Germplasm Repositories for Aquatic Species

Robin: …this version of the kits even encourages users to 3D print components themselves, allowing for further customization, which could revolutionize genetic preservation in aquatic species. Jack will also share how 3D printing plays a crucial role in creating cost-effective devices, like sperm-counting chambers, and tools that streamline cryopreservation. Let’s jump right in.

Hemdeep: These kits and this open hardware include a range of modules and devices. I read up on a couple: the strand cryopreservation shuttle system, and the single-sperm counting chamber. How did you develop those, from brainstorming through to something close to a commercial object?

Jack: Let’s start with the single-piece sperm counting chamber. That came from a need to give people a way to count sperm and determine concentration; knowing concentration and motility is important quality information. There are commercial options: a hemocytometer, developed for counting blood cells, and a Makler chamber, developed specifically for counting sperm; those cost around $300-$700 each. If we can make something 3D-printed that costs ten or twenty cents, that someone can print anywhere in the world and use to count sperm repeatably and consistently across labs, that’s wonderful. That’s where the single-piece sperm counting chamber came from: the need for something low-cost and accessible that could be packaged into a kit like this.

The strand cryopreservation shuttle system came from a need we recognized while developing cryopreservation processes for Aplysia. When we work with outside groups on that project, we work closely with the National Aplysia Resource, based at the University of Miami. Sometimes we (or others) build a device we think a community will find useful, hand it over, and hear back, “We don’t need this,” or “This doesn’t fit our workflow,” or “This would cost us another person.” So we work closely with these national centers through user-centered design: building relationships, asking questions, visiting facilities, making observations, and figuring out what hardware would genuinely help them, especially as they adopt cryopreservation.

The strand cryopreservation shuttle system is a perfect example. We had many meetings with the National Aplysia Resource team, visited their facilities, made observations, and brought that back to the AGGRC, keeping their needs and environment in mind while developing cryopreservation processes for Aplysia.

To give you a sense of how difficult it is to work with Aplysia egg masses: imagine a coffee straw, and a small piece of wet noodle you need to package into it, over and over, repeatably.

Robin: How many eggs, or noodles, do you need per straw?

Jack: Say you want one one-centimeter piece of noodle per straw, a hundred straws, and ten minutes to do it: that’s basically not possible. And a single one-centimeter piece of noodle in a twenty-centimeter straw is a huge waste of space, so wouldn’t it be better to fit multiple noodles per straw? But ask someone to package three noodles into a hundred straws in ten minutes, and they’d say a hundred wasn’t possible in the first place: now you want three hundred?

So we built a device where you push down and load ten egg-strand pieces at a time into cassettes, or “shuttles.” I can take those ten and put them back in seawater, or load up a hundred strands ready to go, without the ten-minute clock running yet. Once I’ve packaged a hundred strands, I arrange my straws in the same spacing as the shuttles, and when the timer starts, I just snap ten in at a time, fitting three noodles per straw, with packaging efficiency going way up. That’s the basis of the system: efficient sample handling and packaging.

We also developed a way to seal the straws. Traditionally you’d use an ultrasonic sealer, using high-frequency vibration to melt the straw shut without heat, but those cost around $10,000. Without one, people might heat a pair of tweezers over a flame instead, but heat isn’t great for sperm. So we made 3D-printed sealing balls that work with this system: ten straws, spaced appropriately, ten sealing balls, sealing ten at a time and rapidly cutting the time it takes to fill, seal, and prepare straws for cryopreservation. All printed on a $300 consumer-scale printer with basic resin; it opens the door to so many possibilities, not just for Aplysia, but for other animals around the world.

Robin: For these devices, did you go straight to 3D printing, or try other fabrication methods first?

Jack: While I’ve been there, we’ve only used 3D printing. It came out of my brain one night: it goes back to the idea that the limit of 3D printing is your imagination. You can print pretty much whatever you want. It’s incredible.

Robin: Even so, 3D printing isn’t at its peak yet. Are there still limitations for your research? What are the main ones?

Jack: For cryopreservation, reducing the thermal mass around your sample matters a lot. If a printer can only print something one millimeter wide, that millimeter next to your sample holds a certain amount of heat, and if there were a way to cut that in half while keeping the same structural integrity, that’d be great. That’s a lot of what we run into: reducing material while maintaining structural integrity. That’s when we bring in engineers: I’m not one, but they’ll say, “Add some cross-links here, and you get the rigidity back while reducing material.” Metal and polymer 3D printing are advancing rapidly, especially at smaller scales: metal is great because it’s a heat sink, pulling heat away from a sample much faster than resin. There’s also work incorporating materials like graphene, mixing resins to improve cooling speed or structural integrity.

Then there’s toxicity: of resin, or any material, even the plastics used in fused filament fabrication. You just have to test it. A lot of the time your sample is only in contact with a device for a couple of seconds, or you’re using a sub-sample that doesn’t need to remain viable afterward.

