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“With this approach, you could theoretically just thaw the cells and then inject them, without any extra processing steps,” says Ana Jaklenec.
Image Credit: MIT News; iStock
(CC BY-NC-ND 3.0)
Scientific Frontline: Extended "At a Glance" Summary: CAR-T Cell Cryopreservation Using Sugars
The Core Concept: A novel cryopreservation technique utilizing nontoxic antifreeze sugars, such as trehalose and sucrose, to protect CAR-T cells during freezing and thawing without requiring extensive chemical removal prior to patient infusion.
Key Distinction/Mechanism: Traditional methods rely heavily on dimethyl sulfoxide (DMSO) to prevent ice crystal formation, a compound that is toxic and must be removed before the cells can be administered, a complex process that most hospitals cannot perform. The new approach introduces sugars into the cells via electroporation (applying a small electrical current to create temporary pores in the cell membrane), allowing the sugars to stabilize proteins and prevent ice crystals, significantly reducing the required amount of DMSO so that it no longer necessitates removal before treatment.
Major Frameworks/Components:
- Chimeric Antigen Receptor (CAR) T cells: T cells isolated from a patient, engineered to express CAR proteins to target specific cancer cells, and multiplied before being transfused back.
- Cryopreservation: The process of freezing biological material to preserve it for storage and long-distance transport.
- Dimethyl Sulfoxide (DMSO): The conventional cryoprotectant that prevents ice crystal damage but requires specialized removal to avoid toxicity to the patient and damage to the cells during the removal process.
- Antifreeze Sugars: Trehalose and sucrose, naturally occurring sugars used by organisms like North American wood frogs to survive extreme cold by preventing protein denaturation and ice crystal formation.
- Electroporation: A technique using an electrical field to increase the permeability of the cell membrane, allowing the large sugar molecules to enter the CAR-T cells.
Branch of Science: Bioengineering, Oncology, Cellular Biology, and Immunology.
Future Application: Could enable hospitals and cancer treatment centers without specialized cell-washing facilities to store, thaw, and directly inject CAR-T cell therapies and other biotherapeutics, such as mesenchymal stem cells.
Why It Matters: Currently, only about 5 percent of hospitals in the United States have the capacity to process and deliver CAR-T cells due to the complex DMSO removal requirement. This new method increases post-thaw cell viability and functionality, demonstrating higher survival rates in mouse models of lymphoma and glioblastoma, and could democratize access to this critical cancer immunotherapy.
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| MIT researchers developed a way to protect CAR-T cells from damage that can occur when they are frozen for storage and shipment. In this research image, the green dots are CAR-T cells containing fluorescently tagged trehalose and the blue dots are nuclei stains (DAPI). Image Credit: Courtesy of the researchers (CC BY-NC-ND 3.0) |
Immune cells engineered to attack cancer cells, known as CAR-T cells, are used to treat some types of blood cancer. However, only about 5 percent of hospitals in the United States have the ability to generate and deliver CAR-T cells to patients. For many patients, this means the cells must be frozen and shipped long distances.
To help make this type of therapy accessible to more people, researchers at MIT have developed a new method to protect the cells from the damage that can occur when they are frozen for storage and shipment. Their technique significantly reduces the use of a chemical preservative currently used to protect the cells, which should make it easier for more hospitals to provide this treatment option to patients.
Instead of treating the cells with a cryoprotective chemical that must be removed prior to treatment, the researchers successfully preserved them using a nontoxic antifreeze sugar.
“With this approach, you could theoretically just thaw the cells and then inject them, without any extra processing steps. We think that could allow a lot more cancer treatment centers to be able to give CAR-T cell therapy,” says Ana Jaklenec, a principal investigator in MIT’s Koch Institute for Integrative Cancer Research and a lead author of the study, which appears this week in Trends in Biotechnology.
In the study, the researchers demonstrated that cells preserved using this process had higher survival rates and could be successfully used to treat lymphoma and glioblastoma in mice.
Robert Langer, the David H. Koch Institute Professor at MIT, is also a senior author of the paper. MIT postdocs Amy Lee and Khanh Tran are the paper’s co-lead authors.
Preserving Cells
To manufacture CAR-T cells, clinicians isolate T cells from patient blood samples. These cells are then engineered to express a protein called a chimeric antigen receptor (CAR), which can be designed to target specific proteins found on cancer cells.
