Hemdeep (00:15)
Hi there welcome to big ideas at micro scale my name is Hemdeep, i am co founder of creativeCAD works CADworks3D and Resinworks3D my co host Robin, Robin how are you.

Robin (00:29)
Good, I'm good. Like you said, I'm Robin. I'm also a co-host and I'm the technical writer on the market team at CADworks3D.

Hemdeep (00:38)
And so today we've got a team ⁓ from a institution that's very close by. In fact, ⁓ I used to work downtown for the better part of close to 20 years and used to always walk by up and down the street that would be adjacent to this place. was it was St. Michael's Hospital has had ⁓ new name changes and I had the opportunity to while I was there, had an opportunity to first talk to Dario, ⁓ who leads the microfluidic manufacturing division at what used to be St. Michael's, and I think it's now Unity Health. there's, I'm sure Dario is going to sit down and explain all the inconsistencies and all the changes ⁓ that has been happening there. And along with him will be joined by Stephanie and Niloofar, and they are part of the team from ⁓ Dr. Scott Sye's lab and they've got some very interesting research that they're doing on, I think I'll have them describe it. So why don't I bring them on board. Dario, Niloofar, Stephanie, welcome.

Stephanie (01:53)
Thank you so much for having us.

Dario (01:54)
Hello. Thank you.

Hemdeep (01:56)
Yeah, so Dario, why don't you sort of give us your CV and what you do at this lab and I guess everything ⁓ pertaining to microfluidics.

Dario (02:08)
I work as micro fabrication specialist at the research core facilities department at St. Michael's Hospital Research Institute. Yes, we are part of Unity Health Toronto, but still St. Michael's Hospital. Also, we have a partnership with Toronto Metropolitan University and I-BEST ⁓ Institute. So we offer facilities, ⁓ expertise ⁓ and training. To our users here at the research core facilities. So the core that I'm representing is called Microfabrication Core. So specialized in microfluidics device ⁓ design, development and small scale manufacturing. Also we have analytical equipment. We also offer some 3D printing capacities, laser cutter. That's what we have here.

Hemdeep (03:07)
I think the first time that we spoke was in 2019 or so. And I think at that time, you had come to our office when we were downtown. there was, think, what was it ⁓ at that time that you were looking at? Was there a need that you guys were seeing at that time? Or was it just a curiosity of how 3D printing could work for you?

Dario (03:32)
It was both. was both. That was early stage of the core ⁓ that I'm representing here. We still in kind of construction phase. We were looking for new equipment. ⁓ at that point, ⁓ 3D printing for microfluidics was at a very, very early stage. But it sounds like something that is coming as ⁓ big and promising in the field. ⁓ So yeah, I found ⁓ your company here and it was basically neighborhood ⁓ and that's how it started. ⁓ There were not that many companies in the field at that time.

Hemdeep (04:16)
Yeah. Yeah, that's true. NilooFar, how about you?

Niloofar (04:27)
So I'm, as you said, Niloofar. I'm finishing up my PhD in biomedical engineering. Just finished my fourth year and in a few weeks or less, less is going to be my PhD defense. I haven't studied yet, but ⁓ from next week, I'm going to study on the material and everything. But so far, no worries. So my project, my ⁓ supervisor is Dr. Scott Tsai, as you said, and our lab is at I-BEST, which I think we are really ⁓ grateful. And I always say that we shouldn't take for granted that our lab is at I-BEST because I-BEST kind of have everything that and also like some really amazing people. And because there are lots of like different labs that they are working on different things, it's really amazing. in a way that you can collaborate with different people and have a really good project because everyone is from different expertise. yeah, so for the first time with Dr. Scott Tsai, I started working on microfluidics and Dario was the one that's like... ⁓ first taught me how to use clean room, to make microfluidic device using soft lithography. And again, Dario was the one that introduced 3D printing in our project.

Hemdeep (06:05)
Amazing. So how about you, Stephanie? What is your experience and how do you fit into, I guess, IBEST and the lab there?

Stephanie (06:14)
Yeah, for sure. ⁓ like you said, my name is Stephanie. I'm a recent graduate from the Biomedical Engineering undergraduate program at Toronto Metropolitan University. I graduated just this past April and ⁓ I just recently began my master's degree as well under the supervision of Dr. Scott Tsai. I'm also co-supervised by Dr. Michael Kolios at TMU as well. And I'm the same. So Dario was the first person that introduced me to like clean room operations. So photolithography, soft lithography. And recently he's trained me on how to use 3D printing for microfluidic applications as well. So yeah, in Dr. Scott's lab, we do a lot of microfluidics work. So my project also focuses on using microfluidics. So is it that IBS is kind of like the

Hemdeep (06:59)
amazing.

Robin (07:03)
what is the connecting point between TMU and St. Mike's Hospital? Is that how it works?

Dario (07:08)
Yes, that's partnership between St. Michael's Hospital and TMU.

Robin (07:14)
Okay, so you're training a lot of students in that case, Dario.

Dario (07:17)
We have quite a few groups here that are interested in microfluidics and microfabrication.

Robin (07:24)
And so then Stephanie and Niloofar, you're saying that the first time that you were introduced into 3D printing that was through Dario, right?

Niloofar (07:33)
Yes, exactly. also, he was the one that ⁓ talked about ⁓ getting a grant for 3D printing and ⁓ asked for my help to make some fibers using ⁓ the 3D printed device that sample that he had to write the grant. And ⁓ he was successful in getting the grant and bought this amazing 3D printer that helps lots of people right now, the students at Ibus.

Robin (08:01)
Were you using a different technology with 3D printing? For example, Niloofar, you're doing ⁓ fiber fabrication. Stephanie, you're doing... Is it micro bubbles or nano bubbles? is it both? 

Stephanie (08:10)
Yes. We micro fluidically generate micro bubbles, but these micro bubbles shrink down to nano bubbles. The final product is batch of motor dispersed nano bubbles. A little bit of both. mean, they're hand in hand really. Yes.

Niloofar (08:23)
So both.

