June 1, 2014 (Vol. 34, No. 11)

Alcohol-Free Controlled-Rate Cell Freezing Offers Comparable Results to Programmable Freezers

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. Cells are cryopreserved to avoid loss by contamination, to minimize genetic change in continuous lines, and to avoid transformation in finite lines. Successful cryopreservation of cells requires that a standardized and reproducible protocol be followed, although each protocol may require optimization for a given cell type or line, to achieve maximum viability upon thaw. Mammalian cells that are cryopreserved include immortalized cell lines, primary cells isolated from tissues, and stem cells.

For optimal cell viability post-thaw, the following factors must be taken into consideration:

  1. Media selection: Most commonly, the freezing media contains a cryoprotective agent, such as 10% DMSO. Alternate media options include serum-free protein solutions and standard blood serums.
  2. Freezing methodology: The optimal freezing rate for cryopreservation of most cells down to –80°C is –1°C/minute.
  3. Temperature control management: During the entire process from cell handling and preparation to freezing and thawing, the cells must be maintained at specific pre-determined temperatures.

Challenges with Current Methods

Conventional methods of cell cryopreservation rely on step-down programmable freezers, which up to now have been considered the gold standard, or passive isopropanol (IPA)-filled containers placed in a –80°C freezer. Some methods even call for unstandardized Styrofoam boxes as the “freezing device.” The IPA-filled containers and Styrofoam boxes do not provide uniform freezing rates across all cryogenic vials and may not be reproducible across freeze runs due to variability in the IPA that accumulates with each freeze run and differing Styrofoam density.
Isopropanol-filled containers have a stated freeze rate of –1°C/minute, but performance is dependent on vial position and continuous isopropanol replenishment. Isopropanol is a variable in each freeze run, thus hindering reproducibility. Long wait periods for the IPA to equilibrate to room temperature between freeze runs effectively limits throughput to one run per day.

Programmable freezers are reproducible and documentable; however, they are also expensive to purchase, difficult to operate and maintain, and require a large footprint. Complex design limits the device to a single use and malfunction is common. Programmable freezers require manual installation of at least two thermocouples in the freezing chamber and on the samples that are to be cooled. If these sensors are not connected appropriately, the control system will not receive accurate data and will respond with incorrect inputs of liquid nitrogen and subsequent cooling.

Due to the irreproducibility of IPA-filled containers and the high cost, complexity, and difficulty in implementation of programmable freezers across multiple sites, researchers are looking for other viable options.

The ideal solution for researchers is a controlled passive device with a small footprint that combines the ability to be deployed across multiple sites and is standardized to ensure optimal cell viability upon thaw. The CoolCell® containers from BioCision were developed to specifically meet these needs (Figure 1).

Figure 1. BioCision’s CoolCell Portfolio

Reproducible Controlled-Rate Freezing

CoolCell alcohol-free, controlled-rate cell-freezing containers provide a consistent and reproducible –1°C/minute freezing rate and deliver results comparable to a programmable freezer.

CoolCell containers are made from a closed-cell polyethylene foam that is highly insulative and has superior material properties at cryogenic temperatures. The radially symmetric vial distribution ensures identical heat-removal profiles for each vial while a solid alloy thermal core balances and fine-tunes the freezing profiles, ensuring reproducibility (Figure 2). The single-block base construction is durable and provides nearly indefinite product life-cycle without changes in performance, resulting in a consistent, reproducible, and standardized alternative to programmable freezers for cell cryopreservation.

Recently published papers in Nature Protocols advocate the use of BioCision’s CoolCell alcohol-free controlled-rate freezing container in the preservation, storage, and recovery of sensitive stem cells. According to John Gardner, senior project leader at Roslin Cellab, “controlled freezing with the CoolCell container greatly increased the reproducibility of the freeze process, with increased cell viability and cell growth post thaw.”

In addition, ATCC also now recommends the incorporation of a CoolCell container into their iPSC stem cell handling guide. Kevin Grady, product line business manager at ATCC, says “the device is elegant in its simplicity and ease of use and offers researchers a method to cryopreserve cells in a standardized fashion with great reproducibility and little variability in performance.”

Standardized cryopreservation techniques are also an important aspect of developing cell therapies. Following the expansion and/or manipulation of cells in culture, some cell therapy products are stored in liquid nitrogen until immediately prior to patient administration. A recent independent example of product utilization in the cell therapy space was published in the Journal of Autoimmunity. Researchers cited the use of a CoolCell freezing container for studying the role of T cells in myasthenia gravis. The CoolCell freezing container was used to gradually freeze cells isolated from the blood of both patients and healthy donors for long-term storage while sample collection was underway.

Figure 2. CoolCell reproducibility. The freezing profiles of five consecutive freeze runs in a –80°C freezer were recorded and graphed to demonstrate the consistency and reproducibility of the CoolCell cell freezing containers.

Additionally, TxCell, a French cell therapy company, recently integrated CoolCell freezing containers into its production of an autologous antigen-specific Treg (Ag-Treg) cell-based immunotherapy for the treatment of patients with severe refractory Crohn’s disease. Researchers successfully demonstrated the CoolCell freezing container yields increased post-thaw cell viability over programmable freezers (Figure 3), while lowering costs and increasing scalability compared to dedicated, expensive cell-freezing machinery.

Given the speed and increasing importance of cell cryopreservation for research, biobanking, and cell therapy among other applications, choosing the best cell cryopreservation method is crucial. CoolCell freezing containers, available in a variety of configurations for cryogenic vials or closed-system injectable ampules, have been validated as a viable and cost-effective alternative to programmable freezers for effective cryopreservation. Carefully controlling every aspect of how cells are handled, including the temperature at which they are stored and the process by which they are frozen, is the best way to ensure that the full potential of any cellular research study or clinical trial is realized.

Figure 3. Effects of freezing on antigen-specific Treg (Ag-Treg) cell therapy products. Ag-Tregs (n = 6) were frozen at a concentration of 1 to 10 × 106 cells/mL using the CoolCell freezing device or controlled-rate freezer (CRF, freezing rate of –1 °C/min). Viability and absolute viable cell count of thawed Ag-Treg cell therapy products were evaluated by flow cytometry. Data demonstrates that CoolCell performs comparably to a programmable controlled-rate freezer in both post-thaw viability and cell yield.

Maria Thompson, Ph.D. ([email protected]), is vp of scientific affairs at BioCision

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