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Cell Types & Culture Characteristics

Cook Book Sept 2010 Volume 12, Fundamental Techniques in Cell Culture Laboratory Handbook-2nd Edition

5.1 Primary Cultures

Primary cultures are derived directly from excised, normal animal tissue and cultures either as an explant culture or following dissociation into a single cell suspension by enzyme digestion. Such cultures are initially heterogeneous but later become dominated by fi broblasts. The preparation of primary cultures is labour intensive and they can be maintained in vitro only for a limited period of time. During their relatively limited lifespan primary cells usually retain many of the differentiated characteristics of the cell in vivo. Important Note: Primary cultures by defi nition have not been passaged, as soon as they are passaged they become a cell line and are no longer primary. ‘Primary’ cells sourced from most suppliers are in fact low-passage cell lines.

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5.2 Continuous Cultures

Continuous cultures are comprised of a single cell type that can be serially propagated in culture either for a limited number of cell divisions (approximately thirty) or otherwise indefi nitely. Cell lines of a fi nite life are usually diploid and maintain some degree of differentiation. The fact that such cell lines senesce after approximately thirty cycles of division means it is essential to establish a system of Master and Working banks in order to maintain such lines for long periods.

Continuous cell lines that can be propagated indefi nitely generally have this ability because they have been transformed into tumour cells. Tumour cell lines are often derived from actual clinical tumours, but transformation may also be induced using viral oncogenes or by chemical treatments. Transformed cell lines present the advantage of almost limitless availability, but the disadvantage of having retained very little of the original in vivo characteristics.

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5.3 Culture Morphology

In terms of growth mode cell cultures take one of two forms, growing either in suspension (as single cells or small free fl oating clumps) or as a monolayer that is attached to the tissue culture fl ask. The form taken by a cell line refl ects the tissue from which it was derived e.g. cell lines derived from blood (leukaemia, lymphoma) tend to grow in suspension whereas cells derived from solid tissue (lungs, kidney) tend to grow as monolayers. Attached cell lines can be classifi ed as endothelial such as BAE-1, epithelial such as HeLa, neuronal such as SH-SY5Y, or fi broblasts such as MRC-5 and their morphology refl ects the area within the tissue of origin.

Figure 2. Examples of Cell Morphology

The cell lines most commonly ordered from ECACC are listed in Table 1 .There are some instances when cell cultures may grow as semi-adherent cells, e.g. B95-8, where there appears to be a mixed population of attached and suspension cells. For these cell lines it is essential that both cell types are subcultured to maintain the heterogeneous nature of the culture.

Table 1. Commonly used cell lines of each culture type

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5.4 Phases of Cell Growth

It is important to know and record the growth characteristics of the cell line of use before starting any experiments. An alteration in cellular growth can indicate a signifi cant problem within the cell line and if undetected can have detrimental effects on experimental results.

A typical growth curve for cultured cells displays a sigmoid pattern of proliferation. The growth phases associated with normal cells are defined as:

  1. Lag Phase – at this stage the cells do not divide. During this period the cells adapt to the culture conditions and the length of this phase will depend upon the growth phase of the cell line at the time of subculture and also the seeding density.
  2. Logarithmic (Log) Growth Phase – cells actively proliferate and an exponential increase in cell density arises. The cell population is considered to be the most viable at this phase, therefore it is recommended to assess cellular function at this stage. Each cell line will show different cell proliferation kinetics during the log phase and it is therefore the optimal phase for determining the population doubling time.
  3. Plateau (or Stationary) Phase – cellular proliferation slows down due to the cell population becoming confl uent. It is at this stage the number of cells in the active cell cycle drops to 0-10% and the cells are most susceptible to injury.
  4. Decline Phase – cell death predominates in this phase and there is a reduction in the number of viable cells. Cell death is not due to the reduction in nutrient supplements but the natural path of the cellular cycle.

Note: Different cell lines have different timescales for each phase, this graph is provided as a general example of a typical growth curve.

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5.5 In Vitro Age of a Cell Culture

Two terms are predominantly used to defi ne the age of a cell culture: (i) passage number - indicates the number of times the cell line has been sub-cultured and (ii) the population doubling (pd) number - indicates the number of cell generations the cell line has undergone i.e. the number of times the cell population has doubled. The in vitro age of a cell culture is particularly useful to know for cell lines with a fi nite lifespan or unstable characteristics that change over time in continuous culture.

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Did You Know?

The cell population is at its most viable during the log growth phase.

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