3-4

Surface area, cell yield, and final volume of growth medium for commonly used culture vessels

Adapted from Freshney ( ).

Citation: Garcia L. 2010. Cell Culture Techniques: Serial Propagation and Maintenance of Monolayer Cell Cultures, p 31-39. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.3

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