3DbioNet

Collaborative Challenge update: First Progress Report from Dr. Adedamola Olayanju

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)

Mini-brain organoids: a Post Card from John Hopkins

Dear 3DbioNet,

Many thanks for the Collaboration Challenge award. I visited Professor Thomas Hartung’s laboratory at Johns Hopkins University, MD, USA from 8th to 14 th April 2019.

Thomas Hartung’s team at John Hopkins

During the visit, Dr Lena Smimova showed me how to culture mini-brain organoids (spheroids) from iPS-derived NPC using a shaker protocol.

The shaker in a CO2 incubator and Dr Lena Smimova at work

I also did one set of my own culture and follow it for 5 days. At the same time, I did immunofluorescence staining for fixed spheroids provided by Dr Smimova. It was an enjoyable and successful trip for our collaboration.

Left: my spheroids in day 5 from NPC; right: spheroid stained with tubulin in green and GFAP in red. Blue is Hoechst.

At the moment, I am setting up the culture system at Sheffield Institute for Translational Neuroscience (SITraN) and will compare it with a protocol from Professor Jens Schwamborn at University of Luxembourg by using the Quasi Vivo millifluidic system from Kirkstall Ltd.

I have contacted Simon Butterworth at Kirkstall to arrange delivery the system in the near future.

Sincerely yours,†††††††

Ke Ning

Senior Lecturer in Translational Neuroscience

A productive workshop in Sheffield

The second 3DbioNet workshop took place in Sheffield on the 6th of June 2019, hosted by John Haycock and the University of Sheffield. The day combined lectures, technology highlights, an early career researcher led speed-dating session, the launch of our first call for pump prime grants, small group brain storming on 3DbioNet challenges, and poster sessions culminating with the announcement of poster prizes (1st Joseph Leedale; 2nd, Amy Harding; 3rd Lynne Bingle). We thank our sponsors (Manchester Biogel, Merck, the National Measurement Laboratory, the Electrospinning company) and all the speakers, poster presenters and attendees who contributed to the success of the meeting.

First call for pump-priming projects

This is the first 3DbioNet call for pump-priming projects. The aim is to build on discussions and networking that took place in Liverpool (Launch Workshop, January 2019) and Sheffield (2nd Workshop, June 2019). The budget for individual projects is capped at £80K and we envisage funding approximately 5-6 projects in this call.

Scope and eligibility

  • Applications must address the remit of the network and aspects of the challenges identified during the 3DbioNet workshops.
  • The projects will be evaluated by 3DbioNet’s advisory board based on the quality and originality of the science, the fit with the remit of the call, the added value of the collaboration, and the potential for sustainability and longer term funding.
  • A minimum of 2 institutions should be involved in each project. One of these could be an industrial partner.
  • Applications must be led by a UK-based investigator, who attended at least one of the 3DbioNet workshops.
  • We expect to fund a range of projects. We have a budget of £240k for this call and expect to fund 5 to 6 projects, with a maximum budget of £80k per project.
  • Projects will be funded at 80% FEC.
  • All projects must be completed by 31st December 2020 (at the very latest).
  • If a grant is awarded, then the applicants commit to providing a final report by 30th January 2021 including a one page summary suitable for sharing on the network blog.

How to apply? Simple: download, complete and send the application form.

Key dates

Deadline for Submission: Friday 12th July 2019

Notification of outcomes: Monday 29th July 2019

Submit your proposal to 3dbionet@liverpool.ac.uk

3DbioNet Peeriodical – discussing 3D cell biology

A Peeriodical is a website that facilitate discussions of any published journal article that has a DOI. We have set up a 3DbioNet Peeriodical as a platform for scientists with interest in 3D biology field to review and discuss recent or older publications. We are facilitating this journal club because we believe that open discussion of science is essential, especially in an interdisciplinary field like ours. We hope this space for scientific discussion will benefit all the 3DbioNet network members in their current and future research, to improve the robustness of research outputs, identify problems, and share new ideas/protocols to improve our working practices.

Have a look at the first three reviews:

Blended electrospinning with human liver extracellular matrix for engineering new hepatic microenvironments (2019); Rhiannon Grant, John Hallett, Stuart Forbes, David Hay, Anthony Callanan – Reviewed by Raphaël Lévy and Jonathan Temple

Familial Alzheimer’s disease patient-derived neurons reveal distinct mutation-specific effects on amyloid beta (2019); Charles Arber, Jamie Toombs, Christopher Lovejoy, Natalie S. Ryan, Ross W. Paterson, Nanet Willumsen, Eleni Gkanatsiou, Erik Portelius, Kaj Blennow, Amanda Heslegrave, Jonathan M. Schott, John Hardy, Tammaryn Lashley, Nick C. Fox, Henrik Zetterberg, Selina Wray – Reviewed by Sandrine Willaime-Morawek

Aggregated P19 mouse embryonal carcinoma cells as a simple in vitro model to study the molecular regulations of mesoderm formation and axial elongation morphogenesis (2009); Yusuke Marikawa, Dana Ann A. Tamashiro, Toko C. Fujita, Vernadeth B. Alarcón – Reviewed by David A Turner

You can participate to this journal club in several ways:

1. Comment on any of the existing reviews (just hit “Reply” at the bottom of a review; you will have the option of staying anonymous or of signing your comment)

2. Contact us (email or Twitter) to suggest a paper you would like us to review.

3. Write your own review of a paper and send it to us to share with the wider community.

Collaboration Challenge

3DbioNet announced the #CollaborationChallenge, its first call for funding in January 2019. This is to facilitate new collaborations between partners.

