Hemdeep (00:13)
Welcome to Big Ideas in Microscale, the podcast where we explore groundbreaking research happening at the microscale where micro innovations makes a big impact. We're excited to showcase the incredible work being done by our users from around the world who are pushing the boundaries of microfluidics, lab on a chip, organ on a chip, and beyond.

Through these conversations, we hope to learn from their experiences, uncover their insight, and bring their big ideas to wider audience. So whether in a lab, on the go, or just curious about the future of microtechnology, join us as we dive into big ideas at Microscale.

Welcome back to Big Ideas in Microscale. I'm your host, Hemdeep, co-founder of Creative CADWorks, CADWorks 3D, and ResinWorks 3D.

Robin (01:19)
And I'm Robin, co-host as well as the technical writer within the marketing team. Last week, we had the pleasure of chatting with Jack Kosh, an assistant professor at the Aquatic Germplasm and Genetic Resource Center, or AGGRC for short, who gave us a glimpse into his groundbreaking work on cryopreservation and germplasm repositories for aquatic species.

Hemdeep (01:42)
From his early childhood fascination with marine life to his innovative research combining biology and technology, we had a fascinating discussion on the preservation of genetic material. If you don't know what we're talking about, go back and listen to our previous episode.

Robin (01:57)
This week, we're diving even deeper into the world of germplasm repositories. Jack will return to talk about how these genetic banks are essential for preserving the sperm, eggs, and embryos of aquatic species to support breeding and conservation programs. We'll explore the challenges of preserving aquatic species and cutting edge cryopreservation techniques that are emerging to address these hurdles. So, let's jump right in to big ideas at Microscale.

Hemdeep (02:26)
is it in terms of milestones? Obviously, the number of species that you could sort of foresee yourself itemizing and then having a repository could be very large. Do you guys internally have sort of a number or the type of species or do you classify species that are important for this type of safeguarding?

Jack (02:47)
We don't, don't have any internal classification of species, nor do we have like a number because there's a lot of aquatic species out there. We work pretty closely with the United States Department of Agriculture, National Animal Germplasm Program in Fort Collins, Colorado. And that's a world, a state of the art germplasm repository for.

animals and I think they also have a plant section there as well. The plant germplasm repositories are distributed in a network across the United States, whereas animals are centralized there in Fort Collins. They probably have a more curated goals of what they would like to see in the collection there. I know that more recently people have been concerned about two camps of people like

just collecting samples just to have them. a high number of them versus the quality of the samples. So collecting samples that are of strategic importance to whatever field that they may be relevant to. If we take oysters as an example, again, there's many types of lines of oysters because people grow them in different areas. You might, you know, different farms, different hatcheries. We could ask each of those hatcheries to send us a couple of oysters and we freeze their.

germplasm and store it there. And now we have hundreds of lines of oysters. Or we could say, Hey, send us the five oysters in the United States that are super disease resistant. And so now we only have five lines, but the quality of those lines might be perceived as being a lot better than the hundred lines that could be out there. I don't think there's any right answer between quantity and quality. It's all about what the mission of that particular project is.

Because in biology, lot of times quantity is a good thing, especially when you're talking about genetics, because you can get things like genetic bottlenecking where you take a hundred genes and you're doing a lot of breeding and you end up with two genes. Your chances of like inbreeding go way up. So I don't think there's any one right answer between quantity and quality, but it is something that you have to think about.

Robin (05:05)
Going back to barriers for why it didn't advance as far as has as of yet, is there drastic changes in the methodology to cryopreserve certain materials or between animal species? I guess it's different for the sperm versus the egg versus the embryo, that sort of thing.

Jack (05:25)
We like to focus on different levels. So there's like a protocol level, which would be like add five milliliters of cryoprotectin A to your sperm sample. So very specific. But then there's a level above that called the process level. And at the process level, most cryopreservation is the same, whether you're doing it for sperm, eggs, embryos, larvae. There's some differences, but

At the process level, a lot of things are the same. And it's just a matter of fitting that process to whatever environment you're working in or what, what material you have available to you. Now those specific types of germplasm sperm is the most advanced because it's a single cell. It's small and it's abundant, right? Usually when you collect sperm from something, have millions of billions of sperm and

If you lose half of them during the crop preservation process, like it's not a big deal because you still have millions more. Eggs are really far behind because they're typically pretty rich in lipids. They're pretty fatty. so freezing them is just very difficult. And then larvae and embryos are rapidly advancing in our technology and ability to, to crop preserve those. And even bigger things like whole organs, people are figuring out how to freeze like

livers and kidneys, things like that, that will advance the medical field a lot. But it's a slow process to figure things out like that.

