Celvivo culture characteristics

The CelVivo system creates an environment which promotes the growth and maintenance of large 3D tissue mimetic structures, whether they are spheroids, organoids and other aggregates.

There are several advantages when using a clinostat for cell culture, they are explained in depth here. The main benefits of the clinostat are low shear stress and active diffusion. These benefits manifest in the cell culture by physiological changes within the cells and reestablishment of  the way they communication compared to cells cultivated in 2D. Below is some of the physiological changes observed in the cell culture when cultivated with the CelVivo Technology. The observations presented are all reviewed and published observations.

Characteristics of cells cultivated in ClinoStar

These observations are made usign the immortal C3A strain of HepG2 hepatocytes (ATCC CRL-10741) cultivated for at least 18 days in the first generation of the ClinoStar. In general we see the cells need 18 days to mature before they reach a pletau in physiologicl reovery. After reachign this plateau the cells remain stable and can be used for experimetns for at least 24 days, probaly longer. In a pilot experiment a culture have been maintained in the system producing viable spheroids for 302 days (Wrzesinski et al. 2013 )

Proliferation

By following the morphological changes and evaluating the protein content, the growth rate of the spheroids was evaluated. The proliferation of the cells dramatically decreases their proliferation rate to >60 days and a size of 1400µm after 42 days in culture. To compare immortal C3A strain of HepG2 hepatocytes grown in classical standard cell culture conditions, proliferate rapidly and after 5 days reach and pass 100% confluency.


Spheroid viability

Cell viability and metabolic activity can be correlated with ATP activity. In HepG2 spheroids the ATP content increase as the spheroids in the culture matures. The cell death as estimate by adenylate kinase release is essentially constant (data in publication). As the proliferation rate decrease on could argue that the cells have died or decreased their metabolic activity, but this is not the case, as the cells are alive and viable.

Physiological performance

To have a good model the physiology functionality and structure of the cells used should resemble its parental tissue. Functionality of culture was evaluating by three normal physiological functions of the hepatocytes in the liver: Urea, cholesterol, and glycogen production. In a standard flat culture of HepG2 cells the production of urea, cholesterol and glycogen are close to non-existing.

Though out the maturation period the level of urea and cholesterol is increased to in vivo concentrations in the spheroids. Around day 21 the level of both urea and cholesterol starts to plateau and remains stable until day 42, when the experiment was terminated.

Another and important function of the liver is the storage and release of energy in the form of glycogen and glucose. Glycogen synthesis in spheroids was evaluated by evaluating the incorporation of radioactive glucose into glycogen supplied in the media.

Ultrastructure

Examined by electron microscopy, cells and spheroids exhibit all the ultrastructural organelles normally seen in eukaryotic cells (nuclei, mitochondria, rough endoplasmic reticulum, etc. (N, M, RER respectively). Both cells grown in 2D and 3D spheroids exhibit tight junctions (TJ) that compartmentalize the plasma membrane, allowing different receptors, transporters and enzyme systems to be sequestered to specific regions of the plasma membrane. However, the tight junctions are more distinct and more common in the spheroids. In addition, spheroids exhibit several features that are infrequently seen when the C3A cells are grown under 2D cell culture conditions. These features include the formation of bile canaliculi-like structures in the microvilli-free inter-hepatocyte faces. In vivo, in the liver these canaliculi merge and develop into bile ducts leading to the gall bladder. Large numbers of microvilli can also be seen in the sinusoidal like channels that in the liver in vivo, together with an incomplete layer of endothelial cells, form the space of Disse. Spheroids also exhibit glycogen granules characteristic for the storage of glucose in the liver.

Epigenetics

Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties. Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture.

Metabolic reprogramming

Cells cultivated in ClinoStar as spheroids experiences complete metabolic and architectural alterations after 21 days of culture, when comparing to a regular flat 2D cell culture. A proteomic study of the same cell type, grown in the same media, in the same incubator but as either a 2D (flat) or 3D (spheroid) culture showed a significant metabolic reprogramming resulting in structurally in changes in actin organization, increases in microtubules while keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in G1/Go. Transport around the cell – along the microtubules, through the nuclear pore and in various types of vesicles is prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated.