Webinar Q&A: Automated Hepatocyte Counting

Yes, absolutely. That was a feature that we spent a lot of time talking to experts about since hepatocytes tend to be very clumpy looking to begin with. We spent a lot of time making sure that we were accurately counting clusters versus possibly large either dividing or blebbing hepatocytes.

Since the algorithm was trained on a large variety of sample types, it does a good job with most species and extraction methods. We talked to some hepatocyte suppliers and hepatocyte isolation companies while we were developing the algorithm, and they provided us with a comprehensive list of sample types and methods that we should include. So all of that optimization has already been done for the user, they should just need to load and go.

Yes, we do have both of those options available. 21 CFR is available for purchase, and users can activate it through a simple software download. We also have IQ-OQ available that takes the users through the steps of ensuring your instrument is meeting its specifications.

No, this is a fluorescence app only and is not going to be available on CellDrop brightfield models. The reason for that is because hepatocyte counts are complex, and we’ve found that the AO/PI gives the most accurate results. However, if customers have a brightfield instrument and want to upgrade to fluorescence, we have options for making that possible. If you’re interested in upgrading your unit, contact us and we can discuss that with you.

There are a couple of options. First, the device was designed to be able to fit inside of a sterile hood. It can plug in and operate inside the hood just like it would on a lab bench. 

For cleaning the surfaces and making sure that the surfaces are sterilized, you can treat it as you would any other piece of lab equipment. Use 10% bleach, spray it on a kim wipe or a paper towel, and wipe down the surfaces. The sample surfaces are made of sapphire, so they are very robust and non-reactive to chemicals. We recommend that users don’t spray the instrument directly, but spraying solution on a wipe and using that on the surfaces isn’t going to damage anything. 

Lastly—since we know that there are labs that require this—the CellDrop is designed to also work with disposable slides. All you have to do is switch the instrument into “Slide Mode,” and then it’ll adjust the chamber height to accommodate for slides.

The hepatocytes app is specifically trained to count hepatocytes, so I wouldn’t recommend trying other sample types on it. For PBMCs, we do have a really good option—our Primary Cell AO/PI app. It uses our traditional fluorescence algorithm, which works really well for those sample types.

After your hepatocytes are prepped and you’re ready to plate, you’ll need about 20 seconds to mix the sample with the dye and load it onto the instrument. It’ll take a few seconds to make sure all the cells are settled. The count itself will be around 30 to 40 seconds. So in total, we’re talking about a minute from when you have your sample sitting in the tube to having an answer in front of you on CellDrop.

That’s part of the machine learning algorithm we developed. In addition to quantifying live and dead hepatocytes, we also trained it to detect additional objects in the sample. A live lymphocyte fluoresces green, so the CellDrop uses a combination of the green fluorescence from the AO/PI dye and the object detection algorithm to determine that it’s a lymphocyte.

If a cell has green fluorescence, it is a live cell. That’s just the nature of how the dye works, and there is data in technical notes and literature to prove that. Cells can exhibit both green and red fluorescence because the Acridine Orange dye does have a slight red fluorescent emission spectrum itself. But if the cell has any green in it, then it is a live cell.

We have a technical note that compares the CellDrop to manual counts on a hemocytometer, which you can access here. We find that the CellDrop’s results are very comparable to trained researchers doing manual counts on a hemocytometer.

The advantage of automated systems is that using the same algorithm will standardize how samples are being counted between researchers and across different labs. We’ve run some of these tests in our lab, and we find that even among well-trained scientists, there is some variation person to person.

For samples like hepatocytes, which are quite irregular and odd looking, different researchers might be counting things slightly differently. By using one algorithm to count samples, you’ll have a lot more consistency with results and decrease the learning curve for counting.

That’s going to depend on the size of the mouse, and if you’re doing the whole liver in a single perfusion. For the particular sample we used in our live demo, I resuspended in 2 mL. That’s something that would need to be determined empirically by each lab, or it might come in the procedure for the hepatocytes isolation kit that you’re using.

Yes, you would need to upgrade from the CellDrop FL to the CellDrop FLi to use the Hepatocytes app. The processor on the CellDrop FLi is higher powered and capable of running the more advanced algorithm we’re using for hepatocytes. In addition to getting the updated processor, the upgrade will also include an improved fluorescence excitation system. If you’re interested in learning more about upgrading to the CellDrop FLi, you can contact DeNovix or your local distributor.

Yes, we did use cryo-suspended hepatocytes to train the algorithm. We’ve also had customers that have used this algorithm on cryo-suspended hepatocytes. We’ve also used freshly prepared liver perfusions, where we’ve done hepatocyte isolations from those. That was all used to train and test the algorithm, and as I mentioned, a variety of species was also used.

If it’s a fresh liver sample, then we would do a perfusion. One option that we’ve had success with is using the Miltenyi Biotec gentleMACS™ kit and their liver perfusion system.

Users will not have to do any machine learning or programming themselves. All that is required is loading the prepared sample onto the CellDrop, then pressing the Count button.

Since the cells are being mixed with a fluorescent dye, they generally will not be able to be used again in a downstream process. However, the amount of sample required for counting is only 5 µL along with 5 µL of the dye. The total sample size is 10 µL. Typically, you wouldn’t have an issue with needing to recover and reuse that small of a sample volume.

The accuracy of the cell count is based on the accuracy of the chamber height, which we have tested to be within 1 µm. Additionally, allowing the customer to be able to visualize what’s being counted on the screen and compare it to how they would have counted it is what provides an important opportunity to manually confirm the counts.

If users want to test out the instrument in their lab before purchasing, we offer a Free Trial program. If a lab decided to purchase the instrument, one of our DeNovix application scientists would be available to help them set up the unit and provide additional training over a video call if required.

The CellDrop is essentially plug-and-play so once it is unboxed and powered up, it will perform a system verification and is ready to use. The CellDrop hardware and algorithms are fixed, so the data will be standardized across all users in the lab. All the data is automatically saved to the device and can be reviewed after the count if needed, which is another benefit over manual processes.