Department of the Science of Food of Animal Origin, Faculty of Veterinary Medicine, Utrecht University, PO Box 80.175, NL-3508 TD, Utrecht, The Netherlands.
Introduction
In experimental veterinary research intact animals are often
employed. Although this will remain important, both basic and applied
research may benefit from well-chosen and well-designed model systems,
which range from isolated perfused organs to subcellular fractions.
Cell and tissue cultures of organs of euthanised companion animals and
slaughtered production animals have been used only infrequently in veterinary
science. However, like no other method, cell culture systems offer possibilities
to screen for effects of compounds like hormones and drugs in a controlled
way and under a wide variety of conditions. Application of new technologies
commonly termed "functional genomics" will help to identify (cell-specific)
target molecules. Thus, cell culture systems may contribute considerably
to knowledge in the veterinary sciences. Here is a brief overview of
the potential of species-specific primary cell cultures.
Isolation of viable cells for culture
Over the last three decades, cell and tissue culture methods have been
refined and have now become an essential tool in biomedical research.
Animal welfare concerns may recently have played a role in this development,
but the main reason was to develop systems that allow the study of single
cellular functions under controlled environmental conditions (see Fig.
1).
In vitro systems share the characteristic that they exclude the influence
of other organs and of the circulatory and immune system, thus providing
the possibility to study direct effects on a cell population. Today's
cell culture systems are based on mechanical and/or enzymatic disaggregation
of the tissue to single cells. Tissue samples are mostly obtained from
laboratory animals that are killed for this purpose, and from embryonated
chicken eggs. Biopsy specimens or samples from surgically removed material
are another source, but their use is limited by irregular availability,
small volume, and difficulties in standardisation due to variations
in sample origin (genotype, strain/breed, age etc.).
Primary cell cultures vs. cell lines
Two types of cell culture should be distinguished:
1. Primary cultures, which are obtained directly from an animal
and can keep the differentiated state for a short period (days to weeks).
Functionally differentiated primary cell cultures have a limited life
span, and although maintenance of the differentiated properties has
been improved by additives to the culture medium, components of the
extracellular matrix or by different forms of co-culture (see Figures
2 and 3), cell specific functions will eventually decline.
2. Permanent cultures (e.g. HeLa, 3T3, MDCK), which have an unlimited
proliferation capacity and which originated from embryos, tumors or
transformed cells
Cells can proliferate and/or differentiate, both with different limitations,
depending on the cell type studied (e.g. neurons). Numerous publications
provide protocols for the isolation of different cell types, their culture
conditions, and for the evaluation of the degree of differentiation.
Much attention is presently given to stem cells (see our News section).
Primitive pluripotent embryonic stem cells derived from post-mortem
foetal tissue have been used in cell therapy studies to replace diseased
tissues. Until recently it was difficult to isolate and culture stem
cells from adult tissues other than bone marrow. However, the cell therapy
field is rapidly expanding, and it appears that many tissues harbour
adult stem cells that possess a much greater plasticity than previously
thought. Adult stem cells may obviously also be used as a model system,
and it is to be expected that they will receive more attention also
in the veterinary sciences.
Primary cell cultures from farm animals
When searching through the literature, it is quite easy to find cell
culture systems for any desired tissue. However, not all published protocols
are accepted as standard procedures, and there is a large variety of
culture conditions. In general, care should be taken when comparing
methods and extrapolating a system from one species to the other. There
are only a few publications dealing with primary cultures derived from
veterinary relevant species (as compared to those from laboratory animals).
It was the use of laboratory animals, which has resulted in the development
of standard procedures - but these must be adapted for each species.
It can be expected that species-specific cell culture systems will become
important to complement results obtained by genome and proteome analysis,
both in human and in veterinary research.
Nevertheless, primary cell cultures of several tissues have found their
way into basic and clinical veterinary research. The choice of animal
and organ is often made on practical grounds, like availability of tissue
or slaughterhouse procedures. For example, isolation of porcine pneumocytes
from slaughterhouse material will be difficult, as many lungs are affected
by the scalding procedure. On the other hand, kilograms of bovine adrenals
can be collected in the slaughterhouse, from which pure adrenal cortex
cells are easily prepared that are viable for some weeks and maintain
differentiated functions. Fig. 4 shows bovine adrenal cortex cells cultured
for six days in serum-free medium and treated with ACTH, cAMP, dexamethasone,
cycloheximide, and tunicamycin.
Endocrine tissue
For these technical reasons it is not surprising to find high-ranking
publications using bovine adrenal cortex cultures [5,10].
The protocols leading to a standardised procedure have been developed
in the late 70ies [14, 18].
More information concerning optimal culture conditions of these cells
can be found in the literature [9,
24],. Other endocrine tissue has
been successfully cultured, like bovine and porcine thyroid cells [2].
A detailed description of cell cultures from porcine pancreas has been
published [20]. Primary cell cultures
from the porcine reproductive system have also been described, like
endometrial cells [6], cells from
the ovary [11], and Leydig cells
[3, 13].
Bovine mammary epithelial cell culture was employed to study the synthesis
and secretion of IGF binding proteins [12].,
and bovine pituitary cells for the analysis of signaling pathways [22].
Liver
The isolation of cells from the liver has been a challenge for nearly
two decades. The main problem is the rapid onset of cell death post
mortem. However, the importance of this tissue led to large efforts
in method development to maintain viable and differentiated cultures
from large animals. Bovine, canine and sheep primary hepatocytes were
studied for their hormonal responsiveness under serum free conditions
[7, 8,
16]. Adult chicken hepatocytes
were analysed for their differentiation state under serum free conditions
[23]. The optimisation of differentiation
was also the focus of studies using porcine hepatocytes [15,
19]. These refinements are of great
importance, as improvement of the quality of primary cultures makes
them a viable alternative to in vivo experiments, particularly for pre-screening.
Thus liver cell culture systems were employed to study aspects of inflammation
in bovine hepatocytes [1, 17].
A methological option that needs more attention is the preparation of
cell cultures from animals that had died in veterinary clinics. In a
recent publication the development of equine adipocyte cultures from
expired animals has been described [21].
Skeletal muscle
Skeletal muscle is also easy to obtain and to process into viable cultures.
It can be obtained from a variety of animals, and used even many hours
post mortem. A recent review [4]
summarises isolation and culture protocols from animals of veterinary
interest. In the case of skeletal muscle culture it is important to
realize that these do not result from outgrowth of existing fibers.
It is the dormant stem cells, satellite cells, which are isolated and
which fuse in vitro to form new myotubes.
It has been the aim of this mini-review to briefly summarize the principles
of primary cell culture and to show that protocols are available for
various species of veterinary relevance. The above list of tissues as
a source of primary cell culture is by no means exhaustive; cartilage,
bone, skin, kidney, spleen, blood derived cells, lung and intestinal
tissues have also successfully been processed for primary cell cultures.
Future Developments
The development and standardisation of species-specific primary cell
cultures will likely become more important in veterinary research. We
can expect to get new insights while exploring and modulating metabolism
and function at the cellular level. The diversity of the physiological
and pathological features of companion and farm animals is guaranteed
to reveal the equivalent on the cellular level. Perhaps the best feature
of primary cell cultures is the possibility to modulate the metabolic
and regulatory pathways of cells of interest and to delineate the physiological
effects of various compounds and drugs in a controlled way. The refinement
of this experimental tool can be expected to accelerate species-specific
research. Furthermore, cell cultures can be employed as screening systems
in various fields of veterinary science, thereby reducing the need for
live animals.
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