3D Cell Culture in Microgels: A Novel Screening Platform for Disease Modelling and Drug Testing

Abstract

Cancer is the second leading cause of deaths in the world with colon cancer being one of the deadliest types of cancer. However, only 7% of new anti-cancer drugs make it through clinical trials and are brought to the market. This low success rate has been attributed to a lack of relevant cancer models. New drug candidates are usually tested on 2D cancer cell lines adhered to a plastic surface, which neglect many aspects of a tumour in vivo, such as cell-cell and cellmatrix interactions. In 2011, the first successful long-term culture of epithelial cells from primary tissue was developed by the Clevers group. The culture of these so-called ‘organoids’ was first developed from mouse small intestinal stem cells and then expanded to human healthy and cancer intestinal epithelial cells. The development of the organoid culture system opened the road for new cancer models which represent the cancer in a more natural setting by including a hierarchical cell organization in a 3D system with cell-cell-interactions and extracellular matrix components. Despite their advantages over 2D culture and the use of cell lines, organoids are not widely used for drug screening by the pharmaceutical industry due to reproducibility issues and lack of standardization of organoid culture systems. Organoids are cultured in a basement membrane extract (BME) purified from mouse sarcoma (Matrigel, Cultrex, Geltrex), which is not fully defined and exhibits batch-to-batch variation. Thus, an important step towards a more robust and standardized organoid culture system is the replacement of this BME extract by a fully defined hydrogel. This thesis describes the development of a new hydrogel for human colorectal cancer (CRC) organoid culture that consists of low-melting agarose and a peptidebased gel (peptigel). Five patient-derived CRC organoid lines were characterised in this gel and compared to culture in Cultrex. Light microscopy, immunofluorescent staining and RTqPCR revealed comparable growth, structural organization and gene expression profiles of organoids in both gels. Furthermore, this hydrogel was used to encapsulate CRC organoids in microgels via droplet microfluidics to enable new assays applicable for drug screening. To make the organoid-microgel system applicable to combinatorial drug screening, I developed a barcoding method to couple treatment information to the microgels. By using biotinfunctionalized agarose, barcodes can be immobilized onto microgels during drug treatment to encode a specific drug. This can be used for screening multiple drug-combinations at the same time via a split-and-mix approach. This system was used for a proof-of-principle experiment iv with MCF7-spheroids and additionally for a small screen of different combinations on encapsulated CRC organoids. 3D cell culture in microgels can also be applied to study cell-cell-interactions and cell migration towards a signal. By re-encapsulating microgels in an outer layer of cells and hydrogel, two compartments with different contents can be created. This approach was used to study the interaction of neurons and microglia in microgels. Another application of microgel culture is the incorporation of sensors into the microgel to study certain cell behaviours. A MMP9-sensitive peptide was incorporated into biotin-agarose microgels to investigate cell invasiveness. In this way, MDA-MB231 cells were shown to secrete more active MMP9 than MCF7 cells, which was also confirmed by RT-qPCR of both cell lines encapsulated in microgels. In Summary, I developed a fully defined hydrogel which can be used to culture patient-derived CRC organoids. Moreover, this gel is applicable for droplet-microfluidics to create microgels for 3D cell culture. Development of a spheroid and organoid culture system in microgels enabled the design of new assays for drug screening and analysis of cellular features such as invasiveness or cell proliferation.