Optimizing cryopreservation: Serum-free cell freezing medium

Cryopreservation is an integral part of biomedical research and clinical applications. However, maintaining cell viability and function during freezing and thawing can be challenging. Serum-free cryopreservation media formulations are advanced solutions that can be used instead of serum-containing freezing media to maintain cell function while ensuring reproducibility and standardization — read on to learn why.

What is a serum-free cell freezing medium?

Serum-free cell freezing medium is a ready-to-use solution for freezing cells that does not contain serum, proteins, or other ingredients of animal origin. Unlike traditional freezing media that rely on serum — usually fetal bovine serum — serum-free formulations eliminate the need for animal-derived components.1 Instead, they harness synthetic or plant-based substitutes to protect cells during freezing and thawing. Common ingredients of serum-free cryopreservation media include dimethyl sulfoxide (DMSO) and methylcellulose.1

Why use a serum-free cell freezing medium?

A key reason for using serum-free cell freezing media is to minimize the risk of contamination and reduce batch-to-batch variability. The composition and quality of serum may differ considerably from batch to batch.2 In addition, mixing cells with traditional freezing media can introduce bioactive factors, antibodies, or toxins from the animal serum.3 These factors can have unpredictable effects on the physiology of the cells and influence the results of downstream assays.2,3

Advantages of serum-free cell freezing formulations

In today’s fast-paced world of cell research, the choice of cell freezing medium can have a significant impact on the success of experiments and the integrity of cells.3 Serum-free cryopreservation media can overcome various limitations of traditional freezing media (Figure 1). Unlike traditional cryomedia, serum-free cell freezing solutions provide:

  • Low risk of contamination: Fetal bovine serum (FBS) or other animal-derived serum components used in traditional cryopreservation media can contaminate cells with viruses, mycoplasma, or other pathogens.4 By eliminating animal-derived products, serum-free formulations minimize the risk of contamination.5
  • High reproducibility and standardization: The composition of serum can vary between batches, leading to inconsistencies in results.2 Serum-free media allow for better control and standardization of the cryopreservation process.6
  • Defined composition: Compared to serum-containing media, serum-free cryopreservation formulations have a more defined chemical composition. By offering precise control over the cellular microenvironment, serum-free media can lead to enhanced and predictable cell performance after thawing.
  • Enhanced cell recovery: Serum-free cryopreservation media can lead to better cell recovery after thawing than traditional serum-containing media because they provide an optimized environment for cell survival.7,8 This is particularly important when working with sensitive cell types or valuable primary cell samples.
  • Low immunogenicity: Inflammatory cytokines in the animal serum can trigger immune responses in cultured cells.2 By eliminating serum-derived immunological triggers in cell cultures, serum-free solutions improve cell recovery rates and overall experimental success.
  • More ethical approach: Serum-free cell freezing media overcome the need to use animals for serum and other animal-derived products. This offers a more humane and ethical alternative to traditional cryopreservation media.9
Key advantages of serum-free medium over traditional methods, including reduced risk of contamination, low immunogenicity, and no animal use.
Figure 1: Differences between serum-containing and serum-free freezing media.
The scheme illustrates the key benefits of using serum-free cryopreservation media over traditional methods.

Cell type compatibility

From primary cells to immortalized cell lines, serum-free cryomedia are designed to support the cryopreservation of a wide range of cell types.1 In addition to established cell lines commonly used in research, such as HEK293, HeLa, NIH/3T3, serum-free cell freezing formulations are ideal for the following sensitive cell types:

  • Primary cells: Primary human and animal cells, including fibroblasts, epithelial cells, and endothelial cells, are particularly susceptible to changes in their environment.10 The defined composition and enhanced performance of serum-free media ensure optimal preservation of primary cells.
  • Stem cells: Maintaining the properties of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and neuronal progenitors during cryopreservation is critical for the success of downstream applications.11,12
  • Specialized cells: Cells derived from specific tissues or organs, including neurons, cardiomyocytes, and hepatocytes display a notable responsiveness to fluctuations in their environment.13 Cryopreservation in a defined and controlled environment can be key to their survival during long-term storage and their functionality in post-thawing assays.10

