Ronawk Webinar 1/27
AJ Mellott: [00:00:00] All right. Well, let's go ahead and get things started here. So welcome everyone. We are really excited to have you this morning. For our first family and friends webinar from Ronawk
We just a few housekeeping things. So first off we have a really diverse audience. We have friends in the science community that are on, we have some financial experts that are joining us.
We have general members of the public, and then of course, friends and enthusiastic related to Ronawk. So we really appreciate your time and coming to, to watch today. A few other things is as we go through the presentation here if you have a question there is a Q and a panel. If you'll click on that and you can type your questions out there.
And when we get to the end of the webinar today, we're going to address as many of those questions as we can. And we certainly welcome questions. And we just want to share with you today. [00:01:00] What's going on with Ronawk, we're hitting a really exciting inflection point. And so let's go ahead and kick it off and get into the bio block universe.
So we'll start with introductions. I'm AGA Malott the co-founder and CEO of Ronawk. I'm also the inventor behind the bio block universe, which we'll talk about today. My background is in tissue engineering. My PhD and graduate work focused on manipulating a variety of different types of cells. In addition to co-founding roadblock, Heather Decker, one of my closest colleagues is the other co-founder of Ronawk.
She is an expert when it comes to analytical imaging and Heather is actually the one that has come up with a lot of the exciting applications that the T blocks and the other blocks we're going to talk about are used for today. And so it's important to acknowledge her and her contribution, and she is a wonderful member of this team.
I'll go ahead and let Tom now introduce him. Hi, [00:02:00] I'm
Tom Jantsch: Tom Jansen. I'm the president and COO for Ronawk. And my education and training is, is, is as a CPA. And I have twenty-five plus years of financial and operational experience with a particular expertise in startup companies which I find that to be quite enjoyable.
I like the chaos of the startup environment and have been fortunate to weather multiple exits. That's turned out well and I'm very excited to to join Grenache and the team here, how they're an AIG and Scott just the potential behind this technology that we're going to talk about today is, is extraordinary.
And I find it quite exciting. So I'm very excited to be here.
Scott Leigh: And my name is Scott Lee on the chief revenue officer here at Ronawk and with 25 years in the biotech industry and sales, marketing, branding management. Many of you, I met in the labs years and years ago this technology, I'm just amazed at what we're going to be able to do together, how we set you guys free remove parameters for so growth for you.
This really is about making you all the heroes within whatever project you're working [00:03:00] on.
AJ Mellott: So with that, let's get into it. So what are the bio blocks? Well, simply the bio blocks are hydrogels substrates on which cells category. So for those of you that are not necessarily scientists, what ourselves, what does that mean?
Why is that important at all? Well, all biology starts with cells and cells are the smallest unit of life. And they're wonderful because they are these amazing little factories that produce all kinds of biologics and other things that help us live and heal and do everything that we are able to do. The challenge is when we are studying different diseases and whatnot, trying to grow these cells outside of the body can be a bit challenging.
I mean, we've got great ways to do it. However, getting the cells to behave like they do in the body has been the big challenge. And so here at Ronawk, we're [00:04:00] using the technology to actually. The, to allow cells of virtually any tight grow environments that are more closely related to the environments from which they arise in the body.
So our bio blocks I mentioned are hydrogels substrates. So these are cross-linked polymer networks where 70% of their mass or more is just, wow. And so because of that, it gives us this wonderful ability to tune different parameters from mechanics to infusing the blocks with different chemicals, changing diffusion, gradients, all these wonderful scientific things that allow us to make an environment in which the cell grows that is more closely tuned to the original environment within the body.
So you'll notice that these blocks have a puzzle piece shaped to them and that's on purpose that allows the blocks to actually connect together so that when cells outgrow the T block or the one of our other [00:05:00] blocks as a container. Instead of having to dissociate those cells, like normally would be done in traditional cell culture.
Another block has just added on and the cells keep growing much. Like what happens in the body? Furthermore, you'll notice in the middle top picture, there's a, there's a picture of the top of one of our blocks. And there are all these kind of nice little dots or holes on top. And those are the actual micro channels that those are the entrances that permeate through the block.
And so what we've been able to do is we have been able to take all this massive surface area and we've been able to compress it into this cubic centimeter block, which we'll explain a little bit more later. In addition to that, just to give you a reference on size, the top left picture shows. Six well plate, which is a common consumable used in most labs for doing different scientific experiments.
And we actually culture the blocks in these six well plates. So they're not very big. The blocks themselves also fit. If you look in the [00:06:00] top right corner into 12 well plates, and when we finish actually processing them and when they're ready to go, they're actually translucent and the blocks are really good at taking on the color of whatever da or whatever liquid or media they are submerged in.
So in the middle, it's red for media on the bottom, it's yellow from some sterilization agents we use. So this is just to show you different views of the block in the bottom center picture. What you'll see is kind of a nice side profile of the block, and that shows you the texture. It shows you the exits of some of the micro channels.
So that fluid can flow through the block and cells have access to more surface area. And then. In the bottom left, there is a picture that was submitted to us by one of our collaborators where you can actually see cells growing around the central pores and channels in the block. So let's go to the next slide,
Tom Jantsch: just really briefly about how, the, how, how do you, how do you make
AJ Mellott: the bio block?
