Designing Microgels for Cell Culture and Controlled Assembly of Tissue Microenvironments

Abstract

Micron-sized hydrogels, termed microgels, are emerging as multifunctional platforms that can recapitulate tissue heterogeneity in engineered cell microenvironments. The microgels can function as either individual cell culture units or can be assembled into larger scaffolds. In this manner, individual microgels can be customized for single or multi-cell co-culture applications, or heterogeneous populations can be used as building blocks to create microporous assembled scaffolds that more closely mimic tissue heterogeneities. The inherent versatility of these materials allows user-defined control of the microenvironments, from the order of singly encapsulated cells to entire three-dimensional cell scaffolds. These hydrogel scaffolds are promising for moving towards personalized medicine approaches and recapitulating the multifaceted microenvironments that exist in vivo.

2. Introduction

Hydrogels are highly water-swollen polymer networks that can be used to recreate critical features of the extracellular matrix for in vitro cell culture and in vivo cell transplantation. Features include mechanical properties similar to many soft tissues,^[1–4] the ability to incorporate cellular adhesive sites,^[5,6] and facile diffusion of exogenous or cell-secreted growth factors,^[7–9] which together recapitulate components of the extracellular microenvironment. However, creating multifunctional hydrogels with properties that can be controlled on multiple size and time scales remains challenging. Many bulk hydrogel properties are highly coupled and dependent on the network crosslinking density. As such, increasing the crosslinking density not only increases the modulus, but also decreases diffusivity of macromolecules and increases the concentration of any tethered ligands. Thus, the design of advanced materials where multiple biochemical and mechanical cues can be presented to cells in a spatiotemporal manner continues to be an area of interest.

In this regard, classic polymerization and processing methods are readily applied to synthesize micron-sized hydrogels, so called microgels, from materials traditionally used for bulk cell encapsulation. Single microgels may function as individual cell culture units, where cells are either encapsulated within or seeded onto the surface of the microgel. As the diameter of the microgel begins to approach the size scale of the cell (e.g., 1–10 um), differential cellular responses to physical and chemical features within the material microenvironment can begin to emerge, especially compared to responses to similar material chemistries processed in the bulk. In particular, individual microgel characteristics can be manipulated to suit distinct cell types, allowing for co-culture models in a single microgel (Fig. 1A). For example, multifaceted or Janus microgels can be designed where microgel composition, mechanics, and biochemical cues can be isolated to distinct zones within an individual microgel. Microgels can further be designed and tuned as cell-carriers, creating very high surface area to volume ratios ideal for cell expansion for a variety of cell types (Fig. 1B). This cell expansion platform is highly tunable, where microgel characteristics can be optimized for specific cell types. Functioning as a highly injectable platform, microgels with loaded cargo (e.g., exogenous cells, cytokines, small molecules) are also widely explored for therapeutic in vivo purposes (Fig. 1C). These can extend circulation times of transplanted cells, and allow for controlled release of delivered drugs and bioactive moieties.

Figure 1:

Microgels provide numerous design parameters that can be tuned for various cell culture applications. Microgels can be tuned individual to direct cells during 3D encapsulation (A) and cell expansion (B). Cellular or molecular cargo can be included within formulation and delivered from individual microgel subunits into the circulation (C), or from assembled scaffolds (D). Finally, microgels can be designed with “lock and key” type shapes (E) or complementary molecular binding pairs (F) to create higher ordered scaffolds.

Beyond controlling the size and material properties of microgels, fabrication strategies (e.g., microfabrication, controlled aggregation, microfluidics, and additive manufacturing) enable hierarchical assembly of multiple types of microgels. As such, multifunctional scaffolds that mimic the heterogeneous, extracellular microenvironment may be achieved, as microgels may serve as individual units for tuning mechanical and biochemical properties.

While in each of the previous cases distinct microgels can be tailored to specific applications or cell types, multiple microgels can further be assembled to form macro-scale cell scaffolds, creating highly user-defined environments. For example, microgel drug carriers can be assembled into a larger scaffold to function as a local drug reservoir. Adding a layer of complexity, however, individual microgel carriers can be designed separately, and scaffold can be tailored to deliver several growth factors over independent time-scales (Fig. 1D). Similarly, just as individual microgel units can function as co-culture models, “lock and key” designs allow experimenters to create clusters of several cell types, creating unique organ- and tissue-specific models in vitro (Fig. 1E). As a compilation of these design levels, entire scaffolds can be designed with distinct microenvironments (multiple cell types, chemistries, biochemical cues, etc.) (Fig. 1F). Combined, these allow the user to tailor multiple aspects of cell-culture independently, creating precisely regulated biomaterial platforms.

In this review, we highlight emerging strategies for the design and implementation of microgels as individual biomaterials units that can serve as building-blocks for assembling novel stem cell and tissue-engineered microenvironments. We discuss the selection of chemical and physical properties for altering microgel design and examine how microgels may be used as “building blocks” for encapsulating cells and assembling scaffolds. Finally, we highlight how microgels may be leveraged now and in the future for developing novel cell delivery systems for in vivo applications.

