Dr Iestyn Pope’s team at Cardiff University was recently awarded £53,950 to take the following project forward:

Incorporating deuterium into 3D organoids to identify cell types and track cellular metabolic rates using CARS microscopy

Organoids are 3D microtissues that reproduce more accurately how a tissue behaves compared to a 2D culture on plastic. This is because it allows the cells to interact with each other and to organise their own microenvironment. Recently, we and others have shown that patient-derived cancer organoids retain the cellular diversity that matches that of the tumours from which they are derived. Thus 3D cell culture has the potential to revolutionise technical approaches in personalised medicine and drug discovery.

Typically, identification of different cell types within cancer organoids, and many of the experiments undertaken on them are performed by first tagging the cells or molecules of interest with a fluorescent marker, which can then be visualised on a microscope. However, using this technique there is always the potential that the addition of the fluorescent marker alters the behaviour of the cell or molecule you are interested in following.

Over recent years, Coherent anti-Stokes Raman Scattering (CARS) microscopy has emerged as a powerful label-free way of rapidly imaging live cells and tissues with high 3D spatial resolution and quantitative chemical information. Rather than looking at a fluorescent marker, CARS directly probes the bonds of the molecules that make up the cells and tissues of interest. One such bond that is frequently probed is the bond between carbon and hydrogen (CH2) since it is present in large numbers within biological components. Recent work has demonstrated how the addition of deuterated water (also known as heavy water, since the hydrogen atoms that make up the water contain an extra neutron compared to regular hydrogen) can be used to generate carbon–deuterium (C–D) bonds in molecules within cells. Owing to the heavier isotopic mass, deuterated bonds can be easily distinguished from other cellular signatures when imaged with CARS microscopy.

We hypothesise that this will allow us to perform two crucial experiments on 3D cell models:

1) As different types of cell make different biological molecules, we should be able to tell the cells apart simply by the way in which they use the deuterium from the heavy water.

2) Different types of cells metabolise material in different ways, and this heterogeneity could potentially affect their susceptibility to therapeutics. By studying the turnover of deuterium we should be able to understand the cells metabolism in more detail without using fluorescent markers.

In this study we intend to use CARS microscopy to image organoids that have been cultured in media containing deuterated water. The aim being to identify the different cell types that make up the organoid structures without the need for fluorescent markers. Similarly by feeding the cells with deuterated compounds we will be able to observe the uptake or metabolic rate of the individual cells and what effects different drugs have on this rate. This will allow us to study these complex systems with minimal interference or modification, allowing long term studies of the effect of therapeutic drugs on cell development and metabolism – a current unmet need within therapeutic design.

3DbioNet received 21 eligible applications in total, and had a final ranking review call with our advisory board on Friday August 30th, the outcome of which we are pleased to share. We were only able to fund four projects in this round, there are many more we would have liked to have funded. The four successful projects are as follows:

Award amount: £45,750
Principal Investigator: Dr Ipsita Roy 
Institution: University of Sheffield, Material Science and Engineering
Project title: A natural and sustainable biomaterial-based 3D model of healthy cardiac tissue

Award amount: £63,980
Principal Investigator: Prof Ruth Cameron
Institution: Cambridge Centre for Medical Materials / Materials Science and Metallurgy
Project title: Directing respiratory organoid development via tailored three-dimensional macromolecular environments

Award amount: £57,906
Principal Investigator: Dr Eirini Velliou
Institution: Bioprocess and Biochemical Engineering group (BioProChem), Department of Chemical and Process Engineering
Project title: On the design of a biomimetic 3D printed metastasis prototype of primary ovarian cancer – towards Personalised Healthcare

Award amount: £53,950
Principal Investigator: Dr Iestyn Pope
Institution: Cardiff University, School of Biosciences
Project title: Incorporating deuterium into 3D organoids to identify cell types and track cellular metabolic rates using CARS microscopy.