We received six applications and three of them will receive the award of upto £2000 upon completion of their project. The selection criteria for the award were the scientific excellence, the quality of the interdisciplinary collaboration and the fit with 3DbioNet’s remit. At least one of the applicants must have had attended 3DbioNet’s Launch Workshop in Liverpool (14-15 January 2019). Participation from industry partners and Early Career Researchers was encouraged.

The winners of the first collaboration challenge are:

  1. Dr. Ke Ning, senior lecturer in Translational Neuroscience at Sheffield Institute for Translational Neuroscience at Sheffield University. The group was also the winner of the Quasi Vivo millifluidic system from Kirkstall Ltd.

Project description: The project will be on the investigation of the neurons and glia interactions in organoid disease models of Parkinson’s disease (PD).  The collaborators will include Professor Jens Schwamborn at University of Luxembourg, and Professor Thomas Hartung, Johns Hopkins University, MD, USA. The expected outcome is that pathways involved in the neuron-glia crosstalk inside the hMOs would be identified as potential therapeutic targets for drug screening for PD.

2. Manohar Prasad Koduri , PhD student, University of Liverpool and NTHU, Taiwan, Dr Jude Curran (School of Engineering, University of Liverpool) , Dr James Henstock (Institute of Ageing and Chronic Disease, University of Liverpool), Professor John Hunt (Nottingham Trent University), – Professor Fan-Gang Tseng (Engineering and System science, NTHU, Taiwan). The group were also the winner of theBiogelx Discovery kit from Biogelx Ltd.

Project description: Tri-Functional Nano-Particle Biosensors for Real Time Monitoring of Physiological Conditions for 3 Dimensional Tissue Engineering Applications. Within this system, they propose to develop a novel 3D cell culture system, which will monitor and control the spatial resolution of selected cell types within a construct using a combination of hydrogels (Biogelx), Optical sensors and human stem cells. Nanoparticles can be used to tether fluorescent based oxygen, pH and glucose sensors onto one bead, and therefore providing spatially relevant information regarding the cell environment inside hydrogel. The hydrogel/cell/Nano particle spheres will be produced by an electrospray method (developed at NTHU) and culture conditions prevalent to the longevity of human cells will be optimized in vitro (Liverpool).

3. Dr. Adedamola Olayanju, an early career Post-doctoral scientist at Northwick Park Institute for Medical Research (NPIMR).

Project description: The use of PeptiGels as an alternative source of ECM in the development of GI organoids. M in the development of GI organoids will be investigated. GI crypts or single cells will be 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 will be assessed by 3D morphology using microscopy and immunostaining techniques. In addition, a pilot study will be done to compare organoids grown on PeptiGels to those grown on conventional ECMs such as Matrigel. Resulting organoids will be phenotyped by PCR for selected markers. This will be a collaboration between the NPIMR and Manchester BIOGEL.

FUTURE FUNDING CALLS

There will be more calls for this type of funding in the future. The next funding call will be for pump priming projects, to be launched on 6th June 2019 at the workshop in Sheffield. Click here to REGISTER for this workshop.

Call for articles: special issue on 3D microtissues

The 3DbioNet team is inviting you to contribute articles for a special issue on the interdisciplinary challenges associated with the development and adoption of 3D microtissues. The special issue will be published in the Royal Society journal Interface Focus (information for authors). Read more about the scope of the special issue below.

Conventional two-dimensional (2D) cell models are a poor representation of human tissue anatomy and physiology; this can result in pharmacological and toxicological responses to pharmaceutical agents which are not relevant to humans.  Increasing awareness of these issues has led to the development of 3D cell culture models of human tissues, but many of the routine research methods do not easily translate from 2D to 3D.  Exploitation of the new 3D models therefore requires the development and application of new technologies that can control cell growth conditions to better recapitulate the anatomy and physiology, enhance reproducibility, and enable the complexity of the cellular structures to be monitored as they develop. This special issue welcomes reports, research articles and reviews that define and address challenges associated with the development and adoption of 3D microtissues. The focus is interdisciplinary science for 3D microtissues, from the development of biomaterials and bioengineering processes for cell culture, to tools that can perturb and record thousands of individual cells and their biochemical responses, and advanced mathematical and computational models that will assist in the design and understanding of these systems.

Contact us to express your interest before Monday 4th of February 2019 (publication in this special issue is by invitation only).

Submission deadline: Monday 1st of July 2019.


One week to go before we launch

3DbioNet wishes you a happy New Year 2019.
2019 will be our first year of operation, starting in one week with the launch workshop in Liverpool (14-15/01/2019). Registrations are now closed. Whether you join us or not, we hope you contribute to the discussions via social media, including comments on this blog and posts on Twitter #3DbioNet.


Organoids: 2017 method of the year.

Nature Methods has selected Organoids as their method of the year 2017 for:

… their immense potential as tools to study human biology in health and disease.

You can find the collection of article here and the Editorial here.

 

Become a member of 3DbioNet

Whether you are in academia or industry, if you are interested to become a member of our network, please register your interest here.