Robin (08:31)
first started the research, did you kind of already immediately know that about 3D printing and that maybe this is the direction that you were gonna take it or was that you just came to Dario for example and said like, this is what I'm working on, what are my options? 

Stephanie (08:45)
For my case, was, my supervisor was actually the one that suggested that we try using 3D printing because the traditional mechanism of making our microfluidic devices uses the photolithography, soft lithography, the process is a little bit tedious. So we thought it might optimize the procedure a little bit more if we could actually translate it into like a 3D printed platform.

Niloofar (09:07)
Yeah, so for me, it was Dario that suggested that. also the way that, so for me, I guess like the case was different and the way that I spent the first two years of my PhD on making that microfluidic device. And most of the time I wasn't doing the experiment, I was making the device. For days I was just filing some glass capillaries and stuff. So ⁓ it helped me so much. For me it was just like make the device fabrication easier.

Dario (09:40)
Yeah, the idea comes from the project that Niloofar is doing. And ⁓ there was very labor intensive ⁓ process of making these devices. So we thought, how about we incorporate 3D printer in general, even before knowing about those ultra high resolution 3D printers used for microfluidics and maybe combine it with capillaries ⁓ to assemble device. And that's how it started. And then we ended up actually using the ProFluidic printer that we can print the entire device without ⁓ combining it with ⁓ capillary, which of course is one step further. And now we can do entire device, including even the connectors ⁓ and everything that we need ⁓ to use it to extrude the fibers. So simply just connecting it to the external pumps. and tubing and start experiments. think that helped and in a look far and also reduced time for making device significantly. Also enable people to, without previous experience, with photolithography to start making microfluidics devices, which is also very important aspect of having the 3D printer such as ProFluidics.

Hemdeep (11:11)
So now, in terms of, like I do want to touch on all of the research that you guys have been doing, but if we were to take one step backwards before knowing this is an existing 3D platform, what was the original steps like? What was involved in order to make these capillaries and any of the devices that you were working with?

Stephanie (11:35)
can speak to ⁓ my experience first. So ⁓ for my devices, I didn't really need to integrate any capillaries or anything like that. It's a little bit more of a simplistic process, I would say, than the Neloofar's devices. But making the microplate device so first incorporated like photolithography to actually fabricate like a silicon wafer where the microfluidic channels were kind of patterned onto it. So this was like a clean room procedure. It takes a lot of time. So there would be a CAD design that would be fabricated onto the wafer. And then following that, so I would be able to use that one wafer like time and time again to make my actual like PDMS based devices. And the PDMS based devices were made using like soft lithography. So a liquid PDMS would just be poured over top of the wafer, cured, and then peeled off. And I was left with the ⁓ channels transferred onto a PDMS block.

Hemdeep (12:24)
And then, so now ⁓ if you were to look at in terms of time, how much time did you put into ⁓ a few days, a couple of weeks?

Stephanie (12:29)
Yeah, like. Yeah, so in terms of the fabricating the wafer itself, that's something that usually takes like ⁓ about a day in the clean room to fully complete. ⁓ from just fabricating the wafer and soft lithography is something I do need to repeat every single time I run an experiment. So I can't really reuse one single device. I have to ⁓ continuously make new ones. And we also have to bond them to like glass slides that the channels are enclosed. I have to make my channels hydrophilic as well for my purposes. So I require like additional treatment that way. So that's something that needs to be repeated like every single time I run the experiments. And so about an hour before the experiments. Wow. You said like, it's a little bit more of like a simplistic design, but did you find that you needed to have a lot of different like small design changes and like changing the silicone wafer and everything like that? And that did that kind of like delay a lot of things for you? Yeah, partially. Like the design I use was something that actually ⁓ is kind of standardized for my protocol. So previous students in my lab. for that reason, was kind of, I'm kind of grateful that I didn't have to go through a lot of iterative design like that. ⁓ So yeah, for me, it was a little bit easier to, I don't have to do a lot of CAD iterations, but I know with photolithography, it can be pretty tedious when you do have to do a lot of iterations of a single device. Obviously you'd have to repeat that photolithography. over and over until you get it right. So that can be a very time consuming. For you it was fine. That's great. Oh, could you actually explain a little bit about your device design, its applications, that sort of thing? Of course. Yeah. So my device design, so it's essentially a flow focusing geometry. So I have two inlet channels in my device. One is going to be like a dispersed phase that is a gaseous phase. And then I have also like a continuous phase of my device, which is going to contain like a lipid solution. So the bubbles that are generated from the, they kind of pinch off of the flow focusing orifice and they're micro bubbles and they're shelled by this lipid solution. And we use a gas composition of like a low solubility and a high solubility gas mixed together. And that influences or facilitates ⁓ dissolution of the high solubility gas. And it allows my micro bubbles to shrink to very small nano bubbles. And because we're using microfluidics, it actually allows the final size distribution to be very monodispersed in size uniform. which is ideal for applications. ⁓ So the reason we make these bubbles are actually to be used as in contrast enhanced ultrasound. So they can be injected into the bloodstream to actually enhance the contrast of the blood pool in the body. Nanobubbles are not quite clinically implemented yet, but micro bubbles, actually, those are already clinically implemented. So the reason why we're interested in like mono dispersed nanobubbles is because in the body, can actually, they demonstrate potential to ⁓ enter extravascular space and expand applications into a different area beyond the bloodstream. So that's kind of the purpose of my research as well and the work that I do.

Hemdeep (15:38)
And so by a factor of how much does this these bubbles shrink by from the micro to the nano size?

Stephanie (15:47)
So we actually find that there's kind of like a critical diameter that they need to be initially to facilitate complete nanobubble dissolution when it's above this critical diameter. it depends on a lot of factors. It also depends on like the concentration of the solution we're using and things like that. When they are ⁓ like a lot bigger bubbles, they will kind of shrink by like a known factor. Like so by 10 times or something like that, we can predict it. When it's below that, like it's kind of a critical diameter, we call it. they will always shrink down to like 200 nanometer size, like in diameter nano bubbles. So that allows us to facilitate like really great mono-dispersity since we see a really, really tight size distribution centered at like 200 nanometers, which is great for controlling like their, response of these bubbles under ultrasound as well. Does the channel of the size, the flow focusing channel size, does that affect kind of like how much it shrinks by anything like ⁓ Not so much the actual channel itself, but the flow focusing orifice, that plays a big role because ultimately the size that we can make the micro bubbles at is kind of dictated by the width of that flow focusing orifice.

