Animal-free cell culture

We would like to highlight animal-free alternatives to traditional cell culture components with a focus on serum-free media, 3D-cell culture, and alternatives for detachment and freezing of cells.

Cell cultures as in vitro models often replace animal experiments; however, they are usually not entirely animal-free. Many components used in a traditional cell culture to maintain cell growth are derived from animals and sometimes animal experimentation is required to produce cell culture components. Here we highlight animal-free cell culture components and useful resources for serum-free media, 3D-scaffolds and components for handling of cells in vitro.

Serum-free media

Fetal calf serum (FCS) was initially introduced to cell culture in the late 1950’s as a way to keep cells viable and promote proliferation outside of an organism, in a cell culture. FCS is derived from unborn calves, including mature fetuses in late stages of gestation, through direct heart-puncture without anaesthetics – a procedure that raises ethical questions. Use of fetal calf serum in cultivation of human cells has been long debated, since it introduces uncharacterised and foreign animal components to the human cell culture. Another disadvantage is batch-to-batch variation that can result in reproducibility issues. Thus, chemically defined media with known components have been developed. Some of them can be reproduced in the lab setting (components are defined and published) whereas others can be purchased (components are defined, but not published open source).

Defined serum-free media

Defined serum-free media have a known chemical composition and their formulations are available through publications along with information about which type of cells they can be used for. Sometimes these media are defined as xeno-free serum-free media, which means that they are free from “foreign” components and comprised of human-derived components only. Xeno-free media are especially important for clinical applications.

Attempts to find a medium composition that is serum-free are dated back to the 1960’s, less than a decade after serum was introduced to cell culture. First publication by Ham on clonal growth of mammalian cells in a chemically defined medium was released in 1965. Since then, numerous articles have been published where different cell types are cultured in defined media of various composition. One thing in common for many FCS-free media is that they contain human platelet lysate (hPL). The main advantage of hPL is that it is of human origin, improves human cell culture and replaces animal-derived serum. However, the nature of hPL is still not fully understood and batch-to-batch variability might affect cell cultures. Here we summarize both media with hPL and serum-free media that are fully defined and do not contain hPL.

Where can I find serum-free media recipes?

Here is our selection of recent and original papers with the disclosed composition of serum-free media along with the cell type and cell culture they are suitable for.

Publications with serum-free media composition

Cell type



Breast cancer cells (CAFs, JIMT-1, MDA-MB-231)

Pancreatic cancer cells (MiaPaCa-2)

Human colon cancer cells (CaCo-2)

Human keratinocytes

Rafnsdóttir et al. (2023)

A new animal product free defined medium for 2D and 3D culturing of normal and cancer cells to study cell proliferation and migration as well as dose response to chemical treatment.

- Full recipe available!

- Universal cell culture medium for both normal and cancer cell lines.

- For use in both 2D and 3D culture (spheroids).

- Fully defined, NO human platelet lysate (hPL).

Human pluripotent stem cells

Liu et al. (2021) Chemically defined and xeno-free culture condition for human extended pluripotent stem cells.

- Full recipe available!

- Chemically defined and xeno-free culture system for maintaining and deriving human extended pluripotent stem (EPS cells).

Human induced pluripotent stem cells (iPS)

Saleh et al. (2021)Xeno-free cultivation of human induced pluripotent stem cells for clinical applications. Methods in iPSC Technology.

- Use hPL in media.

- Table of references to xeno-free cultivation of iPS cells.

Human induced pluripotent stem (iPS) cell differentiation to authentic macrophages

Vaughan-Jackson et al. (2021) Differentiation of human induced pluripotent stem cells to authentic macrophages using a defined, serum-free, open-source medium.

- Full recipe available!

- Defined, serum-free, open-source medium for the differentiation of iPSC-derived macrophages (OXM medium).

- This medium is compared with commercial alternatives.

Human mesenchymal cells and fibroblasts

Figueroa-Valdes et al. (2021)A chemically defined, xeno- and blood-free culture medium sustains increased production of small extracellular vesicles from mesenchymal stem cells.

- Full recipe available!

- Based on Dulbecco’s modified Eagle’s medium (DMEM), high glucose and includes hPL.

- Media tested for exosome production.

Mesenchymal stromal cells

Wu et al (2016)Serum and xeno-free, chemically defined, no-plate-coating-based culture system for mesenchymal stromal cells from the umbilical cord.

- Full recipe available!

- Defined media based on Iscove's modified Dulbecco's medium and includes recombinant hPL-derived growth factor.

Mesenchymal stromal cells

Tozetti et al. (2017) Expansion strategies for human mesenchymal stromal cells culture under xeno-free conditions.

- Human serum (AB HS) from human plasma for culture medium supplementation during expansion and cryopreservation to enable a xeno-free bioprocess.

Human retinal pigment epithelium (fRPE)

Shen et al. (2019) A novel xeno-free culture system for human retinal pigment epithelium cells.

