Alginate Microencapsulation for Three-dimensional in vitro Cell Culture

Abstract

Advances in microscale 3D cell culture systems have helped to elucidate cellular physiology, understand mechanisms of stem cell differentiation, produce pathophysiological models, and reveal important cell-cell and cell-matrix interactions. An important consideration for such studies is the choice of material for encapsulating cells and associated extracellular matrix (ECM). This review focuses on the use of alginate hydrogels which are versatile owing to their simple gelation process following an ionic crosslinking mechanism in situ, with no need for procedures that can be potentially toxic to cells such as heating, the use of solvents, and UV exposure. The article aims to give some perspectives, particularly to researchers who typically work more with poly(dimethylsiloxane) (PDMS), on the use of alginate as an alternative material to construct microphysiological cell culture systems. More specifically, this review describes how physicochemical characteristics of alginate hydrogels can be tuned with regards to their biocompatibility, porosity, mechanical strength, ligand presentation, and biodegradability. A number of cell culture applications are also described, and these are subcategorized according to whether the alginate material is used to homogeneously embed cells, to micropattern multiple cellular microenvironments, or to provide an outer shell that creates a space in the core for cells and other ECM components. The article ends with perspectives on future challenges and opportunities for 3D cell culture applications.

Graphical Abstract

1. Introduction

Microscale three-dimensional (3D) cellular systems allow the study of biological processes that involve the interplay among cells, their surrounding environment, and different external physical and chemical stimuli.^1–3 In multicellular organisms, cells in nearly all tissues (and organs) reside in complex 3D networks of macromolecules, known as the extracellular matrix (ECM), that provide structural scaffolding and regulate intercellular communication. The ECM modulates a cell’s dynamic behavior, and is therefore important for morphology, differentiation, proliferation, evolution, and function of the cells and tissues.^4,5 In addition to the complexity of ECM components, it is also important to appreciate the diversity among different tissues, as cells of different types require significantly different microenvironments. Although microscale 2D cell culture systems, often comprised of poly(dimethylsiloxane) (PDMS) devices, have considerably improved the understanding of basic cell functions, cell growth and manipulation in 2D can be insufficient to provide physiologically accurate representations of the in vivo microenvironment of many cells and tissues.^6–8

3D cell culture systems are often better in vitro models for investigating aspects of the in vivo microenvironment compared to their 2D counterparts.^9–11 Table 1 summarizes some representative differences between 2D and 3D cell culture systems. Gene/protein expression is different between 2D and 3D cell cultures and can give rise to differences in morphology, proliferation, cell fate, and other functions. The main goal of 3D cell culture is to provide a realistic microenvironment in which cellular mechanisms are comparable to conditions in vivo.^12,13 3D cell cultures have increased our understanding of (1) the ability of cells to sense and respond to signals from the ECM in terms of surface topography;^14,15 (2) the effect of mechanical properties (e.g., stiffness, viscosity, and elasticity) on cellular differentiation;^16,17 (3) the role of ECM composition in development and morphogenesis;^18–20 (4) new cellular signaling pathways;^21,22 and (5) the biochemical signaling processes for regulating stem cell differentiation.^23–25

Table 1.

Representative differences in cellular properties between 2D and 3D culture systems.

Cellular properties	2D	3D	Refs.
Morphology	Stretched and Sheet-like cells in monolayer	Spheroid and aggregate structure	225,226
Proliferation	Frequently proliferate at a faster rate than in vivo	Proliferate at a faster/slower rate compared to 2D culture	227,228
Response to soluble factors	Cell monolayers are equally exposed to nutrients and growth factors	Nutrients and growth factors may not be able to fully penetrate the 3D structure	229
Gene and protein expression	Gene and protein expression levels often vary from in vivo	Often display gene/protein expressions similar to in vivo	230,231
Nutrient limited physiology	Cells are not nutrient limited	Some cells are quiescent, hypoxic, or necrotic	134,232

