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Good afternoon, everyone.
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So I'm going to tell you everything about what we do.
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So we are a small company based in Lyon in France and we develop disruptive technology to master our cell biology.
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That's kind of what encompass all our activities.
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We started a few years ago with the first device called CELLEN ONE, it's widely used in the field of single cell isolation and in particular single cell proteomics for sample preparation.
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And today what I'm going to focus on is on our latest device called SPHERO ONE which is used for sorting and isolation and dispensing of 3D cell models.
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So you can put any type of steroids organoid or tomorrow it's with and I'm going to describe to you how this works.
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So this is a very general introduction.
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I will not teach you anything there.
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I think you're all specialists in this field.
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So it's not obviously difficult to manipulate those 3D models.
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Imaging can be tricky.
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The throughputs, depending on the method that you're using can be challenging as well.
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And what's makes this image, the implementation of those technology, more particularly difficulties the heterogeneity that you're obtaining after generating those With the silent one, what we hope to answer is the ability to select specifics of population in your 3D model so that you can improve the reproducibility of your assets.
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How does it work?
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So we have a glass capillary which we call a nano dispense capillary with a different size of opening and they we generate tiny droplets in the nanolitre range which are very reproducible and basically.
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So if you are just to dispense your spheroids or organoid with that or you see whatever you will get at the end would be following personal distribution.
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So you would have like different numbers in the different well.
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But what we do is we add a bit of intelligence to it if you want.
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We developed an image-based technology.
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So we're going to image the tip of the capillary, and we segment the tip of the capillary into two zones, the ejection zone, which is the equivalent to the volume of one droplet.
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And what we have above is a sedimentation zone just to account for the fact that this 3D model will sediment while being idle in the in the capillary.
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So after doing each droplet, what we do is we image the tip of the dispenser whenever we detect a particle that fits the criteria that you have defined, then the capillary will move to your plate and dispense the droplets containing your 3D, your 3D model.
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Obviously not all of the droplet will contain a single particle.
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Many droplets will be empty.
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We discard those.
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Some droplet will have two spheroids coming together for example.
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We don't want that typically, so we discard that as well.
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And also the algorithm will discard whenever we have a particle in the sedimentation zone as well, just to make sure that whatever it is isolated is a droplet containing a single particle.
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The imaging that we developed in order to achieve this, we use a dark field imaging because so if you just use a bright field image on a cylindrical capillaries, you get a lot of edge effect.
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Sorry about that.
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And so the best modality that we could develop in order to facilitate this imaging is using dark field.
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So basically what we collect is a reflected light from your object.
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What is this useful?
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Well, you can start from a very heterogeneous population of spheroid organoids, and you just tell our device, I want to select all the organoids around 200 Micron, and you will just exclude everything and isolate 1 is the one that you want.
3:45
You can do like selection - I just wanted a large one or the small one for example.
3:52
So yeah, you can see the starting population in blue, isolation of just the largest one and then isolation of the smallest thyroids.
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In this experiment, we collaborated with a company called Biopredic.
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They developed aparg cells which are very widely used liver epatoblast model used in the pharmaceutical industry for metabolic studies.
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And with them basically we developed a method for high support production of those ferroids.
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So we used micro cavity plates like the L Plaza or the Google Meyer, generated hundreds and thousands of ferroids and then loaded that on our device from the L Plaza plates.
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You do get, you're supposed to get something very homogeneous.
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In practise it's not so bad, but you still have like variation in size from about 150 to 350.
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And basically, we wanted to have something very reproducible afterwards.
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So we just selected the one that were between 200 and 250.
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Then loaded straightforward plate with one ferret in each well, and then you could undertake a number of different characterisation working with bio predicts we verify like the functionality of those ferrets.
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Did some aluminium secretion ATP assays and the results were very similar to what they get on their 3D models.
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Typically you can control biomass.
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Obviously you can put one, but if you want to put more than one, you can put three, you can put five.
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It's really down to you.
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The dispenser is being mounted on a very precise axis system.
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We can dispense those in tiny features so we can go into microwells.
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So in this example at the top you have the one sphere weights in a microwave of about 400 Micron in diameter.
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At the bottom, we've been working with different company like aim bio tech from Singapore or Protein Fluidics in the US which have like dedicated plates in order to bring flows to those to those ferroids.