3. Microgel design

Similar to bulk hydrogel materials, there are a variety of fabrication strategies for creating microgels (Figure 2) with distinct mechanical, structural, and biochemical features. In this manner, microgels can be synthesized with a specific cell type in mind, matching the microgel characteristics to a tissue microenvironment. Building-block networks can also be created by designing microgels with complementary reactive groups to allow for subsequent assembly. When assembling microgels into macroscopic scaffold materials, numerous microgel units with varied properties (e.g., size, drug-loaded, crosslinking density) can be introduced to control the final porosity, chemistry, and mechanical properties. For example, a 1 mm³ gel synthesized from 10 μm diameter microgels could contain over a million different microgel formulations. Bulk hydrogel materials synthesized from molecular precursors, on the other hand, primarily depend on the initial reactant mixture. Subsequent modifications can then be introduced by incorporating biorthogonal chemistries ubiquitously within the polymer network, or through post-fabrication patterning of specific features and moieties. In microgel assembled networks, biomaterial scaffold properties can be modified using distinct building blocks to create an array of heterogeneous materials, in which mechanical or chemical characteristics are specifically tailored to elicit a desired cellular response with greater control. The difference is in the size scale of the control (molecular to mesoscopic) – for many cellular applications, the micron size scale is particularly beneficial. In this section, we will detail some of the parameters that can be manipulated to create distinct microgel building blocks and cell culture units.

Figure 2:

Designer toolbox for controlling microgel parameters. A) Microgel composition, size and stiffness can be varied to create mechanically distinct building blocks. B) Dynamic chemistries can be utilized to create responsive microgel subunits through use of enzymatically-, photo-, or redox-responsive crosslinks, while both covalent and non-covalent interactions can change particle elastic and viscoelastic responses. C) Microgels can be polymerized using suspension and emulsion type polymerizations, as well as formed through microfluidics, microdropletting techniques and micropatterning. D) Particle assembly can be engineered through simple physical jamming, as well ascomplementary reactive groups or shapes. E) Bioactive microgel units can be created through the inclusion of either tethered or releasable moieties. Distinct “zones” can be created to study cell-interface interactions or polarization potentials.

Mechanical Properties:

A key aspect of microgel mechanical properties derives from particle composition, where polymer characteristics, crosslinking, and volume fraction can be modified to control microgel size and modulus (Fig. 2A). A variety of crosslinking mechanisms exist for the creation of traditional hydrogel networks, where polymerization type (i.e., step-growth or chain growth) can be varied to render unique mechanical properties to the material.^[10,11] Dynamic environments can also be created through the use of reversible chemistries^[8,12] or stimuli-responsive materials^[13–15] to modify cellular responses during culture in bulk hydrogels. In particular, the range of hydrogel elasticity/viscoelasticity in these materials has been shown to produce lasting phenotypic and genotypic effects on cells.^[1,16–18] These approaches can then be applied to microgels, where microgel mechanical properties can be modified to elicit specific cellular responses. Polymer fraction or cross-linking density can be modified during microgel synthesis to synthesize a range of high or low modulus materials, while polymer type can also be chosen to introduce dynamic or reversible linkages.^[19] Microgel mechanics can be varied to control spreading and clustering^[20,21] or differentiation^[22] of encapsulated cells. The chemical toolbox available to researchers allows for the creation of varied microgel mechanics (e.g., stiff vs soft, elastic vs viscoelastic), which can then be subsequently assembled into macroscopic scaffolds. Using these strategies, the micro-environmental modulus can be varied independently from the bulk scaffold properties, allowing for the creation of soft, deformable materials that maintain a high local modulus for cell signaling.^[23] These alterations allow one to assemble heterogeneous microenvironments in which cells can interact and alter their phenotype. In this manner, tissue interfaces (e.g., tendon-bone, cartilage-bone) can be recreated within a single hydrogel scaffold through heterogeneous mechanical properties. Alternatively, the porosity, overall scaffold strength and toughness can be similarly tuned through variations in the individual microgel building blocks and their packing efficiencies, which can influence cell spreading and proliferation.^[24] Overall, this degree of freedom gives the user a high level of control over matrix mechanical properties, as matrix moduli can be tuned to suit specific of multiple cell types.

Crosslinking chemistries:

Engineering specific cellular interactions within microgels requires careful control of the microgel chemistry. Similar to bulk hydrogels, microgels can be fabricated using a variety of macromolecular precursors (e.g., synthetic or natural polymers, multifunctional monomers) and crosslinking methods (e.g., guest-host interactions,^[25] covalent crosslinking,^{[21,24,26,27]} physical interactions,^[28–30] and ionic interactions^[31,32]) (Fig. 2B). Incorporation of degradable crosslinks can be key for in vivo applications, where scaffold degradation allows for tissue in growth and remodeling. This is often achieved through the incorporation of protease degradable^[33–35] crosslinks within the microgels. Microgel degradation can also be utilized to achieve drug release in response to specific stimuli (e.g., redox conditions^[36] or enzymatic activity^[37]). Similarly, microgels can be functionalized with targeted moieties (e.g., heparin,^[38] or specific antibodies^[39]) to sequester specific therapeutic proteins. As untargeted therapies often rely on supraphysiological drug concentrations,^[40] targeted microgel platforms can improve drug efficacy and loading formulations. These approaches can also be used to create formulations that allow for on-demand release of encapsulated cells.^[41]

Fabrication techniques:

