Tunable and dynamic soft materials for three-dimensional cell culture

Abstract

The human body is complex and hierarchically structured, composed of cells residing within the extracellular matrix (ECM) of tissues that are assembled into organs, all working together to complete a given function. One goal of current biomaterials research is to capture some of this complexity outside of the body for understanding the underlying biology of development, repair, and disease and to devise new strategies for regenerative medicine or disease treatment. Polymeric materials have arisen as powerful tools to mimic the native ECM, giving experimenters a way to capture key aspects of the native cellular environment outside of the body. In particular, dynamic materials allow changes in the properties of these ECM mimics during an experiment, affording an additional degree of control for the experimenter. In this tutorial review, the basic cellular processes of cell migration, proliferation, and differentiation will be overviewed to motivate design considerations for polymeric ECM mimics, and examples will be given of how classes of dynamic materials are being used to study each cellular process.

Fig. 1. Complex cellular processes key time and size scales. The formation of pre-cartilage condensations illustrates the complex nature of cellular processes and the need for control of the cell microenvironment over multiple time and length scales for examining them. The cell microenvironment is highly dynamic, and cells migrate, aggregate, and begin synthesizing ECM proteins within the first week of embryonic development (chicken wing model). The cells migrate in response to chemotactic agents (represented by stars) or physical gradients. Once the cells aggregate, they begin differentiating, accompanied by changes in gene expression and protein production. Cells interact with the matrix they synthesize through ligands (squiggles) and cell receptors (curved lines on cell border).

Fig. 2. Enzyme responsive materials for examining cell migration. (a) Cells migrate through enzymatically degradable materials (enzymatically degradable units depicted by circles) in response to chemical or physical gradients. Cells migrate along gradients, such as from regions of low chemotactic agent concentration to high chemotactic agent concentration, cleaving proteolytically degradable units, such as GPQGIWGQ. (b) Hadjipanayi et al. studied the migration of human dermal fibroblasts in response to a gradient of matrix stiffness in a collagen hydrogel. Cells preferentially migrated towards the stiff region of the material. In their study, the stiff region also had a higher density of integrin-binding ligands. Reprinted from ref. 37 with permission from John Wiley and Sons. Copyright (2009). (c) Ehrbar et al. studied the migration of mouse preosteoblastic cells in synthetic enzymatically degradable PEG hydrogels. At low moduli, cells can migrate in degradable (white triangles) and non-degradable hydrogels (grey diamonds). At higher moduli, proteolysis and matrix degradability are required for migration, and migration decreases as modulus increases. Reprinted from ref. 46 with permission from Elsevier. Copyright (2011).

Fig. 3. Growth factor presenting dynamic materials for controlling cell proliferation. (a) Cells need nutrients, mitogens, and space to proliferate, providing design criteria for biomaterials to direct and study this process. For example, cells can receive signals from mitogens (stars) to promote proliferation, but without the space for additional cells, they will not proliferate (top). In contrast, while cells may have space to proliferate, they will not proliferate without appropriate mitogen signaling (middle). Cells given both mitogens and space proliferate (bottom). (b) There are several methods by which growth factors can be dynamically presented by biomaterials towards regulating cellular processes such as proliferation. Growth factors can be electrostatically attracted to a moiety incorporated within the material (top), sequestered by non-covalent interactions with an affinity molecule (middle), or covalently immobilized within the biomaterial through a degradable linker (bottom). (c) Tae et al. released vascular endothelial growth factor from hydrogels with heparin, controlled via electrostatic interactions. Approximately 40% of the growth factor was released over the course of about three weeks. Reprinted from ref. 63 with permission from Taylor & Francis. Copyright (2006). (d) Shen et al. measured proliferation of endothelial cells in collagen hydrogels in response to vascular endothelial growth factor. The authors saw significantly greater proliferation when the growth factor was immobilized in the hydrogel compared to control hydrogels without the growth factor or hydrogels where the growth factor was presented in soluble form. Reprinted from ref. 67 with permission from Elsevier. Copyright (2008).

Fig. 4. Photoresponsive materials for probing cell differentiation. (a) Stem cells, such as mesenchymal stem cells, show multilineage differentiation potential and can differentiate into a variety of different cell types. (b) Cell differentiation can be regulated by soluble factors (stars), solid-phase integrin-binding peptides (squiggles), and mechanical cues transduced by actin filaments (depicted by hollow circles). (c) DeForest et al. used photopolymerization to pattern RGD in specific regions of an enzymatically degradable PEG hydrogel. The authors demonstrate that significant cell spreading is only observed where RGD has been added. Reprinted with permission from Macmillan Publishers Ltd: Nat. Mater., ref. 70. Copyright (2009). (d) Khetan and Burdick used sequential crosslinking to demonstrate the importance of matrix mechanical cues on stem cell differentiation. The authors synthesized a hyaluronic acid hydrogel and cultured cells inside of the gel with (+UV) or without (–UV) additional crosslinking at a later timepoint. The light-mediated addition of crosslinks limited cell spreading (+UV), causing the cells to differentiate down an adipogenic lineage (marked by oil red o). The cells were better able to spread without additional crosslinking (–UV), causing the cells to differentiate down an osteogenic lineage (marked by ALP). Reprinted from ref. 98 with permission from Elsevier. Copyright (2010).

Matthew S. Rehmann

April M. Kloxin