The cells then spend several weeks proliferating until there are enough to infuse into the patient. A small number of hospitals are equipped to generate and administer these cells, but most CAR-T cells are generated at centralized laboratory facilities. Once ready, the cells are frozen and shipped to a hospital or cancer treatment center.
To protect the cells from ice crystals that can damage their membranes, they are treated with a chemical called dimethyl sulfoxide (DMSO), which prevents ice crystal formation. This compound must be removed before the cells are infused, but most hospitals do not have the expertise to do this, limiting their ability to provide CAR-T cell treatment.
The process of removing DMSO can also harm cells, reducing the number of viable and effective CAR-T cells. In the new study, the MIT team sought a way to reduce or eliminate DMSO in the process, making it easier for these cells to reach more patients.
“We looked at this cell-manufacturing process to see if there are ways to improve it, to increase the efficacy, and, hopefully, eventually get to the point where these cells can be easily distributed to treatment centers,” Jaklenec says. “Our goal was to eliminate adding this chemical and really focus on safe excipients like sugars.”
The researchers employed two sugars that scientists have previously used to help cells survive cold temperatures. These sugars—trehalose and sucrose—help cells naturally combat the cold by protecting proteins from denaturation and preventing the formation of ice crystals. This antifreeze mechanism is found in many Arctic organisms, such as North American wood frogs (Lithobates sylvaticus), and helps them survive extreme subzero temperatures.
To get sugar molecules into the cells, the researchers used a technique called electroporation. By applying a small electrical current to the cells, researchers can briefly create pores in the cell membranes, allowing large molecules, such as sugars, to pass through. They found that they still needed to add a small amount of DMSO, but not enough that it had to be removed later.
“We believe that our cryopreservation strategy can truly improve cell therapeutic accessibility because, with our strategy, you don’t need to remove the cryoprotectants. You could use the cells upon thawing,” Lee says.
More Effective Therapy
The researchers tested this technique on CAR-T cells, as well as on mesenchymal stem cells, which can differentiate into many other cell types and hold potential for use in regenerative medicine. For both types of cells, a higher percentage survived the freezing and thawing process when sugars were used as the main cryoprotectant instead of DMSO.
They also used thawed CAR-T cells to treat non-Hodgkin lymphoma and glioblastoma in mouse models. Mice treated with CAR-T cells preserved using the new strategy had higher survival rates than mice treated with cells preserved using the conventional DMSO approach.
“Preservation methods for living biotherapeutics have seen limited innovation, remain poorly characterized at scale, and often compromise cell viability and function after thawing,” Tran says. “We believe that our findings underscore the importance of thorough characterization and optimization of every stage of cell therapy manufacturing, which could have dramatic impacts on treatment efficacy.”
The researchers now hope to work with hospitals to explore whether their new technique could be easily integrated into the process of producing and thawing CAR-T cells.
“If that’s successful from a cell viability and functionality standpoint, perhaps we will do a small trial with patients,” Jaklenec says.
Vijay G. Sankaran, a professor of pediatrics at Boston Children’s Hospital and Harvard Medical School and a Howard Hughes Medical Institute Investigator, who was not involved in the study, says he is excited by the potential applications of the research.
“As a pediatric hematologist and oncologist, many of the cell therapies we use, including CAR-T cells and blood stem cells, require us to collect and freeze a substantial number of cells so that enough healthy cells are available after thawing for when patients need treatment. This work suggests an innovative approach that could help more cells survive the freezing and thawing process, potentially making these powerful therapies more reliable and effective. Of course, further work will be needed to validate these results in settings where this approach can be clinically applied,” Sankaran says.
Funding: This work was supported by postdoctoral fellowships from the Ludwig Center at MIT’s Koch Institute and the Convergence Scholars Program at the MIT Marble Center for Cancer Nanomedicine.
Published in journal: Trends in Biotechnology
Title: Cryopreservation of cell-based therapies with enhanced cell viability and therapeutic efficacy
Authors: Amy H. Lee, Khanh T.M. Tran, Alexander Hostetler, Kewen Lei, Jinbi Tian, Sevinj Mursalova, Alicia Lau, Gary W. Liu, Charles Gorelick, Darrell J. Irvine, Robert Langer, and Ana Jaklenec
Source/Credit: Massachusetts Institute of Technology | Anne Trafton
Edited by: Scientific Frontline
Reference Number: beng081926_01