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Immunofluorescence image obtained after thawing and immunostaining human airway organoids with for DAPI (nucleic acid stain; blue), KRT5 (basal cell marker, red) and phalloidin (F-Actin, green).
AMSBIO cryopreservation medium, CELLBANKER® 1, enabled human airway organoids to be frozen and imaged post-thaw, demonstrating immunofluorescent expression of key genes: DAPI (nucleic acid, blue), KRT5 (basal cell, red), and phalloidin (F-actin).
AMSBIO, courtesy of Clevers Lab, Hubrecht Institute

Simplifying and Standardizing Organoid Cryopreservation

Latest cryopreservation media are convenient, reliable, and follow regulatory guidelines

Published:Jun 29, 2022
|3 min read
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Headshots of William Hadington-Booth, PhD (left) and Jelte van der Vaart, PhD (right)

William Hadlington-Booth, PhD, (left) is the business unit manager for regenerative medicine and advanced therapies at AMSBIO, where he is responsible for a fully integrated range of products that allows easy translation from research to GMP applications. He is particularly interested in the extracellular matrix.

Jelte van der Vaart, PhD, (right) is a postdoctoral researcher in the lab of Madelon Maurice, PhD, at the University Medical Centre Utrecht in the Netherlands. Jelte’s research focuses on the development of model systems for human disease using adult stem cell-derived organoids of the intestine and adrenal cortex.

Why would a clinical laboratory need to cryopreserve organoids?

Organoids facilitate biobanking: rather than repeatedly harvesting cells from patients, you can generate a lot of patient data using the same sample to grow different kinds of organoids. Cryopreservation is essential to creating a stable model system that researchers can always go back to, while enabling biobanking of diverse cell types in both control and disease conditions. This facilitates high-throughput and personalized drug screening, as well as the continued development of cell therapies. Eventually, the goal with organoid research is to create healthy organs for transplantation.

What are the key considerations for labs performing organoid cryopreservation?

Labs consider both the convenience and reliability of cryopreservation and of thawing organoids. Unlike in vitro cell cultures and other biological material, full organoids can be very dense with up to 20 cell layers and different cell types, requiring reagents that preserve organoids into to their core and help maintain that cellular diversity. In addition, in pharmaceutical and diagnostic settings, there are regulatory considerations, where you may be required to use serum-free reagents that adhere to good manufacturing practice (GMP).

What methods have laboratories traditionally used for organoid cryopreservation, and what are the pitfalls of these methods?

Traditionally, you cryopreserve cells and organoids with a self-made medium of fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO), followed by slow gradient cooldown in cryogenic containers. This requires time and effort, and since FBS can have batch variations and DMSO can undergo freeze-thaw cycles, it translates to batch variation every time someone in the lab makes cryopreservation medium. FBS can also affect downstream clinical applications since it is an animal-derived product.

How does the latest cryopreservation technology address these issues, and why should clinical labs adopt this technology?

The latest technology, like AMSBIO’s line of cryopreservation media, is ready-to-go and designed with regulatory conditions in mind, making it more convenient, reliable, and preferable for developing therapeutic applications that have stringent requirements for experimental control and documentation. From the AMBSIO products, CELLBANKER®1 is the simplest and most economical solution for the cryopreservation of cells and organoids. Then, we have the serum-free variant CELLBANKER®2, which means that it contains no animal-derived products that can impede clinical use. Our serum-free GMP-grade media include STEM-CELLBANKER®, and the latest addition, STEM-CELLBANKER® EX, where each component is approved for intravenous administration.

Other than organoids, what are some important applications of the latest cryopreservation techniques?

With organoids, you can have sampling error or variation in how organoids develop, so it is useful to also cryopreserve samples of the original tissue or individual cells isolated from it. Biobanking the original tissue allows for a more faithful recapitulation of the patient’s biological state. Along with the use of patient-derived material for drug screening and future therapy, clinical applications for patient-derived material will continue to require reliable, serum-free, and GMP-approved cryopreservation.