Robin (06:56)
Okay, because you have two papers, or not, you have way more than that, but I'm going to refer to two. Juan Cryopreserva- preserving the, my god, I don't know how to say this, but Hydrocitinia Sim- Symbiolongucarpus. Who needs tongue twisters when you have scientific names?

Jack (07:13)
Hydractinian.

Robin (07:20)
Hydractinian. Okay, so that sperm and the California seahair eggs. So if beyond a certain level, the cryopreservation is similar, like what aspects did it differ between these two specific species that you worked?

Jack (07:40)
Hydra are pretty cool. are like miniature. Have you guys seen Finding Nemo? Yeah, of course. You know the sea anemone that Nemo lives in? Imagine that sea anemone but like a couple millimeters tall and imagine that there's like hundreds of them on a single microscope slide. Microscope slides like this big, right? That's how big these Hydra actinia are. And so

Collecting sperm from an individual is very difficult because they only produce like a couple of microliters. And so for that process, that's the sperm process and sperm is relatively easy. We had to address the barrier of how do we collect enough material to actually freeze. And so we created a little open hardware device that you can put multiple slides together and when they spawn, you have a nice soup of sperm.

You get a couple of milliliters of sperm instead of one microliter. The hydractinian is the sperm side. Aplasia are interesting because they're simultaneous non-selfing hermaphrodites and they do internal fertilization. So let's unpack that. So simultaneous hermaphrodites mean that they have both male and female reproductive biology at the same time. Non-selfing, they can't take their own sperm and fertilize their own eggs.

and then internal fertilization. they're not like broadcast spawners. They actually, they copulate with each other. They form these chains of these mating chains. You can get like 20 individuals where the first one is the female for the second one, which is the male, who is the female for the third one, who's the male and so on and so forth. For Aplasia, collecting their sperm.

Robin (09:26)
Wow.

Jack (09:31)
And then figuring out how to do internal fertilization was one option. The other option was working with their egg masses. And it looks like a ramen noodle or a big wad of cooked spaghetti. It doesn't taste like spaghetti, but it has the consistency of cooked spaghetti. If you zoom in on a single spaghetti strand, it's not just a bunch of embryos living in there free-floating. There's capsules within the egg strand.

Hundreds of capsules, thousands of capsules within one of these egg masses. And each capsule can hold up to a hundred embryos inside of it. So one egg mass, like the size of a baseball or like a fist, has millions of babies in it, millions, which is all part of their reproductive biology, where you make lots of babies and only like 1 % of them make it to adulthood.

Robin (10:23)
So this egg mask, is that from the entire chain of the sea hairs or is that like from one pair for example?

Jack (10:32)
That's from one individual mixing egg mass. Yeah.

Robin (10:37)
lot of eggs.

Hemdeep (10:39)
I can see how the whole notion of having a camera in the middle of a pond waiting for these things to spawn would not serve you well. You would never know what on earth happened.

Jack (10:52)
We're lucky because Apligia are intertidal. So they live in the zone on the coast where low tide goes out and then high tide comes in. You know, during low tide, you can go out there and watch them. But you know, for like animals that live in the middle of the ocean, what are you going to do? Hope that your boat is in the right place when they're spawning. I mean, there's plenty of stories of animals like great white sharks for the longest time. We didn't know where they gave birth because the ocean is massive and

good luck, you just have to be there at the right time or have the right technology to track them. So we're lucky in that they're in the intertidal and people have a pretty good understanding of what's going on in the intertidal zone.

Robin (11:35)
So these open hardware devices that you're developing, it goes across the entire process. So from collecting the genetic materials to, I guess, loading it or preparing it for cryopreservation, right?

Jack (11:49)
Yeah. So it's really cool. If you look at the evolution of our open hardware technology through time, at first we were creating a device, a single device that would address one step in the crowd preservation process. And now we're introducing a kit concept. you, have either of you gotten like HelloFresh or Blue Chef? You know, they, they delivered food kits to your door. Imagine that for crowd preservation.

Robin (12:15)
I'm aware of what it's yeah

Jack (12:20)
where you get a kit to your door, it has everything you need to cryopreserve your sample. And that's what we're working on right now, creating these, we're calling them capability kits right now. They provide you the capability to do cryopreservation. Our first one will be for axolotl salamanders, I think. But one of the great things about open hardware is that anything we create is pretty generalizable to any species. So we're not just creating a piece of hardware for axolotls and a piece of hardware for frogs and a piece of hardware for...

a please to see hairs, we're creating one piece of hardware that can be used for all of them.