A wide variety of fabrication techniques (e.g., suspension and emulsion polymerizations,^[23,42,43] precipitation polymerization,^[44] microfluidics,^[45,46] electrospraying^[47,48]) also exist for processing polymers into microgels (Fig. 2C). The microgel chemistry is typically selected for ease of fabrication, functionalization, and compatibility with the fabrication process. For instance, synthetic polymers can be readily functionalized with tethered bioactive moieties, while some naturally-derived materials like alginate can be cheaply and readily cross-linked via divalent cations, leading to simple fabrication in microfluidic or electrospraying techniques. Substrate shape and curvature provide additional instructional cues to cells, with particle curvature and edge characteristics affecting cellular growth and development.^[49] While spherical microgels are most common, distinct shapes and geometries can be obtained using micro-molding^[50,51] or micro-patterning^[52,53] techniques. Beyond shape control, anisotropic microgels have been created, using multicompartmental, Janus, and core-shell designs, which allow for multiple chemical environments within a single culture platform^[54]. Amphiphilic polymers can be assembled to create microgels with hydrophobic pockets,^[55] while multi-channel microfluidic devices can be used to create rod shaped microgels^[56] or even multi-compartment microgels out of a single polymer type.^[45]

Directed assembly:

Importantly, assembly mechanisms can be pre-programmed into microgels through the inclusion of surface reactive groups (Fig 2D). While unmodified particles can partake in scaffold assembly through physical interactions and particle jamming,^[57–59] complementary reactive moieties or binding pairs can also be included on separate microgels for subsequent assembly into macroscopic structures.^[23,34,48] Through variations in ligand and microgel density, unique structures can be designed (e.g., 90% microgel type A, 10% microgel type B). In this manner, users can assemble permutations of unique microenvironments for cell culture. For example, microgel carriers have been designed with lock and key designs to create highly ordered microgel assemblies,^[60] allowing for precise spatial regulation of included cells and growth factors. Here, microgel populations are photopolymerized into complementary shapes, allowing for microgel assembly into user-defined patterns. Similarly, multiple orthogonal binding pairs (e.g., DNA complementary sequences) can be used to create specific patterns of microgel assemblies.^[61] Additive manufacturing techniques can also be used to form microgel assembled scaffolds. Microgel “inks” may be designed and then 3D printed to create user-defined constructs^[48,57,62]. In some instances, scaffold structure is subsequently stabilized by a secondary cross-linking mechanism^[35,48] or surrounding support hydrogel.^[63,64]. Direct printing of microgels into complex structures is a valuable tool for controlled assembly, allowing for the creation of distinct tissue environments.

Inclusion of bioactive cues:

Functional microgel units are readily designed through the inclusion of bioactive moieties and dynamic materials. Small molecules,^[65] peptides,^[33,34] and proteins^[27,58,66] can be loaded within the microgel for either transient or permanent presentation of biological signals (Fig 2E). Incorporation of cues can be achieved through non-covalent (e.g. physical entanglements^[22,47,58], electrostatic interactions^[67,68]), or through covalent tethering.^[69,70] The incorporation of cargo into microgels through physical entanglements is relatively facile, as the cargo can be embedded without chemical modification or ligation. Release rates of these transiently expressed signals can then be tuned by altering the material composition^[33,35] or polymer density.^[58] Similarly, microgel composition can be altered to include affinity ligands (such as heparin)^[71–75] to control cargo release rates. However, the incorporated cargo can also be modified with specific reactive groups (e.g. click chemistry pairs^[23,27,76]) to allow for tethering to microgels or into microgel networks. This approach has previously proven advantageous in bulk hydrogels in achieving spatiotemporal control over cue presentation.^[8,77–79] Similar to the incorporation of biochemical cues, responsive materials (thermo-responsive polymers^[80] or degradable motifs^[34,65]) can be included to create dynamic cell culture environments. Through control over microgel shape and isotropy, researchers can precisely dictate both the chemical and mechanical environment that cells experience, and control microgel organization within aggregates and assembled scaffolds. Thus, beyond simply serving as structural sub-units, microgels can also be designed to serve as signaling mechanisms in cell culture.

4. Cell incorporation

Microgel carriers are useful as either two-dimensional substrates (i.e., cells are seeded on the microgel surfaces), or as three-dimensional microenvironments (i.e., cells encapsulated within microgel units). To date, numerous literature examples demonstrate how microgels are used to modify responses in cell aggregates (e.g., embryonic stem cell spheroids, neurospheres, and islet beta cell clusters), and these examples have shown how microgel incorporation can be used to alter cellular growth, differentiation, and secretory properties.^[81–86] This section will focus specifically on reviewing the use of microgels as compartmentalized culture platforms for cellular expansion and differentiation protocols.

Cell expansion and differentiation:

Effective regenerative cell therapies or engineered immune cell therapies currently rely on the rapid generation of large numbers of cells for transplantation; thus, high density expansion techniques are required to meet this need. Compared to traditional two-dimensional cell culture, microcarriers have a higher surface area to volume ratio, providing increased space for expanding cells in culture.^[87] For instance, commercially available dextran microcarriers (Cytodex® 1) at 3 mg/mL can provide the same available surface area in 1L as in close to two hundred 75 cm² culture dishes.^[88] Microcarriers composed of stiff materials (e.g., polystyrene spheres) can also be used, but recent results have documented that cells maintain an irreversible mechanical memory of stiff substrates,^[16] potentially biasing cells toward osteogenic lineages and altering multipotency. Conversely, by using hydrogel materials that more closely replicate the physiological microenvironment of many stem cell niches, cells can be grown on microgels while maintaining multipotency,^[27,89,90] making them promising platforms for stem cell expansion (Fig. 3A). Report show that human embryonic stem cells (hESCs) can be rapidly expanded on microgels without loss of multipotency genes (e.g., NANOG, OCT4) and with comparable proliferation rates to 2D plate culture,^[91,92] providing an efficient platform for stem cell expansion while maintaining regenerative capacity.