Sure. So actually that's one of the things that's [00:07:00] really fun when it comes to our bio blocks. All of them are 3d bioprinted here at Ronaldo. So we put all the engineering and the design to the structure, and then we utilize a couple of different bio printers to actually build these blocks here at Ronawk in our production area.
So then with that, let's talk about the three different types of blocks we actually have. So the first block is what we call the IE block, which is short for extracellular matrix block. And this block is actually a standardized block where the parameters are all uniform across all each blocks. And this is meant to be kind of the starter blocks.
So if you're new to growing cells in 3d and whatnot, or you just want to try the product without having to optimize anything, then the E block is the block to start with. And what's great is there's no coding that's required. The, the block itself is already made from materials for which most cells will.
After the E block, we [00:08:00] have the T block, which stands for tissue block. And the T block is our original block and the bio block universe. And this block is really special because of the fact that it was engineered to behave like tissue. It can be processed like tissue. So when cells are growing or tissues are growing in it, this block can actually be fixed.
It can be embedded in a media and then sectioned and stained using histological stains or antibody labels. Now with the T block. The other thing that is really exciting about it is the fact that we can customize different parameters of the T block to more closely mimic a specific tissue. So if we want to change the mechanical properties, if we want to change the diffusion gradients, et cetera, like I mentioned before then the tissue block is really there for that type of customization, where an environment is needed to grow a very specific type of cell.
Then finally we have the X block, the X block is [00:09:00] our newest product, and that stands for degradable or collapsible block. And so the X block is really for end users that are growing a large volume of cells, but then need to extract those cells for terminal assets, for example, flow cytometry, RNAC, or any other type of bio assay to procure some type of data specifically from cells and kind of a free form.
And what's great is actually all three of these blocks fit together. So an end user can start with, say a T block, grow a specific cell type, and then needs to extract cells add on an X block. Have those cells migrate over into the X block and then take off the X block, degrade it with our proprietary reagent and use those cells where terminal assets and keep the original culture and the T block going.
Tom Jantsch: what type of cells can you grow in bio blocks?
AJ Mellott: So you can grow virtually any type of cell that you can grow in any standard cell culture, [00:10:00] plastics. So anything like a T flask, a roller bottle bioreactor, et cetera, these T blocks or these bio blocks can take any of those cells. So any adherent and any cells that need fluid flow.
So fluid actually flows freely through the blocks as well.
Scott Leigh: Now, one of the things I think that's really important about this is if you're a tech, you know, how difficult cell culture is you, risk contamination, you risk of cross-contamination and then just keeping everything consistent as a challenge. Now, if you're on the other end, you're the.
Director or your principal investigator you're somewhat bound by the biological opportunities. Well, this block changes all that. And I'll explain that over the next few slides. So let's talk about from just how easy this is to operate is number a step one. You're going to marinate the block in the coding of your choice.
We recommend fiber act and collagen. [00:11:00] And quite honestly, if you're going to go to something that is not in our protocol, consult with us first about that, we have a lot of tips for you. So you code the block. That's an easy process. Anybody that can pipe that knows how to code a block. Second step is you're going to just add yourselves.
And this is a couple pipettes up and down just to get the, the cells to started hearing within the block. And the block starts to act like a sponge to those cells. And then every couple of days you're going to change out your media. No more subcultures. Let me say that twice. No more subcultures for you all.
That's going to be a huge advantage. All you're going to do is aspirate out eight and a half mils per well, and put back in eight and a half mils. It's so simple. And then it goes straight back in the incubator. So instead of that disassociation the spin down the labeling, the making sure you're not touching the wrong thing at the wrong time, just pipette, but at the back of the incubator, step four is your, is [00:12:00] you make the choice.
Do we need to expand our culture? Or is it time to start extracting cells? And again, no subculture. So you can start with one block and add on second, third, fourth, fifth block, or you start with five blocks and just monitor that growth. And going back to the X block, if it's time to harvest, all you're going to add is our proprietary reagent to the block, the block degrades down and you're left with over 90% of yourselves to do whatever you need to do.
qPCR westerns Eliza's flow, cytometry, whatever you're going to do now, all those downstream applications. Now the other thing is if it's a tissue block or an evil. Now you can move into sectioning, doing whatever you want to do, just like you would do a tissue.
AJ Mellott: So then microscopy, because if you want to do stains on the block after they've been sectioned, you're good to go.
Or even while you're growing, you can still look into the top or the [00:13:00] bottom of the block he sells in real time.
Scott Leigh: Absolutely. Absolutely. And when we get into this harvest, this is really an interesting concept. Here is what you see in this graph is we're not accelerating how fast a cell splits the cell is doing what it does in nature.
That because you're growing across the X, Y, and Z axis, you're getting unfettered growth. And that's where the exponential growth is. Now, if you need massive amounts of cells, like your a CDMO a CRO, your pharma, and you just want to grow as much as you can for bio baking, whatever. This gives you an accelerated rate to do it.