Robin (12:54)
Does the high-writings be tweaked in any way to cater to specific species or exactly the same standardized across all sorts of species?

Jack (13:05)
standardized across, let's say, species.

Robin (13:07)
Okay.

Hemdeep (13:09)
What is the key characteristics that you use to standardize across all these species? Like what is the key characteristic that each of these species have that allow you to sort of use that one template right across that entire range?

Jack (13:24)
There's a couple of ways that you can generalize or standardize by. You can do it by, by germplasm form. So whether you're working with sperm, eggs, embryos, egg masses, tissue, right. You could, you could split by that. There are some devices that in theory could work with any type of germplasm. The other thing that you could split by is the type of cryopreservation that you do. And there's two major types of cryopreservation that people use.

There's a slow crowd preservation that the freezing rates are anywhere from like one degree C per minute to 60. And then there's ultra rapid crowd preservation called vitrification and the freezing rates or the cooling rates for vitrification are a thousands to tens of thousands degrees per minute. So it's very, very fast. And so there's probably some, could split these devices into two categories based on that as well.

There's probably other ways that you could standardize devices by different categories as well.

Hemdeep (14:26)
when you talk about the rapid freezing and those two techniques, I the thought that crossed my mind was, ⁓ there are humans that want to be cryopreserved. And what does that look like? Which one would they do? Wasn't Walt Disney cryopreserved? Or wasn't there someone very famous as cryopreserved?

Jack (14:46)
Possibly. There's a whole field. I'm not sure. I'm not versed in that field. So it's called cryonics, I think. And so there's whole groups of people that are interested in that. But yeah, I don't have much that I can say on that.

Hemdeep (14:49)
benefit. that I do get these moments of just epiphany and I need to sort of sometimes just vocalize it out.

Robin (15:12)
So can you actually explain more about what's exactly is in your cryo kits and like have you already started distributing it to you know other people in the research community?

Jack (15:23)
Yeah, so if you just imagine it's a cardboard box and it has different modules in it and each module addresses a certain set of steps in the crop preservation process. So there's a module for collecting time and temperature data, which is very important for crop preservation that time and temperature data is quality management so that we can make sure that everybody that their samples didn't get too hot.

or that they cooled at the rate that they said that they cooled them at. So many people do crowd preservation, they don't record the cooling rate. And you're like, you just have to trust them that they said, you know, my samples were five centimeters above the liquid nitrogen, but you know, the box size matters and whether there's anything between the liquid nitrogen and the samples, that all matters. And so providing someone with a tool that they can actually send us a piece of data to be paired with the sample.

now makes that sample that much more valuable. So there's a module for time and temperature. There's a module for filling and sealing and labeling French straws. French straws sort of look like coffee stirrers. They were created by the dairy bull industry. A lot of what we do relies on all the technology that's been created by the dairy bull industry. We're not reinventing the wheel. And when we want to scale up, right, if we want to

freeze a hundred straws or a million straws, there's a lot of technology that we can use from that field. There's also a special box that is customized for each species or each group of animals that the kit is for. So one of the animals that we work with right now with the National Institutes of Health is the axolotl or the abyss them of salamanders. You may be familiar with them from how to train your dragon.

the key dragon, I don't remember, toothless. Toothless is based on an axolotl. And now if you go look up axolotl and then go look up a picture of toothless, you'd be like, wow, they are remarkably similar.

Robin (17:24)
His little ears. I don't know what they are. Ears. I'm just calling them that.

Jack (17:30)
And then Minecraft has also like exploded popularity of axolotls. But anyways, I digress. There's a special box in there that would help you collect germplasm from axolotls in a standardized way. There might be a special device that we create for oysters or for salmon that we could package in there. It's a customizable module. And then there's the meat of crop reservation, which is the freezing or the cooling process.

And there's a module that it provides you a device to help you cool your samples again, in a standardized way, in any laboratory, in any field setting. There's a module that let you do that. And then we were really fortunate to find that there is a pretty accessible shipping device. It's called a dry shipper. And the way a dry shipper works is you pour liquid nitrogen into it and it has absorbent material in it.