Figure 3:

Microgels can function as platforms for cell expansion and culture. A) Microgels can function as substrates for cell expansion in suspension (top). Embryonic stem cells cultured on microgel carriers can be rapidly expanded (bottom), with or without the presence of exogeneous growth factors (right). Reprinted (adapted) with permission from^[27], Copyright 2013 American Chemical Society. B) Co-culture models can be created within single microgel subunits through the creation of dual-faceted microgels. Microfluidics can be used to create “core-shell” type carriers (left). This approach was used to model hepatocyte-fibroblast interactions, where coupling the two cell types within individual microgels significantly upregulated hepatocyte activity.^[110] Images were reproduced with permission from Royal Society of Chemistry.

While there is a growing appreciation that precise combinations of mechanical and biochemical cues play a role in guiding stem cell fate decisions, their presentation must be carefully regulated during culture to achieve a desired phenotype. Microgel chemistry, mechanics, and dimensions (curvature) can all be modified to affect and control the proliferation and differentiation of seeded cells.^[49,93,94] For instance, gelatin based microgel carriers have been shown to maintain human mesenchymal stem cell (MSC) multipotency more effectively than in 2D plate culture,^[95,96] and laminin coated cellulose microgels have been reported to significantly bias human embryonic stem cells (hESCs) towards cardiomyocyte lineages compared to traditional embryoid body culture.^[97]

Finally, microgel carriers have been functionalized to capture and separate specific cell types from multi-cell solutions.^[98,99] Inclusion of antibodies for specific cell surface markers allows for capture and isolation of a target cell type from a heterogeneous population without the need for traditional cell sorting techniques. In one instance, researchers were able to isolate either human mesenchymal stem cells or endothelial cells from a solution of both cell types with relatively high specificity.^[98] Microgel carriers have also been used to model alveolar cysts in culture, where degradable particles are used to template hollow epithelial spheres representative of their native morphology in the lung.^[100] Thus, by modifying the composition and structure of microgel carriers, users can isolate specific cell populations, control their proliferation and fate, and create physiologically relevant structures in culture. Further development of these types of systems could help bridge the gap between cell isolation and transplantation, creating a single cell culture device capable of isolation, expansion, differentiation, and transplantation.

Cell encapsulation:

Compared to platforms where cells are seeded on the surface of microgels, cells may be encapsulated directly within microgels to generate compartmentalized 3D scaffolds. Microgel carriers are advantageous due to their favorable mass transport characteristics, owing to their high surface area to volume ratio (compared to bulk materials),^[101] allowing for higher seeded cell densities, thereby increasing expansion efficiencies. Beyond serving as simple platforms to support cellular development, microgels can be tailored to precisely control the cellular microenvironment for promoting cell survival, promoting or suppressing cell-cell versus cell-matrix interactions, and guiding cell fate decisions. While there are many well-known protocols for directing stem cell fate decisions, they are often guided by precise combinations of cues, both mechanical and chemical in nature. Thus, microgels provide additional advantages as delivery depots that can be designed to instruct cell function locally, as opposed to adding soluble signaling factors to cell culture media often circulating through bioreactors. Microgels provide a platform to isolate stem cells (encapsulated singly or in cell aggregates) within distinct 3-D microenvironments to control their growth and development.

Just as changing the size of microgel carriers affects cell proliferation and differentiation when cells are seeded on the surface of these materials, microgel size can affect cell growth and fate decisions when cells are encapsulated within them. The degree of cell-matrix associations on this scale can affect differentiation potential, and as such, single-cell encapsulation provides a unique method for tracking and manipulating cell fate. For example, individually encapsulated MSCs exhibit increased osteogenic potential compared to cells encapsulated with one or more other cells^[32], likely due to a higher density of cell-hydrogel interactions. Notably, cell density within microgels has also been demonstrated to affect MSC secretory properties, with a significant increase in secretion of pro-angiogenic cytokines with increasing cell density.^[102] Microgel chemistry can also be tailored to pause^[103,104] or amplify^[22,105,106] the differentiation of encapsulated cells, serving as “controllers” to manipulate cell fate. Controlling cell multipotency and differentiation capacity in the absence of feeder cells and exogenously delivered growth factors can allow for more efficient cell culture systems, and can assist in large-scale, reproducible and high-quality stem cell expansion. Beyond stem cell culture, these materials can also create more accurate disease models compared to bulk materials or cell aggregate culture,^[107,108] and could be used to fill unmet needs for certain tissue models (e.g., lung, lymphatics) that are difficult to achieve with bulk materials or aggregate cell culture.

Co-culture:

Multiple cell types can be co-encapsulated within the same microgel for improved paracrine signaling between cell types. A significant gap of knowledge exists in understanding both how different populations of cells interact with each other and how these interactions vary between in vitro and in vivo models. Including complementary cell types within cell culture scaffolds can more accurately recreate a cell’s native environment and provide more accurate in vitro environments that more closely recapitulate in vivo conditions. The resultant paracrine interactions within microgel culture can be used to provide support to and enhance cell function.^[109–111] In one instance, the activity of encapsulated hepatocyte spheroids was first improved by altering the material chemistry of the microgel culture platform, and then improved further by co-encapsulating endothelial progenitor cells with the hepatocytes^[109]. Furthermore, cell-cell interactions can alter cell phenotype and the effectiveness of exogenously delivered signals.^[112,113] For example, the Mano group encapsulated endothelial cells with adipose stem cells within microgels and demonstrated that the inclusion of endothelial cells biased the stem cells towards osteogenic lineages, even in the absence of osteogenic media.^[113] While co-culture systems can be designed in traditional hydrogel environments, microgel platforms provide a more isolated system that provides precise control over cell-cell interactions, proximity and density of cell types, and time scales and distributions of cell secreted factors.

