The 3D'omics Sample Preparation Work-flow
First, we ran small-scale proof-of-concept trials to i) obtain samples for technique development in WPs 3:5, ii) obtain first-hand biological information on effect sizes of features of interest to better design the trials scheduled for WP6 and WP7, and iii) obtain intestinal/faecal samples for the in vitro tests in WP3, WP6 and WP7. Second, we employed multiple approaches to identify the most optimal procedures to sample, fixate, and microdissect intestinal tissues in a way that maximises nucleic acid and metabolite yield while enabling high quality imaging procedures.

Specifically, we conducted two Proof-of-principle poultry trial(s). In Copenhagen and Vienna, we ran small-scale Salmonella Infantis challenge trials similar to the larger trial to be conducted in WP6. The trials, consisted of three treatments, one pen replicate per treatment and 15 chickens per pen for a total number of 45 chickens. The three treatments were Treatment(T)1: PoultryStar® ME application from day 1 onwards, T2: an infection control with no synbiotic product, and T3: negative control with no synbiotic product and no infectious agent. Birds were infected with S. Infantis on day 21. Five animals per pen will be sampled just before the infection, five more one week after infection and the last five animals three weeks after infection. We further conducted one Proof-of-principle swine trial, which mimics the larger trial to be conducted in WP7. The trial was carried out with single sex and breed. The data gathered from the Proof-of-principle trials were used to test and refine the most optimal sampling and sample fixation strategies. To be able to implement the protocol for large scale trials, and preserve the DNA, RNA and native proteins, we prepared frozen sections. Tissue was dissected post euthanasia and immediately submerged in precooled carboxymethyl cellulose gel (CMC) (Sigma-Aldrich) and snap-frozen in liquid nitrogen. Samples were stored at −80 °C until tissue sectioning and use. We also performed trials with tissue embedded in precooled Tissue-Tek OCT Compound (Sakura) and TFM-Tissue Freezing Medium (GD Healthcare).

Task 3.4 >> Continuous sampling and fixation from in vivo models | Leader: UCPH-V | M1-M18 The proof-of-concept swine trial will be also used to assess the possibility of generating longitudinal 3D’omic data from lower intestinal biopsies from alive pigs. One piglet from the control group will be subject to a continuous sampling Page 18 of 48 of the colon intestinal tissue through colonoscopy to assess the possibility of obtaining longitudinal samples for 3D multi’omics. Biopsies will be obtained through colonoscopy from anaesthetised pigs. The procedure will be conducted at three time points, one week before weaning and one and four weeks after, respectively. Task 3.5 >> Sampling from in vitro microbiota models | Leader: ETH | M1-M29 ETH, BGU and NMBU will perform preliminary in vitro assays following different strategies to optimise procedures for sampling and fixating in vitro samples for generating 3D’omic data. Microbiota samples will be derived from the trials described in T3.1 and T3.2 and cryopreserved following validated protocols. ETH will run in vitro experiments that will mimic the trials designed for WP6 to test different procedures to sample and fixate PolyFermS-based in vitro spatially structured microbiota samples (1-2 mm polymer beads), while BGU and NMBU will use their anaerobic microbiology facilities to pre-evaluate the prebiotic fibers and to prepare the procedures for the larger trial to be conducted in WP7 with the experimental prebiotic fibres. Task 3.6 >> Simulations for optimal slicing and laser microdissection | Leader: CRG | M1-M18 Different host tissues, microbial communities and omic information will probably require different slicing and microdissection strategies to be efficiently implemented. For example, the resolution required for capturing gene expression differences (transcriptomics) is expected to be larger than the resolution required for identifying bacteria (genomics), as many bacteria are expected to be found forming clonal aggregations that while being genetically identical can be expressing different genes depending on the surrounding environment. CRG will work along with KUL, ETH and BGU to develop a series of methods to computationally assess multiple of such scenarios based on the available knowledge of microbial organisation in the analysed tissues, which will be used to guide prioritisation strategies when testing different slicing and microdissection approaches in T3.5. Task 3.7 >> Tissue sectioning and microdissection | Leader: UCPH-M | M4-M24 UCPH-M will develop the tissue sample sectioning and laser microdissection procedures for obtaining appropriately preserved slides and microsections for downstream multi-omic analyses. For this, UCPH-M will test a range of strategies to find the optimal solution. The embedded tissues will be sectioned at 4 to 10 μm thickness using the cryostat (Leica Biosystems) at a chamber temperature of −35 °C and object holder temperature of −22 °C. Individual sections will be thaw-mounted onto different types of MembraneSlides (i.e. PPS, polyphenylene sulfide; PEN, polyethylene naphthalate; PET, polyethylene terephthalate; POL, polyester; FLUO, fluoropolymer) and subsequently frozen in the cryostat chamber. Slides with tissue sections will be stored in slide containers with silica granules to prevent air moisture condensation on the tissue after removal from the freezer. The preliminary tests will include varying slicing angles (e.g. cross-section vs. longitudinal), multiple slice thickness (e.g. 4 μm, 10 μm), multiple microdissection grid sizes (e.g. 10x10 μm, 50x50 μm, 100x100 μm) and different automatisation strategies. These tests will aim to identify the technical limitations, as well as to acknowledge the most optimal procedures to obtain relevant biological information in an efficient manner. These experiments will be conducted in the dedicated 3D’omics lab UCPH will accommodate with the entire infrastructure (i.e. cryostats tissue sectioning, laser microdissection microscope for microdissection, storage units) needed to prepare samples for 3D’omic data generation.