1.3.1. Mimicking Cellular Microenvironment Biomechanics

There have been many diverse approaches to capturing the complexity of the ECM and the biomechanical cues directed by it via in vitro hydrogel models. Tissue specificity is a crucial factor to consider in developing an ECM-mimetic hydrogel-based cell culture system, since the cells of each tissue produce and degrade matrix constituents, leading to variable ECM composition, bioactivity, and mechanics amongst tissues [129] (Figure 3). In the bone, the ECM is calcified to provide structural support to the tissue, while in tendons it is structured to provide tensile strength [130,131].

Most natural ECM mimics in vitro have been hydrogels composed of ECM components such as collagen, fibronectin, or hyaluronic acid. Such hydrogels have been significant in studying cellular behavior in 3D environments as well as cell response to controlled mechanical properties such as stiffness, elasticity, and rigidity [63,132–139]. Photo-crosslinking, chemical crosslinking, changing fiber density, and the development of 'smart' hydrogels that are responsive to external stimuli (e.g., changes in pH, temperature, and light) have all been used to fine-tune hydrogels to mimic the cellular microenvironment mechanically [140–151]. For example, photo-crosslinking of gelatin methacryloyl (GelMa) hydrogels alters hydrogel stiffness based on the light intensity, exposure time, and concentration of photo-initiator used in photo-crosslinking [143]. Nanocomposite smart hydrogels have been produced to change volume, Young's modulus, and breaking strength based on applied chemical and physical stimuli [148,150]. *Gels* **2022**, *8*, x FOR PEER REVIEW 10 of 36

**Figure 3.** Young's modulus of hydrogels. The reported Young's modulus (kPa) values of various hydrogels are plotted with an image of the tissue that has similar Young's modulus. It is crucial to choose a hydrogel model that corresponds to the in vivo mechanical properties of tissue that is rel-**Figure 3.** Young's modulus of hydrogels. The reported Young's modulus (kPa) values of various hydrogels are plotted with an image of the tissue that has similar Young's modulus. It is crucial to choose a hydrogel model that corresponds to the in vivo mechanical properties of tissue that is relevant to the work. (Created with BioRender.com, accessed on 14 July 2022).

### evant to the work. (Created with BioRender.com, accessed on 14 July 2022)*.* 1.3.2. Recapitulating Cellular Microenvironment Heterogeneity

1.3.2. Recapitulating Cellular Microenvironment Heterogeneity While the above-discussed methods are useful to study the effects of semi-isolated mechanical conditions on cells, they often disregard the biological complexity of the ECM. In vivo ECMs can also serve as a medium through which cells communicate with one another via secreted growth and signaling factors in vitro. Several protocols have been developed to decellularize ECM isolated from animal or human tissue, or to harvest ECM secreted by fibroblasts in vitro [152–155]. To better capture the biological complexity of the ECM, hydrogel models have been developed to incorporate fibroblasts, growth factors, and more diverse components of the ECMs specific to the cell type being studied. Incorporating fibroblasts in a co-culturing system with the cell type of interest is a relatively newer approach, which allows for fine-tuning mechanical properties using a hydrogel model while adding biological complexity to the culture through the addition of the secreted matrix from fibroblast cells [156,157]. Lee et al. developed a 3D hydrogel sys-While the above-discussed methods are useful to study the effects of semi-isolated mechanical conditions on cells, they often disregard the biological complexity of the ECM. In vivo ECMs can also serve as a medium through which cells communicate with one another via secreted growth and signaling factors in vitro. Several protocols have been developed to decellularize ECM isolated from animal or human tissue, or to harvest ECM secreted by fibroblasts in vitro [152–155]. To better capture the biological complexity of the ECM, hydrogel models have been developed to incorporate fibroblasts, growth factors, and more diverse components of the ECMs specific to the cell type being studied. Incorporating fibroblasts in a co-culturing system with the cell type of interest is a relatively newer approach, which allows for fine-tuning mechanical properties using a hydrogel model while adding biological complexity to the culture through the addition of the secreted matrix from fibroblast cells [156,157]. Lee et al. developed a 3D hydrogel system for coculturing human liver-cancer cell spheroids with fibroblasts on a micropatterned fibrous

tem for co-culturing human liver-cancer cell spheroids with fibroblasts on a micropatterned fibrous scaffold, thereby modeling the three dimensional structure of tumors and

also developed methods of controlled growth factor release into hydrogel-based cell cultures [158–160]. It is crucial to combine the biochemical components of the ECM with the

Hydrogels are defined as hydrophilic polymer networks that form a three-dimensional structure [161]. A critical property of hydrogels is their ability to swell with water without dissolving due to hydrophilic functional groups present on the polymers of the hydrogel. Their high water content makes them flexible and resemble soft tissue, implicating their use in biomedical studies [162]. There are many polymeric substances that can be classified as hydrogels, and they are generally composed of one or more natural or

mechanical properties to better mimic the cellular microenvironment in vitro.

