Your Questions Answered: Q&A From Our Recent GenomeWeb Webinar

Key Insights from the Webinar:

1. Why Advanced 3D Models Matter
   The webinar explored how matrix-embedded 3D cell models replicate the tumor microenvironment, offering researchers better biological and phenotypic relevance. These models enable more accurate compound testing compared to traditional 2D systems.
2. Simplifying Complex Workflows with RASTRUM
   Professor April Kloxin from the University of Delaware demonstrated how the RASTRUM platform streamlines the creation of customizable 3D models. This automation enables reproducible results at scale, allowing researchers to focus on insights rather than experimental complexity.
3. Applications in Drug Discovery
   3D models powered by RASTRUM address key challenges in disease research, from oncology to interstitial lung diseases. Models can mimic complex cell behaviors and serve as robust tools for drug screening.

Webinar Q&A

This transcript has been edited for clarity and brevity. Some portions of the conversation may have been rephrased or condensed to enhance readability.

[GenomeWeb]: So the first question, one of our attendees would like to know what your main considerations were when you were moving from the hydrogels fabricated in your lab to the bio inks available from Inventia.

[April Kloxin]: Ya so how did we think about that: we were really excited in terms of looking at the Inventia bio inks, to see good complementarity to the types of building blocks that we had been utilizing before. And so that made the transition for us really smooth in that context. They have a platform that a bio inks, inspired by different tissue types, and that achieve different matrix density and stiffnesses in addition to different biochemical content.

I think for us, it was really straightforward then to directly start bioprinting very similar synthetic ECMs to what we had been using before. But it's also very seamless if you're not someone who's been utilizing these types of synthetic ECMs, to then pick specific composition based on the tissue type of interest and to start utilizing them in your cultures.

[GW]: Okay, and the change in throughput was pretty remarkable that you mentioned! I mean, how does that change your day to day lab life?

[AK]: Yeah, it's transformative. For example, the intrepid graduate students that started the breast cancer dormancy projects, right, would spend multiple months making all the building blocks and characterizing them and stockpiling. And then you'd spend all day preparing the three dimensional cultures in addition to having the 40 days of culture, right? And so overall, the lead time for a particular experiment, then would be six months in the making. Once you got a system off the ground strategically, we would start to stagger experiment start.

So we still do that, right? So, like that you need to have an experiment that starting every week and you always need to be Making new batches of material benchmarking them versus the old batches. All of that is going on as well. And so now it really allows us to shift our focus, more to the types of biological questions that we're asking and towards the evaluation of therapeutics, because we're not having all of that, you know, three months of building block preparation and characterization piece of it.

And then in terms of our teams that we still work in collaborative teams. But now we have folks that are focusing on the bioprinting, Folks that are focused focusing then on the cellular response aspect in addition to people who want to be at that interface. So ya, it really lets us focus on the biology and the drug discovery in a way that we're really excited about and so more and more of our lab members are utilizing the the RASTRUM system, in their studies, even if they're developing New synthetic ECM modalities. They are using that the RASTRUM system in a complimentary way for asking the biological questions. So ya, we're really excited about it.

[GW]: One of the questions was within the 3D models, how are the metastasis cells getting their nutrients to maintain them and which media are you using to give them nutrients?

[AK]: Yeah, that's a great question. For both our manually prepared system and then for the Inventia printed system, the path links for diffusion, is at 500 microns. And we find that ends up being really important for these highly metabolically active cells for us not to see differential responses of the cells in the Z direction. Which would indicate that they have insufficient access to nutrients. We do change out the media every two days as well. With one having a sample that has controlled thickness, that's not very thick, then frequent media changes. So say on the order of every two days, and then in terms of media selection, I think that this is an ongoing, you know, area of research.

One could see it as a challenge or also as an opportunity within the field, right? As we try to move to more well-defined systems. The breast cancer dormancy work that I showed you here was with more traditional media that do have, FBS, and those media and then for co-cultures, then we'll do a mixing of medias. One for the bone cell types and one for the breast cancer cell types.

We are now moving as we go into different tissue types and in general, moving to more well-defined media, starting to use some of the serum-free media, and integrating then the bullet kits that they provide, for example, for having, you know, some, appropriate nutrients for your specific cell types while still not having the potential batch to batch variability that's introduced with FBS. So hopefully that gives some perspective. But I think that this is an ongoing conversation that we need to have as a community as we drive into these more complex, but well-defined culture models.

