A Method to sort heterogenous cell populations based on migration in 2D and 3D environments
Posted on: 2 July 2020
Preprint posted on 7 May 2020
Categories: biophysics, cell biology
Background
Cell migration plays a pivotal role in all stages of the life of a multicellular organism. Important homeostatic processes requiring cell migration include tissue morphogenesis, wound healing and immune responses. Conversely, aberrant migration of diseased cells, such as that displayed by cancer cells, can result in metastasis. Phenotypic assays, including Boyden chambers and wound healing assays, have been developed to study the migratory potential of cells at the population level. Equally, quantitative high-resolution imaging has allowed the study of the molecular basis of collective cell migration. Approaches to study cell migration in 3D are less popular due to the technical challenges of imaging. Despite their utility, although various of these 2D and 3D assays reveal heterogeneity of migratory behavior within a cell population, the quantitative analysis usually provides only a migration index averaged over the entire cell population, and many of them do not provide easy means to sort and retrieve cells from within the population. Altogether, very few methods exist that can sort subpopulations of cells based on their migratory behaviour from an initial heterogeneous pool. In their work, Arora et al present a new approach to sort migratory cancer and immune cells, based on their spontaneous migration in 2D and 3D microenvironments (1).
Key findings and developments
In their work, Arora et al propose readily implementable methods to separate a faster migrating sub-population of cells from a heterogeneous population based on migration in 2D or 3D environments. Initially unsorted groups of cells are locally confined in a series of scattered predefined regions. They are then left to migrate spontaneously away from the original confinement zone to another substrate (for 2D or 3D as described below).
2D sorting device and proof of concept
The 2D migration sorting assay (2D-MSA) relies on a multi-layered PDMS micro-well device, in which arrays of holes are perforated, using a layer cutter. Cells are seeded at 70-80% confluency, and some of these cells will fall into the perforated cavities, and will adhere within a short time. Cells are then allowed to migrate up the cavity walls, to reach the top collection layer initially devoid of any cells. The authors optimized the diameter and height of the cavities, as well as the spacing between cavities, to accurately study cell migration. Ultimately, the collection layer will be enriched with fast migrating cells, while the base layer will be enriched in slow migrating cells. The authors validated the method using a 1:1 mixture of cell lines MCF7 and MD MB 231. Both are breast cancer derived cell lines, however, they have different motility characteristics: while MCF7 maintains an epithelial state and lacks the ability to metastasize, the latter is mesenchymal, with extensive migratory capacity. Both were differentially labeled for recognition. Quantification of both cell lines using image analysis revealed a significant enrichment of MDA MB 231 cells on the top layer of the device, as expected. This demonstrated the 2D-MSA to be useful for separating a heterogeneous cancer cell population into a more homogeneous population based on migration. The authors went on to further test the method with patient samples, and performed downstream analyses on the different sorted cell populations.
3D sorting device and proof of concept
The 3D sorting uses hierarchical hydrogel systems consisting of collagen micro-gels suspended in degradable bulk hydrogel. The surrounding matrix is rapidly cross-linked while mixing to minimize the sedimentation of the collagen beads. The way this method can be used to separate cancer cells in 3D, is that cells are left to migrate for several days from the collagen microbeads into the cleavable matrix. The cleavable matrix can then be selectively digested to release all the migrated cells, and a strainer can be used to separate the collagen beads from the less mobile cells. Each fraction can then be cultured separately. As for the 2D device testing, the authors used a 1:1 mixture of cell lines MCF7 and MD MB 231 as proof of concept, and again demonstrated that this method allows separation of both populations. They went on to test their method on cell types for which 2D migration assays are challenging, such as primary leukocytes, in this case, T cells. Using 3D sorting, they achieved a substantial enrichment of fast migrating cytotoxic T lymphocytes. Downstream analyses showed that these faster migrating T cells had significantly higher killing efficiency as compared to slow migrating, and unsorted cells. This further supports the potential of the method, for functional sorting of CTLs in the context of immunotherapeutic applications.
The authors further emphasize the easy implementation of this method, its high throughput, and the fact that it allows downstream genomic, molecular, phenotypic and functional tests.
What I like about this preprint
I like the out-of-the-box approach for solving a methodological problem that has existed for a long time. It’s ingenious, uses the cell’s own biophysical properties, and allows downstream analyses in the sorted cells. It overcomes many of the limitations of other existing assays, and is in many cases complementary. I like the proofs of concept, and the fact that the authors included a very detailed materials and methods section which allows reproducibility of the devices.
References
- Arora A., et al A method to sort heterogeneous cells populations based on migration in 2D and 3D environments, bioRxiv, 2020.
doi: https://doi.org/10.1242/prelights.22587
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