3D Cell Culture Primer: Freezing, Culturing, and Measuring Organoids | Corning

Working with 3D cell culture — organoids, especially — can be fascinating and enlightening. Organoids are a vital tool in disease modeling and drug discovery because they better mimic the composition and functionality of organs, resulting in miniature in vitro versions of in vivo material.

Organoid applications have virtually limitless potential, but they're much more complicated than 2D cultures or 3D spheroids. Don't let that complexity intimidate you from diving into this emerging research field, though. We know a few tips, tricks, and best practices about freezing, culturing, and measuring organoids that can help you dive into 3D cell culturing right away.

Proper freezing can mean the difference between cells that will last for the long haul and those that die right away. According to Nature Protocols, a good methodology follows these 10 essential steps.

1. Cool a freezing container to 4°C. Have at least one or two confluent wells ready for each cryovial, depending on whether you're using 6-well or 48-well plates.
2. Piipette the media up and down using a 1,000-µL pipette and between 500 µL to 1,000 µL of basal medium to break up the basement matrix, which contains has the organoids.
3. Move the suspension with the organoids to a 15-mL centrifuge tube.
4. Add cold basal media to the organoids and repeatedly resuspend to remove the basement matrix.
5. Set the centrifuge to between 100 × g and 200 × g at 8°C. Run it for five minutes.
6. Aspirate all but about 2 mL of the supernatant. Avoid breaking the organoids into single cells.
7. Add more cold basal medium to the organoids.
8. Set the centrifuge to between 200 × g to 250 × g at 8°C. Run it for five minutes.
9. Aspirate all of the supernatant and resuspend it in 500 µL of cold freezing medium per one well or two wells, depending on whether you are using 6-well or 48-well plates.
10. Transfer the suspension to 500-µL cryovials and set in the cooling container. Immediately transfer the container to -80°C; keep it there for at least 24 hours before transferring organoids to liquid nitrogen for long-term freezing.

Please note: The freezing medium contains cryoprotective agent (CPA) like DMSO which is toxic to cells at room temperature. For cell viability when freezing, avoid cells need to avoid excessive exposure to CPA at room temperature during harvesting.

You're not freezing single cells — you're freezing organoids, which grow bigger and more complex every day.

"The size of the organoids when frozen can have a big impact on their recovery when you ultimately thaw them out," said Hilary Sherman, a senior applications scientist at Corning Life Sciences. "Typically, smaller organoids survive the freezing-thawing process much better. "

If carefully preserved, cultures can last for a long time — even up to a decade, Sherman says — giving experiments the benefit of long-term utility as new information arises.

"There are many reasons why a researcher would want to store their organoids for that long," Sherman said. "If you're making a library, you might have a potential therapy that you want to test against a patient's sample from many years ago. Or it could be that you're picking up work that someone else started before their postdoc and moved on."

There's more than one way to create an organoid — you can form them in droplet domes, use bioreactors to scale them up for high-throughput use, or from permeable supports and low-adhesion microplates. The method you choose will depend on what you're looking to learn from the experiment.

• Matrix domes place single cells or tissues in a self-contained extracellular matrix dome so that the organoid can assemble. Researchers might choose this method if they don't have a large amount of the organoid material or if they're concerned that scanning images by surface area will take too long.
• Bioreactors encase organoids made from pluripotent stem cells in an extracellular matrix. They're ideal for high-throughput uses, where you need to scale up a large amount of organoids or culture for extended periods of time .
• Permeable supports and low-adhesion microplates mechanically support the organoid with air-liquid interface materials or ultra-low attachment, which prevent cell binding. They're ideal for researchers looking to further differentiate cells or conduct endpoint assays. Precoated plates can save even more time.

Depending on your process, you can grow an organoid in as little as six days. But Sherman recommends maintaining size similarity whenever possible during harvesting.

"When you're breaking up organoids, you're breaking them into all different- sized pieces," she said. "The more consistent you can keep them, size-wise, the more you'll be able to ensure they grow at the same rate for future applications. One way to help reduce that variability is to allow gravity to settle the heavier pieces of organoids to the bottom of the tube so that the smaller organoids remain at the top. That way, you can collect the population that you want for more uniform harvesting."

There's a lot at stake when it comes to measuring organoids. You need to get it right.

"Size is one of several factors that are measured to determine an organoid establishment, so it's very important," said Kyung-A (Katie) Song, Ph.D., a scientific support specialist at Corning Life Sciences. "In addition, organoids can only be grown to a limited size in vitro, due to the lack of a circulatory system and limitation in the transfer of oxygen and nutrition."

Measurements are hard to do manually, given organoids' irregular shape and the challenge of keeping them sterile. Processing programs can help researchers take accurate microscope measurements without breaking the bank.

"As long as you have a basic microscope camera, you can take images of your organoid cultures and process them with free software, such as ImageJ," Sherman said.

But, she notes, such services aren't ideal for large volumes of work.

"If you're instead trying to do high-throughput research," she said, "you might need to invest in more expensive equipment."

Watch our Organoids Master Class Series: Introduction To & Getting Started With Organoids webinar for more best practices and exclusive tips on setting up your organoid culture program.