[GW]: Sort of speaking of conversations in the community, I know AI is a big topic. Are you using that now at all to ask questions of, now that you have this defined system, can you find out what might be the best therapeutics or anything using that kind of discovery approach?

[AK]: I think that's a great question. So, we're starting to have conversations around particularly around like labels-free methods for analysis of images in a more real time fashion for probing cellular responses. And I think that's one area where AI can be particularly helpful, in terms of, really looking in real time, and without necessarily having to do fixing of samples and do immunostaining, for being able to probe how cells are responding in a more deep way, strictly with a bright field image.

I think that the bioinformatics team that we've collaborated with at UD are fantastic as well, where they have a lot of different tools, including machine learning and AI tools to really drill down on those transcriptomic data. And I didn't show it here, but we've done some proteomics work as well, but I definitely see opportunities there too. We're excited to collaborate with folks in that space that are that are using AI as well.

[GW]: Excellent. Another one of our attendees wanted to know: can you recover viable cells from your culture models?

[AK]: Yeah, absolutely! It's exciting to be able to recover the viable cells. They didn't show it here, but we can degrade the synthetic matrix in this case. For our manually prepared systems, we apply collagenase, and we can degrade the synthetic matrix in about 15 to 30 minutes and then centrifuge the live cells and then plate them and then continue to grow them.

We do see them that cells that are dormant in that 40 day culture, if we take and harvest them and now throw them into a tissue culture dish that we'll get proliferating cells again which is really interesting to us, but we haven't been able to probe it on a single cell basis yet. We are also able then do downstream assays that can be on either live or fixed cells then from that point after having retrieved the cells from the synthetic matrix. And for the Inventia bio inks, there's a cell retrieval solution and workflow that that is available. We have utilized that to recover the macrophages and to then be able to do flow cytometry, for example.

[GW]: And the ones that are cultured, not like taken out again, but how long can you keep them alive? What's the lifespan?

[AK]: You know, there's some beautiful work by others in the field. Shelly Peyton's work comes to mind. She's now at Tufts where she's been able to look at really long times of cells out in culture under different conditions. I can comment on it in our own studies. You know, we've gone out to, 40 plus days in three-dimensional culture. And then after we harvest the cells and continue to culture them, out to 60 days. But her work is fantastic. And she has really great work too in dormancy and recurrence. She's gone to much, much longer times. I'll give you our datapoint, but there's some great work in the literature going longer.

[GW]: Excellent. Could you say what other types of cells can be integrated into these co-culture systems? Anything that you're using or anything that theoretically would be also interesting?

[AK]: Ya so in our collaborative work with Cathy Fromen's group, we, integrated, for example, macrophages from a number of different cell sources, both cell lines and from primary cells and see great success with printing of either primary cells or cell lines. We also have been integrating epithelial cells from healthy donor tissue. For example, lung epithelial cells and skin epithelial cells in addition to fibroblasts from different tissue types. That's what we've been proving today in addition to introducing bacteria as well as we start to look at how then the tissue models respond to bacteria.

[GW]: It’s really wonderful work! Somebody wants to know the smallest resolution and the equipment necessary for 3D imaging to detect amino acids of a protein within the cell membrane.

[AK]: In terms of imaging, I think some of the super resolution microscopy techniques can start to get you down to some of those size scales, at least for, we've been able to image the live bacteria or fragments of them. For example, where my colleague Catherine Grimes has the quick chemistries that allow us to label the bacterial cell wall, and then now to be able to image the bacteria or fragments of their cell wall. And then we work then in collaboration with our bioimaging experts in the Delaware Biotechnology Institute, particularly Jeff Caplan, is a faculty member there who's fantastic and has worked with the students on protocols that allow us then to image those fragments. So I would say we're on the sort of 100 nanometer-ish range is where we are resolution-wise. When you're got that range there though, then you are losing the Z sectioning capability. But we do see that what we observe at the interface at the very bottom of the gel with the cells is representative of what we see at the top when we're looking at it with confocal Z sectioning capability at the larger size scales.

And so we think what we're seeing is representative, even though it's at the you know, we're limited to that interface between the plate and then the gel. So hopefully that gives some perspective.

[GW]: Yes. Thank you so much. That is all the time we have for today! We'd like to thank Dr. April Kloxin and our sponsor Inventia Life Science.