The liver microenvironment is proving to be a major obstacle to the success of immunotherapy in solid organ tumours. The liver is characterised by tolerogenic properties that lead to the suppression of the immune system that can potentially benefit tumour growth and evasion. A key cell type in this tolerogenic environment are the endothelial cells that line the liver vasculature termed hepatic sinusoidal endothelium. These cells have distinct mechanisms to engulf and filter foreign antigens and suppress T cell responses.
Most of these studies have taken place in murine models but testing on primary human liver tissue will hopefully drive clinical translation. Through our transplant programme in Birmingham, we have access to human tissue and can isolate our own endothelial and epithelial cells. The possibility to recapitulate the human liver microenvironment using a 3D printed chip with the cells from patients may prove key when investigating cross-talk between resident cell types and the immune system during disease and cancer. This could help set up a platform for testing immunotherapies targeting the liver and support personalised medicine.
Figure 1. Histological section illustrating hepatocytes separated by sinusoids and the cell types involved in general homeostasis
Current experimental techniques using primary LSEC
The current standard technique for storage and shipping of primary endothelial cells Is through the use of cryopreservation in liquid nitrogen (-130°C). In order for novel 3D experiments to take place, our isolated LSEC must be sent to specialist laboratories where this technology is housed, meaning they must be transported in a cryopreserved state. This technique does have limitations including impact on cell viability after freezing. Due to human cells needing to operate within a small temperature range (36-38ºC), uncontrolled transition between normothermic and low temperature ranges causes a variety of damage to the stored cells. The process of ice formation within the cells after freezing can cause damage in two ways: intracellular salts become more concentrated inducing osmotic stress; or extracellular ice puncturing or damaging the cell membrane.
Figure 2. The LSEC isolation process from primary liver transplants using CD31 positive magnetic separation
Our LSEC are isolated fresh from transplanted tissue as seen in figure 2, through positive selection using CD31-coated dynobeads after enzymatic digestion of liver tissue. These cells are then cultured in preparation for seeding into 3D printed chips. Within the chips, LSEC are able to be successfully co-cultured with epithelial cells and primary peripheral blood mononuclear cells (PBMC’s), to recapitulate the microenvironment that can be exploited by a cancerous tumour. For these chips to become clinically personalised, epithelial, endothelial and immune cells must be isolated from the same patient. Making these experimentally available at the same time is challenging, so certain cell types must be cryopreserved to wait for patient bloods or during transport to specialist laboratories.
How CytoStor™ can help
To combat the issue of cryopreservation, our primary LSEC were preserved in CytoStor™ for up to 7 days and retrieved back into culture to determine whether cell viability and function were affected by CytoStor™, as seen in figure 3.. As previously described, LSEC were isolated from donor tissue and cultured to an appropriate cell number. LSEC were transferred to CytoStor™ gels with the total cell number split in two with one cohort incubating in CytoStor™ at 4°C and another cohort, from the same tissue sample, incubated at room temperature. These were subsequently left for a week before the gel was dissociated and the cells were returned to culture. After the week, a sample from the same tissue donor with the same cell number was removed from liquid nitrogen storage and returned to culture also. Cell viability was measured by counting number of live cells that were in culture for each cohort to determine which preservation condition worked best. Furthermore, quantitative and qualitative analysis was performed on the LSEC through staining for key phenotypic markers of LSEC and activation markers after proinflammatory treatment to determine whether CytoStor™ increased expression in comparison to liquid nitrogen storage. Finally, qPCR was performed on each sample to quantify whether activation markers of LSEC were maintained across each sample at a DNA level.
Figure 3. The protocol of preserving LSEC in CytoStor™ at 4°C and Room Temperature and the quantitative analysis subsequently performed
CytoStor™ permitted the transfer of cells from a preserved state to an active one whilst maintaining all key phenotypic and activation markers for experiments and proves an exciting prospect to facilitate the transport of primary LSEC across the country for novel research.
James Kennedy
PhD Student
LifETIME CDT
