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NC3Rs: National Centre for the Replacement Refinement & Reduction of Animals in Research

Moving to animal-free scaffolds for the construction of 3D organotypic models

In vitro scaffolds

The development of organoids, organotypics, micro-physiological systems and other 3D culture techniques allow the recapitulation of complex physiological tissues and organs in vitro. These complex in vitro models (CIVMs) facilitate the study of specific mechanisms or pathways using human tissue or cells in a more physiologically relevant environment than traditional 2D culture methods [1].

CIVMs have diverse applications in research and regulatory testing and are a key technology in moving towards increasingly human-relevant models [2]. Materials used as scaffolds for 3D cell cultures are often derived from animal cells, such as Matrigel, or directly extracted from animal tissue, such as rat tail collagen. The inclusion of animal-derived scaffolds in CIVMs limits their physiological relevance. For example, numerous growth factors, chemokines and biologically active proteins have been detected by proteome array in Matrigel that may have significant implications on cellular behaviour when cells are grown in or on the matrix [3][4].

The reproducibility of CIVMs is hindered by the batch-to-batch variability of animal-derived scaffolds, such as the numerous observations of inconsistencies between batches of Matrigel [3]. Lack of reproducibility may be a significant barrier to entry for the use of CIVMs in regulatory studies in the future.

Constructing CIVMs with scaffolds from animal-free sources such as synthetic materials, human tissue or plant tissue allows the development of highly relevant human models. The different materials available each have their own advantages that can be harnessed to optimise specific assays. For example, synthetic scaffolds have minimal batch-to-batch variability, increasing the reproducibility of CIVMs. Many animal-free scaffold materials are non-reactive and reducing the risk of immunogenic interference with human cells. Certain polymers are useful within specific disciplines due to their mechanical or biological properties, for example, alginate is often used to model bone and cartilage due to its molecular structure and compatibility with bone cells [5].

Using animal-free materials to build CIVMs will allow these models to bridge the gap between discovery and clinical research by improving human relevance and reproducibility, and support standardisation across the in vitro research landscape (see Good In Vitro Practice).

Animal-free scaffold technologies

Animal-free scaffolds appropriate for CIVMs are often either derived from existing structural proteins and polymers in nature, such as those derived from plants or fungi, or synthetically constructed. Each scaffold has specific properties that are useful for certain applications and may provide a scientific advantage over animal-derived scaffolds that are less specialised.

Available animal-free scaffolds and their specific properties

  • Recombinant polymers – polymers observed in nature that can be synthesised using recombinant technologies, for example, procollagen production using bacteria [6].
  • Micro-organism-derived scaffolds – polymers found in micro-organisms offer potential use for the construction of scaffolds due to their wide abundance and flexibility, for example, fungal chitin has been used to construct scaffolds supporting human keratinocytes [7].
  • Plant-derived scaffolds – polymers found in plants, such as cellulose and alginate, can be harvested and used to construct biocompatible scaffolds, for example, cellulose can be decellularized to generate pre-vascularised tissue engineering scaffolds [8].
  • Nanomaterials – nanomaterials can mimic natural tissue conditions, and carbon nanomaterials are useful for tissue engineering due to their biocompatibility, for example, graphene foam can be used as a scaffold to support neural stem cells [9].
  • Synthetic polymers – synthetic peptide substrates are produced to have properties for specific applications, for example, poly(ethylene glycol) (PEG) hydrogels can be used for the fabrication of bioactive scaffolds [10].
  • Human-derived scaffolds – extra-cellular matrix or scaffold components can be derived from decellularised human tissue, for example, scaffold construction using decellularised amniotic membrane [11].

Benefits of adopting animal-free scaffold technologies

There are a wide range of commercially available scaffolds of animal-free origin (see Resources) that offer significant benefits over animal-derived scaffolds.

  • Supply – clear supply chains that are not influenced by unpredictable external factors. 
  • Non-infectious – clear sources or synthetic constitutions reduces the risk of infection considerably, including reduced risk of mycoplasma contamination.
  • Tailoring – defined media can be tailored to specific cell types to avoid differentiation or other phenotypic changes.
  • Reproducibility – high level of consistency between batches.
  • Physiological relevance – only human-derived or synthetic molecules (no xenogenicity).
  • Non-immunogenic – defined media does not contain immunogenic molecules, an important consideration in cell therapy.

Case studies

We have supported research projects to develop CIVMs involving the use of animal-free scaffolds. Examples of these projects are listed below.


Further reading


  1. Kim J, Koo BK and Knoblich JA (2020). Human organoids: model systems for human biology and medicine. Nature Reviews Molecular Cell Biology 21: 571–584. doi: 10.1038/s41580-020-0259-3
  2. Batista Leite S et al. (2021). Establishing the scientific validity of complex in vitro models. EUR 30556 EN Publications Office of the European Union: Luxembourg. doi: 10.2760/376171
  3. Aisenbrey EA and Murphy WL (2020). Synthetic alternatives to Matrigel. Nature Reviews Materials 5(7): 539–551. doi: 10.1038/s41578-020-0199-8
  4. Talbot NC and Caperna TJ (2015). Proteome array identification of bioactive soluble proteins/peptides in Matrigel: relevance to stem cell responses Cytotechnology 67(5): 873–883. doi: 10.1007/s10616-014-9727-y
  5. Venkatesan J et al. (2014). Alginate coposites for bone tissue engineering: a review. International Journal of Biological Macromolecules 72: 269–281. doi: 10.1016/j.ijbiomac.2014.07.008
  6. An B, Kaplan DL and Brodsky B (2014). Engineered recombinant bacterial collagen as an alternative collagen-based biomaterial for tissue engineering. Frontiers in Chemistry 2(40). doi: 10.3389/fchem.2014.00040
  7. Narayanan KB, Zo SM and Han SS (2020). Novel biomimetic chitin-glucan polysaccharide nano/microfibrous fungal-scaffolds for tissue engineering applications. International Journal of Biological Macromolecules 149: 724–731. doi: 10.1016/j.ijbiomac.2020.01.276
  8. Gershlak JR et al. (2017). Crossing kingdoms: using decellularized plants as perfusable tissue engineering scaffolds. Biomaterials 125: 13–22. doi: 10.1016/j.biomaterials.2017.02.011
  9. Li N et al. (2013). Three-dimensional graphene foam as a biocompatible and conductive scaffold for neural stem cells. Scientific Reports 3(1604). doi: org/10.1038/srep01604
  10. Zhu J. (2010). Bioactive modification of poly(ethylene glycol) hydrogels for tissue engineering. Biomaterials 31(17): 4639–4656. doi: 10.1016/j.biomaterials.2010.02.044
  11. Ramakrishnan R et al. (2020). Human-derived scaffold components and stem cells creating immunocompatible dermal tissue ensuing regulated nonfibrotic cellular phenotypes. ACS Biomaterials Science and Engineering 6(5): 2740–2756. doi: 10.1021/acsbiomaterials.9b01961

Further opportunities to replace animal-derived products