**2. Hydrogels and Their Applications**

scaffold, thereby modeling the three dimensional structure of tumors and the cross-talk between cancer cells and neighboring fibroblasts [156]. Several groups have also developed methods of controlled growth factor release into hydrogel-based cell cultures [158–160]. It is crucial to combine the biochemical components of the ECM with the mechanical properties to better mimic the cellular microenvironment in vitro.

### **2. Hydrogels and Their Applications**

Hydrogels are defined as hydrophilic polymer networks that form a three-dimensional structure [161]. A critical property of hydrogels is their ability to swell with water without dissolving due to hydrophilic functional groups present on the polymers of the hydrogel. Their high water content makes them flexible and resemble soft tissue, implicating their use in biomedical studies [162]. There are many polymeric substances that can be classified as hydrogels, and they are generally composed of one or more natural or synthetic materials for use in a wide variety of applications. The three-dimensional structure of hydrogels is preserved despite swelling by chemical and/or physical crosslinks within the polymeric network. Changes in composition, protein concentration, and crosslinking density lead to changes in elasticity, polymer density, and biodegradation rate [163]. These and other tunable properties of hydrogels make them suitable for a wide range of studies on mechanical, chemical, and biological conditions in vitro as well as ideal candidates for use in in vivo clinical applications such as in drug delivery systems [161,162]. Here, we describe the major classifications of hydrogels based on their composition, the general properties of hydrogels, and current and potential experimental and clinical applications of hydrogels with a focus on pre-clinical cancer models.

### *2.1. Types of Hydrogels*

There are several classification methods for hydrogels, such as those based on ionic charge, biodegradability, physical properties, crosslinking, and preparation. They are most commonly classified by their source polymer(s): naturally occurring biomaterial, synthetic bio-mimetic, or a hybrid of these two sources [163]. Synthetic materials are traditionally used extensively due to low biodegradation rate, ease of manipulation, and greater control over biochemical interactions [149,164,165]. Natural biomaterials, however, are preferred due to their ability to biologically mimic the structural and biochemical properties of the cellular niche in vitro, and are responsive to cellular activities in terms of biochemical reactivity and degradability [164]. Numerous studies have presented methods for forming hybrid hydrogels that possess properties of both natural and synthetic biomaterials that are mechanically and biochemically responsive and tunable by the surrounding environment [165,166].

### 2.1.1. Natural Hydrogels

Natural hydrogels are composed of naturally occurring polysaccharides and proteins such as collagen, Matrigel®, hyaluronan, gelatin, and their derivatives, including alginate and chitosan [167,168]. While most studies cite hydrogel formation from one ECM protein, there are several that utilized hydrogels composed of several proteins [167,168]. Either protein concentration or crosslinking density is altered in the hydrogels in order to change the Young's modulus of the gels [167,168]. However, these are not finely tunable or as well-understood as the mechanical properties of synthetic hydrogels [169]. In contrast, the biocompatibility of natural hydrogels and their responsiveness to cellular degradation add an important dimension to biological studies [170].

Many studies incorporate several proteins into biomaterial hydrogels to improve hydrogel stability and similarity to the in vivo cellular microenvironment. Comparing cellular properties in vitro in composite hydrogels versus single-material hydrogels can give more specific information on how cell mechano-sensitivity is influenced by specific cell-ECM protein interactions. A useful representative investigation of the insight obtained by studies undertaken with composite hydrogels is a study by Rao et al. [171] (Figure 4).

They mixed collagens I and III with hyaluronic acid (HA), collagen IV with HA, and used collagen (I and III) as a standalone in hydrogel formation to determine modulus values of the different hydrogels and observe changes in cell morphology, spreading, and migration. Composite hydrogels had much higher elastic modulus values than the collagen hydrogel, and increased HA content correlated to increased modulus values and greater cell spreading and migration [171]. Interestingly, cell morphology differed by the type of collagen used in the hydrogels: cancer cells cultured in collagen IV had a rounder cell shape, while those in collagen I and III were spindle shaped [171]. *Gels* **2022**, *8*, x FOR PEER REVIEW 12 of 36

