This is a preclinical research project and no patient intervention is planned. The project makes use of anonymised human tissue samples discarded during radical surgery. The spleen organ samples included in this project will be from patients undergoing elective surgery for a lesion in the pancreas in the HPB Unit of the Leicester General Hospital. The sample size of 100 (100 enrolled subjects for 42 spleens) has been determined by power calculations and is in line with the number of spleens actually removed by elective pancreas surgery over the past years. No change in the surgical procedure or recruiting results from this study and the use of the samples. All the experimental work carried out on the spleen samples will have no effect on the patient.

Experiments include ex vivo organ perfusion and infection with bacteria or viruses. Biopsies of the perfused 42 spleens are processed for in vitro tissue slice and macrophage cell culture. The resulting primary cell cultures and tissue slices will be stored frozen for future research. All in vitro studies on the 42 organs, and the tissues and cells derived from them, will evaluate the effect of therapeutic interventions (antibodies, receptor antagonists, antibiotics and other molecules) on the capacity of splenic cells to clear invasive bacteria or viruses. The experiments will be carried out each time a spleen sample will be available.

The detailed technical description of the experimental strategy including the strategy for storage and discarding of the human tissue will be as follows:

1. Whole organ ex vivo perfusion: This work is done to stabilise the organ, infect the organ and then to derive from it tissue slices and cells for primary cell culture. We have successfully set up a normo-thermic ex vivo porcine spleen perfusion model. This expertise allowed us to define the parameters for organ collection, transport and perfusion of porcine spleens for up to six hours and importantly develop and infection model. Infection showed conservation of capacity to remove bacteria from blood and good filtering capacity and, essential for this application, that also in the whole organ model specific subsets of macrophages are permissive to bacterial intracellular replication. In this experimental series we propose to perfuse whole explanted human spleens. In contrast to pigs, we will use for perfusion of the human organ synthetic medium in order to neutralise or minimise acquired immunity. Histology, confocal microscopy, FACS analysis aRNAseq of pathogens and host cells and bacterial, parasite and viral enumeration will be the main methodologies to analyse the perfused organ. The aim of this part of the work is to demonstrate that at the whole organ level bacteria and viruses are able to replicate in splenic tissue macrophages pinpointing attention of anti-infective treatments to the prevention of these early steps potentially preventing invasive bacterial and viral disease. Samples from the 42 whole perfused organs will be the source of tissue slice cultures and primary cell cultures.
2. Primary cell cultures: From the 42 human spleens we will derive samples for primary culture of human tissue macrophages and other immune cells. Using the methodology in use for cells from mice and pigs, we will set up primary cultures of human splenic immune cells and in particular macrophages. Immune cells including macrophages and neutrophils will be purified by different methodologies including by percoll gradient centrifugation, FACS and magnetic separation and cultured for up to seven days. Cultures will be tested for multiple parameters including cytokines, surface markers, anybody production and gene expression. To test interaction with bacteria or viruses, the cultured cells will be infected with a panel of bacterial and viral pathogens. Upon other methodology, confocal microscopy, FACS and RNAseq will be used monitor behaviour of bacterial, parasitic and viral pathogens and host cells. Interventions with cytokines, chemokines, drugs, antibiotics and other compounds will be carried out to test tractability of the system. With the aim in mind to identify targets for intervention, the cell culture models are intended to provide detailed quantifiable parameters of the interaction of bacteria or viruses and immune cells in particular tissue macrophages in a controlled in vitro culture system. This is intended to allow the design of medium-throughput assays to test strategies modifying initiation of invasive disease. Cells will be stored in liquid nitrogen for further research.
3. Organotypic slice cultures: From the 42 human spleens we will derive, in addition to cells, also tissue samples for organotypic slice cultures. With the methodology well set up for brain slices and described for spleen, we will culture organotypic human spleen slice cultures. These organ slices are superior to cell cultures, as they maintain the multicellular spatial microarchitecture of the organ, which is particularly important when analysing tissue macrophages. As for brain slice cultures, we will infect the slices with bacterial or viral pathogens and monitor their migration within the tissue, the infection of cells and the reaction of the single host cells. The use of tissue slice culture should allow to specifically study the targeting and replication-permissive nature of specific subsets of immune cells including macrophages. A goal of the objective is to allow for detailed development of infectious foci in the spleen including time laps confocal microscopy of infected spleen slices. Testing in this model cytokines and other host derived molecules are expected to show the potential of interventions targeting specific subsets of immune cells including macrophages. Tissue slices will be stored in liquid nitrogen for further research.

Sample transfer between units: The 42 planned organ perfusion will be performed at the laboratories of the HBP unit of the Leicester General Hospital. In selected cases organ perfusion might be performed at the Department of Genetics and Genome Biology of the University of Leicester. The relative HBA form has been obtained (Hazardous Biological Agents form GEN/45 Cell lines and primary human cell and tissue culture, date 02/10/2017). During and after perfusion, tissue samples for tissue slice culture and primary cell culture will be taken. These tissue samples will be transferred to the Department of Genetics and Genome Biology or Dept. of Infection and Immunity and Inflammation for the scheduled research work and will be stored there in accordance with regulations relative to the Human Tissue Act. Stored tissue slices and primary cell cultures may then be transferred for immunological studies to the University of Nottingham for experimental work detailed above for cell cultures. Conditions for handling, storage and transport are defined by the HBA form GEN45 and transfer will be regulated by an MTA.