Robin: You mentioned the AGGRC has a lot of 3D printers, what does that look like, one giant room?

Jack: I think we just broke a hundred printers. You’ll have to come down to Baton Rouge sometime to see it. We have a printer farm: one big room with maybe fifty printers on shelves. But we have printers in other rooms too; a couple in my own office. We have a training and prototyping space with about a dozen printers, where people learn to break printers, fix them, do their first prints, learn CAD and slicing. Then there’s our industrial space at the other end of the building: a room with six or eight printers, and another with a couple of resin printers and a polymer printer. They’re spread across rooms based on the printer’s level and purpose.

Robin: Tell us more about the AGGRC’s overall mission.

Jack: As I mentioned, we help people and communities around the world form germplasm repositories: thinking through the cryopreservation process, technology development, bringing in other users, deciding where and how to store and track samples, and what equipment and staffing is needed, specifically for aquatic species.

Robin: You mentioned four pillars of the center; can you give an example?

Jack: We support our mission through four major programs. First, basic biological research: studying animals’ reproductive biology, and how different chemicals interact with their germplasm to prepare it for cryopreservation. Second, technology development, split into open hardware and industrial processing. Third, outreach: talking to people across industries, universities, and education levels, both to sharpen our own thinking and to let people know these technologies exist. Fourth, industrial-scale processing: we have equipment that could double or triple the total global aquatic-species germplasm samples in a couple of weeks, given enough sperm to freeze. We use high-level equipment for certain projects and train people on it too, since scaling from ten samples to a hundred, or a hundred to a million, requires different levels of technology, not just more people.

Hemdeep: The center has clearly grown significantly. How did that growth track with the adoption of 3D printing, hand in hand, or was it a necessity? Would the center have reached its goals anyway without it?

Jack: The center would definitely have continued without 3D printing, but growth has been much more rapid, letting us reach more diverse communities in more places. There are commercial cryopreservation technologies we could have used instead. When 3D printers became more accessible (let’s say the center’s first printer cost around a thousand dollars, a big deal at the time since they used to cost much more), they printed something, ran it down the hall, and dunked it in liquid nitrogen to see if it would still flex. It did. Normally you’d expect something dunked in liquid nitrogen to shatter. That was a huge realization at the time: that we could make devices safe to bring near or into contact with liquid nitrogen, bring them to more people, and drive standardization and access; that’s really helped direct our mission ever since.

Robin: Does the community you’re reaching already have 3D printing experience, or is it something you teach them?

Jack: It’s a mix: some know about 3D printing but have never done it, some have printed a thing or two, some have never seen a printer before. But more local libraries have 3D printers now, and universities (especially those with engineering programs) often have 3D printing facilities their students learn on. There are also more student maker clubs with printers. We don’t necessarily teach people 3D printing directly; we try to create an environment where they can succeed and learn on their own, with support available. We can guide people, but we won’t hand them a fixed recipe, because that doesn’t work every time: 3D printing needs different parameters for different things. We also can’t help everyone directly, so we rely on a network approach: we help one person, they help two more, and the shared knowledge spreads.

Robin: I imagine that’s part of why it’s important for your team to push toward commercial scale rather than staying with industrial printers, I think that’s what your latest paper was about. What devices did you test between the two printer types, and what were the results?

Jack: That goes back to the single-piece sperm counting chamber. One reason devices like hemocytometers and Makler chambers are expensive is that they’re highly precise: the volume under the cover slide is exact, which comes down to manufacturing. The coverslip on a hemocytometer is specially weighted so the sample volume underneath is known, allowing for accurate concentration counts.

Trying to 3D print something like that requires hitting a tolerance level that guarantees the volume under the cover slide falls within a known range, which can be difficult on a consumer printer. We used a CADWorks 3D printer, purchased through a seed grant from the Louisiana State University Agricultural Center, to see what consumer-level versus industrial/commercial-level printers are each capable of. We didn’t want to directly compare them, since that would be unfair: we wanted to know where consumer scale covers one range of sizes, commercial scale covers another, and where they overlap.

Not all the sperm we work with are tiny: Ambystoma sperm are absolutely massive; we call them “aircraft carriers,” huge compared to human or even frog sperm. Printing a device for that scale is relatively easy on a consumer printer. For zebrafish, whose sperm are only about two microns, you’d be hard-pressed to use a consumer-scale printer to build an accurate counting device: the gap has to be small enough that sperm don’t stack on top of each other. That’s where a commercial-scale printer might be needed. So the purpose of that paper was finding where consumer and commercial scales overlap, so we can make informed decisions when designing new devices: whether to print something accessible on a consumer printer, or whether a device needs commercial-scale precision, either printed directly for people or as molds they can pour themselves.