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We could load Mimetas plates if Mimetas want to try, you can bring different ferroids to do some co-culture.
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And another example here you can see we've arrayed actually organoids onto a slide in order to have them really well positioned and all in the same plane to facilitate imaging and image at the bottom right.
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We are working with a group in Lyon doing some tissue engineering application.
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So that's pretty challenging.
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They have developed an Electro spin scaffold, so that's what you see here.
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So far, we've just done the proof of principle with the HEK.
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The next step is for them to bring their MSC derived chondrocyte spheroids.
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We can dispense so spirit suspended in fairly non viscous solution, but we can also handle hydrogel.
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So we've done some experiment with UPM with developing the codex.
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So it's a nanocellulose gel.
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There we demonstrated that we can just use the device for preloading the place the well with the codex.
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Then we embed one spheroids into the codex and then add another layer of codex on top.
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Then we can add the media.
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Many of you are going to be working with metric gel.
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So typically the question I get at the end is can I dispense my metric gel?
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No, you cannot dispense your metric gel.
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I'm sorry.
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But we do have a work around.
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So you cannot dispense it because it's too viscous and I just make a mess.
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But in our device, we control the temperature of the plate in which you're working.
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So you just preload your material in your plates, we keep it at 4°, then you dispense your droplet containing the organoids and you will end up nicely embedded in your material.
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And then you continue your experiment.
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Some examples of organoids that we've been successfully isolating.
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So some longer organoids with a group in Lyon and some kidney organoids with a group in Roma in Italy.
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These are an example of stuff that we've handled.
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We've only released the device about a year ago.
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So we haven't tried everything, but we are happy if you send a sample, we are happy to try.
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We are pretty confident we can do things, pretty good things with it.
8:45
So in summary, so we provide like accurate isolation, you select the size of the particle you want, we isolate it one at a time or multiple, one after the other.
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So to have defined biomass, big difference compared to your ULA plate, for example.
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So typically in ULA not all of the cells are going to aggregate.
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So you will end up with lots of debris audience, which can be a problem for your imaging or your asset depending on what you're doing.
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In our case, what you would do is you would prepare them in bulk, you can philtre them easily and then load them in the device.
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When they are isolated, they are in a nanolitre droplet, so there is hardly anything else coming with it.
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So you end up with a very clean well with just your nice, spirited organoid at the bottom of the well.
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And we've also started doing some experiment where we try to select according to the functionality of the organoids.
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So that's the image you have at the bottom.
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If you're interested in our technology, we have a small booth, so don't hesitate to come and talk to my colleagues.
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It's Alice and Gemma on the booth.
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And if you want more information, contact us.
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You have my personal address or contact at cellenion.com.
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Thank you very much if you have any questions.
10:04
Thank you so much.
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Are there any questions from the audience?
10:13
A question that you probably often get, is there an upper limit on the size?
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Yes, absolutely.
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So currently 100 to 600 Micron is our sweet spot.
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We are working to make it larger, but for the moment it's 100 to 600.
10:28
Thank you.
10:35
So can you share a little bit about what the, is it based on light?
10:41
Because it seems like it doesn't matter what cell types there are.
10:44
So is it based on purely bright field microscopy?
10:48
And so we use this dark field imaging, but it's just in order to avoid.
10:55
So when we did, we initially tried with brightfield because we have another device called cell and one in that device, we use brightfield imaging to get the image of the single cell.
11:03
But we are working in this much smaller capillary.
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So we get less of the edge effect.
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When we tried doing the same thing with larger capillaries in the hundreds of Microns, what you are getting is like a big shadow on the site which was preventing the detection of the steroids that were travelling on the side of the capillary, inside the capillary.
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But on the side by using dark fields we don't get any of these edge effects.
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So then you get a nice detection throughout the capillary and yeah, doesn't really matter what cell you put inside.
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As for us, it's an object that we detect and isolate and I guess I can visit your boot later, but so you can buy this whole setup and place it.
11:50
And so what are this like a size requirement?
11:53
Is it a standard like tabletop?
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So you, the American will say it's the size of a European fridge.
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It's about, it's about this.
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It's a bit light higher.
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It's not a bench top system.
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It's a standalone system, right?
12:07
And temperature controlled and all inside, yes.