Stem cell expansion and processing

Cell culture media in the laboratory.

A problem common to all tissue engineering projects is how to culture enough of a tissue to be clinically useful. For example, 0.5cm3 of bone contains 75,000,000 cells – large repairs will therefore require vast numbers of cells. Basing a clinical procedure on a small biopsy from the patient, a very efficient in vitro expansion programme must be developed that allows us to keep cells viable for about 20 population doublings (or ‘passages’).

Research is being undertaken to address this and it has been found that populations of stem cells survive better in vitro than differentiated cells. Derived from the bone marrow of a patient, mesenchymal stem cells (MSC) show great promise for tissue engineering because a number of studies suggest they can be converted into a range of other cell types. They thus offer a potential source of material that will avoid rejection problems, and the ethical problems associated with embryonic stem cells.

Even stem cells can be difficult to culture – 30% of MSCs show signs of senescence by the 16th passage (P16 – by contrast differentiated cell lines are usually in trouble by P8). We are working to overcome this problem and the limitations it places on tissue engineering in vitro. Work is ongoing into determining the genes involved in stem cell ageing, so that genetic engineering approaches can be designed. Additionally, however, studies at Sheffield have shown that some of the problem is due to traditional tissue culture techniques.

For example, cell expansion is traditionally carried out in a medium with very high glucose – 4.5 g/l. Physiologically this equated to extreme hyperglycaemia. This results in hyperactive cell metabolism and overproduction of oxygen radicals – directly contributing to genetic damage and senescence of the cell line. We have found that by reducing the glucose to 0.5 g/l we get faster growth, and less cell damage.

Also cells are usually cultured at 37°C – the normal physiological temperature in humans. We have found that by reducing the temperature to 32°C, this inhibits cell differentiation, so the mesenchymal stem cell lines can be kept going for longer.

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Phillip Wright

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