Advances in 3D cell culture for spheroids: On demand

3D cell culture techniques have continued to receive attention, with many scientists curious about if it is now time to start transitioning from 2D to complete 3D cell culture techniques. 3D models hold remarkable promise for disease modeling, with the potential to help study tissue repair and drug responses.

In this on-demand SelectScience^® webinar, Carl Radosevich, Senior Manager, Scientific Applications and Collaborations, PHCbi, incubation product specialist, Holly Hattaway, also of PHCbi, and Jin Akagi, CEO of On-chip Biotechnologies explore the advances in 3D cell culturing for spheroids, compare 2D and 3D methods and their respective results, and discuss the options available to overcome the limitations associated with spheroid development. Also, find out how to enhance your research capabilities through safer cell sorting, uniquely designed 3D culturing vehicles and smart controls in incubation decontamination methodology.

Read on for highlights from the Q&A discussion at the end of the live webinar or register to watch the full webinar on demand>>

Q: What is the timing of CO₂ detection with a dual wave IR sensor inside the chamber?

HH: The dual IR sensor enables the incubator to read CO₂ in real time and respond in real time. Dual sensors allow you to have one that's self-calibrating to make sure that it's reading CO₂ levels properly and it's not affected by any events like a door opening. Regardless of humidity and temperature, the CO₂ should be brought back up in real time.

Q: Does the dispenser have fluorescence detection?

JA: Unfortunately, the dispenser itself doesn't have a fluorescence detector, it uses the brightfield camera to detect whether there is a cell or not. It can determine the size of the spheroid or the cell and it dispenses according to the sizes or the number that's been aspirated into the pipette tip. We designed it in a way where the purification steps will be involved before doing the distinction. For example, using our cell sorter to purify the sample.

Q: Can you only use phosphate-buffered saline for the sorter?

JA: No, the instrument is unique in the way that you get to choose whatever media you'd like to use to sort your samples, including the spheroid. People often use culture medium to have optimal conditions. Your cells will be happy all the way from when you put it in, to when they come out. That's a unique feature of our instrument that is not something that's attainable using other conventional systems.

Q: What are the benefits of physiological oxygen conditions? And how does it generate truer data for downstream procedures?

HH: That sounds like a question about oxygen-controlled incubators. When it comes to testing in conditions that have limiting factors involved, especially in cancer research and stem cell research, they've found that physiological oxygen levels are much lower than the levels of oxygen that are received in the incubator, (which is atmospheric, about 21%). This creates a hypoxic environment for cells. It doesn't necessarily hold true to in vivo environments. And by producing this more physiologically relevant environment, you can have more significantly in vivo-like results.

When you're dealing with a 3D spheroid cellular culture, you end up seeing these gradients of gases and oxygen. When you're considering the efficacy of drugs, you must consider the oxygen levels that are actually presented in vivo. There is a lot of research that is turning towards oxygen levels representing more physiologically relevant levels like about 5%, because that better represents the oxygen level that gets to the actual tissue in vivo.

Q: Does this system need additional cytokines or growth factors to keep the spheroids growing for a longer time?

HH: In the research I've found that addresses spheroid growth, they didn't report having to use extra antibodies or cytokines, and it could be dependent on your research. The most recent studies concerning stem cells, it's protocol specific, but they haven't needed to add anything. I have read some studies comparing the Matrigel to the 3D spheroid with the low-adhesion plates, they found that there's less intrusive signaling, there's less unwanted signaling from the biologically derived Matrigel. In the protocol, they haven't had to add any additional cytokines comparing it to a scaffolding type of 3D culture.

Q: Do you need different types of culture medium for different types of tumor spheroid?

HH: Research using prime service plates specifically, they haven't needed to add any specific things like that for their culture. That's part of the reason the ULA plates, or the ultra-low attachment plates have been considered one of the easier ways to implement 3D culture in laboratory settings because you don't find these extra hormones or extra markers that need to be added necessarily that don't adhere to your normal protocol.

Q: Can I use a commercial H₂O₂ solution?

HH: We have a specialized formula of the H₂O₂ decontaminant that has been perfected for use in the square footing and the machinery of the incubator. It's enabled us to make it so that the sensors and everything in there just stays in place, but it's perfected under our H₂O₂ solution. Unfortunately, we cannot guarantee the same results using anything other than ours.

Q: Could you give information about your plates for spheroids? Is it easy to use U-bottom plates for observation of the spheroids on this phase-contrast microscope?

HH: All the well shapes have produced great optical clarity for experiments. What it depends on is what your culture seems to do the best in, and the attractiveness of these plates is that you don't have to transfer it to a different type of well shape, there's no transfer step needed. They all provide optical clarity and I haven't heard any specification for one of the shapes as far as optical clarity is concerned.

Q: What are the minimum and maximum sample volumes you can load into a chip at a time?

JA: The minimum is as low as 20 microliters and that's a lot smaller than many of the other instruments. The maximum will be one milliliter at a time. You can always continue to load more and more; you can carry on and keep on loading by stopping the run each time. In one single run, if you do not want to stop, one milliliter is the maximum.

Q: Would you recommend a biocontainment cabinet or equipment to house the 3D technology?

JA: In some cases, people would like to culture 3D spheroids put them back into the body – in these cases you would. In other cases, we like to simply culture them. We do recommend having them inside a biocontainment cabinet or a biosafety cabinet to keep them nice and clean, and if something happens, you can have everything contained. It depends on what kind of downstream application people would like to test.

Q: Do you see spheroids clogging up the nozzle?

JA: No, we do not have the concept of the nozzle anymore, because what we have is a continuous flow of liquid through a channel. Clogging could potentially happen at the intersection where we do all of our sorting, but we usually don't see them. If it does happen because the sample has something very gooey there or you get dust in there, we have a way to de-clog this easily. It's not like the conventional sorting process, where it has a nozzle and then the clog is a big issue.

Find out more about the latest advances in 3D cell culturing for spheroids: Watch this webinar on demand>>