0:39
So I'm going to take the conversation a step back and go through how we can look at screening clones that are producing antibodies.
0:49
So for that I'm introducing our recently launched instrument, the Cyto-Mine Chroma and talk through how it can accelerate clonal selection.
0:59
So what's we really want to do with these instruments is to allow us to our customers to find and analyse and screen their pressure cells faster and with high accuracy.
1:10
And to do that we're using picodroplet technology.
1:13
So we're using picodroplets, which are, as the name suggests, picolitre volume size droplets, encapsulating cells and acid reagents.
1:24
And this picodroplets, as you can see in the picture, they contain cells within them.
1:28
They have acid reagents, they're biocompatible.
1:30
They keep the cells alive during the screening process and make sure that we can select a viable choice for downstream processes.
1:42
So the Cyto-Mine Chroma similar to the Cyto-Mine, the classical Cyto-Mine that came before that has is automated and it's very fast.
1:56
It's user friendly.
1:57
But compared to the other Cyto-Mine that we had has additional lasers and detectors, which means that it can allow users to make Multiplex assays and using fluorescent signals to identify their correct clone based on the functionality and surface expression.
2:17
So you can see the four lasers and the four detectors.
2:20
We'll go through a little bit more in terms of data how to use these lasers and detectors and what we have done with biological assays with the screening of the cells.
2:31
So Cyto-Mine Chroma is fully automated, but what it does really well is to screen millions of cells.
2:39
So it can screen up to 40 million cells within a day, which means that it will free up a lot of time of a scientist.
2:48
And it's by doing that it also reduces the timelines of projects.
2:53
So with antibody discovery and cell line development, we have shown that the time can be reduced by weeks or months of a project that is running.
3:02
But also it does that by reducing the consumable reagents cost.
3:08
The reason that it can do that is because the volumes that the picodroplets have is picolitres, which means that you don't really need much reagents to have an assay to screen millions of cells, which means that the experimental cost will be reduced dramatically.
3:25
And also it comes with a software, integrated software which allows the users to gate sequentially gate cells live.
3:34
So as the cells are coming through in the droplets, they can sequentially gate them and sort the droplets that contain the right type of cells and dispense this into microtiter plates.
3:49
So this is a typical workflow for the Cyto-Mine Chroma.
3:53
So everything really is happening within a consumable called Cyto-Cartridge and you can see that this is the disc like consumable that is available from sphere, and everything starts with counting cells and adding them to this cartridge.
4:09
And this is it.
4:09
This is the well that is really required for the screening.
4:13
The cells are in a mixture with the acid reagents.
4:17
They are added to Cyto-Cartridge and these cells are then encapsulated within the picodroplets and which we can make about up to 2 million picodroplets in the Cyto-Mine Chroma.
4:30
The droplets are then pushed into a chamber, the incubation chamber, where the user decides on the temperature and the incubation time required that it's optimal for the cells to produce, for example, antibodies, secrete antibodies or be functional if you've got two different cell types within the Picodroplet.
4:48
And also it is within this time that the assay is being developed.
4:52
So once the incubation time is completed, the droplets are then pushed towards sorting where the lasers and lasers are exciting the fluorescence ignorance detectors to take them.
5:05
And this is where the users can engage on the population of interest that needs to be sort of pushed towards the dispensing.
5:14
The droplets are then pushed towards dispensing chamber.
5:17
And again during dispensing, the user has the chance to look at the fluorescent signal to make sure that the droplets that have been sorted contain the right type of cell.
5:30
You still get the right type of signal and to make sure that for applications such as cell line development, we can provide the monoclonality assurance.
5:40
Cyto-Mine Chroma takes 5 pictures of each of the droplets that is being sorted to make sure that we can see the number of cells within the droplet.
5:48
And this reassures that you can see whether there's only one cell within a droplet or multiple cells, which is important for cell line development.
5:56
Then these droplets are dispensed into microtiter plates.
6:00
This could be 24 well plates, 96 or 384 well plates.
6:04
And it is possible to see which droplet went into each of the valves.
6:13
So this whole workflow takes up to 8 hours depending on how long the incubation time is.
6:20
So this is the Cyto-Cartridge.
6:22
So that's where the workflow happens.