**Figure 4.** Glioblastoma cell morphologies and migration when cultured on different substrates. OSU-2 glioma cells were cultured on collagen I and III and HA composite hydrogels with different concentrations of HA. Cell morphologies shown in (**A**) were quantified via cell area (**B**), circularity (**C**), and roundness (**D**). Increased HA content led to lower cell area but increased cell circularity and roundness. The scale bar in (**A**) indicates 100 μm, \* in (**B**–**D**) represents a *p*-value < 0.05 compared to 0% HA condition. Figure reprinted/adapted with permission from Ref. [171]. Copyright 2013, American Chemical Society. **Figure 4.** Glioblastoma cell morphologies and migration when cultured on different substrates. OSU-2 glioma cells were cultured on collagen I and III and HA composite hydrogels with different concentrations of HA. Cell morphologies shown in (**A**) were quantified via cell area (**B**), circularity (**C**), and roundness (**D**). Increased HA content led to lower cell area but increased cell circularity and roundness. The scale bar in (**A**) indicates 100 µm, \* in (**B**–**D**) represents a *p*-value < 0.05 compared to 0% HA condition. Figure reprinted/adapted with permission from Ref. [171]. Copyright 2013, American Chemical Society.

rigel®, a mixture of ECM proteins secreted from mouse sarcoma cells, which is commonly used for spheroid formation in 3D cultures [172]. Several mechanics studies use Matrigel® for the formation of spheroids before seeding onto other types of hydrogels or for studies of cell invasion. For instance, Grundy et al. reported that primary GBM cells that were insensitive to rigidity were more invasive in spheroid cultures in Matrigel® as opposed to rigidity sensitive cells [173]. Hydrogels using natural biomaterials are also advantageous, since they allow for the Another common composite hydrogel used in several cell mechanics studies is Matrigel®, a mixture of ECM proteins secreted from mouse sarcoma cells, which is commonly used for spheroid formation in 3D cultures [172]. Several mechanics studies use Matrigel® for the formation of spheroids before seeding onto other types of hydrogels or for studies of cell invasion. For instance, Grundy et al. reported that primary GBM cells that were insensitive to rigidity were more invasive in spheroid cultures in Matrigel® as opposed to rigidity sensitive cells [173].

Another common composite hydrogel used in several cell mechanics studies is Mat-

design of an in vitro extracellular environment that can biologically mimic the in vivo ECM and various cellular conditions. Natural hydrogels have been used for tissue engineering and regenerative medicine because they form structures similar to tissue ECM due to the natural proteins and polysaccharides that they are composed of [168]. While the biological similarity is closer to in vivo when natural biomaterials are used in hydrogel preparation, the mechanical properties are not adjustable or robust compared to those of synthetic hydrogels. Additionally, natural hydrogels can lead to great variation from batch to batch due to the components being sourced naturally [168,174]. Recently, how-Hydrogels using natural biomaterials are also advantageous, since they allow for the design of an in vitro extracellular environment that can biologically mimic the in vivo ECM and various cellular conditions. Natural hydrogels have been used for tissue engineering and regenerative medicine because they form structures similar to tissue ECM due to the natural proteins and polysaccharides that they are composed of [168]. While the biological similarity is closer to in vivo when natural biomaterials are used in hydrogel preparation, the mechanical properties are not adjustable or robust compared to those of synthetic

ever, 'smart' hydrogels, which are robust, have localized mechanical properties, and are responsive to changes in pH, temperature, and light have been developed from natural

plications in tissue engineering, regenerative medicine, and stem cell and cancer research.

hydrogels. Additionally, natural hydrogels can lead to great variation from batch to batch due to the components being sourced naturally [168,174]. Recently, however, 'smart' hydrogels, which are robust, have localized mechanical properties, and are responsive to changes in pH, temperature, and light have been developed from natural materials [148]. Smart hydrogels composed of natural materials have many possible applications in tissue engineering, regenerative medicine, and stem cell and cancer research.

### 2.1.2. Synthetic Hydrogels

Synthetic hydrogels are composed of synthetic materials such as polyethylene glycol (PEG), poly(vinyl alcohol), and poly-2-hydroxy ethyl methacrylate [175,176]. They offer advantages to natural hydrogels in the sense that they offer greater control over gel mechanical properties, have higher capacity for water absorption, do not degrade as rapidly as biomaterial hydrogels, and have great reproducibility [177]. For instance, PEG-based hydrogels are useful in mechanics studies due to stiffness tunability and ability to support long culture periods (several weeks) [178]. Synthetic hydrogels have more utility in studies focused on the effect of isolated mechanical properties on cell behavior without the additional influences of interactions between cells and biochemically active biomaterials [179]. Biomimetic polymers have also been synthesized which may contain a similar component to a natural material, such as a specific amino-acid sequence, which can add biological activity such as degradability or biochemical signaling to the gel in a more controlled environment than if a natural biomaterial were used [180]. Therefore, synthetic hydrogels are extremely customizable and can be synthesized for specific medical or research applications.