If you're a small lab and you're like, well, gosh, we just don't need 350 million cells think of this. As we grow cells, once we freeze them and we only bring them back as we need it, we basically create our own bio bank for our lab. Think about that. You have, instead of just replaying and replaying, replaying, you have [00:14:00] your own bio
Tom Jantsch: bank.
Talk about Scott, you, you, you mentioned I don't know if you've used this word. Good, but supersize talk about some of the numbers that you can get out of a single block, or when you put blocks together, kind of what
Scott Leigh: happens there.
This is, this is a fun image for us to put forward. One of these T blocks can replace up to 120 T 75. So it just that one little centimeter cube. Now the other thing is to think about this is it doesn't remember. It's supersizes every vessel. So instead of going from flash to flash, maybe you're just growing massive amounts of cells in one flask in one roller bottle in one bio-reactor.
And that's where we come up with this, you know, one block could replace 120 or you're, supersizing all that capacity for yourself.
AJ Mellott: Well, and just as a reminder to the audience, you know, we're not doing anything that goes against what naturally happens and nature, what we've done is [00:15:00] normally with cell culture, plastics, you have a flat surface that has a limited amount of surface area.
What we've done is that Scott said we've taken up to a if you grow them to the max, 120 of those flat of those and we have essentially molded and compacted and folded all of that flat surface area to fit into our block with how we've created all the different micro channels and pores and the internal struggle.
Tom Jantsch: And the why is it important to grow cells in 3d
AJ Mellott: as opposed to, because, well, we'll get to that here in just a couple minutes, but when it comes to growing cells in 3d, the, the simple answer is that cells behave more physiologically as they do in the body. When they're grown in 3d
Tom Jantsch: and the bio blocks are meant to more closely resemble how cells grow
AJ Mellott: in the body.
That's exactly right.
Scott Leigh: Absolutely. So as you look through this, you know, five blocks can produce as many cells as five CO2 incubators full of flash right now. So it, it, again, it just is [00:16:00] it's unlocking your potential as well. And potentially reducing some of the actual labor. Now, the great, exciting thing about this is the FDA considers this just like they do any other cell culture environment.
It is, it is five, 10 K exempt. It's a class one device. So regardless of what you're using it for, if you're using a T flask or plate or roller bottle or a cell factory, we have just now giving you the ability to supersize all of those
AJ Mellott: materials. And just to comment further on that part of the reason behind that designation is because of how the cells are being used with the product.
So this is not a product that is meant to be implanted in the body. This is solely for just growing cells outside of the body. And so that's where we're able to get this as a class one device. Absolutely.
Scott Leigh: Now let's give you some numbers. If you just look at the amount of units used the media, the labor.
We're going to bring down costs per cell growth [00:17:00] significantly, but then come over a little bit further to the right, with going smaller and less. You're actually getting more, you know, and as we see with, you know, going from I'm going to show my age and rotary phones down to a cell phone, we're getting, you know, I remember when the first cameras were put in cell phones.
I'm like, why would I want that? Well, now we know we all use that. I can text on my phone. It's now a computer in my hands. That's essentially what we're doing. But even more exciting about this is in these examples. We're going from 63 days down to 14. Had a great conversation with a friend of mine at a CRO the other day.
And she was saying that some of their projects are kicked back as far as 36 weeks, just because they have to grow that many cells before they start the project. So from your perspective, instead of doing all that work before you're doing your analization before you're doing all your downstream applications, we've just shorten that time.
So you can get to the stuff you really want to do. [00:18:00] And that's exciting. The other thing is on the far, right? We're by removing subculture and we're reducing the contamination potential significantly because we're, and we're not disrupting the cells. We've eliminated a cross-contamination potential. And I want to put a thought out there.
We're not there yet. But think about this. If we remove the cross contamination and we remove the subculture and all we're talking about pipetting is ultimately this block can be used for automation to where we, we virtually eliminate the potential for contamination.
AJ Mellott: Yes, potentially. And there's two things I want to touch on that you mentioned.
So when it comes to the contamination risk, the reason the contamination risk goes down is because we are eliminating the human touch points in the process of cell culture. So instead of having to dissociate cells from one flask or a hundred flasks over and over, [00:19:00] we have compressed all that so that there are fewer times that has to be done.
And that's where we decrease the risk of contamination. Furthermore, With that same point because we're not sub culturing. And now we are combining substrates that link together. We are actually preserving the phenotype or the characteristics of the spells for much longer. And we'll show you the data here just a little bit, but that's how we're getting higher quality cells with less work.
So all together, this makes life easier for scientists and it accelerates their time to resolve
Tom Jantsch: the CPA. And me likes the right-hand of the slide or the kind of the cost figures and, and cost is important. And, and there's an old saying time is money and that's exactly right. But when time to result is the most important component and this achieves that.
But because of that time, because you know, less labor, less media it's significantly less expensive to create.
AJ Mellott: Lots more shelves. Yes. [00:20:00] So, and then what I'll say, what's great about this. Just one of the things that I enjoy is the fact that right now, since we bioprint all these we actually are procuring all of our raw materials here from within the U S specifically in the Midwest and manufacture everything here.