6:23
Everything within the Cyto-Cartridge is biocompatible, which means that it keeps the cells alive and the cells are happy and viable at the end of the process.
6:34
And it is a single use only, which means that there is no cross contamination from one experiment to another.
6:45
So I guess to be able to sort cells in Cyto-Mine, obviously you need bioassays and the type of assays that can work in Cyto-Mine are we've divided these into three different types.
7:00
So one is to look at diffuse signal.
7:03
So what we mean by diffuse signal is when you have the fluorescent signal spread within a droplet.
7:11
So here for example, a cell that produces antibodies decrease the antibody within the droplet can be identified as positive if or cell of interest if the antibody binds to the FRET donor FRET acceptor.
7:25
So a type of acid that can be used in Cyto-Mine is a FRET assay and a lot of our I think it's are based on FRET assay.
7:31
So the FRET donor and acceptor bind to the right antibody and that produces a FRET signal and that droplet then is seen as a positive droplet that is sorted and dispensed.
7:44
Another type of acid that can be developed within droplet is to have two different cells the interaction of which would result in production of cytokine for example or release of granzyme B.
7:54
So one being for example a tumour cell, the other one being a T cell interaction induces T cell activation and that results in the production of a molecule and that is then identified by quencher fluorochrome complex formation.
8:11
The other type of signal is, for example, using a reporter cell that has a GFP interaction of a molecule within the droplet induces the GFP activation that is then identified by the by Cyto-Mine as a positive signal and then isolation and dispensing of that droplet.
8:30
Or if you use stain cells.
8:31
So if you have different cell populations that are stained with different membrane dyes, it is possible to have a mixture of cells with different membrane dyes and Cyto-Mine would then be able to distinguish these from each other.
8:43
But something that I think is quite different in Cyto-Mine from other instruments is that Cyto-Mine can distinguish the diffused signals that you can see with the first type of experiments we discussed from localised signals.
8:57
So that is when you would use a bead, for example, that is coated with protein A or protein G binding to an antibody and you have a detection system where the detection antibody that has a fluorochrome binds to this antibody and then forms a complex with the bead, Cyto-Mine can distinguish that signal in the same droplet from the spread of the detection antibody in the same droplet.
9:21
So you can distinguish that there is a complex formed within that droplet and so you don't need to wash away the materials, create acid reagents which you can't do in a droplet.
9:31
So that becomes a positive signal and the Cyto-Mine can then easily distinguish that as a clone of interest, a cell of interest. And the same kind of example with a cell that expresses a targets receptor on the surface that binds an antibody produced within that droplet.
9:52
So I will go through some of the biological assays that we have run in Cyto-Mine Chroma, but I just wanted to show you the sensitivity and accuracy of the selection that Cyto-Mine Chroma provides.
10:05
And we took to do that, we took four different cell populations.
10:11
Each of the cell populations are stained with a different membrane dye and you can see in this table the different dyes that have been used to use to stain these cell populations.
10:25
To just simplify everything, we used colours for the detectors.
10:28
So we had one of the cell types detected with the blue detector with the green, orange and far red and during sorting we are able to sequentially gate the cell populations.
10:39
So we are what we aimed was here.
10:42
This is an example of the gating at the bottom.
10:45
We said we want only the droplets that contain the blue cells.
10:49
So you can see in each of the dot plots we have 3 populations.
10:52
We have a population of droplets that contain only blue, a population of droplets that contain only green, but also droplets that are polyclonal and contain both colours.
11:03
So we wanted to make sure that the only thing we get at the end is only one colour.
11:08
So we gated the blue against green, orange and far red during sorting and we send this to the dispensing chamber.
11:16
And you can see in the origin within the dispensing chamber, the only thing that is really selected are the blue ones.
11:22
So everything else have been sent to the bin in a way.
11:28
And these were then dispensed into, again you are able to gate during dispensing by the there's no need.
11:37
So these are then dispensed in as single cells to the 96 well plates.
11:42
And then we took pictures using microscopy and with the higher accuracy we could see that each of the colours we are able to sort and dispense the right cells even when we have heterogeneous population to start with.
11:58
So we did three different biological assays to test on Cyto-Mine Chroma to make sure sequential gating works and most of these are for applications of CLD and antibody discovery, but these can be used for other applications as well.