Professor Ruth Cameron’s team at the University of Cambridge was recently awarded £63,980 to take the following project forward:

Directing respiratory organoid development via tailored three-dimensional macromolecular environments

Our aim is to produce alveolar organoid structures in highly defined 3D structures. This will allow us to transition away from current spherical organoid systems, typically cultured using Matrigel, to a 3D system that more appropriately reflects tissue organisation. We propose to design ice –templated, collagen- and elastin-based, porous environments to supply the spatial, mechanical and biochemical cues of native tissue.

The self-organisation of alveolar-derived co-cultures in 3D cell scaffolds will be studied in structures possessing a range of density, porosity and composition, identifying conditions that promote cellular organisation to closely match native lung. Critically these tailorable structures permit systematic decoupling and assessment of extracellular matrix (ECM) attributes that are crucial for lung cell organisation which is not possible using current homogeneous supports (e.g. transwell inserts and Matrigel) or decellularised lung tissue. Instead our system will allow the study of cells in a native-ECM structure with systematically altered properties to address specific, hypothesis-driven, questions such as the influence of architecture, biochemical cues and stiffness.

We will use ice-templating methodology to fabricate highly porous, completely interconnected 3D structures of biologically-derived collagen I and elastin with precisely tailored architectural cues. These are highly cell conductive and can be used for the formation of complex self organised cell structures, developed from multiple cell types. We will explore the effects of pore architecture, mechanics, the ratio of collagen and elastin and the use of synthetic peptide motifs to direct the cellular phenotype.

This project will provide proof of concept of a system replicating lung cell self-organisation with the flexibility to decouple, assess and address environmental attributes crucial for lung development and disease progression.

Dr Eirini Velliou’s team at the University of Surrey was recently awarded £57,906 to take the following project forward:

On the design of a biomimetic 3D printed metastasis prototype of primary ovarian cancer – towards personalised healthcare

Ovarian cancer is a silent gynecological killer with late stage diagnosis, relatively low survival rate, high resistance to chemotherapy and high recurrence rate. While research is being conducted to answer, and solve many of its associated problems, the lack of a robust, high throughput in vitro model for the disease and its metastasis puts a dent on research success.

Most studies for ovarian cancer use either animal models or simplistic 2D cell culture systems. However, animal studies are expensive, difficult to reproduce and have ethical issues associated with their use. At the same time, 2D models are unable to mimic the complexities of the human body in terms of cellular arrangement, presence 3D structure, cell- cell and cell-matrix interactions, interstitial flow etc. Tissue engineering and the emerging of 3D tissue culture models can mitigate many of the problems associated with animal models and 2D cell culture. 3D models provide a 3-dimensional growth environment able to mimic spatially and biochemically an actual tissue, they allow for cell-cell and cell-Extracellular Matrix (ECM) interactions, they provide structural integrity and can be incorporated in bioreactors to mimic the interstitial flow. Additionally, they are less expensive and devoid of ethical challenges associated with animal models.

Due to the above benefits, researchers have started using 3D models for ovarian cancer research. However, most models are single cell based and do not capture the biological complexity of a real tumour. Furthermore, the models of omentum, which is the primary metastasis site, are also relatively simplistic and do not capture the complexity of the organ. Overall, to date, there is no model of ovarian cancer that integrates the complex tumour biological structure coupled with a robust omentum structure. Here, we aim to bridge that gap via developing for the first time a robust dynamic multicellular model of a human primary ovarian tumour and its primary metastasis site (omentum). This novel model involves multicellular tumour aggregates from fresh human ovarian cancer tissue coupled with a complete omentum model.

For the integrated model development, a multidisciplinary group of scientists will work together using human derived tissues and techniques such as 3D-printing, novel materials and bioreactors to mimic the interstitial flow. The model will enable advanced studies on the disease progression, treatment response as well as the screening of novel treatment methods. Therefore, it will be of benefit to clinicians, academics and the industry. Most importantly, it will facilitate faster development of personalised treatment protocols, accelerating appropriate individualized therapies for the disease from bench to bedside.