So that gives us some big advantages when it comes to manufacturing and distribution and the availability of our product right now. So let's get into. Why we made the bio blocks in the first place. We've kind of mentioned all these challenges with cell culture. And I have a lot of personal experience with this.
Back when I was a graduate student I've grown up billions of cells and to all my friends that have done cell culture you know how painful this can be, especially when you need to grow up vast amounts of cells to put in a scaffold or for other experiments and use literally spend all day in the lab, just changing out media or sub culturing.
And because of that, you get [00:21:00] tired and the risk of actually contaminating yourself, no matter how careful you are. Goes up out of my frustration about seven years or so ago, I wanted to find a way to bypass this and make it easier, not just because I wanted to get to the analytical part of my experiments faster, but I just didn't want to keep spending weekends in the lab, which most of my colleagues can appreciate.
And so with principles of bioengineering and tissue engineering, this is what led to the ideas that would eventually evolve into making this product or this platform, the bio block universe with these different types of blocks that we can use. Just as an illustration with my former research, I actually, the reason I had to grow so many cells is I was reseller colorizing, these organs that had been decellularized for the purpose of regenerative medicine.
So it took a lot of work. Now, if we go to the next slide, one thing that I do want to acknowledge is that [00:22:00] we're not really reinventing the wheel. And it's important to understand what we're doing here is built off of a lot of phenomenal technologies that have come before us. We are by no means. The first company to grow cells in 3d, or to try something to scale.
In fact, there have been great technologies that have been used over the last 10 to 15 years to create organoids. And spheroids using a hanging drop culture using spinner flask to help grow up huge quantities of cells different types of other hydrogels for making environments that are tailored to how a cell grows and using microfluidics.
So these all share a component of what we've incorporated into the block that we're, we're talking about. And the reason why there's such an emphasis on growing cells in 3d is because we know that when we grow cells in 3d, as I said before, the cells then behave more physiologically like they do in the body they're of a higher [00:23:00] quality.
And because now they can grow in the X, Y, and Z direction, we can get our results faster, not because we've accelerated biology, but because we are getting more cells and this is important because as we get better at growing cells that behave more like they do in their native environments, it allows us to reduce our need, to use animal experiments down the line and potentially even eliminate those.
So that's one of the big implications of this technology. So let's go ahead and go up. Thank you. Next slide. So. When the T block was originally being conceptualized as the first block in the Bible, Chuck universe. One of the things that we took into consideration was the fact that there had to be a way to allow cells to expand like they do in the body.
So when during development, when cells are growing, there's accreting, all these different factors, extracellular matrix, and what's happening is essentially they're building their own scaffold [00:24:00] and growing into a tissue forming organs. And then finally an organism being an animal, a human. And so one of the things that's really hard.
And so culture today with the most techniques is there's not any easy way to expand a substrate. Well, using the inspiration from Legos, we can do this. So we seed cells into our, our block, and then we can connect another one. And what happens is then the cells can basically migrate into that second block.
And from their perspective, it's like the substrate expanded and they can keep growing. They don't have to be dissociated. And part of the reason they actually migrate is when the blocks are actually combined. And then media is added. There's a swelling effect that locks the blocks together. And so it's kind of, it creates a signal to the cells as if you're in a crowd and someone, you know, sets off a firework or something in the distance and everybody looks over and says, oh, what's going on over there?
And so I'll start migrating. And so that's part of how we get that effect. [00:25:00] Also what I wanted to show here, we've talked about a few times already in the bottom, right corner of this figure shows the difference between cells growing on a surface area in 2d and an X, Y plane, versus when you can utilize a whole volume of cells growing in the X, Y, and Z planes and more cells can be grown a higher density in a much smaller area, because there is more space if you will, within the volume.
So that's one of the other innovative features of the T block. We can grow more in a smaller space. So during the
Tom Jantsch: blocks, more closely resembled the human anatomy. Yes. And then are there any cells that grow in 2d in the body?
AJ Mellott: Not to my knowledge.
Scott Leigh: And I think this is an important point that Tom brings up is in my experience.
Sometimes when you, you mentioned 3d. The mind often shifts to organoids and people get stuck on that notion. Yes, you can grow organoids in this block, but it's essentially taking that 2d [00:26:00] environment, which so many people grow it. And now it's this, it's all you're growing in all dimensions. And that's what we also also mean by 3d.
AJ Mellott: Yeah, exactly. To also give you an idea. There are other functions within the body that we had to take into account. So when it comes to all your different blood vessels and whatnot, those blood vessels are going to expand and contract. So we had to make a microchannel system or a poor system that could mimic that and would be able to expand and contract as well.
In addition to. We had to keep in mind what the shape of our microchannels would be for creating additional surface area. So the shape was really important when it comes to how we want the cells to behave and where we want them to move. And then finally, the way in which we organize all those channels together from using a simple lattice to a more sophisticated network, et cetera, those are the things that had to be considered in [00:27:00] engineering these blocks.
So I want to go ahead now and get into some of the data that we have. So one of our first experiments we did was really simple. We took one of our T blocks and we seeded a hundred thousand adult stem cells. These are cells taken from adult tissues, which have the ability to differentiate into different types of tissues like cartilage, bone, muscle, fat, et cetera.