Synthetic hydrogels have been used to study the mechanics of cancer, as reported in several previous publications. For instance, 2D "films" of poly-methylphenyl siloxane with increasing stiffness values resulted in increased cell spreading and migration of glioblastoma cells compared to more compliant films [181]. In developing an in vitro drugscreening platform for cancer, synthetic HA derivatives, HA-aldehyde and HA-hydrazide, were crosslinked and formed into a hydrogel where cells were able to form clustered structures similar to tumors, and had greater drug resistance than in 2D cultures [182]. PEG hydrogels are often used for hydrogel preparation due to their tunable properties and high biocompatibility compared to other synthetic materials. They were used in mechanical studies of lung adenocarcinoma where matrix stiffness alterations resulted in changes in cellular morphology [183].

While there are several advantages to using synthetic hydrogels, they are limiting in their ability to mimic the complexity of native ECM. This highlights the importance of efforts to add biochemical reactivity to synthetic hydrogels for in vitro studies that are aiming to mimic cellular microenvironments [184]. For instance, Lutolf et al. engineered synthetic hydrogels that were degradable by matrix metalloproteinases through crosslinking of synthetic substrates into the hydrogels [164]. Smart synthetic hydrogels have also been studied to engineer stimuli-responsive synthetic biomaterials that change when faced with altered temperature, pH, light, and other stimuli [185].

### 2.1.3. Hybrid Hydrogels

Many hydrogels are composed of synthetic materials that are mixed, conjugated, or coated with biomaterials to provide researchers with insight into the controlled mechanical response and specific cell-protein interactions while maintaining the ability to adjust mechanical properties, and keep a low rate of degradation [186]. In general, a limiting property of hydrogels is their low stiffness and rigidity, which is the opposite of in vivo tissue properties. Hybrid hydrogels have been of interest in studies to improve both the stiffness and rigidity of hydrogels [177,187].

Many types of hybrid hydrogels have been developed for cancer mechanics studies. Fibronectin-coated PA substrates were used to study the invasiveness of different human glioma cell lines with a focus on cell structure, migration, and proliferation [188]. Cells

were rounder and had lower migration and proliferation rates in ECM substrates with lower rigidity. GBM migration patterns in the brain white matter tract were mimicked in vitro with electrospun alignment of nanofibers in a mixture of gelatin, poly-ethersulfone, poly-dimethylsiloxane, HA, and collagen in order to study migration patterns of GBM cells, and the addition of HA was seen to have a converse effect on migration [189]. The addition of HA to gelatin and PEG composite hydrogels resulted in dose-dependent glioma malignancy marker expression changes and cell clustering [190]. Another study combined PA hydrogels with HA and either laminin or collagen I and found that collagen and laminin presence was correlated to mechanical response to substrate stiffness [134].

### *2.2. General Properties of Hydrogels*

Hydrogels are defined by their hydrophilicity, which allows them to store water and swell without dissolving [191]. The bio-responsive properties of hydrogels, such as biochemical activity and degradability, allow for culture conditions to be dynamic and receptive to cellular cues. Conversely, since hydrogels are tunable in mechanical properties such as elasticity, compliance, and stiffness, it is possible to study the responses of cells to microenvironmental mechanics. While synthetic hydrogels are easier to adjust mechanically, natural hydrogels are more bioresponsive. In crafting a study, these are important properties to keep in mind, as they introduce variables to whichever system is being studied.

Two important descriptors of hydrogels that determine many physical properties are ionization degree and crosslink density. Crosslinks in a hydrogel are either chemical or physical, and can be introduced to a gel by methods such as irradiation, sulfur vulcanization, or chemical reactions aided by temperature and pressure [144]. Swelling and elastic modulus values are determined by cross-linking degree and charge densities or ionic strength of the polymers in the hydrogel [192]. Greater concentration of cross-linked polymers and the number of ionic groups cause higher elastic modulus values and greater swelling capacity [193]. Similarly, the distribution of proteins or polymers and cross-links in hydrogels, which are generally non-homogenous, are affected by cross-link density and degree of ionization [194,195].

There are numerous properties of hydrogels that can be influenced by internal factors, e.g., composition, protein concentration, and polymer modifications, and by external factors, e.g., UV radiation and temperature [161]. Chemical and physical reproducibility of hydrogels depends on controlled conditions and utility in research can be greatly enhanced by proper knowledge of hydrogel properties and subsequent unique modifications of the scaffold to better mimic whichever system is being studied [196].
