Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease

There is a growing awareness that cells grown in 3D better model in vivo behavior than those grown in 2D. In this protocol, we describe a simple and tunable 3D hydrogel, suitable for culturing cells and tissue in a setting that matches their native environment. This is particularly important for researchers investigating the initiation, growth, and treatment of cancer where the interaction between cells and their local extracellular matrix is a fundamental part of the model. Moving to 3D culture can


Introduction
The many roles played by the extracellular environment in cancer development and progression are becoming ever clearer 1 . Recently, detailed proteomic-based analyses have added to an already convincing literature base, demonstrating environments to grow and observe cell behavior in vitro, including PDX-derived and other close-to-patient cells 8 , 9 , 10 . However, this 'one size fits all' approach neglects the complex role played by matrix proteins/glycans in cancer initiation and progression.
Recognition of the role of extracellular matrix (ECM) in the control of cell behavior has also encouraged the use of 3D culture in or on hydrogels composed of specific matrix components 11 . Whilst this is useful for investigating specific interactions, these systems suffer from the inability to separate mechanical and biochemical instructions between cells and matrix. They may also be difficult to handle and can give unclear read-outs of cell behavior. Collagen gels are a key example of this problem, since cell-mediated gel contraction can dramatically reduce the ability to visualize cells within the gel 5 . There are also some very elegant, multi-component gel systems, which experts have used to great effect 12 , 13 , 14 . These can incorporate enzyme-sensitive linkers and bio-active motifs but are significantly more complex in their formulation and application than the system described here.
This protocol describes a method for creating fully defined 3D culture models, allowing the roles of the ECM in development and disease to be modeled in vitro. The basis of the 3D model is a peptide gel, which we have previously described as an optimization of a simple self-assembling octapeptide hydrogel 5 , 15 , 16 . By moving away from complex, animalderived matrices this system offers a significant benefit of improved batch-to-batch consistency and improved handling.
In its simple state, the peptide contains no matrix-derived motifs and effectively provides a 'blank slate' onto which the user can build functionality.
We demonstrate how the mechanical properties of the peptide gel can be regulated independently, alongside incorporation of matrix proteins/glycans. The system is highly tunable, allowing the encapsulation of a range of cell types in various formats. Importantly for building a cancer model, stromal cells can also be incorporated: either in direct coculture or separated to allow specific analysis of indirect cancer cell-stroma interactions. Most crucially, the protocol described here requires no complex knowledge of chemistry and can be reproduced in any cell culture laboratory without the need for specialized chemical knowledge or equipment.
We have optimized methods for the study of cell behavior in the peptide gels, including imaging, rheological analysis, extraction of material for PCR 5

Preparation of matrix components for seeding
NOTE: An example calculation for steps 3-5 is shown in Figure 1.
Step 3 and step 4 may be omitted to produce a matrix-free and/or a cell-free gel respectively.

Embedding peptide gels for sectioning
NOTE: Embedding peptide gels in 4% agar is a crucial step prior to paraffin-embedding for immunohistochemistry.
Alternatively, gels may be embedded in 2% agar and sectioned using a vibratome (typically 500 μm sections give good results). This is an optional step, producing hydrated gel

