0:00
To give the shape of tiny U bottom shape micro welds.
0:03
And here you have a transversal view where you can see how this hydrogel is really patterned with many tiny holes.
0:10
And then the second features is the pipetting port.
0:13
So in the on the right of each of the well, we have this inlet, which is called the pipetting port.
0:19
And it's really allowing to change the media or to do the treatment without disturbing the tissues because we're just entering here on the side.
0:28
Now this is just to show you a bit how it works.
0:31
It's really easy.
0:34
So first we come to see it in the main chamber, and we usually recommend 50 micro.
0:40
Then we just let them sediment.
0:42
So the cells are really falling into the micro cavities.
0:44
And then we can add the extra media and in at this point we suggest to add a bit of ECM.
0:52
So for example, a 2% is what we recommend and here is just a brief movie showing up for 48 hours time lapse.
1:02
So you see the cells are slowly sedimenting and this we don't need to centrifuge is just done by gravity and then they're coming together and aggregating in tiny macrotitius.
1:14
Then just to keep our culture, we can just come and pipetting port, remove the media and add on the side the fresh one.
1:24
And this is just an example of mouse intestinal organoids at day five in the culture.
1:29
And we really see all the microtissues here and the pipetting port on this side again.
1:36
Now here is just a comparison, Grid 3D and ECM in the bottom.
1:42
And thanks to those two graphics, we see how they are more closely distributed, and they present a more mature phenotype in the case of Grid 3D.
1:52
And this was analysed with In Carta .
1:56
Now just to show you a bit how we compare with other technologies for a micro well and 3D cell culture.
2:04
This is an example of the study done by Khoei in 2023 and he tested the different plates.
2:10
So we have our plate that we agree 3D and then a comparison with the plate AggreWell in this case.
2:15
And he said that actually the grip with the hydrogel micro well plates have the best optical properties.
2:20
And then here we see a bit the comparison with the imaging.
2:27
Now this is just to show how versatile is our platform.
2:30
So we can really go from like the full plate or only some columns for custom plates.
2:37
We have different sizes of micro welds starting from a really tiny one and going to a really big one depending on application.
2:44
And then we can do either clear or black walls and we have the two different types of bottoms, so the plastic or the imaging depending on the application.
2:54
And here you see the imaging button is an IBIDI polymer and it's really a thin glass like layer allowing for good imaging.
3:05
Then those are just some compelling examples of what we can do in our play.
3:09
So this is again the mouse mole intestine in a 400 microwave plates.
3:14
Then we have a human reaction organoids in a 500, and finally a human blood barrier spheroids in the 600.
3:22
And as you see here, we have more of course, microwells and then here it's going down to less and bigger ones.
3:29
And now, yeah, I leave the stage to Maria, and we continue.
3:32
Yes.
3:33
So the plate can integrate with diverse microscopy solutions, a full panel of 3D assays and many automation solutions.
3:41
And I'll just briefly run through those and how we can develop different assays using those technologies.
3:47
So starting from the imaging, here is an example of cells aggregating in the micro cavities over time.
3:57
These were GFP-HEK293, which could then be fixed and cleared.
4:02
And these are the quality of the images that we're getting once they are cleared.
4:07
So this is stained and imaged on plate with, with DAPI.
4:13
Then we would go here to understand how we're basically going through the different layers of the micro tissue, and we can actually manage to image through the entire micro tissue.
4:25
And so on the that you have the uncleared spheroid, and, on the right, you have the cleared spheroid, and you actually see around a distribution of the of the nuclei, meaning that you can see the bottom just as well as the top of the spheroids.
4:39
These I remind you that these directly on the imaging bottom grade 3D plates, we can also do imaging of organoids.
4:46
So here we're doing the nuclei segmentation of an organoid stained on the plate.
4:53
In this case, it was not cleared and after the segmentation of the nuclei, we can do many interesting analyses.
5:00
So for example, here we focused on two, it was on the left the epithelium thickness and on the right the lumen definition, where we can distinguish the central lumen and the sides lumen, side lumens that are not necessarily interconnected.
5:14
This was all done with the CQ1 microscope from Yokogawa.
5:19
Then going back to how it integrates, we also can integrate with a wide range of robots because it's essentially a 96 well played format that we've just modified to have the pipette import.
5:29
And so here we just have two examples of the Hamilton-STARlet and the Beckman Coulter- Biomek i7.
5:34
Some of our customers have smaller solutions like the Via Flow from Integra and some have bigger solutions like the Tecan Liquid Handler.
5:42
It's really up to you what, what kind of systems you choose to work with, but they're all going to work with the with the plate as long as they're compatible with standard plates.
5:54
Then in terms of 3D assays, we actually have a very nice collaboration ongoing with Promega who are also here on the at the exhibition hall.
6:03
And so here we just developed the 3D cell-based assay workflow, trying to Multiplex as many assays as we could to understand the dynamics of our system.
6:12
So we start seeding the cells and then we're going to do an LDH assay from sampling from the media cell Tox screen to understand the cytotoxicity.
6:21
And then at the very end, cytochrome activity after treatment with certain compounds and the viability assays and then read it with the Tecan Spark Cyto.
6:31
And so yeah, it was these are just the four different assays that we did.
6:36
And so what's actually quite interesting, yes, there you go.
6:40
So these are just the same amount of cells.
6:43
So these were 7500 cells either in degree 3D or in the ultra-low attachment plates.
6:49
So you see at the bottom there's one single spheroid of 7500, whereas at the top we have 55 different spheroids of around 160 cells, resulting in the seven point 5K in total.
7:03
And so then we can actually do comparisons.
