National Antivisection Society
A dynamic multi-cell type culture system as a model for Multiple Organ Dysfunction Syndrome (MODS)
15 February 2008
Sepsis is a life threatening illness, usually caused by the body’s response to infection. The infection can be caused by various factors, including burns, bacteria etc. A frequent consequence of sepsis is a syndrome known as MODS – Multiple Organ Dysfunction Syndrome. MODS results in the impermeable epithelial layers around the organs in the body (kidney, lungs, liver and intestines) becoming permeable, or “leaky”. This causes the organs to fail.
The aim of this project was to bypass the unethical and unreliable use of animals to model this organ breakdown and to look for a cellular replacement. The current cell culture studies are very simplified versions of what happens in the body, which cannot mimic the conditions of diseased human tissue.
This study therefore represented a development of the existing protocols
The aim was to develop a cell culture system whereby different types of human cell could be cultured together, in a dynamic fluid environment to better model the human tissues affected by MODS. Better understanding of the molecular mechanisms which cause the change in permeability in epithelial membranes during MODS will help identify new therapeutic strategies or prophylactics to combat this condition.
For practical reasons some researchers employ mono-layers of a single cell type, but these do not accurately reflect the complex in vivo environs. To use the cells in dynamic systems, they must be cultured under static conditions then transferred to chambers where they are measured under flow. The change in culture conditions is expected to cause artefactural changes in cell behaviour. Other systems allow more than one cell type to be cultured, separated by a membrane, but they are static, so a new system allowing continuous flow based cell culture & co-culture of separated mono-layers of different cell types was the aim.
The group developed an endothelial-epithelial bi-layer that mimics the blood brain barrier (BBB).
This was done using human brain microvascular endothelial cells (specialised endothelial cells) and human epithelial meningeal cells (derived from brain tumours). This model was then exposed to Neisseria meningitides, an organism that can cause septic shock and MODS.
For our projects, the LDF encourages the use of alternatives to FCS (foetal calf serum), also known as FBS (foetal bovine serum). Thus, the first six months of this project were spent adapting the cell lines to grow in serum free (SF) media – this was successful and it was confirmed by monitoring IL-8 (a protein produced when infection is present) that infection had occurred, and the cells were responding characteristically. In addition to this mono-layers and bi-layers were cultured under static conditions, challenged with bacteria and examined using various modes of microscopy. The results showed that, under SF conditions, bi-layers could be successfully cultured. It was the first time that these two cell types had been cultured together in a bi-layer and neither cell line was previously cultured in SF media
Due to problems with the cell system for holding the samples, it was deemed that it was too early to introduce a flowing media to the culture system, so characterising the model under static conditions was made the priority.
The second half of this project entailed testing the bi-layer to ensure it had the same integrity as an in vivo BBB – due to tight junctions, which as their name suggests, stop substances crossing the BBB.
This was done by testing the electric resistance of the layers. The profiles of various chemicals associated with infection were also monitored to ensure that the bi-layer behaved in the same way as a live human BBB.
The results for a chemical called TNF (tumour necrosis factor) differed between the monolayer and the bi-layer.
This project had two important achievements:
- The first, a very important milestone, was that it was the first time that these cell types had been cultured together in a bi-layer. Prior to this group’s work, neither cell line was previously cultured in serum free media. The ability of a group to grow cells in serum free media means that there is less reliance on FCS.
- The results for the tumour necrosis factor differed between the mono- and bi- layers. This was an important finding as it shows that cells behave differently when they are in different layers.
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