Patient-Derived Colorectal Organoid BioBank 
  Generated by Dr Stephanie Burnell
  BurnellS@Cardiff.ac.uk, Stephanie Burnell | LinkedIn

Organoids are miniature, three-dimensional structures grown from stem cells that mimic the architecture and functionality of real organs. These complex models are invaluable for research as they provide a more accurate representation of human biology when compared to traditional cell cultures. Organoids are particularly useful in studying disease mechanisms, drug responses, and personalised medicine approaches. Visually, organoids resemble tiny, self-organizing clusters of cells, each unique to the tissue they represent. 

In our lab, the organoids we have developed have been derived from patient tissue, kindly donated by patients undergoing surgery. We have successfully developed colorectal organoids from both healthy and tumour tissue, which replicate the heterogeneity found in patients. These organoids serve as powerful tools for advancing our understanding of colorectal cancer (CRC), developing effective treatments and for exploration of how the immune system might recognise, isolate and kill malignant but not healthy cells.

Generation of a colorectal organoid biobank. Following informed consent, tissue and blood samples are obtained from patients undergoing surgery and transported to the lab for processing. From the blood sample, peripheral blood mononuclear cells are isolated and frozen in liquid nitrogen for future use. The tissue samples are split – half is frozen in OCT and stored at -80°C for future staining and comparison to original tissue and the other half is dissociated and processed to isolate the colonic crypts. The crypts are then embedded in a matrigel scaffold to encourage the formation of organoids. 

Characterisation of Colorectal Organoids
Dr Stephanie Burnell and Chloe Harris

BurnellS@Cardiff.ac.uk, HarrisC27@cardiff.ac.uk



We are characterising our bank of colorectal organoids to determine the mutational load, HLA status, and morphology and prove that the organoids contain multiple cell types. We are doing this via various methods including whole genome and RNA sequencing (WGS. RNAseq), flow cytometry and confocal microscopy. Stay tuned for updates as we carry out our characterisation experiments. This information, alongside clinical information, will provide researchers with a more specific and clearer picture of the model they are working with. Additionally, we are developing a computational pipeline to estimate the number of cells within an organoid using only a brightfield image, our manuscript is in preparation.

Organoid/T Cell Co-culture Model

Dr Stephanie Burnell, Chloe Harris, Lorenzo Capitani and Dr Yuan Chen

BurnellS@Cardiff.ac.uk, HarrisC27@cardiff.ac.uk, CapitaniL@cardiff.ac.uk, ChenY158@cardiff.ac.uk 

Our co-culture model involves growing CRC organoids alongside T cells, creating a dynamic system to study immune interactions and test immunotherapies. Using the Opera Phenix high-content imaging system, we have captured detailed images showcasing the interactions between T cells and organoids. Co-culture experiments demonstrate effective T-cell infiltration and tumour-cell killing. We invite researchers and clinicians to collaborate with us in utilising this co-culture system to test novel therapies and investigate tumour-immune dynamics.

Replacement of Matrigel in Organoid Culture

Chloe Harris

HarrisC27@cardiff.ac.uk, Chloe Harris | LinkedIn

My PhD is funded by the NC3Rs and is centred on the replacement of mouse immunotherapy models with human colorectal cancer organoids which Stephanie has developed. Our overall aim is to develop a system, which reduces and replaces the use of mice in T cell immunotherapy studies using human organoids in co-culture with genetically engineered T cells. For this purpose, we will combine human organoid technology with our bank of cancer-antigen specific T cells derived from patients with cancer. Organoids represent a favourable alternative to animal models; they recapitulate multiple aspects of real organs, making them relevant to the study of organ development, function and disease

Within this lab alone, it is estimated that 2000 mice will be sacrificed over a 5-year period for adoptive transfer studies, which could be replaced by a human organoid T cell co-culture system. 

We will also be trialling the use of animal free hydrogels to culture these organoids, rather than murine-derived Matrigel. Studies have found that 1 mouse is sacrificed for every 6.3 mL of Matrigel. Therefore, these efforts will also reduce the lab’s animal burden, with the lab currently using over 200mL of Matrigel annually. Kibbey, Maura C. (1 September 1994): 227–30. https://doi.org/10.1007/BF01540656.

Matrigel, a commonly used matrix for organoid culture, is derived from mouse sarcoma cells and contains a complex mixture of extracellular matrix proteins and growth factors. While it supports organoid growth and differentiation, Matrigel's animal origin and batch-to-batch variability pose significant challenges. These include ethical concerns, inconsistent experimental results, and difficulties in standardising research protocols.

We are currently trialling various commercially available synthetic, natural, and human-derived hydrogels as a Matrigel replacement to overcome these limitations. Synthetic hydrogels offer a defined and reproducible environment for organoid culture, free from animal-derived components. These hydrogels can be engineered to provide specific biochemical and mechanical properties that support organoid growth and mimic the natural tissue environment.

Matrigel alternatives and plan of action. We will test different non-animal-derived hydrogels from companies such as The Well Biosciences, PeptiMatrix, Humabiologics and UPM Biomedicals. Initially, we will transfer the organoids from matrigel into the hydrogel and monitor a variety of endpoints. We have several stop points to discard the gel if the experiments are unsuccessful. At the end of the project, we hope to have a successful alternative to matrigel to grow, maintain and experiment on the organoids.

Funding from this project has been received from Wales Cancer Research Centre, Wales Cancer BioBank, NC3Rs, GW4, UKRI Harmonised Impact Acceleration Account, Institutional Strategic Support Fund Public Engagement Award, Future Leaders in Cancer Research Seedcorn Funding.