One difficulty in designing co-culture systems is that different cell types often require distinct cell culture environments, due to differences in nutrient metabolism, proliferation rates, and spatial organization during growth. Traditional cell culture platforms typically use a cell medium biased in favor of one cell type and is likely not optimized for the other cell types used in the co-culture. Multicompartmental microgels can potentially overcome this limitation, when distinct microenvironments are combined within a single cell culture platform (Fig. 3B). Different cell populations can be encapsulated in their preferred culture environments (i.e., matrix chemistry and stiffness, adhesive ligands, tethered growth factors), while maintaining proximity to each other.^[45] In this example, microgels could also be designed using a core-shell structure for this type of co-culture.^[110] Co-culture models may prove especially beneficial for recreating native organ function in vitro, where the presence of supporting cell types can influence functional properties of cells, enabling more accurate models to track disease evolution or screen for drug potency.^[51,110] Importantly, the use of multi-compartmental and core-shell microgels can allow for cell environments and paracrine signaling to be controlled independently to study, finely tune, and alter cellular activity.

5. Microgel assembly

While microgels can function as individual culture platforms, assembly of a collection of units can create larger cell-laden scaffolds and/or tissue mimics. As opposed to bulk hydrogels with a crosslinking density distribution and molecular pores on the order of 10 nm, assembly of microgels lead to scaffolds with mesoscopic pores (1–100 μm), created by physically or chemically associated microgel units. Stated differently, bulk scaffolds can be fabricated with fully formed microgels providing the internal analogous crosslinked polymer structure, rather than the crosslinked polymer chains that are present in traditional gels. This “bottom-up”, multiscale fabrication method allows for the design of microgels (with molecularly defined crosslinking densities and chemistries) that can be uniquely tuned and combined in varying proportions to form a macroscopic structure. Heterogeneous and customizable cellular microenvironments can then be created to ensure more accurate in vitro cell culture models.

Assembly techniques:

Just as many synthetic techniques exist for microgel fabrication, a variety of manufacturing approaches for microgel assembly can also be utilized. Microgel network formation requires both an assembly force, as well as a mechanism for creating interparticle interactions. Microgel assembly can be induced through microfluidic,^[114–116] magnetic,^[117,118] surface tension,^[53] and light-based^[119] techniques. Microgels have been fabricated with specific surface moieties to induce network formation through physical and chemical interactions after assembly. While simple physical entanglements lead to a percolated microgel network^[58], other interactions including electrostatic,^[120] guest-host,^[35,121] and covalent^[53,76,122] are more common. In particular, “bio-click” chemistries are attractive for assembly of cell laden microgel units, because of their rapid, cytocompatible nature.^[123–127] While numerous mechanisms exits to promote this interparticle cross-linking,^[128,129] microgel formulations can be designed to form in the presence of an exogenous initiator,^[76] spontaneously,^[23,122] in the presence of clotting agents in vivo,^[34] or with reversible chemistries to create “self-healing” scaffolds.^[116,130] Creating easily manipulatable formulations is important for biological applications, as they can be used to create injectable or moldable scaffolds.^[34,131,132] While the vast majority of previous injectable formulations have been non-porous and have relied on degradation rates to allow for cell infiltration, the inherent porosity in microgel assembled scaffolds could allow for improved formulations and rapid tissue regeneration. Importantly, microgel assembly and cross-linking can be tailored to specific cell types and applications. These properties can be integrated to create “user designed” scaffolds where material properties are more accurately paired to in vitro and in vivo applications.

Scaffold properties:

Beyond providing a toolbox of methods for network assembly, functionalizing microgels provides the ability to easily incorporate bioactive cues into a scaffold. Microgel modulus can be varied to control cell response within assembled scaffolds. The role of matrix mechanics in controlling cell fate and function is well documented in bulk hydrogel scaffolds,^{[1,16–18,133]} but due to the more recent emergence of microgel scaffolds, less is known about how microgel mechanics can affect cell behavior. Increases in microgel stiffness have been shown to increase cell spreading and result in the nuclear localization of mechanosensitive signaling proteins (such as YAP).^[21] In other instances, an increase in microgel stiffness was observed to increase the rates of cell proliferation and the efficacy of gene transfer^[24] and cellular morphogenesis.^[134] Another instance demonstrated how collagen content and microgel mechanics could be varied independently to control neuronal cell aggregation.^[65] Importantly, these examples demonstrate how scaffold parameters can be decoupled, assessing the influence of mechanics independent from adhesive ligand density and scaffold porosity, respectively. The Segura lab also demonstrated this concept in vivo, where stiffness, degradability, and adhesive ligand concentration could all be varied to control the immunogenic response and cell infiltration into the porous scaffold.^[135] Microgel scaffold modulus has been varied from several Pascals (Pa) for endothelial cell network formation,^[134] several hundred Pascals to match vocal fold tissue strength,^[64] several kPa for differentiation of stem cell spheroids,^[136] to several MPa in doubly cross-linked networks for supporting degenerated intervertebral discs.^[137] The wide range of achievable mechanics, as well as the ability to independently tune multiple parameters, demonstrate how microgel mechanics can be designed to mimic the ECM of specific tissues in order to optimize culture for specific cell types.

