Novel technology in non-invasive 3D ex vivo imaging and cultivation platforms

3D ex vivo platforms have been diligently evaluated as better predictive tools in research and the preclinical and clinical space in the quest for reducing the dependence on animal research.

In this SelectScience^® webinar, now available on demand, Sumeer Dhar, director of scientific alliances, SCREEN Holdings, and Holly Hattaway, product specialist of incubation, PHCbi, discuss how SCREEN imaging technology enables researchers to perform large-scale drug discovery and early development using multiparametric endpoint measurement assays. Their unique infrared laser-based optical coherence tomography (OCT) technology for 3D ex vivo will also be described.

Read on for highlights from the Q&A discussion with Sumeer Dhar and Joe LaPorte, director of projects and regulatory at PHCbi, and find out about how PHCbi’s cutting-edge 3D imaging systems can enhance your research.

I'm using both suspension cultures and spheroids grown in scaffold and I need to measure at time intervals. Does the imager’s stationary plate sampler work for the floating cultures like I'm using?

SD: Yes. The system comes as a stationary stage, so we are able to use both stationary or suspension cultures, as well as in scaffold. The cultures for the cell suspensions do not move because the plate is standing there, and you are able to scan or image the datasets even if you remove the plate and put it back there. It's not going to disturb the cultures, so you will still be sure to image the same culture that you had done in the time-lapse manner.

Is it possible to measure and control oxygen concentrations in these 3D cultures? And what about oxygen gradients?

SD: At this point in 3D culture, because we are imaging the whole spheroid itself, we are not measuring the metabolic activity, such as in the signaling. However, it should be possible. I think you need to have a specific oxygen flow concentration to really follow what's going on with the culture.

JL: If I might add to that, we do have several incubator line designs that are able to control oxygen levels and displace it. So, if you're trying to run down to hypoxy conditions or if you're trying to run down to anywhere between 1 and 20%, we're able to do that with the controls in our incubators.

What happens if I'm running an application that will cause fluctuations in CO₂?

JL: We do have CO₂ control in our incubators. As I mentioned, we do have oxygen control as well. We are able to control the CO₂ levels very precisely. But, if your application is increasing the CO₂ level, we'd have to look at some very specific modifications to be able to bring it back down again.

I'm interested in brightfield and two-color analysis simultaneously. Can the device image the samples both from the monolayer assays and spheroid-based assays?

SD: Yes! The Duos-2 imager has the capability to have up to three fluorescence units mounted there. So, you could do brightfield in addition to up to three colors at the same time and be able to analyze different markers within the assay type, both for the 2D culture cell-based assays, as well as in the spheroid formats.

When faced with a pathology, can morphological changes be observed at the individual cell level?

SD: Yes, that would definitely be possible on the Duos-2 because it has the capability for 2D assays and monolayer assays, as well as for the tissue. What we can also do is use slices from the spheroids, which are embedded in agarose, and prepare, stain, and then mount these under microscopic slides to look at what's going on with the specific biomarker.

My process cultures require tight maintenance of pH. How do your incubators provide pH control?

JL: We don't have specific pH control in the incubators. However, we control the pH through the control of the CO₂ level and, as I mentioned, we can control that very precisely. So, if you do have a specific application, you can reach out to us and we'll see what we can do to assist.

I would like to know more about the deep learning applications as well as the support available for training data files. Is there a contract service that one could order?

SD: Yes, definitely. We offer deep learning in two packages. One is a package with the system and we can also provide plugin packages, where the customer can send us the data, like the case for City of Hope Hospital described in the webinar. They sent us the data and then our bioinformatics scientists prepared the model based on these datasets for researchers to apply and generate additional data from deep learning. So, we can provide the tool as a package or we can provide the services with our expertise in-house. We are also helping customers with the preparation of the model files. So, there are different options available.

What are the different types of well shapes in the prime surface 3D cell culture plates?

SD: There are standard well shapes – such as U-bottom wells and V-bottom wells – and there are also spindle-shaped bottoms. It all depends on the type of cultures that are being used, whether it's immortalized cell lines, cancer stem cells or primary samples. One needs to really see which plate formats would be more appropriate for their specific cell types.

How does the UV light decontaminate without harming the cell culture?

JL: We use a very specific range of the wavelength of the UV light. The incubator is designed to keep the UV light behind shrouds so that we're not allowing it to escape into the chamber and affect cell cultures.
What are all the endpoints that can be analyzed when working on tumor spheroid-based assays?

SD: It all depends on what the whole question is. In drug discovery or lead development, people are interested in the area of the spheroids and the volume of the spheroids, which can easily be done on both Duos-2 as well as Cell3iMager S-tier. Another very important endpoint is the loss of circularity. Because many drugs are targeted drugs, they don't really result in the collapse of the spheroid, they may just, result in the spheroid losing the definition of its circular shape. We take this loss of circularity, as a measure for the drug efficacy.

In addition, these measurements can be done in a label-free manner. This means that you can follow the culture – up to one week for a cell line and two to three weeks if it's a primary sample. For example, lung cancers would need up to two to three weeks but for melanoma models just one week or 10 days would be enough. So, you can follow the increase or decrease in the tumor volume, and then at the end, you can start the assay and do the label-based assay where you are looking for changes in specific markers.

How often does the UV light need to be replaced and how do I know when it needs replacing?

JL: We do give you an indication by tracking the amount of UV radiation and the aging of the bulb. The UV bulbs do age over time, so we adjust to make sure we're getting enough activity. Every time you open the door or close the door, it does come on because you can obviously get contaminants inside the chamber. Depending on how often you're doing that, the UV bulb could last up to 15 years. It really depends on how often you're using it and at that point, it doesn't really become a consumable.
Is it possible to use custom plate types to image samples in the Cell3iMager S-tier?

SD: Definitely, yes. We work on customized projects as well as standard projects. There have been several customers that have specific requirements for the plate type that they were using, and we have successfully managed to have them assess or use the technology, the different plates.

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