Diverse applications of serum-free cryopreservation media

The broad compatibility of serum-free cryomedia underscores their applicability across diverse research domains and clinical applications.7 This ensures that researchers can preserve the integrity of their precious cell populations with confidence. The advanced performance of serum-free cryopreservation media makes them ideal for various therapeutic and translational research applications:

  • Regenerative medicine: Stem cells, engineered tissues, and organoids benefit from the defined composition and enhanced performance of serum-free cell freezing media. This ensures optimal viability and maintenance of cell functionality during long-term storage.14
  • Drug discovery: In drug research and development, precision and robustness are of utmost importance. Serum-free formulations ensure that cells used in drug-screening assays maintain their integrity, without interference from serum-derived factors.15
  • Cell-based therapies: From CAR-T cells to gene therapies, serum-free cell freezing media support the development of advanced cell-based therapies.16 These therapies rely on robust, reproducible cell banks, and serum-free media deliver just that.

Innovations and emerging trends in cell freezing

Maintaining viable, healthy cells after long-term cryogenic storage is critical but challenging. Innovations in cell freezing include novel cryoprotectants, improved freeze-thaw protocols, automated high-throughput cryopreservation systems, and optimized freezing media.14,17

A notable trend is the increasing use of serum-free media to ensure reproducibility, regulatory compliance, and ethical standards. Serum-free cryopreservation media offer an ethical and clinically relevant approach that minimizes immune responses and preserves cell functionality for therapeutic applications.

In addition, the integration of antioxidants into cryopreservation methods is emerging as a promising approach to counteract oxidative stress and improve cell viability and functionality after thawing.18 The combination of serum-free cryopreservation formulations with antioxidants represents a significant advance in cryopreservation, especially for cells that are sensitive to oxidative stress.19

These emerging trends in cryopreservation ensure improved cell recovery and function while remaining consistent with ethical practices and hold promise in advancing research and clinical applications of cryopreserved cells.

Cryo-SFM Plus: our new animal component-free cryomedium

Cryo-SFM Plus allows standardization of freezing protocols while reducing variability in downstream assays by offering a chemically defined formula free of proteins and animal components.

 Key characteristics of Cryo-SFM Plus:

  • Defined composition: With Cryo-SFM Plus, you obtain a precisely defined formulation for consistent results. No more guesswork — just reproducible outcomes every time.
  • Protein-free: Ensure a standardized and controlled culture environment for precious cell samples. Cryo-SFM Plus is free from proteins, eliminating unwanted variability.
  • Animal component-free: Enjoy a regulatory-friendly format that gives you peace of mind. Having no animal-derived components means compliance without compromise.
  • Antioxidant-enhanced: The innovative antioxidant technology preserves cell viability, attachment, and growth.

Key benefits of Cryo-SFM Plus:

  • Optimal cell yield: Experience maximum cell recovery after thawing. Cryo-SFM Plus ensures that your experiments proceed smoothly, whether you’re working with primary cells, stem cells, or established cell lines.
  • Minimal freeze damage: Protect your valuable cell samples from damage during freezing and thawing. Cryo-SFM Plus minimizes freeze-induced stress, preserving cell integrity.
  • Versatile: Cryo-SFM Plus adapts to your research needs and is suitable for various cell types — from delicate stem cells to established lines.
  • Powered by patented technology: Rest assured with our patented* technology. Cryo-SFM Plus is backed by rigorous research and innovation, ensuring top-tier performance.

*Patent issued in Germany and pending approval in other countries.


