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Goodbye Matrigel? Insights From Organ-On-A-Chip Liver Model Using Animal-Free Hydrogels

  • Writer: Mikayla Shelton
    Mikayla Shelton
  • 12 minutes ago
  • 8 min read

Introduction

Every year, millions of animals contribute - often invisibly - to biomedical research, with 2.64 million scientific procedures carried out in Great Britain alone in 2024 [1]. However, even when no direct animal testing is involved, products like extracellular matrix (ECM) extracts are typically harvested from animal tissue to be used for a wide variety of in vitro research methods. For decades, scientists have depended on Matrigel® as the go-to hydrogel for 3D cell culture, a basement membrane extract derived from mouse tumours, containing a vast assortment of proteins and bioactive molecules that can give rise to major drawbacks, including batch variability, poor reproducibility, and ethical concerns [2].


With the growing push to reduce animal-derived materials in labs, increasing numbers of researchers are striving to follow the 3Rs principle (Replace, Reduce, Refine) in their field, asking the question: can we build a better, cruelty-free matrix that is still scientifically robust? A recent study by Nitsche et al. does just that, by investigating animal free hydrogels using a HepaRG™ liver cell line as a model under both static (standard culture) and dynamic (organ-on-a-chip) conditions [3]. This blog post provides an independent summary of this research paper, titled "Alternatives to animal-derived extracellular matrix hydrogels? An explorative study with HepaRG cells in animal-free hydrogels under static and dynamic culture conditions".


This paper includes the use of our PeptiMatrix™ hydrogels, however we are not affiliated with the authors or their research institutions. A link to this paper is included in the reference list below and we encourage everyone to read the original article too.



Paper aims and methods

The researchers took a three-step approach to their overarching aim of determining whether animal-free hydrogel alternatives could support HepaRG cells under static and dynamic culture conditions (Figure 1) compared to Matrigel.


  1. They first reviewed the literature to identify commercially manufactured animal-free ECM alternatives, including synthetic peptide hydrogels PeptiMatrix and PuraMatrix™, synthetic polysaccharide hydrogel VitroGel® Organoid-3, and wood-based polysaccharide hydrogel GrowDex®. Unlike Matrigel, these synthetic and natural hydrogels have the advantage of controlling mechanical and biological properties by adjustment of stiffness, porosity, and controlled addition of bioactive molecules, promising better reproducibility and human relevance.

  2. The researchers then pre-screened each hydrogel in 48-well plates or a microphysiological system (MPS) device to test whether they supported HepaRG growth.

  3. Finally, the team differentiated the cells into metabolically active liver cells, and measured characteristic changes under static (96-well) and dynamic culture (MPS), such as viability, albumin and bile acid production, CYP3A4 enzyme activity, quantitative expression analysis of multiple target genes, and immunostaining. Results showed all animal-free hydrogels tested supported HepaRG cell proliferation in both static and dynamic conditions, and cells grown in PeptiMatrix 7.5 showed promising metabolic competence under perfusion in the MPS device, discussed in more detail below.


HepaRG culture setups under static and dynamic conditions.
Figure 1: HepaRG™ culture setups under static and dynamic conditions. (A) Diagram of static culture conditions, where HepaRG™ cells were encapsulated in each hydrogel, and seeded into 48 or 96-well plates as cell-gel suspension droplets and grown for 7-16 days. (B) Schematic of OrganoPlate® 3 MPS device, consisting of two perfusion channels (top and bottom) and a central ECM channel, separated by their PhaseGuide™ technology. (C) Diagram of dynamic culture condition in MPS device, where HepaRG™ cells were suspended in hydrogel then taken up into the ECM channel via capillary action and grown for 9 days. (Adapted from Nitsche et al., 2025).

Static culture key findings

Static culture pre-screening results demonstrated biocompatibility in all animal-free hydrogels tested, with viable and growing cell clusters over the 7-day culture (Figure 2 in paper). The highest cell viability and lowest LDH release was consistently observed in the gel-free culture compared to all hydrogels tested, whilst VitroGel displayed the lowest viability and highest LDH leakage (Figure 4A + 4B in paper).  The Matrigel–collagen culture was the only condition had no change in viability throughout the entire culture period, suggesting no proliferation, but instead maturation into hepatocyte-like cells. Whilst GrowDex showed the highest initial viability out of the animal-free hydrogels, this was hypothesised to be due to the high gel viscosity causing gel retention in the pipette tip, resulting in a higher initial seeding density than the other conditions.


