Thawing cells – process, difficulties & recommendations

Alexander Fuchs August 9, 2023

Cell thawing is a critical procedure in the area of life science, medicine and bioprocessing. Ensuring a gentle thawing of frozen cells is essential for preserving their viability and functionality. In this article, we will highlight the cell thawing process, discuss common challenges, and provide recommendations for achieving optimal results in cell banking.

The significance of safe cell thawing for preserving the integrity of valuable cell lines and primary cells is undoubtedly high. Cells are either thawed in waterbaths or thawing devices which are considered uncontrolled, due to the possibility of inconsistencies, temperature deviations and the lack of control to apply product-specific thawing. Single-use technologies can emply controlled thawing in lab environments, but also commercial production to perform freezing and thawing to maintain high cell viability while streamlining the process to be automated and sterile, compatible with cGMP standards.

[[ToC]]

Thawing cells protocol

Both cell freezing and thawing have a direct effect on cell recovery rates and cell viability after thawing, two important quality aspects of cell culture. Whether you are working with primary cells, cell lines, or stem cells, it is essential to follow the correct steps to ensure successful thawing.

In this section, we will provide a step-by-step process overview of thawing cells and compare the conventional process using cryovials with an advanced, automated process using a thawing platform and frozen single-use bags.

1. Preparation

Gentle storage of the cryopreserved cells in an ultra-cold freezer and its timely transport to the designated thawing area are essential prerequisites for a smooth thawing process. For long term storage, liquid nitrogen storage is the recommended freezing medium.

2. Controlled thawing

If cells are needed for studies or as part of mAb upstream process intensification, they can be taken from the working cell bank. For this purpose, the cells must be thawed.

For frozen single-use bags, a 37°C water bath or CO2 incubator is commonly used to gradually thaw the cell suspension. In order to reduce the risk of overheating or off-target temperatures, the waterbath temperatures are often at approx. 20°C to 25°C. Cell medium is added to the thawed cells immediately so that the previously added cryoprotectants (e.g. DMSO) are not exposed to ambient temperature but are effectively diluted. To protect the cells from sudden temperature shifts that may be harmful during thawing, such as putting the frozen cells into a warm waterbath, a controlled thawing process is recommended to produce safe results.

OR: Controlled thawing based on freeze-thaw platform

Freeze-thaw platforms offer ideal conditions for both cell bank freezing and thawing. Cells are frozen in single-use bags that are protected by a protective shell. The shell enables plate freezing technology performed with a freezing and thawing platform – where the single-use shells are cooled and frozen with a uniformly controlled temperature drop. When thawing, this process is reversed, with a controlled increase in temperature until the cell substance is in a liquid state again.

The manual handling step as with the waterbath is taken over by the platform in a controlled and automated manner. Human errors and product loss are reduced, while automated and documented processes ensure aseptic processing.

Thawing: Best practices

automated freezing thawing labs single use support

3. Cell viability assessment

Post-thaw, accurate cell viability assessment is essential. Techniques like trypan blue staining can be employed to determine the proportion of viable cells in the suspension.

4. Seeding or Subculturing

Depending on the research requirements, cells can be directly seeded into a culture dish or bioreactor or subcultured to propagate and maintain the cell line.

difficulties-cell-thawing-single-use-support

Difficulties in cell thawing

Thawing cells comes with its challenges that can impact the overall success of research work or the quality of the cell-based therapy.

Some common challenges encountered during cell thawing are:

Cell viability reduction: Too slow or too rapid thawing processes can lead to reduced cell viability, affecting experimental results and the success of upstream processing. In addition, there are studies that advocate shaking after thawing for quality improvement.
Cell count inaccuracy: The process of thawing can result in cell loss, leading to inaccuracies in cell counts. This can impact subsequent experiments that require precise cell numbers.
Inconsistent thawing: Uncontrolled thawing within the cell suspension - with ambient passive thawing or using a waterbath - can result in variability in cell recovery and survival rates, leading to inconsistent results.
Cell resuspension challenges: Thawed cells might be difficult to resuspend uniformly, leading to uneven distribution during subculturing or experimentation.
Manual handling: the more manual intervention in handling cells the more likely is container breakages or contamination. Container closure integrity of single-use bags and bottles is better secured with robust secondary packaging during cold chain.
Trypan blue misinterpretation: Misinterpretation of trypan blue staining during viability assessment can lead to inaccurate cell viability results, impacting data interpretation.

To overcome these difficulties, careful attention to thawing protocols and best practices for specific cell types is essential. Computer-assisted, controlled thawing with automated platform systems ensures a gentle process and avoids human errors.

Safe & efficient thawing in every scale with single-use technology

Single Use Support takes the process of cell thawing to the next level – with innovative freeze-thaw platforms designed to process a wide range of cell culture volumes, from small-scale cell banking applications to large-scale production. Its level of control with single-use technologies for thawing offers numerous advantages for achieving an optimized cell recovery rate and a high cell viability while streamlining the freezing and thawing processes.

RoSS.pFTU, recommended for cells in 2D bags, and RoSS.BLST, recommended for cells in bottles, are both designed to fit into cGMP manufacturing suites, ensuring the highest quality and consistency for cell-based applications in research and medicine. In contrast to waterbath, the controlled thawing platforms bring standardization, product protection and manufacturing flexibility to the process of thawing.

By incorporating Single Use Support's freeze-thaw platforms into your cell culture workflow, operators can significantly enhance the efficiency and success of freezing and thawing processes.

[[featured-products:Our featured products]]

References

Use of liquid nitrogen during storage in a cell and tissue bank: Contamination risk and effect on the detectability of potential viral contaminants, Cryobiology. 2012 Apr;64(2):121-3. doi: 10.1016/j.cryobiol.2011.12.005. Epub 2011 Dec 28
Improving Cell Recovery: Freezing and Thawing Optimization of Induced Pluripotent Stem Cells, Cells. 2022 Feb 24;11(5):799. doi: 10.3390/cells11050799.
Trypan Blue, https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/trypan-blue, Published 2012

FAQ

What is the purpose of thawing cells?

The purpose of thawing cells is to safely recover and revive frozen cells from cryopreservation, ensuring their viability and functionality for research and cell therapy applications.

What are the differences in thawing adherent and suspension cells?

Adherent cells are typically directly seeded onto a culture dish, while suspension cells can be rapidly thawed and resuspended in a culture medium with appropriate cell density. Centrifugation is often used to collect and pellet suspension cells for downstream processing. The thawing method is tailored to each cell type to ensure optimal recovery and viability.

How do cryoprotective agents contribute to successful cell thawing?

Cryoprotective agents like DMSO and FBS protect cells during freezing by reducing ice crystal formation, preventing cell damage. During thawing, they aid in preserving cell viability, ensuring successful recovery for downstream experiments.

What equipment is needed for cell thawing?

In the conventional process with manual thawing a water bath or incubator for controlled thawing, a pipette or pipet for accurate dispensing of reagents, and cryovials or freezing containers for storing frozen cells are used. Cryoprotective agents like glycerol or dimethyl sulfoxide (DMSO) are used during freezing to protect cells from ice crystal formation. However, when using automated platform systems based on single-use technology, single-use bags are used as primary packaging of the cells and single-use shells as secondary packaging. The shells are placed in the platform system and frozen or thawed in a fully controlled manner.

Which cell types are commonly used for cell banking?

Commonly used cell types for cell banking include various mammalian cell lines, primary cells from different tissues, stem cells, and specialized cell cultures. These cells are cryopreserved to create a cell bank for future research, drug development, and medical applications.