  1. Campbell LH, Brockbank KGM. Serum-free solutions for cryopreservation of cells. In Vitro Cell Dev Biol Anim. 2007;43(8-9):269-275. doi:10.1007/s11626-007-9039-z
  2. Liu S, Yang W, Li Y, Sun C. Fetal bovine serum, an important factor affecting the reproducibility of cell experiments. Sci Rep. 2023;13(1):1942. doi:10.1038/s41598-023-29060-7
  3. Jang TH, Park SC, Yang JH, et al. Cryopreservation and its clinical applications. Integr Med Res. 2017;6(1):12-18. doi:10.1016/j.imr.2016.12.001
  4. Pilgrim CR, McCahill KA, Rops JG, Dufour JM, Russell KA, Koch TG. A review of fetal bovine serum in the culture of mesenchymal stromal cells and potential alternatives for veterinary medicine. Front Vet Sci. 2022;9:859025. doi:10.3389/fvets.2022.859025
  5. Duarte AC, Costa EC, Filipe HAL, et al. Animal-derived products in science and current alternatives. Biomater Adv. 2023;151:213428. doi:10.1016/j.bioadv.2023.213428
  6. Germann A, Schulz JC, Kemp-Kamke B, Zimmermann H, von Briesen H. Standardized serum-free cryomedia maintain peripheral blood mononuclear cell viability, recovery, and antigen-specific T-cell response compared to fetal calf serum-based medium. Biopreserv Biobank. 2011;9(3):229-236. doi:10.1089/bio.2010.0033
  7. Filbert H, Attig S, Bidmon N, et al. Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols. Cancer Immunol Immunother. 2013;62(4):615-627. doi:10.1007/s00262-012-1359-5
  8. Kaiser D, Otto NM, McCallion O, et al. Freezing medium containing 5% DMSO enhances the cell viability and recovery rate after cryopreservation of regulatory T cell products ex vivo and in vivo. Front cell Dev Biol. 2021;9:750286. doi:10.3389/fcell.2021.750286
  9. Merten OW. Safety issues of animal products used in serum-free media. Dev Biol Stand. 1999;99:167-180.
  10. Meneghel J, Kilbride P, Morris GJ. Cryopreservation as a key element in the successful delivery of cell-based therapies-A review. Front Med. 2020;7:592242. doi:10.3389/fmed.2020.592242
  11. Kent L. Freezing and thawing human embryonic stem cells. J Vis Exp. 2009;(34). doi:10.3791/1555
  12. Alasmar S, Huang J, Chopra K, et al. Improved cryopreservation of human induced pluripotent stem cell (iPSC) and iPSC-derived neurons using ice-recrystallization inhibitors. Stem Cells. 2023;41(11):1006-1021. doi:10.1093/stmcls/sxad059
  13. Kolaja K. Stem cells and stem cell-derived tissues and their use in safety assessment. J Biol Chem. 2014;289(8):4555-4561. doi:10.1074/jbc.R113.481028
  14. Uhrig M, Ezquer F, Ezquer M. Improving cell recovery: freezing and thawing optimization of induced pluripotent stem cells. Cells. 2022;11(5). doi:10.3390/cells11050799
  15. Kamiloglu S, Sari G, Ozdal T, Capanoglu E. Guidelines for cell viability assays. Food Front. 2020;1(3):332-349. doi:https://doi.org/10.1002/fft2.44
  16. Cui Y, Nash AM, Castillo B, et al. Development of serum-free media for cryopreservation of hydrogel encapsulated cell-based therapeutics. Cell Mol Bioeng. 2022;15(5):425-437. doi:10.1007/s12195-022-00739-7
  17. Cottle C, Porter AP, Lipat A, et al. Impact of cryopreservation and freeze-thawing on therapeutic properties of mesenchymal stromal/stem cells and other common cellular therapeutics. Curr stem cell reports. 2022;8(2):72-92. doi:10.1007/s40778-022-00212-1
  18. Len JS, Koh WSD, Tan S-X. The roles of reactive oxygen species and antioxidants in cryopreservation. Biosci Rep. 2019;39(8). doi:10.1042/BSR20191601
  19. Lee J-H, Park H-J, Kim Y-A, et al. Establishment of a serum-free hepatocyte cryopreservation process for the development of an “off-the-shelf” bioartificial liver system. Bioeng (Basel, Switzerland). 2022;9(12). doi:10.3390/bioengineering9120738


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