Production of key liver marker albumin and CYP3A4 enzyme activity (a cytochrome P450 enzyme important for drug metabolism) were also assessed, where they found a significant increase in albumin production in only the gel-free and PeptiMatrix 7.5 conditions at day 14, before both declining to near baseline levels at day 16 (Figure 4C in paper). The Matrigel-collagen condition induced the highest increase in CYP3A4 levels in Rifampicin treated cells compared to the vehicle control, and a small increase was observed in the gel-free culture (Figure 4D in paper). Whilst there was an increase in CYP3A4 activity in PeptiMatrix 7.5, it did not reach significance, no significant increase was observed in any of the animal-free hydrogel conditions.


Quantification of primary (conjugated) bile acid secretion in the pooled cell-culture medium of statically grown HepaRG cells found that all hydrogel conditions enabled secretion of bile acids (cholic acid, glycholic acid, taurocholic acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid) except for GrowDex, although the gel-free condition exhibited the highest total bile acid secretion, of almost double (Figure 6 in paper). Analysis of gene expression of six different characteristic differentiation genes (Albumin, CYP3A4, CYP27A1, CYP7B1, KRT18, and KRT19) found expression profiles were similar across the different animal-free hydrogels and Matrigel-collagen (Figure 7A in paper), except VitroGel exhibiting reduced albumin gene expression.


Key findings from static culture conditions.
Figure 2: Key findings from static culture conditions. (A) Schematic overview of static cell culture condition timeline. (B) Cellular membrane damage quantified as leaked LDH activity as an analysis of cell health. (C) CYP3A4 enzyme activity under treatment with either vehicle control (DMSO) or rifampicin. (D) Quantification of primary (conjugated) bile acid secretion in the pooled cell-culture medium on day 14. (E) Fold changes in gene expression after 16 days of culture.  (Adapted from Nitsche et al., 2025).

Dynamic culture key findings

Microphysiological system (MPS) devices are advanced in vitro models on a miniature scale, where the dynamic culture conditions (a combination of the 3D architecture and flow) can impact important cell signalling pathways and influence cell mechanobiology to better mimic human organ and tissue function [4]. A closed MPS device with physical barriers separating the compartments was used, the OrganoPlate® 3-lane device (MIMETAS 4004B-400B, Leiden, Netherlands), containing two perfusion channels and one ECM channel, separated by microfabricated passive barriers. The researchers first tested the ability of all the hydrogels to be absorbed into the MPS channel via capillary force before undertaking the dynamic experiments. PuraMatrix was excluded due to the practicality issues of requiring multiple washing steps, and all recommended working concentrations of GrowDex were not absorbed by the ECM channel, so were not suitable. VitroGel and PeptiMatrix 5 and PeptiMatrix 7.5 were efficiently absorbed into the chip channel, making them suitable candidates for further functional tests.


When HepaRG cells were seeded in gel suspension onto the MPS device for in situ differentiation, brightfield images showed that all tested hydrogels facilitated cell distribution and migration across the entire chip 24h post-seed. However, VitroGel demonstrated presence of detached and curled cells, with fewer attached at day 7 compared to other conditions (Figure 3B in paper). Cells accumulated at the top and bottom of chip channels, protected from the shear flow of the media between the PhaseGuides™. On-chip immunofluorescent staining showed expression of CK18 (hepatocyte-like marker) but not CK19 (cholangiocyte-like marker) for all tested hydrogels (Figure 3C in paper).


