3Rs: Tiny amounts of Matrigel sufficient for mammary epithelial cell organoids

3Rs aims to reduce, refine and replace the use of animals in research. Matrigel® and similar basement membrane extracts are widely used in cell culture as an extracellular scaffold support and also provide key matrix proteins. The sacrifice of mice to generate Matrigel is one of the biggest single uses of animals in research. A new method to culture mammary epithelial cell (MEC) organoids shows that Matrigel use can be dramatically reduced or even (at a cost) eliminated.

Compared with conventional (2-dimensional) cell culture, 3D organoids more closely model in-vivo reality. This advantage has driven Matrigel’s widespread adoption. But replacing Matrigel with something that performs as well isn’t straightforward and needs to be addressed for each specific tissue to provide more closely defined conditions. Focussing on MEC organoid culture, research from the University of Manchester has recently found that just small amounts of Matrigel or Laminin-111 mixed with a specific PeptiGel formulation recapitulates pure Matrigel data for MEC culture.

Why do researchers need alternatives to Matrigel?

Matrigel, a commonly used extracellular matrix (ECM) gel in biological research, is derived from the basement membrane of mouse sarcoma cells. The numbers of mice are killed every year in its manufacture is not well documented but likely to run into hundreds of thousands given the popularity of the product: It is widely used for various applications, including cell culture, studies of cell behaviour, and tissue engineering. In addition to animal welfare, there are other reasons why researchers are seeking synthetic alternatives to Matrigel.

Reproducibility and Consistency: Matrigel is a complex mixture of proteins and growth factors, and its composition can vary significantly between batches. This variability can lead to inconsistencies in experimental results, making it difficult to reproduce findings. Synthetic alternatives can be designed to have consistent compositions, improving the reproducibility of experiments.

Human Relevance: Since Matrigel is derived from mouse cells, it may not accurately replicate the human cellular environment. This discrepancy can be particularly problematic in research aimed at understanding human biology or developing treatments for human diseases. Synthetic alternatives can be tailored to mimic the human ECM more closely, potentially increasing the relevance of research findings to human health.

Customizability: Synthetic ECMs can be engineered to have specific properties, such as stiffness, degradability, and the presence of particular biochemical cues. This customizability allows researchers to create environments that more accurately model specific tissues or disease states, facilitating more targeted and relevant studies.

Safety and Clinical Translation: For applications in regenerative medicine and tissue engineering that aim to develop therapies for human use, the use of animal-derived materials like Matrigel poses safety concerns, including the risk of immune reactions and disease transmission. Synthetic alternatives can be designed to be biocompatible and free from these risks, making them more suitable for clinical applications.

Reducing and Replacing Matrigel with PeptiGel

PeptiGel is a family of related, but distinct nature-mimetic self-assembling polypeptide hydrogels (SAPHs) which provide a viable alternative to Matrigel. Each PeptiGel can be fully configured to address specific tissue niches.

Research at Manchester University led by Andrew Gilmore was particularly interested in MECs which they are using to model the development of breast tissue. This area of research requires the development of culture conditions that extend beyond just survival and proliferation of the MECs to actually generating functional phenotypes capable, for example, of generating milk proteins.

The collaborators sought to recapitulate MEC functionality that can be achieved with Matrigel. As a starting point to develop an alternative, the research team selected PeptiGels as these have a variety of specific attributes, such as charge and elasticity, which can be further tailored to specific applications by the addition of tethered functional groups.

The researchers first evaluated the positively charged Alpha4 PeptiGel. This enabled the culture of the non-malignant MEC line MCF10A. Similar to Matrigel, the cells maintained their viability over 21 days, also forming clusters of 3D rounded structures.  But there were key differences: Whereas cells grown in Matrigel underwent growth arrest at day 14, the Alpha4 cells continued growing forming larger structures. Moreover, Alpha4 cells had lower circularity suggesting poorer organization. Further staining studies indicated that Alpha4 supports viability but is unable to recapitulate the MCF10A polarized acinar organization seen with Matrigel culture.

The researchers considered the difference in performance between Alpha4 and Matrigel may be due to their relative elasticity (stiffness). By diluting Alpha4 with PBS to reduce stiffness, they were able to increase organoid viability, but it did not induce the desired polarity.  A proteomic analysis of cells in each matrix was performed. Significant differences in expression patterns were identified. For example, only MCFs grown in Matrigel make and express the basement membrane proteins Laminin-332 and collagen-IV. 

It was previously shown that Lamanin-111, a major component of Matrigel, could be used to generate polarised acinar ECM structures capable of producing milk proteins. So, the researchers evaluated MCF10A cells grown in recombinant Laminin-111 and compared the results with Matrigel. Both hydrogels formed the desired polar acinar structures which express Laminin-332.

Next, they sought to functionalise PeptiGel with Laminin-111 or Matrigel to determine if it would recapitulate the polarised acinar MCF10A phenotype achieved with Matrigel. Mixing Alpha4 with laminin-111 formed precipitates, possibly because of Alpha4’s positive charge. Similarly, Matrigel did not mix well. Consequently, the team switched PeptiGel to the negatively charged Alpha7. Alpha7 produced a similar MCF10A phenotype in Alpha4.  But when combined with 6.1 mg/ml laminin-111 or just 1.5 mg/ml of Matrigel, Alpha 7 faithfully recapitulated the results seen with Matrigel including MEC differentiation, polarization, acinar structure formation and the expression of milk proteins.

These PeptiGel formulations provide an excellent route for researchers to progress to an animal-free culture system for their MEC studies. Although the high cost of Laminin makes the complete replacement of Matrigel cost-prohibitive, a much cheaper alternative to reducing animal use is provided by supplementing Alpha7 with vanishingly small amounts of Matrigen (diluted 400-fold) achieve the same result. It will be interesting to determine if this approach works with other cell types. 

IMAGE: Mammary epithelial cell organoids   CREDIT:  Sumbal, Chiche, Charifou, Koledova and Li  Creative commons

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