Representative Results
The peptide gel fabrication method described here allows the user to define and create a bespoke 3D culture environment.
While the mechanical environment is determined primarily by peptide concentration, matrix components of interest may also be added at controlled densities, as shown by the example calculation in Figure 1. In its simplest form, however, the peptide gel protocol provides a method for encapsulating cells in a matrix-free 3D environment. Figure 2 shows how this approach may be combined with a wide range of cancer models, including fluorescently labeled cancer cell lines ( Figure 2A) and patient-derived xenograft (PDX) material ( Figure 2B,C). Importantly, cell lines and PDX material may both be cultured within the gels in serum-free conditions ( Figure 2C,D), providing a 3D culture system with fully defined composition.
Since the peptide itself does not contain any cell-binding motifs, encapsulated cells typically display a rounded morphology in the unmodified peptide gels. Figure 3A demonstrates this for human mammary fibroblasts in a 6 mg/mL peptide gel, compared with their classic elongate morphology seen in pure Matrigel and a pure collagen gel. Importantly however, the peptide gel protocol allows incorporation of matrix components of interest. Figure 3A demonstrates how addition of 200 μg/mL collagen I can restore the elongate fibroblast morphology in the peptide gels.
Matrix additions can also support the growth and organization of other cell types, for instance MCF10A, as shown in Figure   3B. In this case, addition of 100 μg/mL collagen I to a 6 mg/ mL peptide gel allows acinar structures to form by day 7.
Further complexity may also be introduced by incorporation of a supportive cell layer in indirect co-culture. Figure   3C demonstrates how the combined approach of matrix incorporation and indirect co-culture with human mammary fibroblasts can enhance MCF10A growth and organization.
Another important parameter is the concentration of peptide used in peptide gel fabrication. Figure 4A shows an example of how controlling peptide concentration, in this case between 4 and 10 mg/mL, results in a stiffness ranging between 100s to 1000s of Pa. These gels may be fabricated matrix free or can be created with matrix additions to allow simultaneous control of both stiffness and composition. Peptide gels with matrix additions may be sectioned and stained to allow the distribution of these additions to be visualized. Figure   4B, C show two approaches for doing this: embedding in 4% agar followed by standard tissue processing and paraffin embedding for immunohistochemistry ( Figure 4B) or embedding in 2% agar followed by vibratome sectioning and fluorescent staining (Figure 4C).
When modifying the composition of the peptide gels, it is crucial to ensure that these changes do not impact the mechanical environment initially presented to the cells.

Discussion
We have found the peptide gels described here to be a simple, cost-effective, and flexible solution to support 3D culture of multiple cell types. By providing full control over the concentration of peptide used and the protein or glycan additions made, this method allows the peptide gels to be carefully tailored to their application. This provides a tangible advantage over existing systems, for instance collagen gels, in which parameters that control stiffness also commonly result in a change in integrin binding motifs 20 , 21 .
We have demonstrated the application of the peptide gel for in vitro culture of cancer cell lines and patient-derived material 5 . A suitable precursor will be completely liquid at 80 °C, and self-supporting at 37 °C. These checks are essential to ensure that gelation will occur correctly. Cells and/or matrix may then be incorporated under physiological conditions.
Labs already using 3D matrices will be familiar with the careful handling needed to encapsulate cells in the peptide gels.
Care must be taken to limit the agitation of cells before and during the encapsulation steps. We have found that specific cell types are differentially susceptible to damage during this process and this must be carefully evaluated by the user. The concentrations of the peptide gel described here allow gelation to proceed in a time frame that, for the cells mentioned, allows cells to be encapsulated before they sink to the bottom of the casting well but slowly enough that they are not damaged by this process. It is, however, of note that some sensitive cell types may require more rapid neutralization to avoid prolonged exposure to raised pH. In this case, the addition of 10 mM HEPES to the medium surrounding the peptide gel can be beneficial.
When adopting the method described in this protocol, it is very important to carefully consider the quality of the peptide source. Rather than being used as a functional motif or coating, the peptide here is the entirety of the non-soluble portion of the hydrogel. Therefore, any contaminants or variation in the peptide structure are likely to have a significant impact on the integrity or capacity to support cell viability in the final hydrogel. When moving to a new batch of peptide, care must be taken to ensure that there is good batch-tobatch consistency from the supplier as well as checking the behavior of the peptide when forming the gel precursor.
In summary, this protocol describes a 3D culture system with a crucial focus on the independent control of mechanical and biological properties. The simplicity and adaptability of the method makes it suitable for adoption by any cell culture laboratory, and for a wide range of applications 5

Disclosures
The authors have nothing to disclose.