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How can you thaw and store frozen cells for cell culture?

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Cell culture is a vital technique in bioengineering, as it allows researchers to grow and manipulate cells for various applications, such as tissue engineering, drug testing, and gene therapy. However, cell culture requires careful handling and maintenance of the cells, especially when they are frozen and stored for later use. In this article, you will learn how to thaw and store frozen cells for cell culture, and what factors to consider to ensure their viability and functionality.

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Nambili Samuel

A Certified Physician | Medical Researcher | Bioinformatic Programmer | AI Researcher | Data Scientist | Security…
Joseph Fischer
Ernesto Palacios

Docente en Tecnológico de Monterrey

1 Why freeze cells?

Freezing cells is a common practice in cell culture, as it enables long-term preservation and transportation of cells, as well as backup and quality control. Freezing cells can also reduce the risk of contamination, genetic drift, and phenotypic changes that may occur during continuous culture. However, freezing cells also exposes them to stress and damage, such as ice crystal formation, dehydration, and membrane rupture. Therefore, it is important to use proper methods and materials to freeze cells, and to thaw them quickly and gently when needed.

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Nambili Samuel

A Certified Physician | Medical Researcher | Bioinformatic Programmer | AI Researcher | Data Scientist | Security Analyst living in Turkey
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The process of thawing and storing frozen cell cultures is critical because a scientist needs to ensure a high survival rate of the living microorganisms. It is essential to employ good technique and work calmly during the thawing process. Following the instructions provided with your cell culture and reagents is crucial for optimal results during both the freezing and thawing of the cells. Be careful when transferring cells from liquid nitrogen storage to the incubator. Rapidly thaw the cells in a 37°C water bath for 1 minute, and slowly dilute the cells with pre-warmed growth medium before incubation. Additionally, follow proper aseptic protocols, and properly wearing PPEs, including a face mask or goggles, as essential precautions.

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Joseph Fischer
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In my experience DMSO may not have to be added if the proper solution developed by Cell Preservation Solutions is used. The solution uses complex liposomal proteins to protect the cell from freezing.

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Ernesto Palacios

Docente en Tecnológico de Monterrey
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La principal razón para congelar las células, es la criopreservación de las mismas para su futuro uso y estudio. Existen diversas técnicas que afectan la viabilidad de las celulas, actualmente la mas usada es la criopreservacion con DMSO al 5%, y guardarlas de manera paulatina a -80 oC

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2 How to freeze cells?

To freeze cells, you need to use a cryoprotective agent, such as dimethyl sulfoxide (DMSO) or glycerol, that can protect the cells from freezing-induced injury by reducing the amount of ice formation and osmotic stress. You also need to use a freezing container, such as a cryovial or a cryobag, that can withstand low temperatures and prevent leakage. You should label the container with the cell type, passage number, date, and any other relevant information. You should also use a freezing rate, such as 1°C per minute or a programmable freezer, that can optimize the cell survival and recovery. Finally, you should store the frozen cells in a liquid nitrogen tank or a -80°C freezer, depending on the cell type and the intended duration of storage.

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Ernesto Palacios

Docente en Tecnológico de Monterrey
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La razón principal de usar agentes crioprotectores, es evitar la formación de cristales de agua de forma intracelular, ya que estos, pueden hacer apoptosis a las células. El crioprotector actualmente mas usado es dimetilsulfóxido (DMSO) al 5%, independientemente de que sea un agente citotoxico, esta concentración mantiene la viabilidad celular al 80% en cortos periodos

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3 How to thaw cells?

To thaw cells, you need to work quickly and carefully, as the cells are most vulnerable during the thawing process. You should remove the frozen container from the storage and immerse it in a water bath at 37°C, shaking it gently until the contents are completely thawed. You should then transfer the thawed cells to a sterile tube or flask, and dilute them with pre-warmed culture medium to remove the cryoprotective agent and any debris. You should then centrifuge the cells and resuspend them in fresh culture medium, and transfer them to a culture vessel with appropriate surface area and volume. You should then incubate the cells at 37°C with 5% CO2 and monitor their morphology, density, and growth.

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Ernesto Palacios

Docente en Tecnológico de Monterrey
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Existen diferentes opiniones sobre el descongelamiento celular, donde algunos autores mencionan descongelarlo de forma rápida, para evitar que los cristales de agua formados perforen la pared celular, mientras que los que mencionan que se realice de forma lenta, dicen que los cristales se descongelarían antes de perforar las paredes. La manera en la que yo realizo esta técnica, es de forma rápida, ya que entre mas tiempo de contacto con el DMSO, va a generar una solución citotoxica.

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4 How to store cells?

To store cells, you need to consider the cell type, the culture conditions, and the purpose of the culture. Some cells, such as primary cells or stem cells, may have limited lifespan or differentiation potential, and may require frequent subculturing or passaging to maintain their viability and functionality. Other cells, such as immortalized cells or cell lines, may have unlimited growth capacity or stable characteristics, and may require less frequent subculturing or passaging. You should also follow the manufacturer's or supplier's recommendations for the optimal culture medium, supplements, and additives for your cells, and change the medium regularly to provide nutrients and remove waste. You should also check the cells for contamination, such as bacteria, fungi, or mycoplasma, and use sterile techniques and equipment to prevent cross-contamination.

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5 How to optimize cell culture?

To optimize cell culture, you need to evaluate the quality and performance of your cells, and adjust the culture parameters accordingly. You should use appropriate methods and instruments to measure the cell number, viability, morphology, and function, such as a hemocytometer, a trypan blue dye, a microscope, or a flow cytometer. You should also use suitable assays and markers to assess the cell activity, differentiation, or expression, such as a MTT assay, a fluorescent dye, or a PCR. You should also compare the results with the expected or desired outcomes, and troubleshoot any problems or issues that may arise, such as low viability, poor growth, or inconsistent results.

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6 How to learn more about cell culture?

To learn more about cell culture, you can access various resources and platforms that can provide you with more information, guidance, and support. You can read books, journals, or online articles that can cover the basic principles and techniques of cell culture, as well as the latest advances and applications. You can also watch videos, webinars, or podcasts that can demonstrate the practical aspects and tips of cell culture, as well as the common challenges and solutions. You can also join forums, groups, or networks that can connect you with other cell culture enthusiasts or experts, and allow you to exchange questions, answers, or feedback.

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Ernesto Palacios

Docente en Tecnológico de Monterrey
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La principal fuente de conocimiento sobre el area, son los libros, los libros guía de thermo y artículos científicos sobre las técnicas especializadas. Pero donde se genera mas conocimiento es mediante la practica.

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7 Here’s what else to consider

This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?

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How can you thaw and store frozen cells for cell culture?

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1 Why freeze cells?

2 How to freeze cells?

3 How to thaw cells?

4 How to store cells?

5 How to optimize cell culture?

6 How to learn more about cell culture?

7 Here’s what else to consider

Bioengineering

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