And when we put those cells in the block, we cultured them for five days. After five days, we added on a second block, which is kind of that whitish block you see in panel a, we then cultured the two blocks together for an additional two days. So seven days total on the first block, two days on the second one.
We fixed them. Then we applied different cellular stains so we could see different features of the cells. So when you look in panel B, now we can look at different features of the cells that are inside the block, and you can see the annotations that show you [00:28:00] the edges of the actual block and where the cells are inside.
Panel. See it's, what's actually really exciting because this shows now the scene between the first block and the second block, the black arrows are actually pointing to cells in the first block that are right on the edge. And then that white arrow is showing you a cell. That's already traversed the scene from the first block to the second block and just under 48 hours.
So this is showing this concept that yes, the cells actually do migrate from one block to another. When you connect them together, let's go to, let me ask a quick,
Tom Jantsch: so true. It's really exciting that these blocks can be put together and you see the, the migration of the cells from the first block into the second block.
Is it possible to put one type of cell in one block and a different type of cell and then in the second block and what happened?
AJ Mellott: Absolutely. So your, your venture 30 co-culture that is possible. So let me give you an example of something that could be done. [00:29:00] You couldn't grow cartilage cells in one block and bone in the other.
And if you want it to then combine those blocks to see how they interface in 3d and how they behave. The only thing you'd have to do I'm on the end user side is balanced out if they have two radically different media formulations. But other than that, yeah, you can, you can call culture different types of cells using the blocks and combining them in a very easy format
Tom Jantsch: where people are able to do that today with current technology.
AJ Mellott: It's very limited not, not mainstream technologies that I'm aware of, but there's also plenty of startups out there. They're probably trying that same thing. So now going back to the data, the other thing that's really important when we're growing cells in some type of new substrate is we want to make sure those cells are.
So that same experiment we decided to perform what's called a live dead assay. And what that means is we have these dyes where green means the cells are alive. And if you see red, that means the cell is in the process of dying or it's dead. And so when we imaged the top and the bottom of the blocks, what you'll see is a sea of green cells.
So that's what we [00:30:00] want. They're all healthy for those of you that notice also the blue dots on there. We also stay in the nucleus of the cells using the dye called hoax. And the purpose of that was just to make it easier for us to count cells. Now, as we go to the next experiment, this is the really exciting one and the really important one we did initially, we wanted this whole idea of being able to bypass subculture was really what was pivotal here.
Going to tell us whether we could move forward in developing this technology. So in this experiment, what we did is we actually, once again, we seated these adult stem cells into our blocks. We also see to them onto a 2d culture surface. And what we did is we then grew the cells for up to 60 days. That was the cutoff that we chose for this experiment and what we wanted to see as if we could continue growing the cells without any major deaths or anything in them.
And so the cells that we culture and we, we seeded the same amount of cells in 2d that we did in. [00:31:00] And so the cells on our, our 2d control, we let them grow to full confluence, see before we fixed 'em. And so that's why that ends right there at the beginning. So we could have account. And what we ended up with after about seven days was around the ballpark of 3 million cells.
And then when it came to the T blocks, when we were ready to harvest them at each one of the time points, we would take the. Fix it embedded in paraffin serial section. And then we applied, hematoxylin and eosin staying to each of the sections and we did nuclear counts on every single section to get these numbers.
So what you see then as the exponential growth. Of the cells as we move from 15 days to 30 days to 60. So at 15 days where somewhere around 15 to 20 million cells at 30 days, we're about at 90 million. And the average number of cells in the blocks by day 60 was actually 350 million. So it's amazing how [00:32:00] fast they grow the exponential rate.
And the picture on the left also is there to show that once you start getting into those numbers, if you were to take, if you were to max out the block and grow as many cells as possible, you'll actually start to see it with your own eyes. You won't need a microscope because you will see the block will become more opaque with the cells in there.
But that's when you get to the high end, if you don't want the full block to be filled, because depending on your cell type you'll want to add on another block. But when they do, when the block does get full, you can tell. So then again, the other thing that we did here, as well as we characterize the cells at the end of 60 days, and we applied some stem cell markers, just to make sure that they had the right phenotype or the right characteristics.
And what we saw is after 60 days, our original cells were maintaining those actual markers. And so what we're showing here is really only the positive markers, but the fact that the cells did retain them while growing in the T blocks. [00:33:00] Another really exciting experiment we did that we sort of stumbled into is when it comes to growing primary cells.
What we have done is we have looked at the ability of whether we can wake the cells back up. So most of my colleagues know that when you grow a primary cell and you subculture it, or you passage it multiple times, when you get out there into. 13 to 17 times, typically what will happen with the cell is it'll start there, the cells and the population will start to Snus.
And what that means is they're going to sleep. They're no longer divide. And once that happens, really the cells aren't usable anymore. So what we wanted to see as we took some, we took a cell population that we knew was already senescent. It had been passaged about 15 times or so. And we attempted to, we passaged at one more time and we split the culture in half.