Hemdeep (16:57)
And so on ⁓ using, I guess, 3D printing, how small are you able to get some of these droplets, I guess, in this or?

Stephanie (17:07)
bubbles. So this is one area where I definitely had, I guess, the most struggle at the moment. ⁓ Obviously, so with fabricating actual channel geometry on the order of a couple of hundreds of microns, that's been pretty easy to do using 3D printing. However, the flow focusing geometry itself, typically we like to see that on the order of tens of microns, which is a little bit difficult to fabricate currently, I guess, with current resolution abilities. a couple of ⁓ ways we've been

Niloofar (17:08)
Mm.

Stephanie (17:35)
kind of thinking to get around this is maybe like integrating a microporous membrane or something like that instead of a flow focusing orifice. So it does kind of incorporate another fabrication step, you 3D printing and then also the embedding of a membrane. We haven't done this yet. This is just kind of ideas that we've been shooting out about how to overcome the resolution restraints. ⁓ But yeah, that's kind of ⁓ some thoughts that have been going into how we can actually generate these bubbles and still have them be small micro bubbles with resolution. 

Robin (18:27)
How about imaging ⁓ or even just analyzing the bubbles? ⁓ Do you use microscope imaging or is it a different measurement system? You mean during the actual generation procedure? Yeah, so once you've generated bubbles, I don't know if you've gotten this far, where you've already generated the bubbles and now you need to study them, whether it's the uniformity or what it is. How does that work for you?

Stephanie (18:31)
So yeah, in terms of like while they're being generated, so typically when we have like clear microfluidic resin or even with like PDMS in the past, it's optically transparent. So we can actually like visualize what's happening as the bubbles are pinching from the orifice. But so when they're micro bubbles, we can actually see them under a microscope. When they shrink to nano bubbles, it's a little bit difficult to visualize with like a traditional light microscope or something like that. So there's a couple of ways. So we can ⁓ actually visualize like the bubbles using like TEM imaging. That's one way we can do it. It doesn't really allow us to gather ⁓ population characteristics well. So the way we mostly rely on is actually called resonant mass measurements. So it uses a cantilever that has a microfluidic channel that's embedded inside of it. And it resonates. So as the bubbles pass through, depending on their mass, will ⁓ change its resonant frequency slightly. And we can drive characteristics of the nanobubbles that way. Dario might be able to speak more to this as well, because this is a device we actually have in iBEST as well.

Dario (19:29)
Yeah, that's an interesting analytical instrument that we have and pretty unique. It's based on microfluidics sensor that can detect particles based on their buoyancy. And that's what is preferred when analyzing the nanobubbles because you want to clearly distinguish between the bubbles and the residual lipids. And since this instrument can recognize the difference buoyancy positive or negative, so you can clearly see which, what particles are bubbles because they're positively buoyant, we ask the other particles, negative buoyant particles or those that would sink or float.

Stephanie (20:15)
So it allows us to get around the ⁓ restraints of the fact that they're so small and we can't really see them under the microscope.

Hemdeep (20:23)
So now for Dario, in your lab right now, you're obviously bringing in a lot of tools together in order to help researchers. What additional tools do you have at the lab that an average, typical researcher team will use?

Dario (20:40)
So we have a 10,000 class clean room, so that's ISO 7, ⁓ that is specialized for photolithography. So we have a standard classic mask liner, then we have a maskless aligner tool, ⁓ optical profiler, and of course, wet bench with spin coaters and hot plates. So we have everything that is needed for that ⁓ traditional standard photolithography. ⁓ Also we have a... suit of analytical equipment, we just mentioned the Archimedes. We are focusing on the particle analysis because most of our users are either in the area of nanotechnologies or making including nanobubbles but also lipid nanoparticles or some other type of nanoparticles. So we are providing ⁓ instruments such as DLS. Most recently we got NTA. We have advanced spectrofluorometers, spectrophotometer, culture counters, ⁓ and ⁓ that would be for analytical side of the core. We also offer the 3D printers. We have standard ⁓ Mark II printer ⁓ extrusion type. We have ProFluidics that we got more recently for microfluidics printing. We also have one laser cutter. Just to mention that are also, that Microfabrication Core is actually part of Research Core facilities and we have another ⁓ five cores that are specialized for flow cytometry, imaging, ⁓ histology, ⁓ and we are all ⁓ situated in the same building. So basically we have four floors. And each floor has one or two cores specialized in some area.

Hemdeep (22:44)
And that's all at that, the Likashin building.

Dario (22:47)
in Likashin building downtown Toronto. We very close to Eaton Center.

Hemdeep (22:53)
Dario, in terms of the work you guys are doing, you're obviously helping a lot of researchers through that building itself. When they look at 3D printing as the... Do they look at it as an alternative to cleanroom or do they build their project based on that they are going to 3D print this?

Dario (23:17)
Yeah, we have users that actually use the 3D printer, ProFluidics, as alternative to the clean room. So for a few reasons. So first, doesn't require photolithography skills and knowledge. Then you can definitely reduce the cost of materials. And probably the most important is time, as Stephanie already mentioned. So you can get... ready to use device in a matter of two, three hours. Plus 3D printing is pretty much walk away process. ⁓ And that's probably the important things. But we also have users that are looking for, so which are very experienced with photolithography and all the suspect, they're not really problem, including time. Yeah, time including rooming. depending on your device, could be everything from one to two hours, which is comparable with the 3D printer up to whole day, as Stephanie mentioned. But what people are looking are some aspect of the using 3D printer that you can't do using the photolithography. So you can do some ⁓ geometries that are ⁓ impossible or will be very difficult to do using a standard photolithography process. And that's, for example, case with Nilu Fire device ⁓ using the capillary where we printed like ⁓ 3D shaped nozzles that helped to establish this ⁓ core of ⁓ fiber that is required ⁓ during the flow focusing process to extrude the fiber out of device. And that helped significantly in terms of ⁓ getting device ⁓ Previously, we would need to insert the capillaries ⁓ inside the PDMS device, which would be very tricky and ⁓ repeatability was not so great. Like here we have device in a matter of two, three hours without any additional ⁓ work on device ⁓ fabrication required simply like... ⁓ plug and play device where you connect the tubing. And another also interesting and very appealing aspect of 3D printing microfluidics is that you can print connectors as a part of your device. So you can just connect either simple tubing or tubing with the fittings like threaded fittings with no problem. And we tested that with multiple devices and that worked really well.