- Full recipe available!

- Human AB serum-supplemented medium based on modification of Minimum Essential Medium (MEM).

Human hepatic in vitro line (HepaRG)

Klein et al. (2013) Long-term maintenance of HepaRG cells in serum-free conditions and application in a repeated dose study.

- Full recipe available!

- Comparison study of culturing of human hepatoma cell line HepaRG in standard serum-supplemented and serum-free conditions.

Media for patient derived tumor cells, carcinoma cell lines.

Ince et al. (2007) Transformation of different human breast epithelial cell types leads to distinct tumor phenotypes.

- Full recipe available!

- Based on F12 and M199 media and called WIT.

- Comparison between new culture medium WIT and standard one.

Breast cancer cell line (MCF-7)

Jensen et al. (2003) Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium.

- Full recipe available!

- Chemically defined medium without serum and estrogen.

- Human breast cancer cell line MCF-7/S9 used as a model system.

Breast cancer cell line (MCF-7)

Beaupain et al. (1993) Continuous three-dimensional cultures of MCF-7 cells in serum free medium.

- Full recipe available!

- MCF-7 cells cultured and differentiated in serum-free 3D nodules.

HeLa cells

Sato and Hutchings (1978). Growth and maintenance of HeLa cells in serum-free medium supplemented with hormones.

- Full recipe available!

- Based on serum-free Ham's F12 medium.

- Evaluated on HeLa as a model system.

Adipose-derived stromal cells (ASC)

Naaijkens et al. (2012) Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications.

- Study that evaluates effects of human platelet lysate (hPL) on the cell culture.

Human lymphoblastoid cell line

Minamoto et al. (1991)Development of a serum-free and heat-sterlizable medium and continuous high-density cell culture.

- Full recipe available!

- Recipe for new serum-free and heat-sterilizable medium.

Additional defined serum-free media (non-open source)

Commercially available serum-free media that are chemically-defined, but of which the composition is unavailable (non-open source) are often referred to as proprietary media. These media are on the market through various companies and as the field is expanding more and more become available. There are also many recent publications where commercially available serum-free media are being used and evaluated for different applications.

Where can I find commercially available serum-free media?

There are several resources available that can help you find the right media.

1) A database initiated by the 3Rs Centre at Utrecht University, the Fetal Calf Serum-Free Database. This database contains information about media for a large variety of cell lines of both human and animal origin.

2) A very useful resource that provides a list of companies that offer human-derived or synthetic serum substitutes and xeno-free serum-free media is available at NC3Rs. The products available are usually intended for specific cell types or tissues and for low-scale to large-scale usage.

3) There are publications where commercially available serum-free media are evaluated through use in different cell cultures. Here are some publications describing the use of serum-free proprietary media.

Publications evaluating the use of serum-free proprietary media

Cell line


Human iPS cells and cell lines

Hua et al. (2022) Development and evaluation of a novel xeno-free culture medium for human-induced pluripotent stem cells.

Human embryonic stem cells (hESC)

Rajala et al. (2007)Testing of nine different xeno-free culture media for human embryonic stem cell cultures.

Human mesenchymal stem cells

Tozetti et al. (2017) Expansion strategies for human mesenchymal stromal cells culture under xeno-free conditions.

Human mesenchymal stem cells

Meuleman et al. (2006) Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical a-MEM medium.

Ovarian cancer cell lines

Ince et al. (2015) Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours.

A549 human lung cancer, MCF7 human breast cancer, human fibroblasts, foreskin cells

Usta et al. (2014) Chemically defined serum-free and xeno-free media for multiple cell lineages.

Human dental pulp cells

Fujii et al. (2018)Characterization of human dental pulp cells grown in chemically defined serum‑free medium.

Human gingival fibroblasts, oral cancer cells and mesothelioma cells

Tsugeno et al. (2014) Cell culture of human gingival fibroblasts, oral cancer cells and mesothelioma cells with serum-free media.

A549 human lung cancer cells

Chari et al. (2022) Maximizing the relevance and reproducibility of A549 cell culture using FBS-free media.

Human adipose stem cells

Patrikoski et al. (2013) Development of fully defined xeno-free culture system for the preparation and propagation of cell therapy-compliant human adipose stem cells.

Animal-free 3D-scaffolds

Since 3D-culture support components emerged, they have usually been based on constituents of animal origin. A well-known example is a solubilized basement membrane matrix rich in extracellular protein, with the tradename Matrigel. Matrigel is produced by injecting mice with by Engelbreth-Holm-Swarm (EHS) sarcoma cells which secrete the extracellular matrix. Essentially, Matrigel is an extract of the mouse sarcomas. Extracellular matrix started to be applied to cell cultures in the 1970-80s to facilitate cell attachment and to enable cell growth in a 3D environment. Main drawbacks of Matrigel are its poorly defined composition, batch-to-batch variations and the ethical issue of using animals.