Hemdeep: Given your experience across such a range of printers, what do you see for the future, for yourself, your team, and the repository?

Jack: Standardized devices are one place we’re really pushing, along with making sure the communities using them feel served by them. All the printers in our building are open-material printers, meaning we can use any resin on the market, though we might need to dial in settings, but that goes back to our open-hardware philosophy: we don’t want to be locked into one ecosystem. I think it’d be good to see more companies allow that kind of tweaking, even at an industry level. I understand companies need to make money, and closed-material-ecosystem printers reflect real investment in making things work exactly as expected, but there are also plenty of open-material printers, in both FFF and resin, that work well right out of the box, because they’re workhorses.

Hemdeep: What’s next for the germplasm repository?

Jack: Our mission won’t change much; the number of communities and species out there is so large we could spend decades on it. Where we may change is in how quickly and broadly we enable other communities to build their own processes and repositories for their own species: a more distributed workflow, where we help accelerate the number of groups doing this work, rather than being the only ones. That means more outreach: the vision, really, is a future where students at every level, in any field, know how to 3D print and are familiar with CAD, because it’s becoming a basic life skill.

Robin: Where’s your home base for this wider community, should researchers go through your website, or is there a forum?

Jack: aggrc.com is the best place to start. We also have a community forum: happy to share an invite link for anyone interested in connecting with us and others in the community. Every new open-hardware device we publish gets posted there. We’re also on LinkedIn, Facebook, X, Bluesky, and Instagram.

Hemdeep: That was a fantastic conversation, Jack. We’ll post all the links, articles, and images we discussed in the show notes. Would you like to give a shout-out to everyone who’s made your work possible?

Jack: Thanks, Hemdeep. Thank you to the Louisiana State University Agricultural Center for seed funding relevant to a lot of what we discussed. A big shout-out to the National Institutes of Health’s Office of Research Infrastructure Programs, who support our biomedical work and help national centers across the U.S., including the National Aplysia Resource, the National Xenopus Resource Center, the Ambystoma Genetic Stock Center, the Xiphophorus Genetic Stock Center, and the Zebrafish International Resource Center. Thanks also to our collaborators at Louisiana State University, including Dr. Monroe’s group, collaborators around the world, and funding sources including the USDA and the National Science Foundation. We really appreciate everything those groups do; we love helping people around the world protect the genetic resources of aquatic species.

Hemdeep: Amazing, we’ll add links to all of those teams as well. I’m so glad we had this chance to speak. Thank you very much.

Jack: Thanks, Hemdeep. Thanks, Robin.

Robin: And with that, that’s a wrap on our series with Jack Koch here on Big Ideas in Microscale. Over the past few episodes, we’ve explored the world of genetic preservation and cryobiology research: from Jack’s early curiosity about marine life to the groundbreaking work he’s doing at the AGGRC.

Hemdeep: We discussed the growing need for germplasm repositories to support breeding and conservation efforts for aquatic species, the challenges posed by complex reproductive biology, and emerging cryopreservation techniques helping to overcome those hurdles.

Robin: We also dove into how 3D printing and open hardware are making cryopreservation tools more accessible, affordable, and customizable for researchers around the world. Jack and the AGGRC’s work is an exciting glimpse into how 3D printing is driving solutions to some of the most pressing challenges in conservation and genetic diversity.

Hemdeep: We hope you’ve enjoyed this deep dive into the vital work being done to protect aquatic species. Our next guests are Assistant Professor Veronica Mendez and master’s student Noah Franco, from the University of Waterloo.

Robin: This episode is particularly exciting for us, since they’ve spent the past several months limit-testing and validating CADWorks 3D’s new 3D printing material, the Cyto-clear photopolymer resin. Here’s a short preview of that conversation.

Coming Up Next: A Preview

Hemdeep: So there was a material my brother was working on: he said, “This has amazing properties, but you know how we have to clean our models with IPA?” We were at a large workbench right beside a wall, and there was a very thin, unnoticed layer of IPA on the bench. My brother wanted to light a model, just to demonstrate something, not realizing a drop of IPA had landed on the bench. We didn’t notice anything until a blue flame shot straight up the wall, and we were just like…

Robin: Was this at your mother’s house, or the office?

Hemdeep: No, this was at the office. At my mother’s house, we burned her kitchen.

Robin: Thanks for tuning in to Big Ideas in Microscale. If you enjoyed this episode, follow us to stay up to date. You can listen on Apple Podcasts and Spotify, or watch the full video on YouTube. Follow us for updates and behind-the-scenes content on LinkedIn, Instagram, Bluesky, and X: we’re CADWorks 3D across the board.

For show notes, paper references, and bonus resources, visit our website: cadworks3d.com

Hemdeep: Thank you for tuning in, and as always, stay curious, keep exploring, and never stop asking the big questions shaping our world.