Better cultivation – making the most of stem cell research

Scientists in England have developed novel technology to grow stem cells and other tissue in the laboratory in conditions similar to the way they grow in the human body.

The technology, developed and patented by scientists at Durham University and its spin-out company ReInnervate, is a plastic scaffold which allows cells to be grown in a more realistic three-dimensional (3D) form compared to the traditional flat surface of a Petri dish (Fig.1).

Evidence gathered by the research team shows that the technology is a cheap and straightforward way of cultivating cells in 3D. Using it could lead to more successful drug development programmes and a reduction in unnecessary tests on animals.

A study proving the effectiveness of the scaffold, funded by ReInnervate and the Engineering and Physical Sciences Research Council (EPSRC), is published in the recent Journal of Anatomy.

A large proportion of drugs fail at the testing stage, costing industry millions of Euros in R&D costs and failed drugs trials every year. At the moment, most drugs in development are first tested on cells grown in two-dimensions (2D) in standard laboratory equipment such as Petri dishes or flasks but cells in the human body form tissues and grow in more complex, three-dimensional ways.

The new study tested the toxic effect of a cancer drug called methotrexate (MTX) on liver cells grown in three and two dimensions. Liver cells are frequently used in the drug development industry to test the toxicity of drugs and MTX is known to cause liver damage at high doses.

Tests showed that the structure and properties of the cells grown using the 3D scaffold were most similar to liver cells found in the human body, compared with the 2D cells that appeared ‘disorganised’ when viewed under the microscope.

When subject to doses of MTX, cells grown in 2D died at very low concentrations, whereas 3D cells grown using the scaffold were far more robust and more accurately reflected the behaviour of cells in the human body when subjected to similar doses of the drug.

Stefan Przyborski, a researcher with Durham University and chief scientific officer of ReInnervate, has tested 10 different tissue types on the scaffold, including bone, liver, fat and stem cells from bone marrow, and is marketing the product for commercial use.

The scaffold is made of highly porous polystyrene, is about the size of a ten pence piece and resembles a thin white disc. It has a structure resembling that of a sponge and is riddled with tiny holes which scientists are able to populate with cells that are then cultivated under laboratory conditions.

The technology has potential to be used to grow human stem cells for drug development. Their use may reduce the need for the tests on animals that are usually the next step before progressing to clinical trials in humans.

Growing skin cells

Another current use of the scaffold involves growing skin cells that are being used by the cosmetics industry to test cosmetics.

Przyborski said: “Our results suggest that testing drugs on liver cells using our 3D culture system may be more likely to reflect true physiological responses to toxic substances. Because the 3D cells are cultivated under more realistic conditions, it means that they function more like real tissues.

“Scientists are therefore able to gain a more accurate idea of how a drug will behave in the human body, knowledge which can contribute to improving the efficiency of drug discovery, reducing drug development costs, and may help reduce the number of animals in research.

“There are other ways to growing cells in 3D in the laboratory. However, these approaches are restricted by their variability, complexity, expense and they are not easily adapted to routine use in high throughput screening studies.

“Our technology is essentially a well-engineered piece of plastic that provides a suitable environment for cells to grow more naturally in a 3D configuration. Our product is available off-the-shelf, it is easy to use in routine applications, it is highly adaptable to different tests, it is inert and it is cheap and easy to produce and manufacture.”

Przyborski and colleagues at Durham University play a key role in the North-east England Stem Cell Institute (NESCI), an interdisciplinary collaboration to convert stem cell research and technologies into cost-effective, ethically-robust 21st century health solutions to ameliorate degenerative diseases, the effects of ageing and serious injury.

Stem cells in the US

Regenetech, the adult stem cell company which has built upon NASA licensed technology, has announces a significant augmentation to its cell multiplication capabilities for CD34+38- peripheral blood progenitor cells (adult stem cells).

These new results were achieved through the continued development of cell culture conditions in Regenetech’s proprietary and patented Intrifuge cellXpansion technology.

Developing a significant and reliable technology for adult stem cell growth and multiplication currently represents one of the most significant barriers preventing more widespread adult stem cell therapeutic use.

Company ceo David Bonner said: “We are delighted with the progress that the team has made; this is a significant step forward for our technology, and we are excited about what it will allow us to do in the future in terms of providing doses of adult stem cells to help patients. This development will demonstrate to the wider industry that this technology will have a significant impact on many treatments, and brings us even closer to realising the potential for treating diseases through the use of expanded adult stem cells.”

Under the new, improved growth conditions, the number of adult stem cells increased by 50-200 times the original number, an achievement accomplished in as few as six days.

Compared with other serum-free media, the optimised Regenetech formulation yields considerably higher cell numbers than were previously achievable, significantly enhancing the market competitiveness and commercial potential of the cellXpansion technology (Fig.2).

Regenetech’s technology uses NASA based technologies that were discovered in space experiments in which adult stem cells and tissue are grown in a weightless three-dimensional environment.

The stem cells are harvested from a filtered extraction of the patient’s own blood and multiplied in Regenetech’s Intrifuge bioreactor (Fig. 2) to achieve therapeutic quantities, before re-introducing them back into the original patient. This technology produces therapeutic quantities of adult stem cells rapidly and at reasonable cost.

In another new development, Maryland-based Osiris Therapeutics has received FDA clearance to initiate Phase III pivotal trial for Prochymal as a first line treatment for acute graft v host disease (GVHD). This can be a life threatening complication of bone marrow transplantation and currently has no approved treatment.

Prochymal is a preparation of mesenchymal stem cells specially formulated for intravenous infusion. The stem cells are obtained from the bone marrow of healthy adult donors.

GVHD is a T-cell mediated inflammatory process that results in high levels of pro-inflammatory chemical signals called cytokines. These cytokines cause the unbalanced activation of certain immune cells that result in tissue damage.

Delivered intravenously, Prochymal is able to target areas of active inflammation. Published data indicates that Prochymal is able to down-regulate the production of pro-inflammatory cytokines, including tumour necrosis factor-alpha or TNF-alpha and interferon-gamma. Additionally, Prochymal up-regulates the production of beneficial anti-inflammatory cytokines, specifically interleukin-10 and interleukin-4.

When the stem cells found in Prochymal are delivered into an inflammatory environment, they appear to change the course of the disease by altering the cytokine secretion profile of the dendritic and T cell subsets, thereby resulting in a shift from a pro-inflammatory to an anti-inflammatory state and arresting disease progression. Furthermore, data indicates Prochymal may also promote the regeneration of tissue structures damaged by GVHD.

Commenting on Prochymal’s success so far, Joseph Uberti, co-director of the Blood and Marrow Stem Cell Transplant Program at the Barbara Ann Karmanos Cancer Institute, said, “These data are very impressive. What is really significant for the transplant community is the high proportion of patients that completely resolved their GVHD without the need for potentially lethal immunosuppressants. We know that if we can minimise immunosuppression, patients do better.

“Ultimately, the study demonstrated that Prochymal has the potential to double the complete response rate we typically see in patients suffering from GVHD, and as a result, appears to significantly reduce mortality.”

However, as eLab went to press, the more controversial aspect of stem cell research was again in the US news as National Institutes of Health director Elias A Zerhouni suggested that embryonic research should be expanded.

Stem cells in the US

According to the Washington Post, Zerhouni has called for research to continue on all levels, ‘or there will be no progress’.

His views are directly at odds with those of US President George Bush who has twice vetoed legislation that would have allowed research using embryonic stell cells.

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