Long-term, self-maintaining primary 3D cultures of mouse and human epithelial tissues for reduction and replacement of animals in research

This project aims at disseminating and helping with the application of a state-of-the-art in vitro 3D system developed during my postdoctoral research at the MRC-CU and the Welcome Sanger Institute and optimised in my laboratory at the Gurdon Institute/University of Cambridge, which allows for the production, long-term maintenance and expansion of 3D cultures of primary and tumour epithelial cells, directly obtained from mouse and human tissues, avoiding any previous step of single cell suspensions to set up the cultures.

Its ability to form tissue-like structures, in a unique and easy technique that combines the potential of amplification, scalability and a low cost, (similar to the cost of any cell line culture), with the generation of a complex 3D model that mimics tissue architecture and resembles physiological cell behaviour, makes this system tremendously useful for many laboratories working on epithelial tissues or using mouse and human primary cells for their research projects. It is also of great interest for researchers working on tissue regeneration and cell therapies, as it provides an almost unlimited number of primary epithelial cells with a high reliability and efficiency, ready to be used in their experimental research projects.

The system allows the culture and amplification of mouse and human primary epithelial cells from several type of epithelial and endothelial tissues (oesophagus, skin, bladder, tongue, urethra, breast, uterus, oral mucosa, etc) and enables studies of cell responses, cell competition mechanisms between normal and tumour cells, gene expression and protein interaction analysis, cancer drug screening, and long-term epithelial-immune cell co-culture assays, with high reliability, efficiency and low cost, compared with other methods currently used.

In addition, this system can also be used as an expandable resource of progenitor cells for different purposes (e.g., generation of organoids with higher yield than using traditional protocols that relies on the reduced number of cells within tissues, CRISP Cas9 screening assays using primary cells, regeneration studies and cell therapies).

Another great advantage of the system relies on its passage-free feature, which avoids the necessity of trypsinization and allows for maintaining primary cells in a homeostatic culture for up to a year and perform experiments such as long-term drug treatments and clonal tracing analyses, monitoring the same culture during the entire length of the experiment (something impossible to achieve with the current protocols that most laboratories are using).

This innovative experimental system provides a capability for tissue-like studies to be carried out in vitro during long periods of time and enables a Reduction in the number of mice used in research between 40% and 75%. It also greatly impacts animal Replacement as it allows the generation and long-term maintenance of primary human epithelial 3D cultures, using patient-derived biopsies, minimising the necessity of using mouse models in biomedical research projects.

Given the great proportion of laboratories in the UK and rest of the world requiring mouse primary cells for the successful development of their research projects, I do believe, the wide implementation of this novel technique will reduce the total number of sacrificed mice for research purposes by more than half while maintaining the robustness and reproducibility of their findings.

While many advances have been achieved in epithelial cultures since the first establishment of keratinocyte cell lines, the generation of complex 3D models that replicate long-term self-maintaining capacity of adult epithelia, relies on the use of complicated and long protocols and a great number of animals as source of primary cells. This project aims at disseminating a novel system for the production of trypsin-free, feeder-fee, self-maintaining, long-term 3D culture of mouse and human primary epithelial cells, both from normal and tumour adult tissues.
The ability to form homeostatic tissue-like structures, with no previous step of trypsinisation, in a unique and easy technique that combines the potential of amplification, scalability and low cost with the generation of a complex 3D model, makes this system of great utility for many laboratories working on epithelial tissues as well as for those using mouse and human primary cells for their research projects. It takes advantage of the tissue self-regeneration and wound healing capacities, to set up homeostatic cultures that resembles proliferation and differentiation levels to those from in vivo tissues and can be maintained for long periods with no changes in cell density or tissue integrity and function. It is also tremendously useful as a source of primary progenitor cells, that can be used for several purposes (e.g., generation of organoid with higher yield, CRISPR Cas9 screening assays in primary cells, regeneration and cell therapies studies, etc).
This innovative experimental system provides a capability for tissue-like studies to be carried out in vitro during long periods and enables a Reduction in the use of mice in research between 40% and 75%. It will also impact animal Replacement as it allows the generation of primary human epithelial 3D cultures using patient-derived samples, avoiding or minimising the use of mice in biomedical research and increasing the translational potential of these projects