Several examples also show microgels may serve as structural components as well as carriers of small molecules and growth factors.^[58,66,138] Of particular note, microgels capable of capturing platelet derived growth factor (PDGF) from complex protein solutions were fabricated and assembled with adipose stem cells to create a bioactive cell culture scaffold.^[66] In another example, two oppositely charged growth factors were encapsulated together within a microporous cell scaffold, both maintaining bioactivity.^[58] While these cues were distributed throughout the scaffold, gradients could be created by using a centrifugation-based assembly while varying the buoyancy of the microgel carriers.^[65] Chemical cues, such as heparin which is known for its protein sequestering activity, can also be used to create such gradients in microgel scaffolds.^[38,71] This approach could be applied for recreating tissues in the body that exhibit a gradient of chemical or mechanical properties (i.e., cartilage^[139]). The creation of three-dimensional gradients, which can be difficult in traditional bulk gels, can be created through the use of 3-D printing of microgel “inks” or controlled assembly techniques. Several groups have demonstrated how this strategy can be used to print cell-laden microgel assemblies as scaffolds shaped as entire tissues.^[48,140] This approach can also be used in a layer-by-layer fashion to recreate tissue interfaces (e.g. the osteochondral interface)^[62], or to vary network structure to influence cell behavior.^[134] Mechanically heterogeneous microgel scaffolds have also been created using a layer-by-layer assembly to control cell spreading and morphology.^[46] Other approaches, such as centrifugation and vacuum molding, can also be used to create 3-D gradients of specific cell types (Fig. 4A)^[141]. These layer-by-layer methods for scaffold formation provide powerful tools for designing complex tissue structures in vitro.

Figure 4:

Microgel assembly can be utilized to create a variety of tissue mimics. A) Distinct three-dimensional cell gradients can be created through controlled assembly techniques. In one instance, fluorescently labeled fibroblasts (red or green) were encapsulated within microgels and assembled into continuous gradient structures.^[141] Image were reproduced with permission from John Wiley & Sons. B) Tissue mimics can be created by assembling microgels with complementary structures in a “lock-and-key” mechanism. Here, rhodamine labeled HepG2 (hepatocellular carcinoma) cells and fluorescein labeled 3T3s were encapsulated within distinctly shaped microgels and assembled to create co-cultures to recreate liver function^[53] (Scale bar = 100 μm). Image were reproduced with permission from John Wiley & Sons.

Scaffolds may also be designed to contain inherent porosity, avoiding the need for including porogens or post-fabrication processing. The inherent microgel micro-porosity allows for favorable cellular growth and, more importantly, cell motility within a scaffold and thus can aid in tissue regeneration. For example, cells within porous poly(ethylene glycol) microgel networks crosslinked via an activated clotting factor proliferated rapidly and exhibited rapid network formation compared to non-porous materials.^[34] Similarly, rapid rates of cellular infiltration were observed in these scaffolds in vivo, while they maintained lower rates of inflammation compared to non-porous materials.^[34] Network porosity can also be used to control cell morphology.^[23] As cellular morphology and genetic and phenotypic expression are highly interconnected,^[1,142,143] manipulating network porosity can be a unique tool for directing cell fate decisions. Altering cell confinement or spreading could be used to direct cells to specific lineages. The Segura group has also demonstrated how culture in porous assembled scaffolds can affect genetic transfection of encapsulated cells compared with traditional culture techniques.^[144] Similarly, the group later demonstrated that cell susceptibility to genetic transfer varies with pore dimensions.^[24] This aligns with a growing body of literature demonstrating that microenvironmental structure and porosity can have significant effects on cell function and phenotypic changes in 3D scaffolds.^[145–149] The facile nature of controlling scaffold porosity in microgel assembled scaffolds makes them ideal candidates to further explore these effects.

Systematic control of scaffold porosity has also been useful for rapid deposition of extracellular network components. Gelatin microgel based scaffolds allow for rapid cell spreading, collagen deposition and maturation.^[150–152] The rate of matrix deposition can be controlled by altering the degradation rate of the microgel scaffold, allowing user defined control over tissue properties over time.^[150] Similarly, microgel assembled scaffolds have been utilized for in vitro cartilage tissue formation, with the porous environment allowing for rapid collagen and glycosaminoglycan deposition compared to traditional bulk 3D scaffolds.^[153] In this manner, both cell morphology and extracellular matrix deposition can be tailored to a specific tissue type by controlling network porosity, allowing for proper regeneration in in vivo applications. Microgels allow for rapid tuning of these scaffold properties to achieve both spatial and temporal control over cellular properties. The highly tunable nature of these materials can allow for countless user-defined culture environments.

Tissue mimics:

Microgels can be formed into a variety of different geometries, either to recapitulate tissue morphogenesis or to recreate native organ environments (tissue ‘mimics’). Traditional bulk hydrogels have done well to mimic several aspects of tissues, especially concerning the composition of the extracellular matrix and the presence of bioactive cues.^[154–158] However the development of tissue “mimics” can further this approach, where organ structure and the presence of complementary cell types are also included to create a more accurate ex vivo model. This has been demonstrated in “organ on a chip” models where this approach can more accurately represent native cell behavior.^[159–161] Recently, microgel assembled scaffolds have proven useful in recapitulating organ structure and providing co-culture platforms to create unique tissue mimics. This would be particularly advantageous in drug development scenarios due to the failing of single cell-type cultures accurately predicting drug efficacy in vivo. Better recreation of a native tissue is widely thought to be able to assist in initial drug screening.^[162,163] Cell-encapsulating microgels can be designed with complementary shapes or surface reactive groups to create co-culture models and “tissue” clusters.^[122,164] In a 3-D co-culture system, microgels have been tailored with specific bioactive moieties to cater to the growth needs of a specific cell type. For instance, the adhesive peptide sequence RGDS was included only in microgels containing fibroblasts to promote cell spreading during co-culture with encapsulated human lymphoblast cells representative of tumor nodules.^[164] Cell-laden microgels have also been assembled into larger macro-scale tissues. For example, single or dual cell-laden collagen microgels were assembled into macro-scale molds to create constructs to mimic liver or cardiac tissues.^[165,166] Importantly, microgels can be adapted to function as a 3D printable ink to form complex structures. Larger, cell-seeded scaffolds can be printed on the order of centimeters, while organ-shaped structures can also be fabricated rapidly.^[48] Complex tissue environments can be synthesized in this manner, providing platforms for functional in vitro assays, as well as allowing the creation of custom-made transplantable platforms.