Various methods have been developed for microscale 3D cell culture, such as microwells,^26,27 hanging drops,^28,29 cellular microarrays,^30,31 and microfluidic devices.^32,33 First, microwell systems are extensions of the traditional multi-well plate where the size of wells are reduced, the surface rendered non-adhesive, and the well-geometry varied to allow generation of 3D spheroids with controllable sizes and shapes.^34–36 Second, in the hanging drop-based method, cells are suspended in media droplets that are freely hanging. Cells aggregate at the bottom of droplets, leading to the formation of 3D spheroids often with tight cell-cell adhesions.^37–40 Third, cellular microarrays fabricated using robotic spotting and micropatteming techniques, allow cell-cell and cell-ECM interactions by cell and ECM printing.^41–43 Lastly, microfluidic devices can perform controlled microscale 3D culture by introducing fluid flows, enabling precise placement of cells within geometrically confined microchannels and the entrapment of cell suspensions in specific regions of porous membrane.^44–46 The above microscale 3D cell culture platforms are all designed to address inherent limitations of 2D culture systems while allowing higher throughput and offering more precise microenvironment control. However, these 3D culture devices can have limitations in providing ECM scaffolds or in controlling the physical and biochemical properties of the culture substrate and environment. Moreover, these methods can be labor-intensive and timeconsuming; often require sophisticated instruments and specially trained users for fabrication and operation of the devices.⁴⁷ A more comprehensive overview of the commonly used microscale 3D cell culture platforms and their advantage and limitations are provided in Table 2.

Table 2.

Conventional devices and techniques for microscale 3D cell culture.

3D cell culture	Advantages	Drawbacks
Microwell	- Control over spheroid size and shape - Multiple cell types culture - Screening application possible	- Little work done with ECM molecules - Fabrication of microwells - Requires specialized instrument
Hanging drop	- Reproducible procedure - Control over spheroid size - Multiple cell types culture	- Low stability of droplets - No ECM molecules - Difficult media replacement - Generally short-term culture - Risk of evaporation
Cellular mieroarray	- Screening of cell-cell and cell-biomaterials interaction - Biomatenals and ECM molecules can be added	- Sharing of media - Requires robotic spottmg instrument - Limited substrate materials - Cross talk between samples
Microfluidic device	- Offer physical stress by controlling fluids - Chemical gradient generation - Higher permeability to gases - Real tune, on-chip analysis	- Surface treatment and coating required - Difficult to recover cells from channel - Immunohistochemistry challenging - Can be permeable to water vapor

Compared to the above approaches, hydrogel microencapsulation of cells is a promising technique for performing microscale 3D cell culture, as it can provide more physiologicallyaccurate ECM-containing environments.^48–50 Hydrogels are porous substrates that can provide footings for cell growth, organization, and differentiation within their network structures. The hydrogels are able to mimic the microenvironments of many tissues due to their high water retention capacity and biocompatibility.⁵¹ The versatile characteristics of hydrogels make them excellent scaffolds for 3D cell culture. Thiele et al. published a comprehensive review on desirable hydrogel characteristics for 3D cell culture applications.⁵² Another advantage of 3D culture using a hydrogel is that the cell culture protocols are robust and simple. However, limitations of hydrogels arise due to their indeterminate and variable composition.⁵³ Nevertheless, studies have performed 3D cell cultures using hydrogels of agarose;⁵⁴ carrageenan;⁵⁵ alginate;⁵⁶ chitosan;⁵⁷ gellan gum;⁵⁸ hyaluronic acid;⁵⁹ collagen;⁶⁰ gelatin;⁶¹ elastin;⁶² fibrin;⁶³ and silk fibroin.⁶⁴ Among them, alginate is the most common material for microencapsulation owing to its permeability, biocompatibility, and ability to perform in situ ionic crosslinking of the material.⁶⁵ In addition, alginate microgel encapsulation of cells can be performed in well-defined processes using isotonic solutions under physiological conditions with regards to temperature and pH.⁶⁶ Based on these controllable physicochemical properties, alginate offers ease of modulation in terms of biocompatibility, porosity, mechanical strength, ligand presentation, and biodegradability, which are important for modulation of mass transportation, regulation of cellular signaling, and control of 3D cell organization (Figure 1).

Figure 1.

Schematic illustration of alginate-based 3D cell culture system.

An important question is how modulation of alginate affects the fate of encapsulated cells in microscale 3D cell culture. Although many review papers have previously summarized the use of alginate hydrogels for 3D cell culture in more detail,^67,68 this article aims to provide a short primer for the use of alginate hydrogels, including methods to modify their properties, and as an alternative to use of PDMS in microphysiological cell culture.^69–73 In particular, our focus is to discuss and provide examples of specific features of the alginate hydrogels, including the engineering of mechanical properties, permeability, degradation kinetics, and chemical ligand presentation; features that are complementary to PDMS. This review then describes applications of alginate hydrogels in cell microencapsulation in terms of homogeneous alginate hydrogel encapsulation, patterned alginate hydrogels, and composite alginate hydrogels. Finally, current limitations and future directions for advanced 3D alginate cell culture are discussed.