Dr. Olayanju, one of 3DbioNet’s Collaborative Challenge award recipients, has been investigating the use of PeptiGels in the development of gastro-intestinal (GI) organoids. He sends the following project update:

INTRODUCTION

A major challenge in advancing preclinical studies is the lack of robust in vitro culture systems that fully recapitulate what happens in vivo. Organoids, the 3-dimensional (3D) self-replicating structures are increasingly being shown to be powerful models for ex vivo experimentation in the field of tissue engineering. Organoid formation requires the use of extracellular matrix (ECM) components to form the 3D conformation. However, most of the commonly used ECMs especially Matrigel come from a tumorigenic source limiting their translational validity. Therefore, the testing of alternative ECM sources such as PeptiGel will contribute immensely to the field of 3D cultures.

METHODOLOY

The use of PeptiGels as an alternative source of ECM in the development of GI organoids was investigated. GI crypts or single cells were isolated from healthy porcine tissue and propagated on different versions of PeptiGels (Alpha 1-5) to see which version provides the optimal environment for the culture of these cells. Resulting organoids were assessed by 3D morphology using microscopy techniques. In addition, a pilot study will be done to compare organoids grown on PeptiGels and those grown on conventional ECMs such as Matrigel. Resulting organoids will be phenotyped by PCR for selected markers.

RESULTS

Time course generation of hepatic organoids using PeptiGel Technology

PeptiGel-generated organoid units were maintained in liver isolation media and initially started as single cells (days 1-2), however as they progressed in culture, the cells started to come together to form the 3D structures (organoids) and by day 6, there were organoid-like features in the cultures. Notably, hepatic cells grown in PeptiGel Alpha 5 showed visible organoids by day 6. By day 13, they were fully formed organoids within the cultures. An assessment of the organoids showed that the hepatic cells showed different rate of formation when grown on the different versions of the PeptiGel when compared to the control organoid grown on Matrigel (Figure 1). PeptiGel-generated organoids using Alpha 1 showed organoid-like structures by day 14 but by day 27, they have fully dissociated. PeptiGel-generated organoids using Alpha 2-4 showed similar morphologies to the ones grown on Alpha 1 but those generated using Alpha 2 showed a higher rate of formation compared to those grown on Alpha 3 and 4. Interestingly, the organoids grown on Alpha 5 showed a different morphology with lots of branching hence requiring further investigation (Figure. 1)

Figure 1: PeptiGel-generated hepatic organoids. Time course establishment of porcine liver organoids using PeptiGels technological platform. All original images were at taken at x10 magnification using an inverted Olympus CKX53 microscope.

DISCUSSION & FUTURE WORK

The PeptiGels are expected to support the generation of GI organoids with appropriate morphology and expressing tissue-specific markers. Hence, PeptiGel-generated organoids have been established using porcine hepatic cells. Present investigations on this work are looking to further establish the culture of hepatic cells in PeptiGels by looking at cryopreservation of PeptiGel-generated organoids, the passaging of the organoids generated, and the growth of organoids for an extended period of time. In addition, more robust internal cellular morphological analysis and spatial arrangement of cells using higher magnifications will be carried out. Finally, phenotyping of the PeptiGel-generated organoids using PCR will be carried out to assess the presence of tissue-specific markers.

Synthetic PeptiGels are non-toxic, biocompatible and biodegradable. PeptiGel-generated organoids may therefore enhance translational research and reproducibility. The present findings from this work showed the potential of PeptiGel technological platform as a suitable ECM to some of the currently used ECMs and such a product may have huge clinical applications in surgical interventions.

Collaborators: Prof. Aline Miller (ManchesterBIOGEL), Prof. Chris. Goldring (CDSS, UoL)