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)