We put half on a 2d substrate and half into one of our T blocks. And then what we did is we did a senescent experiment. And what we saw was there was a drop of 77% [00:34:00] of the senescent markers in the cells grown in our T block and why that's a big deal. And what that means is. The cells started waking back up.
They started to divide again. So that's something that's really exciting to us. And we are still probing and exploring, but we don't even know what the full consequences of that will be. But from a regenerative medicine perspective, this means we might be able to take cells that are older in their life cycle.
And by changing their environment, there could potentially be a way to reinvigorate those cells for regenerative medicine. We also wanted to look at how this worked over time. So. We did a temporal experiment where we started with a primary cell line. Again, in this case, we took adipose-derived stem cells, which is basically fat tissue.
So what was great about this is we had some volunteers with the the department of plastic surgery at a local medical [00:35:00] center that were willing to volunteer and donate some of their fat tissue. So we could process it to do these experiments. And what we found is after those cells were taken from the tissue and we started growing them in 2d and 3d, we did a side-by-side comparison where we looked at the expression of senescence.
And what we saw is in the beginning, both 2d and 3d cell cultures showed the same percent of the population approximately of cells that were senescent. And the 2d is shown in blue, the black bars here. The 3d has shown in the turquoise, but as the cells continue to be, passaged in 2d. And we looked at their 3d counterparts.
What we notice is the percent of cells in 2d that are expressing some Nesbit markers starts going up and it becomes really pronounced as we get to a passage 10, where in 3d, in our T blocks, the percent of cells that are senescent pretty much stays flat around 5%. So this was really exciting for us that once again, we show we [00:36:00] are, we are minimizing and Delaine cells going are becoming senescent and going to sleep.
If you will, while they are grown in the tea box and just give a timeframe for this. My colleagues know that cells will divide as fast as they want, but if we use a metric of approximately the cells and 2d end to be passaged about every seven days, then by the time the cells have been passaged 10 times, that means they've been cultured for 70 days straight.
So over the course of 70 days being grown in the T block, we're still seeing only 5% of the cells expressing senescent markers. Then the next thing I want to point out is we're starting to see that some interesting things are happening with what the cells are actually secreted into the media when they're grown in 3d.
So from a wound healing perspective, we have. Project we're working on right now, where what we've done is same thing. Again, we took the cells from fat tissue and we grew them in 2d and we grew them in our T blocks in 3d. [00:37:00] And we collected the media, used to grow both types. And we applied that media to what's called a carer tennis site, which is a type of skin cell that's in the top layer of your skin.
And we wanted to see what the effect of that media collected from those two formats had on those cells in the skin. And what we noticed is our our fat cells grown in 3d, actually increased the metabolism. The condition media increased the metabolism of the keratinocytes. They could increase the proliferation and most excitingly when we did what's called a scratch assay where we grow those keratinocytes on a flat surface and take a pipe that tip and scratch it.
We look to see how long it takes the keratinocytes to divide and migrate and fill in the gap that was made. And when those keratinocytes in that scratch ass, We're treated with media from cells grown in 3d. We actually see significant increases in their ability to migrate. And this is important because this gives us insight to ProTap perhaps closing a move at the benchtop.
So let's go to the next. [00:38:00] And this slide, we are really excited to show off this data from our colleagues in Istanbul that we've been working with for almost the past year. And what's been exciting as they took a a liver carcinoma cell line, and they were able to successfully grow spheroids within different flavors of our tea blocks very easily.
And so they were kind enough to share those photos with us of their beautiful spheroids growing. And not only that, but they were able to grow these spheroids from over the course of 14 days versus only say two or three days. And that is significant because when it comes to organoids, depending on what you're working on, if the cells get too dense and too compact, the inside of the spheroid starts to become necrotic because those cells on the inside can no longer get the nutrients from outside.
So this allows them to do longer pharmacokinetic studies looking at different cancer treatments, different modeling, [00:39:00] because those fear roids are lasting for 14 days instead of three
Tom Jantsch: pictures, only a scientist. Yes.
AJ Mellott: So then what I want to show here real quick is just with our, our initial customers out there, we've shown you all this amazing data, but I just want to paraphrase a few things that they've shared with us. So from one of my academic colleagues her quote, the fact that my researchers do not need to subculture cells anymore is a huge benefit because it reduces contamination, risks and maintains the quality of the cells.
Furthermore, not only are researchers noticing the clinicians are starting to see the potential, another quote from a colleague in plastic surgery stated that this cellular amplification through the T blocks has incredible potential for improving the outcomes of patients undergoing reconstructive surgeries, following cancer, removal, burns, and trauma.[00:40:00]
So. We are not limited to just here at the bench shop. This technology has implications that go from the benchtop all the way to the patient. And I'm going to turn it over now to my colleague, Tom, to talk a little bit more about those implications.
Tom Jantsch: So the advantage of the, of the bio blocks are significant and this chart kind of lay those out a little bit, but most significantly we talked a little bit about time to result and how important that is and how that timers to result lowers the cost to all involved.
But also with any new tech technology or new platform, you want to make sure that it's scalable and can grow as experiments grown. This, this opens up a lot of opportunity to do new experiments and, and, and just because you can grow so many more cells. And so is it, it's a simple, simple workload it's compatible with.