Hemdeep (26:07)
And then, when most of the people are coming in, are they wanting to clear or PDMS? know that you've ⁓ spoken a couple of times where you've had researchers actually building out their own photopolymer with specific characteristic. Is that sort of the type of... ⁓

Dario (26:27)
That's still an ongoing project, but primarily we use the CADWORKS resins, including clear one, which is a favorite one because people get ready to use device, but we do have users ⁓ making them ⁓ PDMS master molds out of, I call it green resin. And that also is one of the ⁓ interesting aspects. And again, I'm saying because ⁓ not only that save your time of making device as when you compare to microfabrication using standard photolithography, but you can easily create multi-layer device, of course, within the resolution limits, but I believe with green one, we are down to X-Y 50 microns. That's something that we tested. ⁓ And that doesn't take any... more time than when you would do a single layer. And everyone who is in photolithography knows that making multiple layer devices is usually a thing that is time consuming and can take you all day in the clean room, especially when you need to do alignment of multiple layers. While here, simply, technically, you don't have, there's not much difference if you have one layer on your master mode or you're making... something that we call multi-layer device. So, and that's, think, also very interesting and ⁓ useful aspect of using PDMS master mold. Of course, there are some limitations and that's with resolution that you still can get using the photolithography. With photolithography, we can easily go when we use SU-8 ⁓ and that's called soft lithography techniques down to maybe five microns some features.

Robin (28:18)
Stephanie, of curiosity, which material were you using to build out your generators? 

Stephanie (28:30)
I was also using the clear microfluidic resin as well. Yeah, just for my purposes, it's really ⁓ helpful for me to visualize the initial size of the micro bubbles under the microscope while they're being generated. So it's really helpful for me if I have a clear ⁓ material so that I can make sure everything is up to par before I start collecting them actually and characterizing them later. But for my purposes as well, it might be beneficial to use like ⁓ maybe like the green ⁓ material or something like that just because of the resolution improvements that we can get with that. So it's kind of for me like a balance between ⁓ the two of those.

Hemdeep (29:04)
How important was it to have and maintain really good laminar flow in your devices, Stephanie, in order to sort of ⁓ standardize the size of bubbles that you were getting?

Stephanie (29:16)
Yeah, it's pretty important just in terms of like the stream of bubbles that's being produced needs to be very like smooth and consistent. Like it's the size that's coming out of the orifice needs to be very consistent. That's also why it's important for me to like monitor what's going on in case there is like an obstruction or something. ⁓ Even the hydro felicity as well of the channels is something that really influences how smooth that stream and monitor spurs the stream of bubbles is coming out. Just because as I mentioned, there is like a kind of a critical diameter. So every bubble below that is going to be able to shrink to a nano bubble. The bubbles above that will not be able to. So if my bubbles are kind of fluctuating in size when they're coming out of the orifice, it's going to be ⁓ like I might not have a very high concentration of bubbles in the end, or I might have like a very ⁓ wider distribution, which is kind of not ideal for my work.

Hemdeep (30:06)
And how you said something quite interesting is the hydrophilicity. How did you sort of modify that on the clear itself?

Stephanie (30:15)
Yeah, traditionally with PDMS, I used to use just like, I still do with the oxygen plasma is able to induce hydrophilicity. So I actually, ⁓ I haven't actually like created bubbles within the resin device yet, like with the hydrophilicity induced and everything. But I did put a block, like just like a device that was pre-made in my oxygen plasma cleaner that I have in my lab as well. For, I think it was just about 30 seconds, I turned on the plasma. And I just put a droplet of water on the surface of the device. And I noticed a big difference in the contact angle. So it did definitely induce hydrophilicity on the surface. And since the channels are open as well, ⁓ would ⁓ speculate that it would also have improved the hydrophilicity of the channels as well, since the inlets are open to the atmosphere. Was there any other surface modifications that you had to do besides the hydrophilicity?

Stephanie (31:09)
For me, not really. I don't really do any cell culturing or anything like that with my devices. So just hydrophilicity is the main thing for me.

Dario (31:17)
Yeah, we have another project where a user is interested in having hydrophobic. mean, the clear resin ⁓ is hydrophobic by itself, but this is something that is required for droplet generation where it's required salinization and get it like hydrophobic in a sense that is... ⁓ that you can generate the droplets using the fluorocarbon ⁓ oils. And that's something that we are also looking into and some ongoing project. based on literature and discussion with CADworks3D is something that is doable and that channels can be salinized ⁓ and...   something similar to PDMS. So make them acceptable for droplet generation using FC oils, which is very common application nowadays in droplet microfluidics.

Hemdeep (32:22)
So Stephanie, you did all your undergrad at TMU.

Stephanie (32:26)
Yes, I did. I began undergrad in 2021, so back during COVID. ⁓ finally, things opened up around my second year, and then I just finished in 2025.

Hemdeep (32:38)
I don't know if you know this person. Her name is Helen Molino.

Stephanie (32:46)
She's in the Dr. Kouyos's lab. She's in, my co-supervisor is Dr. Kouyos.