In terms of cell viability, VitroGel displayed the largest increase, with a moderate significant increase in PeptiMatrix 5 and PeptiMatri 7.5, whilst the cells in Matrigel-collagen maintained a stable viability, suggesting hepatocyte-like cell maturation (Figure 5A in paper). LDH leakage was not detected in Matrigel-collagen, unlike the other hydrogels, however by day 7, the cells in PeptiMatrix and VitroGel showed a reduction in LDH release, suggesting cell adaption (Figure 5B in paper). To further assess the cell maturation status, albumin secretion and CYP3A4 activity were also analysed (Figures 5C + 5D in paper). Cells culture in PeptiMatrix 7.5 and Matrigel-collagen were the only two conditions with significantly higher levels of albumin secretion, and also with significantly increased CYP3A4 activity when treated with Rifampicin. This novel result suggests PeptiMatrix 7.5 has the ability to mature HepaRG to induce CYP activity under flow conditions, possibly due to the enhanced mechanobiology.


Key findings from dynamic culture conditions.
Figure 3: Key findings from dynamic culture conditions. (A) Representative bright-field microscopic images of HepaRG™ cells 7 days post-seeding on the OrganoPlate® 3-lane, illustrating differences in the cell distributions and attachments. (B) Cellular membrane damage quantified as leaked LDH activity as an analysis of cell health. (C) Quantified albumin secretion as a measure of synthetic capacity. (D) CYP3A4 enzyme activity under treatment with either vehicle control (DMSO) or rifampicin. (Adapted from Nitsche et al., 2025).

Conclusions

The discussion of this explorative paper concluded that although all tested animal-free hydrogels supported viable cultures and proliferation, only PeptiMatrix 7.5 appeared to be a promising candidate for enhanced maturation support to use for metabolic studies under flow (Figure 8 in paper). Principally, the induction of key hepatic marker CYP3A4 in PeptiMatrix 7.5 culture under dynamic conditions support HepaRG maturation towards a metabolically competent phenotype, demonstrating its potential for model liver applications, that require functionality, not just viability of cells.


In summary, these novel results mark an important milestone in the transition toward animal-free 3D advanced liver models, offering independent validation that strengthens confidence in our PeptiMatrix platform. Whilst independent validation is a major milestone and a sign of growing confidence in the synthetic hydrogel platform, it is important to note that paper also discussed areas for further optimisation, such as enhancing initial cell attachment and long-term stability.


Since this study was conducted, we’ve been proactively addressing these challenges through innovations like PeptiMatrix RGD, which incorporates bioactive peptide motifs to improve cell–matrix interactions, and ongoing development of ECM protein functionalisation of the gels that allow fine-tuning of nutrient and mechanical support. These ongoing developments demonstrate our commitment to continuous improvement and our desire to establish ourselves as leaders in ethical, high-performance cell culture. As animal-free technologies move from exploration to adoption, we’re excited to collaborate and engage with researchers developing advanced liver models and organ-on-chip systems - together building the next generation of biologically relevant, animal-free research tools.


Heatmap summarising findings and comparing the tested alternatives.
Figure 4: Heatmap summarising the findings and comparing the tested alternative hydrogels with the Matrigel®–collagen and gel-free mixtures under static (*) and dynamic culture conditions. Colour grading was used to compare the application considerations and biocompatibility aspects for creating viable and metabolically competent HepaRG™ cultures. The blank regions indicate that the corresponding categories were not applicable. The factors were evaluated by (i) reviewing the characteristics described in literature, (ii) injecting hydrogels into the OrganoPlate® at different concentrations, (iii) assaying for viability and proliferation, (iv) staining for cell distribution and on-chip population, as well as (v) measuring the basal functions after in situ differentiation under both static and dynamic conditions. (Taken from Nitsche et al., 2025).

Disclaimer

This post reflects the perspective of the PeptiMatrix™ team on the published work by Nitsche et al. (2025) and does not represent the views of the study authors or their institutions. PeptiMatrix supplied materials used in the research. Some figures have been reproduced or adapted from the original article under the terms of the Creative Commons Attribution (CC BY) license.



About the Author

Mikayla is a cancer research scientist who recently completed her PhD at Leeds Beckett University. Her PhD project focused on the bidirectional crosstalk between melanoma and cells of the tumour microenvironment via secretion of extracellular vesicles. This project involved study of a wide variety of cellular phenotypic and expression changes, as well as determination of cargo within the vesicles. She also has a background as a bioassay scientist in industry in a multitude of client projects.


 


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