Why grow cells in 3D?

Most cell culture is carried out in 2D with cells attached to either tissue culture plastic or to a thin coating that may incorporate components of the extracellular matrix (e.g. laminin or fibronectin). However, for the majority of cell types, this is a very different environment to that in which the cells would grow in vivo where cells are embedded in a tissue-specific microenvironment made up of other cells and surrounding matrix.

For stem cells in particular, this microenvironment is highly specialised and specific – often referred to as the stem cell niche. As well as responding to biochemical signals in the form of growth factors, cytokines and small molecules, cells are also able to ‘read’ the mechanical environment in which they find themselves. There have been some extremely elegant experiments that have demonstrated how the relative stiffness or elasticity of the matrix surrounding cells can directly influence their behaviour with many of these studies focussing on stem cells and their progeny.

By providing a 3D culture environment for cells we aim to better replicate their in vivo behaviour. Groups like ours who have a strong interest in cell-matrix interactions are particularly keen to study how cells respond to the different proteins and glycans of the extracellular matrix and how cells are able to re-model their environment.

Which 3D culture model to use?

There are many options for the 3D cell culture already on the market however we struggled to find one optimal for our needs. We were keen to work in a fully defined setting and to be able to adapt the biological components of the matrix independently of the mechanical characteristics. If possible, we were keen to avoid animal-derived products, mostly due to inherent variability but also to avoid animal-associated pathogens. Most importantly, we wanted to be able to apply all the assays we use routinely to study cell behaviour (fluorescent imaging, PCR, protein extraction, metabolic assays etc.) and to have a system that was both reproducible and not prohibitively expensive.

The real challenge was that the cells we work with (human and mouse stem cells) are often tricky to grow well and are not tolerant of many artificial environments. These cells are also expensive to culture so we needed a system that was user-friendly and which we could use reliably without loss of valuable reagents or cells.

Peptide hydrogels

As part of an EU-FP7 project (BIOSCENT) we worked with Prof. Aline Miller and Dr Alberto Saiani (Polymers and Peptides Group, University of Manchester (view site) to take a hydrogel system they were using for the culture of fibroblasts and osteoblasts (in collaboration with Prof. Julie Gough, University of Manchester (view site) and optimise this for stem cell culture. The peptides used in these gels are relatively short (typically between 8 and 12 amino acids in length) and self-assemble in solution due to their alternating hydrophobic and hydrophilic units. Within the peptide, positive and negative charges are typically balanced with gelation occurring when the concentration of the peptide is taken above the critical gelation concentration at a pH close to 7.

The gels are relatively weak but their stiffness can be altered over a narrow range using peptide concentration alone. Beyond that range, the constituent amino acids can be varied or other components incorporated. A significant advantage of these hydrogels is that the short peptides can be used to display bioactive motifs that then decorate the fibres within the hydrogel and are available to interact with cells. Over the course of the EU-FP7 and subsequent EPSRC, UMI3 and industry-funded projects we were able to define conditions for the preparation of hydrogels in which stem cells could be encapsulated, carefully defining conditions for specific cell types in combination with commercial media preparations.

A ‘blank slate’ for stem cell culture

We found that mouse ES cells proliferated rapidly following encapsulation in the hydrogels forming large spheres with no evidence for central necrosis or the formation of a cystic cavity, in contrast to the formation of embryoid bodies in suspension culture. We could use standard immunocytochemistry techniques to image the cells within the gels, either with whole-mount staining or following sectioning of the gel. Usefully, we could also image endogenous fluorescence (e.g. EFGP expression) non-destructively within the gels demonstrating that this system can be used to monitor for switch-on or –off of marker-gene driven fluorescence. We could use a simple method to disaggregate the gels, allowing recovery of cells for subsequent gel-to-gel passage. The lack of matrix-derived pro-differentiation signals is probably significant in helping encapsulated mouse ES cells to maintain a pluripotent phenotype within the gels, in this way we believe the ‘naked’ peptide is acting as a blank slate, on to which biological functionality can be grafted.

Adult stem cells (bone marrow derived or human umbilical cord perivascular cells) were also encapsulated within the gels where they demonstrated different growth patterns dependent on the density of initial cell seeding. Optimal cell viability was obtained with relatively dense seeding compared to mouse ES cells.

We particularly focused on encapsulation of human ES cells, testing a range of cell preparations taken from 2D culture that either required non-enzymatic passage (e.g. the NCL-1 line) or that could be passaged with enzyme digestion (e.g. the HUES7 line). We also tested a variety of common media preparations to ensure that the methodology we have developed is of broad use to the stem cell community. Again, we could monitor cell behaviour within the gels and found that, in contrast to mouse ES cells, the human ES cells appear to undergo differentiation within the gels, in a similar way to that seen in suspension culture of hES-derived clusters or embryoid bodies.

Functionalisation and bespoke gel formulation

The real utility of this peptide hydrogel system is in the ability to decorate the blank canvas with peptides, glycans, small molecules, nucleic acids etc. to influence cell behaviour. By adding motifs, either via covalent attachment to the peptide fibres or by non-covalent entrapment within the hydrated environment of the gel we can add function in a defined, controlled and adaptable way. Taking a bottom-up approach the gels can be modified to better replicate the complex tissue environments within the body or to provide signals to drive specific cell behaviour e.g. differentiation to specific lineages.

Current projects are testing the cell instructive effects of addition of defined, recombinant proteins as well as short ECM-derived cell- or matrix-binding motifs. Addition of proteins and/or glycans to the hydrogels impacts on the mechanical properties (from the same basis as how different ECM compositions in the body define the properties of tissues) helping to make the gels useful tissue realistic environments for encapsulated cells.

Technology development

Would you like to work with us to develop these gels?

This technology has been developed at the University of Manchester, tested for reproducibility with partner groups across the world, and we have applied for patent protection (view site).

We are keen to work with academic and industrial partners to further develop the hydrogel technology with a particular focus on formulating hydrogels for applications such as the tissue-realistic culture of cells for in vitro drug screening, in vitro assays for tumour growth/metastasis, the culture of iPS cells as disease models and various tissue engineering applications. Please contact us if you have an application you would be interested in developing the hydrogels for and we can discuss possible projects.

Simple hydrogel technology optimised for stem cell culture

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