While this type of assembly results in homogenous cell distribution, microgels can also be designed in unique geometries, allowing for non-uniform cell placement within the tissue environment. For instance, different cell types can be encapsulated within microgels and assembled into specific patterns^[50,53,167] (Fig. 4B). Using complementary “lock and key” type geometries allows for user defined cell placement, which can be highly advantageous for designing tissue mimics with conduits for vascularization or for the recreation of tissue interfaces. Microgels with predefined internal microchannels were created to form assemblies with pre-defined vessels to mimic the vasculature.^[167] In this manner, millimeter sized constructs could be fabricated with smooth muscle cells and endothelial cells in a concentric pattern throughout. Pre-fabrication approaches have also been applied to regulate cell signaling in co-culture models, where patterns can be used to alter a cell’s microenvironment and paracrine effects. One limitation of this strategy is that it lacks the ability to create thicker, three-dimensional tissue constructs. Previous groups have functionalized microgels with self-assembling peptides to allow for the creation of thicker constructs with encapsulated fibroblasts.^[130] Self-assembled peptides can be used to create thicker (cm-scale) cell-laden constructs with high cell viability compared to traditional bulk materials. To date, this type of approach has only been successful in creating tissues with homogeneous cell distributions and hasn’t be used to create scaffolds with spatially controlled cell-patterns. However, just as patterning approaches can be used to create chemical gradients within 3-D scaffolds,^[65] they may be used to spatially regulate tissue assembly. Microgel carriers can be assembled through a layer-by-layer approach to create heterogeneous 3-D structures.^[141] While this specific approach uses a single cell type throughout the gradient networks, this method could be expanded to create “layered” tissues with multiple cell types. In this manner microgel scaffolds provide a toolkit for recreating complex tissue interfaces in in vitro cell culture.

6. In vivo applications

While microgels and microgel-assembled networks have been utilized extensively as drug delivery vehicles in vivo,^{[33,168–171]} their highly tunable characteristics provide opportunities to also address cell transplantation and tissue regeneration limitations. In vivo tissue regeneration strategies usually utilize a scaffold-based cell delivery approach, and several opportunities for technological improvements remain. First, growth factor delivery may benefit from a carrier based approach to modulate release rates for enhanced control of cellular proliferation and differentiation.^[172] Similarly, cells often suffer from low cell survival during and after the delivery process, thus necessitating a platform to protect cells during the transplantation and engraftment process.^[173] Finally, healing of chronic wounds may progress more efficiently with a templated material.^[174] While bulk hydrogels have been used to address these limitations, gel degradation rates may limit cell infiltration and tissue regrowth, and cellular viability remains limited post-transplantation using injectable hydrogel formulations. To address these limitations, microgels may serve as individual cell carriers or provide assembled networks for enhanced cellular infiltration upon implantation. Serving as individual units, microgel properties may be readily tuned to support and direct cellular function and enhance cellular residence time at the desired site of regeneration.

Individual microgels:

Individual microgels serving as cell carriers containing one or more cells have been widely explored for cell transplantation applications. Microgel carriers provide a protective coating from the stresses of transplantation, allowing for improved cell viability and engraftment during transplantation.^[175–177] For instance, Blocki and coworkers observed cellular residence times on the order of weeks using ECM-functionalized agarose microgel carriers, while cells were cleared within 48 hours following intramyocardial transplantation when injected as a cell suspension.^[175] Microgel constructs can also shield encapsulated cells from resident immune cells, providing protection and extending cell circulation and residence times.^[47,178] Furthermore, cell-matrix interactions within these carriers affect cell growth factor secretions in vivo,^[32,102] further modulating the effectiveness of cellular therapies. Of particular note, the Weitz and Mooney groups demonstrated that microgel carriers can greatly extend the circulation half-life of MSCs and improve their therapeutic properties, but also that cell density in the microgel carriers affects their circulation times (Fig. 5A).^[179] Proteins and growth factors can also be co-delivered with cells in these formulations,^[39,47] increasing cell survival, cell engraftment, and tissue regeneration. In one instance, monoclonal antibodies (mAb) to bone morphogenic protein-2 (BMP-2) were included within hMSC loaded alginate microgels during delivery to calvarial defects.^[39] Inclusion of the anti-BMP mAbs allowed for the sequestration of endogenous BMP2 in the defect site to induce osteogenesis in vivo and increased bone formation and defect closure. Thus, while microgels provide support and protection for cells during transplantation, growth factors, monoclonal antibodies, and signaling proteins can be readily included in the formulations to improve engraftment and therapy efficacy. Furthermore, the versatile nature of these carriers allows for extensive control over cell residence times, engraftment, and secretory properties.

Figure 5.