7:04
So for example, in terms of viability, the viability in the case of grid 3D was higher than the one red in dual A.
7:11
And as I said before, this is from the same amount of cells.
7:14
So meaning it's we're obtaining higher viability values, and I'll get there in a second as to why this is happening.
7:24
Usually these assays are developed for cells in 2D, right?
7:28
So you can assume everything is exposed to the assay and all of the cells are going to basically be assayed in 3D.
7:36
This is not the case because you have a gradient from the outside towards the inner core and there and, and here in this case, we have a gradient either in each individual microtissue in 3D or in the one single microtissue in ula.
7:50
Now the assay is going to happen at the surface of the spheroids, but because we have so many microtissue, we're actually increasing the surface that is exposed to the assays and by a factor of 11.
8:01
So the surface exposed to the assay on grade 3D is 11 times higher than the one in ULA and actually then in the case of static globe, because it's like it's lytic, we don't see an 11 fold difference.
8:13
But remember, this number here, we're just looking at the inhibitors of CYP3A4 and CYP1A2.
8:21
And what I want you to look at is on the left.
8:24
Actually, I'm sorry, I have the point.
8:26
So here this is the basal activity Ingrid 3D of the same cells as the basal activity here in the in the ultra-low attachment plates and there and then the inhibition of the activity.
8:38
And we see that this is 11 folds what we see in the ultra-low attachment plates, which if you recall from before is the surface difference, meaning that this is really we think that this is due to that the way the assay is working and how that is dependent on surface.
8:52
And in this case, we can increase the surface that is that that that can do the assay then.
9:00
Yeah, in terms of models, we have many models available, which you can all find in our website in different publications.
9:07
I just want to quickly touch on a couple of key models for our customers.
9:12
The first one would be our first publication in Nature Biomedical Engineering on the intestinal and colorectal cancer organoids.
9:21
So here basically we just present the workflow on how the plates work.
9:26
We also explain how we're how we're manufacturing these plates.
9:29
And then what we do is a proof-of-concept screening on colorectal cancer spheroids and patient derived colorectal cancer organoids.
9:37
And we actually see there's some hits that appear in the organised that we could not see in the spheroids.
9:44
And when we do the RNA analysis after so bulk RNA sequencing, we understood that it's actually related to how to the nature of the organoids.
9:56
And so the organised recapitulating the response that we cannot necessarily see in the in the ACT 1116 spheroids because of their different, different phenotype and transcriptomic profile.
10:11
Now here this is another key application of our plates, which is the co-culture with immune cells.
10:17
We've seen a couple of talks already today on the cell therapies.
10:23
Usually if you want to do that with organise, but you add your T cells on top of the Dome and then you need to wait for them to infiltrate.
10:30
Then if you do that in suspension culture, but as Michelle was saying before, you have one single micro tissue.
10:35
And so in grid 3D you can actually have multiple micro tissues and then a direct contact of the immune cells with the organise.
10:41
And this is basically how one of these wells would look like.
10:44
So here in green we have the tumour infiltrating lymphocytes and in this case it's a colorectal cancer tumoroid.
10:51
So it's both derived, so it's autologous and we have then that the interaction between the two cell types in each of the microwells.
10:59
And we're also then, of course, when analysing, we can increase the, the throughput.
11:03
And this is what Luigi from AstraZeneca was saying before about using our plates, that we can really increase the N in our experiments compared to, you know, just looking at the full well as an assay.
11:16
Here is another example.
11:17
Just I spoke before about the Dal stem cell derived organoids.
11:21
These also work for IPS derived organoids.
11:24
So this is the case of liver organoids.
11:27
So if you're interested, you can go through the details, but this is a collaboration with the lab of David Haye in Edinburgh.
11:33
And this would be a full plate of IPS derived liver spirits.
11:38
And then finally, this is a publication from a couple years ago that we did with Roche.
11:43
There was a collaboration with them.
11:44
They wanted to scale up an already existing model.
11:48
And so this was basically a broad blood brain barrier model.
11:53
So we had astrocytes, pericytes and endothelial cells all co-cultured in this self-assembled.
11:58
So we don't need to sequentially add them.
12:02
We just add them all together and they establish a cord and then the surrounding as they would in the, in the blood brain barrier in our own body, but in the inverted way.
12:14
And then basically they use this platform to understand the infiltration of bispecific antibodies in their portfolio.
12:21
And this is a model that has been established now widely at broach and at their partner sites.
12:29
And yes, so I hope that I could convey to you that actually the challenge that we what that that you guys mentioned was key to you was, was reproducibility.
12:37
Here we obtain homogeneous organoids and spheroids that are all located in one plane.
12:42
So we can image them very well.
12:44
They are traceable.
12:44
So we can focus on a single micro tissue.
12:46
And then over time, we can focus on a on on single organised or at the entire well, do immunostainings and understand, you know, the structure and phenotype of our organised.
13:00
And then yeah, this is a high throughput platform because we have many micro welds per well.
13:05
So we can really increase the end of our of our experiments.
13:08
And so our statistics guys will be happy.
13:12
And then it's an automatable friendly platform.
13:16
So here in this case is the via flow.
13:17
That's what the University of Edinburgh was using.
13:20
But as I told you, there's also the bigger robots that are compatible.
13:23
And so these are our collaborators.
13:25
And with that, I just want to say thank you.
13:27
And in case you have any questions or comments, we're going to be here around, and we dropped some leaflets here and in the back.
13:34
And you can also go to Promega booth.
13:36
They have some leaflets and also some organoid shaped cookies.
13:40
Thank you.