Instruments and media that are currently being used in a lab that's significant it's scalable. You can grow huge, just tremendous, unheard of amounts [00:41:00] of, of, of cells up. And you get to by bypass the subculture, which is significant with age, I talked about the de-risking the contamination and, and it's green.
You're going to use less media. You're going to use less plastics. So all very important advantages. I think. So let's go to that next one.
AJ Mellott: So you've
Tom Jantsch: heard this before, but all, all biology starts with cells and the need to grow more cells is a significant roadblock. In biology and in research. And so as AIG has got explained to Rome, Ronawk removes this model of NEC and Ronawk starts in the $19 billion cell culture market, and bio blocks make the painful process of culturing cells effortless.
And so that's exciting, but they also significantly enhanced the production of biologics and biologics, which is a $325 billion market. It is, if you haven't already heard about it, you're going to hear more about it. It's a rapidly growing market and it offers a wide range products of vaccines, blood and blood [00:42:00] components, allergenic, somatic, gene cells, and gene therapy and tissues.
And as AJJ mentioned, Ronawk is currently collaborating with groups working on wound biologics for diabetic wounds and also for the war fighter.
The biologic market leads then ultimately to the organoid market, which AJ touched on just briefly and an organoids as a miniaturized and simplified version of an organ produced in vitro in three-dimension that shows realistic micro anatomy and the development of organ market presents an opportunity to reduce and eventually bypass the animal model.
My past animal models, each of these markets then eventually lead to the personalized medicine and that includes regenerative and also precision medicine. And that market currently is valued at $1.9 trillion markets. And so kind of in some, the bio block is the platform that accelerates All of these markets.
AJ Mellott: Well, great. Thank you, Tom. And we want to turn it over now to the [00:43:00] audience for questions. So I've seen a lot of them come in and we're are going to do our best to address as many of them as we can with the time we have left. But I do want to point out if we don't get to your question, please feel free to email it to.
Info@Ronawk.com. When we finish the webinar, what we'll do is those questions. We didn't answer. We'll put into an FAQ. And then when the recording is ready, we'll send out that FAQ with the recording so we can get to your questions. But one of the ones I see on the screen first that I want to get to, because it gets to some features about the block we didn't mention was, is there a concern for mycoplasma?
And so actually I would say that risk is very low with our bio blocks. The reason being the bio blocks are actually made right now from all synthetic materials, which are sterilized. So at this point for the introduction of any contaminants like that, it's going to come from lab practices where something was introduced in a [00:44:00] media or a cell wasn't completely washed or whatnot, but from the block itself, no.
So I really appreciate that question. Another one here is how. The bio blocks ever been used for IPS cell culture maintenance and or differentiation of IPS to specific cell types. So regarding that question, The answer is yes, the the T block in particular has been used to culture IPS cells that were derived from skin to maintain them.
However we have yet to try differentiating cells in the bio blocks, but we have multiple experiments going on with collaborators. So we are anxiously awaiting those that want to try that. Another question that I think is really important is can you talk about nonadherent cells? So suspension type cells, and I will turn that one actually over to stop.
Scott Leigh: one of the challenges in, in, in any research is, is scaling. And [00:45:00] a couple of different ideas is one. You could easily seed a a T blog or an e-book or an X block with an adherent cell. And somebody else asked did they float? And the answer is yes. So you can get that same. Biologic out of adherence or not same.
I shouldn't say that you can get the biologic out of the adherence cell using it in a roller bottle, in a cell factory, in a bio-reactor, as far as suspension cells, we're looking for collaborators that work have only on that. And we're about to enter our own study to do some tests on that, but, but tangentially, it should work just fine to answer one of these questions about roller bottles, how would they work?
And, and I'm going to use my handy dandy mug here is what you would do and pretend this is a roller bottle is you could line the bottom of this with as many tea blocks as will fit the float in the media. And as that roller bottle just rotates, you're going to be washing the media [00:46:00] through the blocks.
And since the blocks are porous, you're going to get that good flow throughout your whole environment. So And that doesn't matter whether it's a roller bottle or a cell factory or whatever you're using the plastic now is just a vessel to hold the media, to hold the T block. So again, that goes back to that concept of supersize.
AJ Mellott: There's one question that came in that I, I want to address while we're on this topic too, which is how do you change media with different small molecules, growth factors at different stages of cell differentiation? Well, it depends on the vessel you're using, but it's actually really easy. So if we're using a six well plate, the media is going to be aspirated from the side of the well, and then if you actually want to get media out of the block to introduce new media, or if you're doing kind of a staggered media change it's very simple.
You actually will pipette either your, your saline or your new media gently on top of the block. If you're [00:47:00] wanting to get rid of the old media and gently. That through the block. However because the pores are so open and whatnot. A lot of that media, when you put new media in, we'll just flow through and then diffuse through the hydro gel itself.
So it's not very complicated. But you can, you can still watch with, with saline, just if you're going to do a wash to clear all the micro-channels, you'll just want to gently drop wise pipette your, your saline on top to, to flush the, the micro channels.
Scott Leigh: Here's a great question on can you explain how to degrade the.