Hemdeep (32:53)
Okay, okay, okay, because I know her. Well, I well, yeah, I do know her and I know her family. In fact, her, her uncle is our best friend. And so I've, I remember talking to her about what was it last year or so. And she I guess is doing some work at McMaster or something like that, I think. Right, and I said that, if you ever need access to a machine, obviously I've got, and then you guys at iBest also, maybe she is already accessing it.

Stephanie (33:25)
I think, yeah, I believe she said she did her masters at a different school, so it might have been McMaster. And she just, at the same time I began my masters, she came in as a PhD student. So I she's now, maybe she's, I don't know if she's co-supervised by somebody from McMaster, but Dr. Kolios is also her supervisor. I've seen her in the lab this year.

Hemdeep (33:45)
What is the work that he, what does he do? What's the area of research that he's particularly in?

Stephanie (33:51)
So ⁓ he's like in the physics department at TMU. Dr. Koulos's lab does a lot of ultrasound work. So ⁓ I'm like an engineering student. So that's why my primary supervisor is Dr. Tsai, ⁓ an engineering lab. But because my bubbles have like applications with ultrasound contrast enhancements, that's where I kind of go to when I have questions about that kind of thing.

Hemdeep (34:13)
Dario, where ⁓ did you study? Where did you do a lot of your work?

Dario (34:18)
I got my undergrad from University of Belgrade in Serbia. then I also got my master degree, same university. And then I finished PhD from the University of Edinburgh in Scotland. And then I worked here at the University of Toronto as a postdoc with Professor Aaron Wheeler and later on with Professor Milica Radicic ⁓ before I got this job here as manager of core facility. ⁓

Hemdeep (34:56)
I recognize the name Wheeler ⁓ Raticich. He's at U of T, is he?

Dario (35:02)
Yeah, both are at UFT. Aaron is famous for the digital microfluidics. Radicic, their lab works mostly in the field of tissue engineering. Also, they use microfluidics a lot.

Hemdeep (35:25)
They're probably if ⁓ I think Wheeler, Wheeler uses our platform through craft, think. Radichurch, I can't remember.

Dario (35:36)
Yeah, they're both like user set craft. And actually, are, I believe both are co-directors of the craft.

Hemdeep (35:40)
Yeah, yeah, yeah, so. OK, OK. And then, so what was your area of study while you were at Edinburgh before you came here?

Dario (35:55)
micro fluidics, but it was in the area of heat transfer. So I was studying like a small heat exchanger that was built on microfluidics channels in a silicon wafer with potential applications for electronic schooling. and this project was about ⁓ boiling in micro channels. So that was the core focus, like that two phase flow, including the boiling and the channel. So it's quite explosive. I went through many, many chips before I finished.

Hemdeep (36:44)
And so this one, what kind of substrates were you using for your microfluidic devices?

Dario (36:50)
Silicon wafer

Robin (36:54)
When was your introduction into 3D printing Dario? Was it only when you worked at St. Michael's or were you already doing it back in your studies?

Dario (37:03)
During my postdoc, I started using 3D printers like for printing, but those were standard filament FDM printers that we used for every day, some lab applications like. And then for microfluidics, think, ⁓ yeah, this is our first printer for microfluidics, but I got interested that ⁓ very early when I started this position here to manage the microfabrication core. And I was aware of several companies. I mentioned there were not that many at that time. I'm not sure about now. There are more probably coming. Yeah. So we received samples that was probably seven, eight years ago, including samples from Hemdeep that help us a lot to evaluate possibilities and later on even to ⁓ use some of those preliminary data to apply for the grant, which was successful. And that's how we ⁓ secured funding to get printer in the institute.

Hemdeep (38:20)
Yeah, that is true. Neloofar, how about you?

Niloofar (38:23)
The whole thing that the ⁓ besides microfluidic and fiber generation that they are the main field of my PhD, Scott ⁓ is working on ⁓ bubbles and the other one is ATPS, which is aqueous two-phase systems, which is exactly like water and oil. But in this case, both phases are going to be aqueous. So in biomedical engineering for biological ⁓ materials. This is going to be a safe environment, not toxic. And also if you make your ⁓ dry carriers or your scaffolds at the end, it won't need the washing steps to remove all the oil from it. So for the fiber generation, you need ⁓ some thread ⁓ distinct phases to make a fiber which is going to be uniform. So that's why we needed the ⁓ two-phase system. And instead of oil, since Scott is working on ATPS, we were like, okay, why not to make ⁓ fibers? so for the fiber fabrication, one thing that is really, so I'm going into really technical ⁓ part of it, but like, So my PhD, we wanted to make a microfluidic device, ⁓ we wanted to introduce a new technique to make different shapes of fibers easier with only one device instead of like going to clean room, changing the design, having like multiple designs to make different shapes of fibers. So if your goal is to make ⁓ solid fibers, ⁓ you can use the same. ⁓ device. And if you want to make hollow fibers, you can use the same device, just like the inputs in lead solutions are going to be different. And also, like we showed that by changing pressure, you can change the dimensions and the fibers at the end are ⁓ pretty uniform. ⁓ also, the other type that we made was droplet field fibers. So there would be like some droplets inside the fiber. So those droplets can ⁓ have something else inside of them. Like ⁓ we can add some different drugs inside and they can get released over time or you can encapsulate cells. So this was the whole ⁓ idea for my PhD. so for the microfluidic device, of course, we first went with the soft lithography. And so in general, soft lithography is really amazing. And also like one of the things that is good about it is how easy it is under the microscope to like, it's pretty clear. You can see everything very clearly. And, but the issue was with soft lithography is kind of like the whole design is 2D. You can't have a 3D design, complex design in it. And so for me, which I needed coaxial channels to make fibers, ⁓ it wasn't possible to just ⁓ create a mold, just to spend one time at the room and then you're set for your life. It wasn't like that. I had to afterwards, like after making the PDMS pieces, I had to insert glass capillaries, which was the really difficult hard ⁓ because I think we haven't found out any kind of technology that is going to be cheap enough in the lab to say that you want exactly this size of the glass capillary. It's going to give you exactly that size with really fine edges. so I had to measure it with my hands and your nails are

