This month, we sat down with Dr. Johanna Bolander. Dr. Bolander has a PhD in Regenerative Medicine from K.U Leuven and a postdoc at the MERLN Institute of Technology in Maastricht, as well as the Wake Forest Institute for Regenerative Medicine in North Carolina. Currently, she is an Assistant Professor at Charité Hospital in Berlin, as well as a Principal Member of the Technical Staff at imec. Dr. Bolander is an expert in bone and cartilage tissue and uses 3D models to investigate why certain tissues fail to heal. Her joint positions give her the unique opportunity todevelop solutions from both a biological and technical perspective.

Why do certain tissues fail to heal?

Introduce yourself, your professional journey, and how you got into the field of 3D culture. 

Dr. Bolander: I’m originally from Sweden. I studied engineering with a focus on biotechnology at Lund University’s Faculty of Engineering. I ended up in Leuven for my PhD after an Erasmus year in Brussels and the VUB. My original plan was to go into pharma, but during that year in Brussels I had a course on regenerative medicine, and I was completely mind blown. 

During that time, my research was focused on the development of cell-based therapies for critical size bone fractures and especially children with NF-1 mutations. 

I loved it because my promoter was a rheumatologist, and he made sure that my research was always aligned with the clinical setting, it had to be relevant for the patients at the end of the day. It was always clinical translation we had in mind, and it was very interdisciplinary. 

After my PhD, I got a post-doctoral fellowship so I could continue my research independently. This allowed me to go to MERLN in Maastricht, NL and the Wake Forest Institute for Regenerative Medicine in North Carolina where I expanded my research to look at joints, specifically osteoarthritis. With osteoarthritis, you need to look at the immune system as it plays an integral role in pathophysiology. This is when my engineering background came in handy – we can use immunomodulation to steer our regenerative environment. 

I have always been fortunate to work with human primary cells, and the studies at WFIRM allowed us to study specific patient populations, and I saw how relevant the findings were with the right defined patient groups. 

This fascinated me because, before that, I mainly worked with animal models for mechanistic research. And while they’re interesting in one way, you don’t get the same high resolution and dynamic measurements that you can get with in-vitro 3D cultures. 

Using 3D cell models to investigate bone and cartilage tissue repair

What are some of the current research questions that you’re diving into at the moment, and how are you using 3D models to answer them? 

Dr. Bolander: The big question that we have is: “Why do certain injuries fail to heal?” 

This can sometimes be easily explained for certain injuries, because it depends on the injury type and the tissue or the organ they’re affecting. My two favorite tissues are bone and cartilage.  

Bone is one of the unique tissues that can heal without scar tissue formation. You can have a complex fracture, but within a couple of months you will have beautiful regeneration. In an ideal setting, you won’t see that it ever was broken.  

Cartilage, on the other hand, is very limited in terms of regenerative capacity. 

It’s interesting to study these two tissues in compromised conditions and see what goes wrong when an injury fails to heal.  

I have a background in working with preclinical animal models. There you have some ability to get a systems’ understanding, you have an in-vivo environment, but it’s still far away from human.  

Human tissues are always the main tissue of interest. 

The problem is that you can only get human tissues at certain times. This is why I find bone and cartilage very interesting, as certain surgical interventions enable access if you have clinical approval for them and a surgeon that is willing to work with you. 

If you then use these cells to form in-vitro microphysiologial systems or organoid models, you can study the factors that are going wrong. This allows us to take the material and what we know from the pathophysiological process that we’re studying and model it in-vitro, and then to dynamically monitor cellular communication at the single-cell and at the tissue level. 

You need both because they’re typically unique cell populations that are driving certain processes. If you only use histology or immunohistochemistry from animal models or tissue samples, it will be challenging to gain this detailed mechanistic understanding. 

This is why I’m extremely fond of the 3D models. 

What has your research shown are some of the underlying causes for failed tissue repair? 

Dr. Bolander: This can be a patient background. Age, for instance. We know that as we age, we lose our regenerative capacity to a certain extent. This can also be affected by: 

• Lifestyle 
• Gender 
• Genetic background 

It’s something that you can easily stratify when you’re selecting your patient groups. You can look at certain factors and how they affect the cells and tissues’ ability to withstand stress, certain trauma indicators, or replications. 

Could you just run me through one of the routine assays that you perform in 3D? What’s the end point you’re looking for and what challenges come with it?  

Dr. Bolander: One that we continuously do is aggregation. This can be easily done in a 96-well plate using cells from one population, or you can mix different ones. In that way you can use different environmental factors.  

You can use hydrogels to embed the cells or just keep them in the wells themselves and see how they structurally arrange themselves to see if you have spatial organization depending on the environment. 

We use stimulatory conditions to mimic cartilage or bone matrix to see how the different cells play a role in this — how they communicate and interact with each other during either formation, differentiation, or in fibrotic environments. 

Typically, we try to monitor them dynamically. We label the cells and see how they move within the environment during different time points. We use endpoint analyses where we look at: 

• Immunohistochemistry 
• Gene expression 
• Spatial transcriptomic  

…to confirm that what we are seeing in a dynamic setting is true – this is to validate our findings.  

We’re moving towards detailed monitoring by nanotechnology systems. For instance, by impedance measurements and dynamic cytokine and protein monitoring of the secreted factors. I’m really excited about this because it allows you to get a more detailed understanding of what the single cell populations are doing and hodw they’re communicating with each other.  