We actually provide a proprietary reagent that you just put it on the block and the block breaks apart. Then you want to use a cell strainer to remove any extra debris and you're left with yourself. And so again, that is a, we're experiencing over 90% retrieval with that. And so if you're getting a hundred million cells to grow, you're getting [00:48:00] nine 90 million out.
That's a, that's a phenomenal. Nominal numbers.
AJ Mellott: And then here's another question that was asked, are the blocks already sterilized or need to be autoclaved or irradiated. So when the blocks are actually shipped to the end-user, they have already been sterilized and we do all that in-house so you don't need to worry we have our own queue house to check that.
And as we're going into mass production here, we're getting ready to actually outsource the sterilization with another party that we'll do. Some of the more extensive tests, so that in the future we're providing extensive certificates of analysis on the sterility. So that's where we are now and where we're coming, but you don't need to do any sterilization also, just so you know, because of the type of materials we use, these blocks cannot be autoclaved or they will melt, you
Scott Leigh: know, there's a couple of good questions I'd like to jump in on.
Is that a few of you are [00:49:00] asking questions. We don't know the answer to yet because we haven't done that. And as the sales guy in me says, let's get it in your hands. You try it. And we're going to be running a promo for you for specifically that. But
Tom Jantsch: that's an important point and we've been, we tried to make it and then hope it came through is, is, is we're not trying to go grow specific cells.
We're trying to provide a tool for a role w for whatever you want to do and make you the hero advance your research into next generation technologies. And so the bio blocks, there are they're Gnostic to the types of cells. And so it is going to take some collaboration. And conversation and, and we want to help you optimize what you're doing so that you can advance that research.
AJ Mellott: One off of that, a comment that just came through asking about the production of customized Vblocks, according to the scientist's needs. Yeah. So that's what we're working on, but what we've actually done in regard to the tee box, we actually already have parameters that allow us internally to tune those tee blocks.[00:50:00]
Once we know what the cell type is that the end user is using and, and we have a list of those parameters, but our whole goal to what Tom was getting is this is a tool. This is meant to make it easy on the scientists. We're trying to make your job. And as Scott said before, we want you to be the hero of your lab.
So whatever we can do to minimize your time in the lab, minimize handling the cells and being able to get your data, to do your analysis. That's what we're here for. We're here to help you. So if you, if you have
Tom Jantsch: questions, talk to us because you know, we've done a lot of this, but that conversation will help optimize things and get the experiment off on the right foot, right out of the gate.
AJ Mellott: Yep. Then let's see. I'm 56. This is a, this is a great question here. I like, can you stack the blocks? I E the Z direction create a combined block of the two by two by two centimeters. So the current blocks we sell, no, they only go in the horizontal [00:51:00] direct. However, that is actually something we are in the process of developing so that the box can be stacked in the Z direction.
And that is for a product that we will be releasing, hopefully in the future. We don't have a schedule when, but yes, we are on track for that. And I think you'll be pleased when, when that comes out to that
Scott Leigh: point, we want you to be creative with this. You know, think about the first time you got Legos.
It's not the blocks, it's what you do with them. The first time you got an extra sketch, which I never figured out how to use, but there's clearly some artists that can, it's what you do with it. That's the most important thing. And w we really, one of the things that I'm most excited about is we don't know the limitations yet, and we'd love to find out what they are, because I think.
We just opened the opportunities for everybody to do incredible work.
AJ Mellott: So then one of the questions that came up [00:52:00] is are these blocks manufactured under good manufacturing practices? And the answer is not yet, but soon we are working on developing our own GMP facility here over the next 12 months.
So right now the blocks are R and D grade. But because of that designation with the FDA, they do need to be manufactured under GMP before they can be used in any type of a clinical trial and such. So we're working on it and we are, we are quickly growing. So as soon as they are ready, we will let you know.
Scott Leigh: I would add to that point, if you're in, if you need GMP. We will work with you in all the testing that you're going to have to do in R and D first, which is going to take you a little bit of time to validate, put it through your processes and we'll work with that because in some situations, by the time you do all that, now we're GMP and now we're ready to go.
So I would tell you don't wait until that point. Let's get going.
AJ Mellott: Yep. And then I think I'm going to close with one last question that I'm, [00:53:00] I'm really excited about that came in and this is, has anyone done a CRISPR screen using your system? That question is actually come up a few times and the answer is we have not done.
But we would be thrilled to work with someone to do that. In fact, we had a wonderful meeting just the other day, where we were specifically talking about that that application and for mass producing engineered cells. So that is something we're extraordinarily excited about as Scott already claimed use your imagination.
This is a tool for you to make it easier to perform your experiments, accelerate your, your time to result. So I do want to thank our audience again. It has been wonderful having you. Thank you so much for everything. I know we didn't get to all the questions, but as I said before, please feel free. Any question we didn't answer.
Just email it over to email@example.com and we'll create an FAQ and we're here because of you. We [00:54:00] want to make you the heroes in your lab. So it's with our work with you and our collaborations with you, that our products are only going to get better and we're only going to accelerate the development of new generation or next generation therapies.
So thank you everyone, and have a wonderful day. Thank you. Bye-bye.