Niloofar (42:42)
they have to be really short. And you have to spend ⁓ finding that glass capillary that you can't even see. And at the end, you have to wash it carefully to be sure that all those glass particles are not inside ⁓ your channels. And then you have to insert that glass capillary yourself. You can't use a... ⁓ tweezers because they are glass and you shouldn't apply much pressure. So you have to use your hands and then with tweezers you're going to put it inside and move it a little bit around. so at the end the nozzle won't be exactly the size that you want for all your devices and the resistance in your channel depends on that. And afterwards you have to use... you have to use a silicone paste to secure that opening that you created to insert those glass capillaries, which was ⁓ really difficult because if you're going to add less silicone paste, it would be loose and there would be leakage. And if you're going to apply a lot, it might kill out the whole channel with the silicone paste. So after like spending some days making the devices, I had to throw away 50 % of the devices that I made. So was really frustrating. And then by the first experiment, of course, microfluidic is really prone to clogging. this is, ⁓ I think we really need to, someone needs to step up and spend their PhD on fixing the clogging of the glass. I was thinking.

Robin (44:10)
It's crazy. so close.

Niloofar (44:32)
of like those heart surgeons that they use those ⁓ angiography things that they can insert something in the channel. maybe open the channel something like. Yes, thanks. Yeah. Like because it's really creating some issues. And I think like it's the PDMS issue too. It's ⁓ really prone to.

Robin (44:44)
That's it.

Hemdeep (44:44)
extent

Niloofar (44:57)
like all everything to attach to it, especially for me that I was using Alginate, which at the moment that it gels, if it's going to touch the surface, it would attach and you have to wash it carefully afterwards to be reusable. So that was all the issue that I had that I was trying to really ⁓ change my, like the next project to not use this device. like use something else, create some new ideas, which thankfully we ended up with a 3D printer that helped me a lot.

Hemdeep (45:33)
So when you look at 3D printing, you're trying to replace the current system that you had only because it was painful, right? And it sounds like you really struggled getting class capillaries in place. What was the, ⁓ I guess, what was the base minimum feature sizes or did you need to have a 3D printer deliver for you in order for you to mimic what you are already getting using ⁓ CleanRing.

Niloofar (46:07)
The only important thing was having those nozzles that they're going to act as glass capillary that are going to like the next at the junction when the other liquid is the solution is coming. There would be something that is going to prevent the solution to touch the walls. That was the only important thing. even though ⁓ the glass capillary is going to be a round ⁓ channel, but even though it was ⁓ square, but because of the pressure and everything, at the end it would end up as ⁓ a rod, a fiber kind of thing. And the only thing that mattered to me was those nozzles.

Niloofar (47:04)
⁓ to be exactly like the one that we had with glass capillaries. And because we are using pressure with pressure by increasing the pressure of like one of the ⁓ inlets, you can squeeze your fiber. So it wasn't an issue to like if we can't have channels like smaller than 100. It won't be an issue for me because if I want really thin fibers, I can increase the pressure. So the only thing that mattered for me was the nozzle.

Hemdeep (47:38)
And so as you were increasing the pressure, was laminar flow or having very good laminar flow very important or ⁓ could you overcome that issue?

Niloofar (47:47)
Yeah, laminar flow is really important and still like at the range that a 3D printer is going to give me, it's still is fine because for my device I need really big channels like with my even soft lithography the height was around 600 micron and the width was like 450. And so here we tried around less than I think 200, 150 event channels, the first channel, guess, if I'm not trying, but that was it for me. So that's why it was really helpful for my project. But I could say that also another really advantage of it that I found out later during the experiment was that it clocks less. material it is, ⁓ the alginate ⁓ fibers when they gel, they won't attach on the surface. It's like it was way easier to wash the whole device than the PDMS one. The only issue, of course, like not everything is perfect, ⁓ that under the microscope, it wasn't always clear to see the phases. And for me, I really have to have a control over the ⁓ face and see that the thread is stable and what size it is. So like sometimes some of the devices, they were really kind of opaque and had residues ⁓ on the surface that was hard to see. But overall, ⁓ I guess the whole thing had more advantage for me than just like that.

Robin (49:32)
Did you end up doing any like surface modifications in order to help you see clearly in the device?

Niloofar (49:36)
Not yet. It was the case of like making multiple device and sometimes one of them ⁓ wasn't clear enough. But clear it has like some edges that was, it made me like, I wasn't sure if the things that I'm seeing here is my like... ⁓ the distinct phases of my solutions, or it's just some edges of that. But the interesting part was, I ⁓ think most of the time they were clear enough to see my phases and be able to work.

Hemdeep (50:19)
What was in terms of links, what kind of links are you trying to produce these threads in?

Niloofar (50:25)
The device with fibers. If you're talking about the diameter, ⁓ it can be really like, ⁓ it depends on, I haven't tried more than 500 micron, but I think like that's even achievable if you are going to, like that's the limit of the channel. If you are going to have like ⁓ wider channels, then yeah, we can have that ⁓ or really, ⁓ The Fiber Story thin fibers by applying more pressure. But in case of lengths, something the fiber generation is ongoing. So it's like one thread all connected. So you can cut it at the time that you want, but the whole fiber, when it runs, it's running and it's just one thread. So the length is pretty.

Hemdeep (51:19)
⁓ And what are the potential applications for this technology that you've created?

