How did it all start with your company CryoCapCell? 
During my PhD at EMBL Heidelberg, under the supervision of Claude Antony, my project required to develop an innovative method to CLEM Xenopus laevis mitotic spindles using High Pressure Freezing. I invented a manual assembly of flexible parts that somewhat worked from time to time (the so called 'gummy ring' method). I quickly realized that the reproducibility was not sufficient and needed to manufacture a single solid element that would combine the various observations I made with the gummy ring. At the same time in France, my father invented a novel technology to inject microvolumes of melted plastic. Our discussions led to a technological collaboration between our labs and ultimately to the invention of the CryoCapsule.
Finishing my PhD, I got recruited as a post-doc at Institut Curie, under the supervision of Graça Raposo who gave me a wild card to establish CLEM at the IC facility. We figured that in order to commercialize the CryoCapsule, we would need a company. This is how we decided to found CryoCapCell.

 
High-Pressure-Freezing (HPF) is a very specialized, highly complex and resource intensive sample preparation method for EM. A tough one to start a business with, isn’t it?
Indeed, and this wasn't the initial plan. During my PhD, developing the CryoCapsule, I had the chance to meet the engineer in Chief of Abra Fluid, Markus Frei, manufacturer of the HPM010. He offered me his help in finalizing the product. As my post-doc project progressed, we decided to create our own HPM. We naturally turned to Markus to co-design the HPM Live µ. He shared his knowledge in manufacturing high quality HPMs, and we designed the automated transfer system, invented a novel high speed pressurization technology (worldwide patent) and modernized the whole with GUI and high-end automation. At that time, we were only the three founders of CryoCapCell: me, the biologist, my father, the mechanical engineer, and Frederic Eyraud, the programmer. Martin Belle joined us during that creation, aiming to become the future CEO of the company. This is how it all started. Five highly determined minds. None of us expected it would be so challenging and so rewarding to make my scientific dream, a routine reality.
 

What are the main advances of HPF vitrification versus conventional RT resin embedding of (biological) samples?
Vitrification by High Pressure Freezing takes advantage of the water phase diagram to reduce natural crystallization of water. The water remains in an amorphous state, where water molecules stay in the disordered orientation they have in liquid state. After vitrification, it is possible to replace the solid water by a solvent at negative temperatures: this so-called freeze substitution consists of diluting solid water (at -90°C) in a near solid solvent (acetone freezes at -96°C). This erosion is taking place slowly and limits collapses of protein structures and subsequent remodeling. To complete the process, the solvent comes loaded with some fixatives and contrasting agents that are inert at -90°C. Once the substitution is patiently done (a couple of hours in general), the temperature is slowly raised (typically -45°C). There, the fixatives and the contrasting agents become active and do their jobs of stabilizing and coloring the membranes and proteins. I like using the image of a Trojan horse that is already in place when the remaining water molecules are still asleep. When you finally reach embedding temperatures, everything is already fixed and physical distortions are contained.

In contrast, RT embedding is taking place with freely moving water molecules (20°C or 4°C at best). When dehydration (successive ethanol bath) is taking place, although fixation is already achieved, we exchange freely moving water molecules by freely moving solvent molecules. This unavoidably causes conformational modifications. The best intermediate solution is the Progressive Lowering Temperature strategy. This consists of passing below the freezing point while exchanging the water by the solvent to enter in a more gentle exchange of reduced dynamic water versus liquid solvent. Morphologies are better preserved than in conventional RT embedding, although not as nicely as with FS.
 

How many HPF systems do you have installed on how many continents until now?
Until now, we have installed 20 instruments worldwide. 13 in Europe, 3 in Asia, 4 in the USA. And we work hard to have more!
 

And now you have developed and launched a new Freeze Substitution System. How did that come?
As often with our technologies at CryoCapCell. We invent what we need. We are hosted in an INSERM research infrastructure of Université Paris Saclay. In exchange for benefiting the research infrastructure, we provide free access to our technologies to the building members. As we conduct our own research, we require all the equipment to run RT electron microscopy analysis. The freeze-substitution apparatus we had at hand were giving us quite some difficulties: liquid nitrogen consumption, safety issues with fume extraction limitations, no agitation, impractical consumables. We therefore started to look into developing our own equipment. Funny enough, one day, Roger Wepf came to visit us, and he mentioned he had developed his own FS machine, LN2 free. We shared ideas and figured we came to the same technological analysis. He confirmed we were in the right direction, and he kindly shared his own drawings to help us to progress faster.
 

What are the main benefits of your FS unit?
CryoCapCell has been in the business of freeze-substitution since its creation (including the invention of the R221 electro-conductive embedding resin). I kept an eye on all recent developments, including Quick Freeze-Substitution by Kent McDonald and Rick Webb. The idea of agitating the sample during freeze-substitution isn't so new, but their work literally shook the field in 2011.

This is why it was one of the key element that our machine was meant to support on its original design. Liquid Nitrogen is also becoming a major health and safety concern. We had to get rid of it. Last but not least, highly toxic fumes are one real problem in FS. Normal handling of toxic chemicals must be done under a fume hood. These three main constraints are the three pillars of our FS Unit: bench top to fit a fume hood, liquid nitrogen free to run extreme long protocols without refilling, compatible with most conventional lab supplies (culture well plates), and capable of agitation to run Quick Freeze-Substitution protocols with high reproducibility across diverse laboratories.
 

How do you organize technical service for your systems?
The HPM Live µ are massive and heavy equipments. Maintenance must be conducted on customer's site. To optimize our maintenance, we have remote control of each apparatus, and we run constant monitoring of each individual equipment. If we notice any change in the HPF curves, we can diagnose the issue and take actions accordingly. This reduces downtime and limits our on-site intervention. When possible, we train one local internal expert to run primary maintenance. In general, our customers seem happy about this strategy, as they feel we share trust in our system.

The FS unit is a bit different. It's a bench-top equipment, is exposed to toxic chemicals. To simplify the maintenance, we propose direct shipping back to our factory, where we can completely dismantle the system in full safety for our engineers. This accelerates also the maintenance as we do not have to organize on-site travel.
Finally, we are creating a dedicated 'Discussion group' with our customers to facilitate the live maintenance or operation of each of our instruments. This also reinforces the mutual trust for each of our customers and reduces any potential down time.
 

Well done. Good success!
Thanks!