Hemdeep (00:09)
Welcome to Big Ideas in Microscale, the podcast where we explore groundbreaking research happening at the microscale where microinnovations makes a big impact. We're excited to showcase the incredible work being done by our users from around the world who are pushing the boundaries of microfluidics, lab on the chip, organ on the chip, and beyond. Through these conversations, we hope to learn from their experiences, uncover their insight, and bring their big ideas to wider audience. So whether in a lab, On the go, we're just curious about the future of microtechnology. Join us as we dive into big ideas at Microscale.

Hemdeep (00:59)
Welcome back to Big Ideas at Micro scale. I'm Hemdeep, your host and co-founder of Creative CAD Works, CAD Works 3D, and Resin Works 3D.

Robin (01:09)
And I'm Robin, the co-host and technical writer on the marketing team. In today's episode, we're diving deeper into the groundbreaking work of Alex LeBlond, a master's student in chemical engineering at McGill University.

Hemdeep (01:22)
Last week, we explored how Alex is developing biodegradable packaging using mycelium, discussing everything from mold design to challenges of large-scale colonization's and why this fungi might be the key to sustainable packaging.

Robin (01:37)
If you missed that episode, be sure to go back and check it out. We'll be right here when you're ready to sprout into this one. This week, we're looking at where Alex's research project began on a 2D agar plate. We'll explore how plug size and spacing impact mycelium growth and how results led the team to integrate microfluidics in their later approach.

Hemdeep (01:58)
We'll also hear how Alex tackled the challenges of scaling up using a clever oil-based emulsion method to create optimally sized mycelium microbeads and how accessible tools like 3D printing help prototype and test everything along the way.

Robin (02:15)
So, let's jump right back into big ideas at microscale.

Hemdeep (02:19)
If we were just to sort of take one small step backwards and sort of go through the actual work that you did, I think the work you did wasn't on a three-dimensional mold, but actually effectively on a 2D plate, correct? Yes. What did that work look like? What was the challenges that you sort of faced and what kind of ⁓ optimizations did you guys find?

Alexandre (02:41)
Yeah, so the first kind of question that we had to be able to see, what could be the reason why it's causing this lengthy process is the first thing that we thought was, it the size? So I don't know if you have plants at home, but if you have ever tried to split your plants and to start a new colonization of your plants. Exactly. If you want to try to propagate your plants, I don't know if you've seen, but if the new plant that you're taking is too small,

Robin (03:01)
When you're propagating the plant.

Alexandre (03:11)
is going to take some time to be able to grow back. And if it's too big, it also will take a longer time. Comparatively to when it's just in the right size, it's really able to grow well. I tried to use this idea for the mycelium. we're like, is there an optimal size for the mycelium that we can split into and put it into those 2D pattern agarose agar plates, I'd say? see if that makes a difference in the overall colonization. So what we did is we tested with three different sizes of plugs. So basically, we used biopsy punches, which is basically like fancy cookie cutters of 3 millimeter, 2 millimeter, and 1 millimeter to make these little cookies of agar mycelium that we put at different distances from each other. What we saw is that, well, first, if they were further away from each other, it kind of resulted in this patchy.  overall conversation, which is not optimal, which is not what we want to see when we have a full material. We want it to really be homogeneous.

Robin (04:13)
Sorry to pause you there. Do you know what the distance was between the mycelium cookies, as you called it?

Alexandre (04:21)
Yeah, it was a couple of millimeters that they were apart. So for example, the closest that they were together was approximately two to three millimeters. And the further away was approximately six millimeters, approximately between the different cookies. From there, when they were a little bit further away from each other, it resulted in this patchy growth, which is not what we saw when they were a little bit closer together. That was our first observation. But also is that we made a defined area of the overall agar, which was approximately 34 millimeter overall, so basically the size of a well plate. And we saw that there was not a big difference between the plugs that were 1 millimeter and the plugs that were 3 millimeters. And that's when we were like, this is very interesting, because we had 18 on those little 34 millimeter little disks, 18 plugs at different distances from each other. The smaller ones, they cover the overall same area, the overall 34 millimeters in diameter, at the same time as the 3 millimeters, which was very interesting to us since there was not the same amount of initial biomass. And this is where we're like, oh, so the size do seem to have an impact on the overall colonization. If, for example, we were to apply this to a larger surface at equal biomass, the 1 millimeter plugs, which would be a lot times more, so nine times more if you do the math in terms of the circle area. It would cover a lot faster and a lot more than at equal biomass, the three millimeter plugs. So if it was smaller and more dispersed, we would be able to colonize much faster than if it was bigger particles, fewer bigger particles. This is kind of what we saw with this 2D essay at the beginning.

Hemdeep (06:08)
So was that a function of not the biomass, but actually the surface area of each of your plugs and the contact point to the actual wood chip in this case?