Niloofar (51:26)
So my background is tissue engineering for ⁓ my master. Specifically, the field was tissue engineering. And in tissue engineering, the main component to ⁓ make ⁓ or engineering new tissue, make a functional tissue, would be the template or the scaffold. ⁓ So that ⁓ scaffold need to mimic the environment that cells are going to have in ⁓ the body, in their home. If you want to make a liver like a tissue, you have to give them the same kind of feeling or texture, structure. And most of the cells ⁓ in our body, besides the blood cells, they are surrounded with extracellular matrix. And exotelar matrix is filled with like fibers, collagen fibers, elastin fibers. So they are, they really like the environment, the scaffolds that they are in the shape of fibers. So that's why there are lots of research that ⁓ and techniques in tissue engineering that they try to make fibrous scaffolds. And so you... could use those scaffolds based on the type of chemical that you're using. Here I'm using alginate or you can make like ⁓ dextromethacryl fiber, gelatin met acryl fibers, and based on the application it would change. Or you can change ⁓ the dimension, the size, the shape of the fiber based on the final application. For me, the whole goal was to introduce a new technique, but in general, ⁓ Those fibers can be used ⁓ for wound healing, because alginate is ⁓ one chemical that has been used a lot in ⁓ wound healing. In case of ⁓ hollow fibers, ⁓ one interesting research that I encountered was they co-cultured those fibers, added ⁓ endothelial cells in the core of the hollow fiber and then co-cultured those fibers in an environment that had ⁓ muscle cells. So at the end, those endothelial cells would sit on ⁓ at the inside of the surface of the hollow fiber and then the other one would be at the outside. And over time, those fibers would degrade. So ⁓ they kind of show that they can make ⁓ new vessels and show that ⁓ angiogenesis can be possible using this way. But ⁓ the whole purpose is to make a fibrous scaffold for the cells.

Robin (54:33)
So then when you are fabricating the fibers, is biocompatibility in your device an important characteristic? then, okay, so then how did you had to do anything in order to make sure the device was biocompatible? And did you do like any biocompatibility tests, that sort of thing?

Niloofar (54:41)
⁓ Yeah. actually know and I'm interested why no one in our review paper like the papers for revision they haven't met

Hemdeep (55:06)
Okay, we will not release this podcast at all until you defend it so they no one figures out these questions that they have to ask you

Dario (55:14)
But I think here that the way you make your fiber basically because you shield them with a two-phase, so that core phase that eventually could come into contact theoretically actually doesn't get into contact with the material of the device itself. Plus the time, the resident time of the material inside the device is relatively small. So I don't think there is possibility of significant or any contamination. And second thing we haven't tested like any, we are planning to do, but we haven't done any self work with these fibers. That would be next step here. Just finishing a paper that hopefully will be published soon. But that's only about ⁓ creating the fibers as a proof of a concept using these 3D printed devices.

Niloofar (56:09)
Yeah, so and also with the previous one, the PDMS device that we made, I encapsulated cells inside the fiber, but as Dario said, even if ⁓ it's going to be a little bit toxic, if it's gonna depend on the residence time in the channel, ⁓ it would be really slow and like maybe like in way less than a second, milliseconds, it would be ⁓ really fast. It would be like shielded with the next phase. ⁓ Even though if you're going to like put them in the like at first when they are coming to the like channels, might like, the cells might touch the surface, but ⁓ it would be really fast. ⁓ but yeah, that's something that we have to of course do in future.

Dario (57:04)
We also plan to test different kinds of resin that has biocompatibility or better biocompatibility than current resin while making the same transparent device with the same level of features that is possible to achieve.

Hemdeep (57:25)
Yeah, I think the cytoclear would be perfect for that.

Dario (57:27)
So that would be probably next step.

Hemdeep (57:34)
So I think we could easily say that, Niloofar, you did not fully defend your PhD quite well here.

Robin (57:43)
This is why this is the practice. Good timing. ⁓

Hemdeep (57:45)
This is your practice round.

Niloofar (57:47)
Exactly, I was thinking.

Hemdeep (57:53)
In terms of a so yeah, you also mentioned a couple times you were able to is it correct you were able to change the shape of the the fiber itself meaning if you wanted a triangle or for some from Yeah, answer looked like no

Niloofar (58:11)
No triangle like everything like how we can make that. I don't think that ⁓ having a nozzle with different shapes is going to help because I think like that should be exactly at the moment of like extruding and like gelation. Like ⁓ when they are coming out at exactly at that time, they have to be in contact with the crosslinking. So they keep their shape. But here what we are doing, we are using those nozzles. just like keep them out of the wall. And after that, because of the pressure, they would form a round shape. ⁓ yeah, not with my ⁓ device, ⁓ with different shapes. mean, the ⁓ hollow fiber, core shell fibers, we made Janus fibers, and we made droplet filled fibers. ⁓ like, besides core shell, like it could be like, Holocaust show, like all of them, have to be around.

Hemdeep (59:11)
You all have to be around. And then you're also encapsulating, you said cells. What other items, what other, I guess, ⁓ particles could you encapsulate inside there and what would be the application?

Niloofar (59:22)
Anything like ⁓ any drug, any kind of thing like that can be encapsulated inside. And one interesting thing about ⁓ like having, for example, Janus fibers that reach one side is one material and the other side is another material that we created that with the ⁓ 3D printer with a Y-shaped channel. made Janus fibers. So you can add one type of ⁓ drug in one of them and another type in the other ⁓ half. And the interesting part would be like, ⁓ I recently read a paper that for the wound healing, because in wound healing, we have different stage of like the wound healing that in the first stage, you need to add these kind of drugs to promote those kind of cells to, I don't remember exactly the exact stages, but yeah, the first stage you need to add these kind of drugs, and then it's going to take one, two, three days, and after one to three days, they are going to go completely in a different stage. So you need to release a different kind of drug, you have to be in different environment. they, with... ⁓ core shell ⁓ particles. Here we made core shell droplets, sorry, core shell fibers, but we are able to make core shell droplets, which is my next step. They made core shell droplets, which in the shell, they loaded it with one type of drug. Then in the core, it was another type of drug. They control the drug release ⁓ and the degradation of the shell and then the core in a way to be optimized ⁓ with the stages of wound healing. So for the first three days, the shell would continue to degrade and the content of the shell would release. And then after the three days, ⁓ it would reach to the core and then we are in a different kind of, we are having different materials and different kind of drug which be. release at this point. ⁓ So that could be one of the applications.

Dario (1:01:47)
I think that's very important aspect, versatility of fibers that can be made. So not just only one. First, ⁓ we can tune the diameter of the fiber by, as Niloofar mentioned, by using different flow rates and pressure settings on our ⁓ supply materials. But also it's possible to make ⁓ hollow core fibers droplet-embedded fibers and those Janus fibers, which for those who are not familiar, maybe they resemble like those multicolor toothpaste. So we basically can make the fiber that is in a few colors to say so at the range of 100 microns or so or a few hundred microns.

