Our ambition is to provide cells with conditions which allow them to develop functionality which closely mimics that seen in the intact organism.
To achieve this, we have developed a system which obviates the need for scaffolds, gels or any type of additives which might perturb gene expression away from its natural baseline equilibrium. Furthermore we have very significantly reduced contact with plastics and reduced shear forces. It is possible to use the culture media that you normally use for cell culture. We would suggest reducing glucose levels to 1 g/L glucose, which is more relative to those seen in healthy animals.
In general, it is repair of the damage caused by trypsin (collagenases or other similar enzymes used to dissociate cells) that is important. That means rebuilding the surface proteins so that cells can communicate, establish junctions and then make all the internal adjustments.
If spheroids or organoids are grown under static conditions, the formed constructs do not experience shear stress. This is essential for avoiding modifying gene expression which would prevent them mimicking the in vivo tissue. However, the static conditions come with a price: a large depletion zone surrounding the spheroids/organoids. This depletion zone will starve (nutrients), poison (metabolic waste) and suffocate (O2) the cells, thereby reducing their longevity. The short lifetime will not provide the cells the time needed to recover structure, communication and especially O2 gradients which are essential for the spheroids/organoids to develop tissue-like functionality.
‘Active diffusion’ is needed to minimise the depletion zone. This should be obtained without creating shear stress. The best way to do this is using the clinostat principle. Here, the spheroids/organoids stay in an almost static orbit, i.e. essentially not moving relative to the ClinoReactor. The very gentle tumbling of the constructs in the media washes away most of the diffusion-depleted zone, increasing the steepness of the gradients – to the benefit of the constructs’ longevity.
The technology CelVivo use for culturing cells is based on the clinostat principle (also referred to as a simulated microgravity system).
This approach has the advantages that the spheroids or organoids formed are exposed to very low shear forces and have excellent gas and nutrient exchange. This allows them to grow up to about 1 mm without showing necrosis or apoptosis in the core.
Spheroids formed using microwell plates and are very similar in size (after 21 days in culture (which would amplify any differences) they still only show a 21% variation in protein content.
The CelVivo system creates an environment which promotes the functionality of large 3D tissue mimetic structures, whether they are spheroids, organoids, acini and other aggregates.
A simpified version of the forces that act on the cell constructs, when using the clinostat prinicple for cell culture.
The advantages of using clinostats for culturing cells are:
Large spheroids are created with high uniformity in size
Excellent for co-cultures and tumor studies
The cells experience very low shear stress
Active diffusion which allow good nutrient exchange.
The function, architecture and ultrastructure of 3 week old spheroids mimics that see in vivo
Spheroids or organoids can be maintained functional, in some cases, for at least a year
Metabolic Reprogramming and the Recovery of Physiological Functionality in 3D Cultures in Micro-Bioreactors
Krzysztof Wrzesinski and Stephen J. Fey
Published: 2018 March 05
After trypsinisation, 3D spheroids of C3A hepatocytes need 18 days to re-establish similar levels of key physiological functions to those seen in the liver
Krzystof Wrzesinski and Stephen J. Fey
Microgravity spheroids as a reliable, long term tool for predictive toxicology
Stephen J. Fey and Krzysztof Wrzesinski