Alexandre (06:19)
So for here, we just did it on agar. So there was not any wood on this. It needs to have this balance between the nutrient and the overall surface area. But also, I think there's probably some sort of inhibition in between the mycelium at the beginning, which causes it to have some problems being able to fully expand and result in this patchy growth, even though that when it recognized that it's the same mycelium, it's able to fuse and really form one. So this is how we're able to obtain this one full block at the beginning is even though there's many, for example, different pieces of mycelium, when they recognize that it's the same exact DNA, they're to fuse and form and become one overall mycelium. So this is what we're seeing, but it seems that even though they are further away from each other, they're not able to fuse properly and form uniform coverage, whereas when they are closer together, they are able to fuse well and form this uniform coverage.

Robin (07:16)
So then now that you had this idea of how the different plug sizes work and the distance, did you manage to find the optimal size? Or is it 1 millimeter, for example?

Alexandre (07:28)
That's a really good question. So unfortunately, we don't have biopsy punches that are going smaller than one millimeter. And it would be very difficult, even if we had to be able to take those little very, very tiny plugs and put them at different distances to each other. So we had to change a little bit our approach. And instead, we decided to go with microfluidics. So what we did is we took a little piece of mycelium, put it on the exterior of a microfluidic chip. And we let the mycelium be able to colonize little channels inside PDMS microfluidic chip. So what we had is just a very basic design that had channels that were 100 micron tall by 100 micron wide. And they were 8 millimeter longs. And we were able to also put inside those little channels, were able to put agarose, so some sort of nutrient inside. The microphilic chip so this way the mycelium was still able to get some nutrients. And when the mycelium was able to slowly start colonizing the chip, we decided that we would be cutting the chip after two days. So once the mycelium was far enough into the chip, we would cut it and measure what's the size of initial mycelium that is inside that chip. This way we're really able to get very small fraction of mycelium that's inside and be able to measure if the size do have an impact on the colonization speed on the rest inside ⁓ the channel. So when we're cutting it, we're really able to see either very small, medium, or larger fragments of mycelium and measure the amount of biomass that increases over, for example, the course of one or two days and see if smaller pieces gain much more biomass than bigger pieces. And we saw that exactly like plants. When they were too small, there was almost no growth. It's like if, for example, there's not enough cellular material to be able to start growing back. And just after that, there's just a very big peak that there's this very optimal size that very grows that made up to 10 to 15 times their biomass in just two days, which is absolutely crazy. As soon as we were going to bigger and bigger sizes, it was, for example, only doubling or going up to 1.5 times their initial biomass inside the PDMS chip. So we did find an optimal size in our chip, was an initial biomass of approximately, I'd say, in micrometers cubed. It would be approximately 60,000 micrometer cubed. So it doesn't tell you much, but this is approximately the size that we were able to identify that was able to get consistently very high rates of biomass growth.

Hemdeep (10:28)
I know that you used the 3D printing and you created your masters. How did you separate your final device out of the mold itself? Was there a special process that you needed in order to get the agarose off the mold itself?

Alexandre (10:43)
So for this one particularly, we used PDMS for the mycelium chip. With the 3D printer from CalWORKs with the green resin, we are able to pour our PDMS, and we just remove it from the mold. And that's pretty much it. It was very easy to be able to just start with PDMS with this. So we used in the other experiments with a 2D layer with the different plugs. That's where we used agarose. We used 3D printed molds. We poured agarose on top of it, and we just removed it easily and there was not even a problem, not even extra process. We just remove it from the mold. It was extremely easy.

Robin (11:18)
the process for the agarose was pretty much the same as what you've been doing for regular PDMS devices.

Alexandre (11:24)
It was exactly the same.

Robin (11:25)
And then for the, you know, when you test the growth of the mycelium, right, the microfluid device, because you incorporated agar as a source of nutrients, right? So like, what did you do there? Did you kind of like insert into the channels or, you know, what was the process there?

Alexandre (11:43)
Yeah, so this is one part that took us quite some time to be able to fully get. And it was just trials and error. And what we saw is after making our full chip, so we have PDMS, and we put it onto PDMS as well after plasma bonding, we immediately put liquid agarose inside the chip. And the agarose was able to fully enter and colonize all the channels. And we just let it solidify. And that's it. The agarose was able to stay there. So since with the plasma bonding, it was really able to fully go into all the channels and we didn't have any problems integrating the nutrients.

Robin (12:19)
And did you kind of test this device with other fabrication methods or you went straight to 3D printing?

Alexandre (12:25)
straight to 3D printing. It was very easy for us. was in the lab. We said, okay, let's try it. And we tried with the 3D printing and it was working perfectly.

Hemdeep (12:34)
And have you used a 3D printer before using the ProFluidics?