Niloofar (1:02:39)
The Janus name is coming from the origin is the Janus Greek god, which had two heads. so in the nerds scientists for the droplets and nanoparticles and also like with the fibers whenever they have a like a nanoparticle that exactly half of it is something else, the other half is something else, they would call it Janus, like from the Greek god.

Robin (1:03:14)
Ha ha.

Hemdeep (1:03:16)
Okay, so we've gone full circle. We've gone from Korean barbecue, Japanese barbecue, some other type of barbecue to Janice.

Hemdeep (1:03:27)
Yes, Greek mythology, Janis Joplin.

Niloofar (1:03:32)
Proof that we're not just scientists, we have a life too.

Robin (1:03:37)
personalities outside of them.

Niloofar (1:03:39)
Sometimes touch the ground and go back to the lab

Robin (1:03:44)
Hahaha

Dario (1:03:46)
Yeah, think using 3D printer now allows people spending more time outside instead of being in the clean room.

Niloofar (1:03:57)
Yeah.

Robin (1:03:58)
They have more time now.

Dario (1:03:59)
I finally can have some social life

Robin (1:04:01)
I think you guys already touched on the next steps for you ⁓ here and there. I guess, does 3D printing still fit in there? Is there kind of like certain advancements you would want in 3D printing in order to kind of like help you bring you to that next step? Yeah, I can, that you could think of. I can kind of speak to this stage.

Stephanie (1:04:30)
So yeah, as I mentioned, like for my devices, like the flow focusing geometry is kind of the most difficult thing to fabricate, like to completely translate from soft lithography to 3D printing. So, know, in the future with like resolution improvements and things like that, I really think that like it will really, really help with accessibility to like the kind of research that I do with the microfluidic bubbles. and generating these devices in a much more efficient way. And we can of completely eliminate the clean room reliance, which would be the ultimate goal. Because for my work as well with microfluidic nanobubbles, so there's a lot of different ways that nanobubbles are generated. Another more common way is taking a vial and literally shaking it like crazy, it's called agitation, and it makes a bunch of bubbles. But they're poly dispersed, they're very different size. ⁓ They can range from like micro bubbles all the way to like tiny, tiny nanobubbles. So which is not ideal. So microfluidics offers this amount of diversity, but ⁓ it's not super widely studied. And I think that one of the biggest reasons is just because, you know, the access to the clean room, they very like long tedious procedure versus just shaking a vial and making bubbles. So I think if 3D printing can kind of completely replace like these photolithography, soft lithography dependence here, that will really be ⁓ really important for advancing this field into like preclinical clinical studies with the bubbles.

Niloofar (1:05:51)
I think for me too, if ⁓ one issue that we had, we couldn't put the channels really close to the surface. They had to have like, I think half a millimeter or so. I'm not sure exactly, but ⁓ they couldn't be really close to the surface. So with the inverted microscope and we want to like look at the and solutions, ⁓ the flow from ⁓ bottom of the device, ⁓ the objective needs to get close to the channels and that was making an issue. ⁓ also ⁓ if they could be more clear, to more transparent and to see inside of the channel, that's one thing that would... helped specifically me with that I have like multiple phases in ⁓ inside the channel. We recently made a microfluidic device with five inlets. So inside I would be having one, two, three, four phases and I need to see the interface of each one of them. And it's kind of hard and sometimes I have to guess ⁓ but Overall, because I don't have to like cut the glass capillaries, I'm still okay with it, but if you can fix that, I would be happy.

Robin (1:07:22)
Very important question. How many glass capillaries did you break? So many. So many. I need a number.

Niloofar (1:07:33)
No. But I still like, think like when you buy glass capillary, it comes with so many of them and you need a teeny tiny for each one. So even if you're going to break like multiple of them, it's still going to be okay. And you have to be careful for them not to go to your

Robin (1:07:53)
You're also underestimating cost of stress. Exactly.

Niloofar (1:07:58)
Like ⁓ for that whole time, the thing that I was doing, because I was just like sitting next to the microscope and for the whole day, I'm not like exaggerating for the whole day. I was grabbing a glass cap that you couldn't see that I'm holding and I was just like doing this and one time because like all my friends and family, are from like in different countries, different cities. So always. we don't call them during these kind of like tasks and works that I have. So my friend was talking to me and they were saying, like, what are you doing? We were talking for two hours and for the whole two hours you're just doing this. So that was my whole life for two years.

Dario (1:08:40)
I mean, Niloofars project is an excellent example of using a 3D printer for applications where it would be very difficult to use standard ⁓ soft lithography or photolithography processes. And that's something that we are looking into future to identify those projects that are difficult or impossible to do other ways than 3D. printing and I think that's great opportunity here.

Hemdeep (1:09:15)
I remember seeing the, I think the first draft of that STL when it was sent over for us to take a look at and I was very curious as to exactly what on earth this thing is supposed to do and how it all fit. And I'm so glad to have had this opportunity even, know, how it comes full circle. It really is amazing how the world works, doesn't it? No. I think... we have come to the end of this conversation. And it's not really an end of the conversation, but I think this phase of the conversation ends now. But I think I'm so glad that I had a chance to talk to all three of you. You highlighted the type of research that is being done and that a lot of people don't know about. I know me as part of the, on the outside definitely doesn't realize that this is the type of complex research that's being done. It's amazing that you guys had a, that we had a chance to listen to it, about it and all the struggles you had and the successes you had from ⁓ your hard work as well. Thank you very much for coming.

Niloofar (1:10:24)
Thank you so much for having us. 

Stephanie (1:10:26)
Yeah, thank you for having us.

Dario (1:10:27)
Thank you for having us.

Hemdeep (1:10:29)
And that will end today's ⁓ episode of Big Ideas at Microscale. My name is Hemdeep Patel, and my co-host Robin, will say goodbye for now.

Robin (1:10:52)
Hahaha