I think we’re going to learn a lot from it. 

Merging biology and technology to make 3D cell models accessible

You wear two different hats: Your Assistant Professor position in Berlin and your position as Technical Staff Member at imec. 

What do those positions entail, and how do they overlap or complement each other? 

Dr. Bolander: I live a dream life. If you are in the area of 3D models, you know that both biology AND technology are crucial, and typically people’s focus is restricted to one of them. 

Imec is one of the world-leading institutes for nanotechnology solutions with a big arm in life science and health technology. Charité, on the other hand, is one of the largest hospitals in Europe that has a heavy focus on clinical research. 

The clinicians that work there are research-minded and interested in collaborating. They don’t mind walking the extra mile so we can get tissue samples. They’re also interested in the outcome, so we make sure that we can use the findings we produce for translational purposes so that one day we can go back to the patient. 

With these two settings, I have a team that is focused on technology development and a team that is focused on the biology that can collaborate. We have meetings together so that both teams can learn from each other. This makes us strong because both groups can have their focus, but they have a natural way of meeting each other. 

How do you think the requirements for working with 3D models differ between a pharmaceutical drug development setting and your regenerative medicine setting? 

Dr. Bolander: I think it’s very similar to the situation with biology and technology. At the moment it’s two very different things. I think we are learning that biology is not so easy, so drug screening needs to be done on more complex models. Not necessarily complex 3D bioreactor systems, but even if it’s just 3D aggregates or organoids, they need to represent the right compartments from physiology. 

On the other hand, we also need biological models to be relevant so that we can gain enough input from different patient populations, because we know that what happens in one patient may not be the case for another. 

High throughput and added complexity are needed in both cases. We need to find a match between the two aspects. 

The moment we can monitor physiologically relevant organoids in high throughput is when we will get a better understanding of why certain individuals respond to certain therapeutics, to trauma, and other events. 

The benefits of using 3D cell models when studying bone and cartilage tissue repair

When it comes to your cartilage and bone models, what information do 3D models give you that you can’t get in 2D? 

Dr. Bolander: The complexity. Both cartilage and bone are structured tissues. If you just have one layer, it’s just one part of the tissue. In biomechanics, this plays a huge role. This is true anywhere in the body, but especially for bone and cartilage because they are weight-bearing organs. 

This is something you can’t replicate in the 2D model. You need the 3D structure, and we see that this affects healing and homeostasis. It’s something that you can absolutely not ignore in my mind. 

In your opinion, what are the main obstacles that are still in the way of the full-scale implementation of these models in the clinic? 

Dr. Bolander: I think ethical guidelines are a big reason. There’s a lot of interest in creating biobanks and established pipelines so that organoids and tissues can be available to researchers. But it’s not clear what role the patient is playing in that, because they are contributing a lot. 

I think this is where a lot of work is needed to find a working model for how these kinds of materials can be used for research and also commercial purposes, because this is where it will lead. 

Another thing is capacity. You need to: 

• Isolate the tissue 
• Prepare it 
• Process it 
• Have the ability to culture 
• Drive the protocol  
• Do the analysis  

This isn’t something every lab can do…  

Also, robustness from the first point of isolation to the readout. We need to know what we’re actually looking for and make sure that what we’re doing makes sense. 

Another crucial aspect is regulation. With organoids and 3D models, we typically have a local tissue that we’re looking at. This is a very, very micro environment. The human body is regulated by the central nervous system through the peripheral nervous system. We have the vasculature, then we have the lymph nodes. 

We are still far away from being able to replicate this in an in-vitro model. I think AI will be very crucial to understanding how we need to model the data that we have to include the influence of these regulatory systems. 

What kind of partnerships or collaborations are you looking for right now?  

Dr. Bolander: I like to collaborate with researchers that are not afraid to walk the extra mile and investigate the unknown. People who want to contribute to our understanding of why tissue regeneration fails independent of organs and tissues. 

What I’m trying to understand is how regeneration is regulated. So, anyone who is interested in teaming up to further our understanding here is very welcome to connect with me. 

What’s one thing that you do in your work that you love? 

Dr. Bolander: I love my days. I can start one morning with discussing clinical samples and end the day talking about how different conducting polymers can be used to enhance the sensitivity of our in-vitro technologies. 

They’re extremely varied and they’re very intense. 

I have a lot of amazing students that I’m supervising, but that makes every day different and interesting. I love it. 

When it comes to different papers, events, newsletters, or even specific people or groups, where do you look to stay up to date on the field of 3D culture? 

Dr. Bolander: I go to a lot of conferences. I think it’s absolutely the best way to learn about new trends and new data. You get a good indication about where the field is moving. 

I attend niche conferences, like the Osteoarthritis Research Society International (OARSI). But also European Organ-on-Chip Society (EUROoCS) and at the MPS conference are both nice because there you get a lot of information and trends. Last year, there was the first World Organoid Research Day, which was a fantastic conference. It was small, but everybody was so enthusiastic and open to sharing.

Final thoughts? 

Dr. Bolander: It’s great to see this area of research really growing. Especially that everybody’s very passionate about it. There was a moment after the FDA came out with their Modernization Act a few years ago where they said that the immediate models can replace animal testing. This really excited people. 

Development has gone slower than we hoped, but I think in the long term it’s going to be promising and also be of great benefit to patients. 

Want to connect with Dr. Bolander? You can find her here:

LinkedIn: Johanna Bolander