So it absorbs the liquid nitrogen into that absorbent material. And rather than there being liquid in the bottom of it, it's just stored in there, but it keeps it cold. And you can ship this anywhere in the world. If you don't have access to liquid nitrogen, you can charge it up, ship it somewhere and do your cryopreservation. But they usually cost, I'd say more than $1,500. Again, that can be pricey for some parts of the world and having something that

is cheaper, more accessible to more people is powerful. And so we found a dry shipper that is a couple hundred bucks and we've adapted some of our open hardware technology to be usable with this dry shipper. And so altogether, this kit delivered to your door, the vision for it is that it's going to let you do crowd preservation wherever you are in the world. If you're in a laboratory or in the middle of the jungle somewhere.

Hemdeep (19:23)
is amazing. So now in terms of the timeline when you and your team are hoping to get it out is there for a lot of the other researchers what does that look

Jack (19:34)
We're in the beta testing phase right now. So we've sent some kits out to people that we've collaborated with in the past and we're asking them to break it. We want to know like what doesn't work, what does work. The biggest thing that we've been working on right now is the instruction manual. And I mean, crowd preservation is not like the easiest thing in the world. It seems like the best way to help people understand crowd preservation and to make sure they feel confident.

is to make sure that they understand why they're doing the processes, understand the physics and the chemistry of crowd preservation. And that's really powerful because you are empowering them so that if they run into any problems, they can troubleshoot and make decisions that are smart. And so how do we have a instruction manual that conveys the necessary information in detail that people want or people don't want? That's been one of our biggest pushes right now is trying to get.

some instructions out to these beta testing groups. So timeline, I'm not sure, but if you're interested, let us know. I think we might be working on a, ⁓ like an interest form where we could find out what species people are working with, where you are in the world, what your experience level is so that we can gauge what the need is. And I think in the, in the future, we're not a huge group. There's eight professional.

staff members there. And then at any one time we have like 40 undergraduate student workers that help us on variety, different research, husbandry, maintaining the building tasks. And so we're just not capable of printing hundreds of these kits. And so eventually I think the vision is that it'll be commercialized in some way. We won't be the one sending the kit to people. We'll focus on.

developing the biological background, new devices to go into the kits and have somebody else distribute them to people.

Robin (21:34)
How does this kind of fit in with your open hardware? Because the open hardware is a digital file that they're able to 3D print themselves. So is there kind of like plans to be able to turn it into a kit that you can just fully 3D print everything by themselves?

Jack (21:51)
Yeah. So that is probably one way that we would provide the kit to people. If we go back to the example of the microfluidic devices, we can print the microfluidic devices themselves for people, which would be potentially very difficult. We could send the molds to people and let them mold as many devices as they want. We could send the files to the people and they could just print.

and modify them there. The same thing can be applied to the capability kit and that we can send you a kit and that has everything ready to go. You don't have to do anything. We can send you the files for the kit and you can go print it. Right. So absolutely. Open hardware is very tightly interwound with this kit and there will likely be many different ways that people can access the kits.

Robin (22:51)
As much as myself and Hemdeep want to dive further into the kits you have designed, we're gonna have to end our conversation here today as we've come to a wrap for this episode of Big Ideas of Microscope.

Hemdeep (23:02)
A huge thanks to Jack Koch for joining us and sharing his insight. We explored how genetic banks are vital for preserving sperm, egg, and embryos to support breeding and conservation programs.

Robin (23:14)
Jack also sheds some light on the challenges of preserving aquatic species, especially given their complex reproductive biology. And then we talk about emerging cryopreservation techniques that are shaping the future of genetic preservation.

Hemdeep (23:29)
If you enjoyed today's conversation, make sure to tune in next week when we dive into how Jack and his team are developing open hardware kits to standardize cryopreservation across species. We'll learn how these innovative tools are helping to make cryopreservation more accessible and efficient for researchers worldwide.

Robin (23:48)
Thanks for tuning in to Big Ideas of Microscale. If you enjoyed the episode, make sure to follow us and stay up to date. You can listen on Apple Podcasts and Spotify, or watch the full video on YouTube. You can also follow us for more updates and behind the scenes content on LinkedIn, Instagram, Blue Sky and X. We're Cadworx3D across the board. Let's spell C-A-D-W-O-R-K-S-3D.

show notes, paper references, and bonus resources on today's topic, visit our website, catworks3d.com. That's spelled C-A-D-W-O-R-K-S 3D dot com.

Hemdeep (24:32)
Thank you for tuning in and as always stay curious, keep exploring and never stop asking the big questions that are shaping our world. Whether you're in a lab or on the go or just curious about the future of technology, join us as we continue to dive into big ideas at Microscale.