Alexandre (12:38)
That's a good question. it was in Chris's class in my undergrad. We also had another project before that, and that's where we started using the 3D printer. But it's just the classic resin 3D printing, and I absolutely loved it. It was so fun to really be able to just have something in your mind and just take it, and it creates exactly what you need, the size that you want. So that's why as soon as we saw we had many 3D printers that had extremely high resolution, I was extremely happy to be able to ⁓ keep going and do even more of that and create crazier devices and everything.

Robin (13:14)
And so I guess the main difficulty was getting those initial small sizes of biomass. So I think that leads on to your second part of your research was how do you continuously obtain this very small fragment of mycelium?

Alexandre (13:30)
Yeah, exactly. So as you mentioned, it's very good to obtain this size. But then if we want to expand for larger scale production, since we need to make a lot of those very optimal sized particles to be able to make this full raw material. So how can we do that? And when we take the volume that I mentioned that didn't tell you much at the beginning. So this is basically equivalent to a sphere of 50 micron in diameter. When we saw that, we're like, could we also adapt it and try to encapsulate this mycelium into an alginate bead. So what it is basically, we put this mycelium particle of this optimal size? Can we put it into this protective shell of optimal size? And what we saw is in the literature, there's a lot of different people having proposed to, for example, do smaller particles. And some people have seen, for example, just basically put it into a blender. ⁓ It's kind of mechanical breaking the mycelium and it really forms this inconsistent particle size. It damages is a lot. So there's other people that have shown that encapsulation can be done, but it's still making some very large particles because they are using external gelation of alginate, which consists of a dripping the alginate. So this is the material that is going to be used to make this protective shell around the mycelium. And we're still. Having some really large particles, which is not optimal for us since we need to go down much smaller in sizes for the mycelium. So there's another technique which is called external gelation that you're able to also encapsulate the mycelium bead, but it kind of encapsulated as soon as you put it into this solution. So you mix the alginate, which is what is going to create this like kind of shell around the mycelium and you drip it into a solution. of ⁓ calcium chloride, which is basically going to immediately gel the bead. So you have really poor control over the size and the shape of the bead, which is not very what we want at larger scale, since when we want to do a large scale, we want it to be consistent and always of the right size. And typically what is done is done with the dripping process. So it takes quite some time as well. It's very hard to industrialize at larger scale. So there's other techniques that have been developed to encapsulate ⁓ So for example, we have a lab here as well at McGill that are encapsulating mammalian cell for type 1 diabetes, and they're using internal gelation, which instead of dripping it into the solution directly for the encapsulation, we mix the alginate in ⁓ oil basically. And we're kind of making kind of this vinaigrette, this salad dressing. And the more you mix this salad dressing, the more the water particles in the oil phase are going to become smaller and smaller and smaller and smaller. And this is exactly what we want to use to make those optimally sized and small enough particles for the mycelium. So we want to make them as small as possible when making your salad dressing. So it's the same thing as home. The more you shake your salad dressing, the more the water particles are beginning smaller and smaller. And this is what we want to try to do to have the optimal size of mycelium in those particles.

Robin (16:48)
It kind of shows how some great solutions can emerge from simple concepts, especially when applied with precision.

Hemdeep (16:53)
And with that, that's a wrap for today's episode of Big Ideas at Microscale.

Robin (16:57)
Huge thank you to Alex LeBlond for taking us through how your research approach has grown.

Hemdeep (17:02)
Literally, we explored how this team is tackling the challenges of creating consistent, scalable biomaterials and learned how techniques like emulsification in oil can create sizably large microbeads to optimize the rate of colonization, paving the way for more practical industrial scale applications of mycelium material.

Robin (17:22)
It's a great reminder that behind every major material breakthrough is a series of small, well-tested innovations. And Alex's work sits right at the intersection of biology, material science, and technology.

Hemdeep (17:35)
Make sure to tune in next week when we look at how Alex and his team refine their methods even further. We look at colonization's using 3D setup, bringing them another step closer to live regenerative material. You won't want to miss this.

Robin (17:51)
Thanks for tuning in to Big Ideas in Microscale. If you enjoyed the episode, make sure to follow us and stay up to date. You can listen on Apple Podcasts and Spotify, or watch the full video on YouTube. You can also follow us for more updates and behind the scenes content on LinkedIn, Instagram, Blue Sky and X. We're Cadworks3D across the board. That's spelled C-A-D-W-O-R-K-S-3D. For show notes, paper references, and bonus resources on today's topic, visit our website, catworks3d.com. That's spelled C-A-D-W-O-R-K-S 3D.com.

Hemdeep (18:32)
Thank you for tuning in and as always stay curious, keep exploring and never stop asking the big questions that are shaping our world. Whether you're in the lab, on the go or just curious about the future of technology, join us as we continue to dive into big ideas at Microscale.