Standardization of Primary Cells and Tissues

Where tissues are used for assays, comparison of data from various time points may be difficult, as the tissue will be derived from different individuals and may therefore respond differently [13]. It is important to establish that each tissue preparation used meets a certain minimum specification for the testing schedule, and where primary cell cultures are derived from the tissue and cryopreserved it may be possible to provide identical cell preparations for a series of experiments included in the associated application for ethical approval [5].

Differentiation, in its broadest sense, describes the development of a cell from embryonic to adult status as well as the maintenance of specific cell functions in vivo or in culture. Generally speaking, the genome of the cell in a culture holds the capacity to deliver a broad repertoire of differentiated states. The differentiation state is dynamic, depending on endogenous processes and exogenous factors, and will vary for individual cells within a culture. There are certain prerequisites to evaluate the differentiation state and validity of a cell culture, and these are characteristics that include: (1) species; (2) the stage of development; (3) a correlation offeatures with the original tissue ("lineage"); (4) the position of the cell within the identified cell lineage; (5) cellular transformation; (6) stability/variability of the cell phenotype; (7) a correct provenance and identity of the cells; and (8) the individual characters of a sub-cloned cell line or product of hybridization.

The choice of culture conditions is aimed first at sustaining the vitality of cells and, second, at achieving a certain a differentiation status representative of the in-vivo situation. Any change in culture conditions will potentially change the differentiation state as a result of adaptation of the living material to its environment. Thus, any change in culture conditions must be evaluated for its effect on the endpoints under study, as well as the overall differentiation status of the culture. It is important to recognize that cells cultured in vitro are rarely cultured under homeostatic conditions, and the frequency of changes of culture media will influence availability of nutrients and accumulation of metabolites, which will influence differentiation over the course of culture.

Accordingly, it is important to have indicators of the differentiated state of a culture that may include:

• morphology, histochemistry;

• enzyme content, isoenzyme pattern, gene expression;

• capacity to synthesize, secrete, and eliminate or accumulate substances;

• growth characteristics (e.g., growth curve, population doubling time);

• vitality and sensitivity towards toxins;

• cell functions which can be stimulated (e.g., by signal molecules);

• surface markers or antigens;

• nature of cell-cell interactions;

• nature of adherence to extracellular matrix.

The differentiation state is a combination of these functions, some of which are vital for the cell and will be expressed to some degree, or in some other cases may be absent. Notably, the increase in one parameter can influence negatively, or be accompanied by, changes in other parameters. An important example in this context is that growth rate and differentiation of cells are often opposing parameters, with cells that show a high degree of proliferation generally having a lesser degree of differentiation. Changes in one parameter often impact on others, and are part of complex feedback and control mechanisms of the cell.

Before starting a study, suitable parameters to monitor the differentiation status should be defined. In general, the endpoint of the experiment itself should not serve to control differentiation; rather, this parameter should be controlled over the course of the study in a series of experiments.

The desired differentiation state will usually - though not exclusively - mirror the in-vivo situation. To achieve this, culture conditions should approximate the in-vivo situation. Optimally, organotypic culture conditions ("state of the art") are employed, if they can be achieved with reasonable effort. It is desirable to establish reference tissue culture banks with materials for comparison of known differentiation state.

The most important means of standardization is control of the culture conditions, to assure a comparable differentiation state in all parts of the study. It is advisable to restrict the number of different potential conditions by choosing well-defined medium supplements, extracellular matrices, etc. The adequate documentation of all factors affecting cell function is crucial, as is the choice of monitoring culture parameters, the time point of their measurement, and maintenance of culture systems within acceptable limits.

In order to better understand this complex area of cell differentiation for in-vitro processes, the reader is directed to review by Coriell [14], Freshney [15, 16], Harris [17], Hill [18], Oshima [19], and Watt [20].

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