12:14
So in the first experiment, we looked at antibody production using FRET assay and also labelled the cell population.
12:21
So we wanted to make sure that we can sequentially gate on antibody producing one specific cell type.
12:28
The next one was to look at antibody binding to specific target receptor and also being able to distinguish different membrane dyes, again different cell populations.
12:39
Here in the third experiment, we looked at the antibody production in viable cells.
12:46
So we wanted to make sure that cells can be excluded from dispensing.
12:55
So the first experiment that we ran this was the antibody production and population of cells that were labelled with membrane dyes.
13:03
So here we had a cell line A that was detected in the far red and the cell line B that was detected in the blue and we had both cell lines producing antibody and that was detected by FRET signal.
13:17
So our orange laser, so the 561nm laser had to be switched off because of the accepted FRET signal, but we gated first on the population of the cells that were producing antibodies.
13:31
So that is the first dot plot.
13:32
So gated only on antibody producing cells.
13:37
Within that population, we get both cell lines A and cell line B and droplets are contained both cell types, but we said we just wanted cell line B.
13:46
So we gated on the cell line B during sorting and dispensed the population as single cells in 96 well plates and we could see what looked on the microscope and we could see that with 100% accuracy we were getting the right cell populations selected.
14:04
In the second experiment, which is quite related to antibody discovery, we wanted to show that an antibody secreting cell can secrete antibody, and we can detect the binding of that antibody to a target cell.
14:18
So we use the target cell labelled detectable in far red that expressed the target receptor on the surface binding to the antibody that was secreted.
14:27
Antibody secreting cell was labelled and dissected within the blue detector channel and during sorting we gated on the population that had the antibody secreting cells to the blue and also showed the detection antibody.
14:45
So when you look in the no antigen binding, you can see the detection antibody with fluorochrome green and that is dispensed.
14:53
This is everywhere.
14:55
Whereas with when there is antibody in the presence of the right secreted antibody, detection antibody forms a complex on the target cell, we get a localised signal.
15:06
Now Cyto-Mine can distinguish that there is an antibody produced there.
15:10
So we gated on the green positive as well as blue antibody secreting cells.
15:16
And then within that gate, we said we only want the droplets that contain the target cells, so the whole complex that is required for positive signal.
15:25
And we dispensed the droplets that had the positive signal and again checked under the microscope.
15:33
So you can see for example, there's an example of one of the bars where we had three cells per droplet.
15:40
Two of them were target cells that were red and the two target cells were labelled with the detection antibody.
15:46
So that's the only place that we found the detection antibody as you expect.
15:51
And the antibody secreting cell was also present.
15:54
So that shows that this is a whole complex formed with the primary antibody, and this was with the accuracy of 92%.
16:08
Next we looked.
16:10
So this is related to cell line development.
16:12
Next we wanted to look at selecting cells that are viable.
16:17
We wanted to exclude anything that is non-viable, apoptotic, necrotic, dying within the droplets and but they are antibody secreting.
16:27
So in this system, we're using a dye, a viability dye that would bind to anything that is apoptotic or dying.
16:35
And we also use thread probes that would be able to detect the antibody production within the droplet.
16:42
So where there is apoptotic, we would get the viability dye binding to the cells, and we can distinguish that from a live cell population.
16:51
So in the dot plot within, again this all happening in Cyto-Mine, we are able to gauge the far red.
16:58
So the viability dye shows off as the far red.
17:01
We are able to gauge on the far red cells and distinguish those from the live cells.
17:07
We pulled dispense the apoptotic cells and the viable cells from this system and grew them for about two weeks.
17:15
We looked at the cell growth within the two weeks and what we found was that the cell populations that appeared to be apoptotic did not proliferate at all with low viability.
17:26
Whereas the cells that were not apoptotic they proliferate a lot because a lot of a huge cell population after 13 days as was expected.
17:36
And it is possible to then distinguish whether these are antibody producing as well or not.
17:45
So this article is coming out together with Chroma in April and this our current road map for the current assay kids that we have and can be used with Chroma and assay kids that are coming out in 2025 and 2026.
18:07
So next, we're working on antigen specificity as an assay kid as well as bispecific assay kid to come up with within 2025.