Since then, a variety of synthetic alternatives have emerged that provide a 3D environment for culturing of cells and organoids. These novel components include different chemically derived scaffolds, biomaterials and hydrogels. Based on rigidity of the scaffold they can be categorised as solid scaffolds (fibers and extracellular matrix proteins) and soft scaffolds (hydrogels).

Where can I find animal-free 3D-scaffolds?

1) Here are some published examples and overviews of recent advances in animal-free 3D components. There are many more products available as the field is constantly expanding.

Recent advances in animal-free 3D-scaffold components

3D-scaffold components


Matrigel alternatives (commercial and non-commercial)

Aisenbrey and Murphy (2020) Synthetic alternatives to Matrigel.

Overview of different 3D-scaffold components

Duarte et al. (2023) Animal-derived products in science and current alternatives.

Hydrogels for organoids (review)

Kim et al. (2022) Tissue extracellular matrix hydrogels as alternatives to Matrigel for culturing gastrointestinal organoids.

Plant-derived hydrogels (review)

Andersen et al. (2015) 3D Cell Culture in Alginate Hydrogels.

Polycaprolacone-based scaffold

Rafnsdóttir et al. (2023) A new animal product free defined medium for 2D and 3D culturing of normal and cancer cells to study cell proliferation and migration as well as dose response to chemical treatment.

Recombinant spider-silk proteins

Collodet et al. (2023) Development and characterization of a recombinant silk network for 3D culture of immortalized and fresh tumor-derived breast cancer cells.

Hjelm et al. (2023)

In vitro Blood–Brain barrier model based on recombinant spider silk protein nanomembranes for evaluation of transcytosis capability of biomolecules.

Hirabae De Oliveira et al. (2023) Silk Assembly against Hydrophobic Surfaces─Modeling and Imaging of Formation of Nanofibrils.

Examples of different commercially available hydrogels

VitroGel® Hydrogel Matrix is a xeno-free hydrogel system that mimics extracellular matrix (ECM).

GrowDex® Hydrogel extracted from birch and sourced from forests.

PeptiGels® fully synthetic peptide hydrogels for 2D and 3D Cell Culture.

3D Bioimprinting components for seeding cells into 3D hydrogels.

Examples of different commercially available solid 3D-scaffolds

ECMatrix™ Laminin substrates for human pluripotent stem cells based on laminin protein.

Bio-Spun™ Scaffolds - synthetic cellular 3D matrices.

Cellevat3d NanoMatrice™ nanofiber-based platform that resembles ECM.

HumaMatrix is a native, human-derived ECM.

2) Another very useful resource that provides detailed classification of animal-free scaffolds and a list of biotech companies that offer animal-free scaffolds is available at NC3Rs.

Animal-free components for handling of cells in vitro

Cells growing in a cell culture need to be regularly handled through detachment and attachment cycles and also cryopreserved through freezing. Even cell handling relies on components that often are of animal origin. Here is our guide to animal-free components used for handling of cells.

Animal-free products for cell detachment

In order to detach cells from cell culture dishes, a specific enzyme is usually added to the cells, for example trypsin. Trypsin is a biproduct from the food industry and is derived from pigs. However, there are many animal-free alternatives to animal-derived enzymes commercially available. These include both recombinant enzymes produced in corn or completely enzyme-free alternatives for example composed of a mixture of chelators. These alternatives are often more gentle to cells and allow them to re-attach quicker to a new surface.

Resources to support the use of animal-free enzyme technologies and replace animal-derived enzymes and cells for in vitro research can be found through NC3Rs.

Non-enzymatic cell dissociation solutions are also available from ATCC (American Type Culture Collection) - a non-profit, global biological resource centre.

Animal-free products for cryopreservation of cells

Fetal calf serum is usually used as a component in a culture medium for cryopreservation of cells. However, there are many serum-free alternatives available for this purpose.

Here is a selection of publications describing the use of serum-free, xeno-free alternatives for cryopreservation of human cells. There are as well various non-animal origin freezing media available from different commercial manufacturers.

Serum-free alternatives for cryopreservation of human cells

Cell cryopreservation component


Cryopreservation in defined medium with 10 % human serum and 5-10 % DMSO

Rafnsdóttir et al. (2023)A new animal product free defined medium for 2D and 3D culturing of normal and cancer cells to study cell proliferation and migration as well as dose response to chemical treatment.

Xeno-free chemically defined cryopreservation medium

Miki et al. (2016) Biological impact of xeno-free chemically defined cryopreservation medium on amniotic epithelial cells.

Biological serum-free cryopreservation

Corsini et al. (2004)Biological Serum-Free Cryopreservation of Five Mammalian Cell Lines in Either a Pelleted or Suspended State.

Ecotin as a serum-free alternative to dimethylsulfoxide

Grein et al. (2010)

Alternatives to dimethylsulfoxide for serum-free cryopreservation of human mesenchymal stem cells.

Revision date: 2024-04-04

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