Are you in the Matrix? Using Matrix vs Matrix-free assays in ex vivo culture platforms

TumorGraft3D models are Champions’ three-dimensional (3D) ex vivo tissue cultures that mimic the structure and function of tumors in vivo. They are innovative tools for studying tissue physiology, pathophysiology, and drug discovery. These models can be derived from various tissues, such as the brain, liver, intestine, etc., and can self-organize into complex structures resembling their respective tumor locations and are cultured in a matrix-free environment. The establishment and growth of 3D tissue culture models require the models to interact with the myriad of cellular and structural components within the tumor microenvironment. In this blog, we will explore the differences between matrix and matrix-free conditions in 3D tissue cultures.

Traditionally, 3D tissue culture models have been cultured using a matrix-based approach, where the supportive extracellular matrix (ECM) is derived from natural or synthetic sources. Commonly used matrices include Matrigel, laminin, collagen, and hyaluronic acid. Matrigel is the most commonly used matrix for 3D tissue cultures due to its ability to mimic the natural basement membrane of the tissue. However, using matrices can also pose several challenges, such as batch-to-batch variability, limited scalability, high costs, and difficulty in downstream analysis. Additionally, animal-derived matrices, such as Matrigel, can introduce xenobiotic components that may impact the reproducibility and translatability of the results across the different species.

Matrix-free approaches, on the other hand, offer an alternative to matrix-based assays. These approaches allow the self-organization of 3D tissue culture models without the use of an exogenous matrix. Matrix-free approaches are based on the principle that extracellular matrix components can be provided by the models themselves, where the cellular component of the 3D structure secretes and organizes the matrix components in a more physiological way generating a supportive microenvironment. There are several matrix-free approaches available, including hanging drop and rotary culture. These approaches offer several advantages over matrix-based assays, such as scalability, reproducibility, and compatibility with downstream analysis. In addition, this technique overcomes the species-specific differences between the cells and the matrix which will negatively influence the human translatability of the findings.

Hanging drop cultures involve the formation of 3D tissue culture models in a droplet of culture medium suspended upside down. This method provides a low-adhesion environment for the models to organize and secrete their own matrix components. Rotary culture is another matrix-free approach that involves the constant rotation of a culture vessel, allowing the models to grow and form a 3D structure without the need for a supporting matrix.

Both matrix-based and matrix-free assays have pros and cons for 3D tissue cultures. Matrix-based assays mimic the ECM of the tissue providing an artificial microenvironment for model growth. Beyond batch-to-batch variability, limited scalability, and high costs; including matrix in your ex vivo cultures can also represent a biological problem. Given the artificial architecture of the matrix, it imposes unnatural stiffness and pre-defined structures that will influence the hierarchical organization of the cells during 3D structure generation. Matrix-free assays offer an alternative that eliminates the need for exogenous matrix components and provides a low-adhesion environment for the 3D tissue culture growth for a fully human and more physiological microenvironment. While matrix-free approaches are promising, they are not without their challenges. Specifically, there is the need for ad-hoc development of various protocols for each different tumor type, including rigorous procedures for optimization and standardization to ensure results are reproducible.

In summary, the decision to use matrix or matrix-free culture conditions in 3D tissue culture assays can influence the downstream translatability of your assay.  Researchers should evaluate the differences and understand how using a matrix or matrix-free culture conditions can impact their research.  

References:

1) Loewa, A., Feng, J.J. & Hedtrich, S. Human disease models in drug development. Nat Rev Bioeng (2023). https://doi.org/10.1038/s44222-023-00063-3

2) Proietto M, Crippa M, Damiani C, Pasquale V, Sacco E, Vanoni M, Gilardi M. Tumor heterogeneity: preclinical models, emerging technologies, and future applications. Front Oncol. 2023 Apr 28;13:1164535. doi: 10.3389/fonc.2023.1164535. PMID: 37188201; PMCID: PMC10175698.   

3) LeSavage BL, Suhar RA, Broguiere N, Lutolf MP, Heilshorn SC. Next-generation cancer organoids. Nat Mater. 2022 Feb;21(2):143-159. doi: 10.1038/s41563-021-01057-5. Epub 2021 Aug 12. PMID: 34385685.

4) Kozlowski MT, Crook CJ, Ku HT. Towards organoid culture without Matrigel. Commun Biol. 2021 Dec 10;4(1):1387. doi: 10.1038/s42003-021-02910-8. PMID: 34893703; PMCID: PMC8664924.