Microgels can function as delivery vehicles for cell-based therapies, extending cell residence time. A) Cell-matrix and cell-cell interactions affect therapeutic efficacy of cells in microgel carriers. The Mooney group has developed a system to create both singly encapsulated and multicellular hMSC alginate microgels. The observed that both cell density, as well as capsule integrity, can affect clearance times, with increased residence time using multicellular microgels compared to singly encapsulated cells.^[179] Images were reproduced in accordance with licensing procedures of PNAS. B) Microgel scaffold properties can be tuned to alter biological response. The Segura lab has demonstrated that microgel scaffold properties (i.e., degradability, adhesive ligands, stiffness) can greatly affect the function and therapeutic efficacy of transplanted cells. Here, scaffold properties affected the retention of transplanted MSCs (CD29⁺).^[135] Images were reproduced with permission from John Wiley & Sons.

Importantly, microgels serve as individual units to provide instructive cues to cells from isolation to implantation. For instance, co-culture microgel capsules can be designed to provide instructive cues, such as complementary cell types, to stem cells upon encapsulation, without the need for pre-treatment with exogenous growth factors.^[180] Microgels have been designed with specific adhesive domains to isolate stem cells from bone marrow isolates, culture and expand them in vitro, and deliver them to a wound site with high efficiency.^[99] Thus, while bulk materials can be designed to be injectable and provide support for cell engraftment during and after injection, microgels can be modified to function as a highly specialized platforms for both cell culture and transplantation. Together, microgels can precisely regulate cellular and growth factor presentation in vivo, while providing a single material capable of isolation, growth, and transplantation.

Assembled microgels:

Microgel assembled networks contain an inherent porosity, which may be advantageous for wound healing applications. For example, acellular assembled microgel scaffolds increased the rate of epidermal wound healing over two-fold in a mouse model compared to no treatment, or treatment with a non-porous bulk scaffold.^[34] The inherent porosity in these microgel-scaffolds allows for rapid cell infiltration and complex tissue formation into the wound site, facilitating wound closure. However, while an increase in overall cell infiltration was observed, there was a decrease in overall inflammation, as indicated by levels of infiltrating CD11b⁺ inflammatory monocytes. A controlled inflammatory response allows for proper healing while mitigating the foreign body response to the implanted material. Porous scaffolds can also be used to mediate inflammatory reactions compared to non-porous materials.^[34,181] Microgel networks that are both fully injectable and porous can provide rapid tissue regrowth and regeneration without being limited by bulk gel (or porogen) degradation rates that can limit the rate of tissue regeneration. Scaffold properties (e.g., stiffness, degradability, and ligand presentation) can be modified to tune the regenerative properties and biological response to the material (Fig. 5B).^[135] As microgels serve as individual building blocks in assembled scaffolds and may be tuned and functionalized independently, this approach may be exploited to include multiple different cues within a single scaffold for rapid wound healing. While microgel assembled networks have been used sparingly for in vivo applications, their inherent porosity can provide immediate advantages for regenerating tissue more effectively compared to bulk injectable materials.

7. Outlook

The inherent complexity of the cellular microenvironment necessitates innovative cell culture materials to provide specific and complex instructions to cells and appropriately recapitulate the heterogeneous cellular microenvironment. Microgels uniquely provide highly tunable microenvironments for manipulation of encapsulated cell phenotype, as well as a building block-based assembly approach which provides the opportunity to create complex culture environments to recapitulate tissue heterogeneity. Tuning the physiochemical properties of microgels allows for the creation of defined microenvironments, which can more accurately recreate a cell’s native environment. For instance, co-culture models have long been troublesome as the two cell types can often require different culture conditions (e.g., media formulations). Microgels may serve as promising co-culture platforms, with the ability to include multiple cell types within a single microgel environment and provide compartmentalized heterogeneous cues to each cell type within the microgel using multicompartmental and core-shell fabrication strategies. Finally, microgels may improve residence times and regeneration efficacy of transplanted cells in vivo. Taken together, microgels provide a powerful tool for directing cell fate both in vitro and in vivo by offering a manipulatable platform to recapitulate complex cellular microenvironments.

While microgels provide a wealth of tunable parameters, open questions remain regarding the use of microgels to simultaneously deliver a heterogeneous set of cues to recapitulate native microenvironments. For example, including various bioactive moieties within microgels provides more robust cellular differentiation, however, more work is needed to demonstrate how cues can be sustained or altered over time depending on the application. Current co-culture models do well to better recapitulate native cell function, but important aspects of individual organ microenvironments may still be lost. Moving forward, new co-culture models need to better recreate the native organ structure for an individual cell type in vitro to more accurately model its function. The creation of accurate in vitro organ models will require precise regulation of cell-matrix signaling, but the inherent versatility of microgel platforms will allow them to rise to the task.

In the future, careful design and control of microgel chemistry, mechanics, shape, and cellular encapsulation can be combined to create microgel libraries for precision medicine applications. Precision engineering of both the physical and chemical properties of microgel building blocks could allow for the creation of personalized biomaterials, with cell-therapies tailored for a specific diseased environment within a patient. For example, uses of microgels as unit operators such as “separators” or “controllers” can enable the generation of patient-specific devices where specific bioanalytes may be captured for diagnostics.^[182] We also posit the facile nature of microgel assembly techniques will allow for the creation of cell culture scaffolds that can robustly expand therapeutic cell populations in situ (e.g., CAR T cells). Synergistic cues can be combined for more accurate cell-matrix signaling, and the facile nature of assembly can be exploited to recreate tissue microenvironments. As more is learned about the cellular requirements for a specific biomedical application, the use of microgels as tools to assemble stem cell and tissue engineered microenvironments remains promising.