2. Properties of alginate beneficial for 3D cell culture

2.1. Tunable mechanical properties

Alginate is a natural hydrophilic polysaccharide derived from the cell walls of brown algae (Phaeophyceceae), and is composed of β-(l-4)-linked d-mannuronic acid repeats (Mblocks) and β-(l-4)-linked 1-guluronic acid repeats (G-blocks) (Figure 2a). These blocks are linked by glycosidic bonds and form either homopolymeric block copolymers (M- or G-blocks), or alternative copolymers (MG-blocks).^74,75 The M-blocks possess a linear and flexible configuration, whereas the G-blocks introduce steric hindrance around the carboxyl functional groups. Because of this configuration, the G-blocks form folded and rigid structural feature that governs the overall alginate mechanical property.⁷⁶ Therefore, M-to-G ratio is a major determinant factor for mechanical properties of resulting hydrogels. For instance, a large proportion of G-blocks (i.e., M/G << 1) would result in production of a strong and rigid hydrogel, whereas alginate with a high M/G ratio (>> 1) produces a soft and elastic hydrogel.⁷⁷ The distribution of the M- and G-blocks is mainly dependent on the source and type of algae, and its purity depends on extraction processes.

Figure 2.

Characterizations of alginate, a) Chemical structures of D-mannuronic acids (Mblocks), L-guluronic acids (G-blocks), and copolymer repeating unit; b) Two ionic gelation mechanisms (internal and external) of alginate hydrogels and “Egg-box” formation in presence of crosslinking divalent cations such as Ca²⁺; Schematic illustration of alginate modifications: c) interpenetrating polymer network (IPN), and d) chemical modification for ligand presentation.

Alginate hydrogels can be formed in the presence of a variety of divalent cations such as Ca²⁺, Ba²⁺, Sr²⁺, Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺, as these form ionic bonds with carboxyl groups.⁷⁸ During the crosslinking process, these hydrogels shrink and increase the stiffness of the material relative to alginate in solution. The ionic crosslinking generates a 3D network by coordination between four-carbonyl functional groups of G-blocks and divalent cations, giving an “egg-box” arrangement.⁷⁹ The chemical composition and distribution of active G-blocks in the alginate chains play a major role in the formation of ionic gels with diverse properties. Furthermore, divalent ions can determine the strength and stability. The crosslinking strength of divalent cations to carboxyl groups increases in the following order of Mn²⁺ < Zn²⁺, Ni²⁺, Co²⁺< Fe²⁺ < Ca²⁺ < Sr²⁺ < Ba²⁺ < Cd²⁺ < Cu²⁺ < Pb²⁺.⁸⁰ Therefore, the mechanical properties of alginate hydrogels can be modulated with different divalent cations. For instance, Ba²⁺crosslinked gels are stronger than that of their Ca²⁺ crosslinked counterparts,⁸¹ and have been investigated for cell encapsulation.^82–84 Furthermore, Brady et al. have demonstrated that additional factors such as alginate molecular weight, chemical composition, and concentration determine the mechanical properties of alginate hydrogels.⁸⁵ However, it is important to note that many divalent ions show high toxicity, particularly Pb²⁺, Cu²⁺, and Cd²⁺, which should be taken into considerations for the gels to be used as 3D cell culture systems.⁸⁶

The ionic crosslinking process can be by internal or external gelation (Figure 2b).⁸⁷ In the internal crosslinking mechanism, gelation occurs in a controlled manner by first reacting insoluble forms of divalent salts with the cations-of-interest with alginate solution, then subsequent acidification induces exchange of ions, hence the release of cations into the solution causing gelation. The main advantage of this method is that it provides better control of gelation kinetics for homogeneous gelation. On the other hand, in the external method, gelation occurs gradually over space and time as cations diffuse throughout the alginate solution. In contrast to the internal gelation method, gelation kinetics are dictated by diffusion of cations, and generally produce inhomogeneous hydrogels with a gradient of the extent of crosslinking; the outer surface is most crosslinked and the interior is least crosslinked.^88,89 To summarize, many methods have been developed and implemented to modulate mechanical properties of alginate hydrogels. This mechanical tunability is one key feature that makes alginate an attractive solution to mimics ECM scaffolding in microscale 3D cell culture applications.

2.2. Semipermeable and degradable encapsulation

An important consideration for 3D hydrogel networks used for cell culture is permeability to gases and biological macromolecules. In the in vivo cellular microenvironment, cells obtain essential nutrients and eliminate metabolic wastes via diffusion, convection, secretion, excretion, cellular uptake and filtration. The porosity of hydrogels enables exchange of O₂, CO₂, nutrients, and metabolic wastes without disrupting the physical structure or mechanical characteristics of the material.

Alginate hydrogels can swell. Swelling is defined by the ratio of hydrogel sizes before and after swelling, and depends on crosslinking density, solvent polarity, and affinity between solvent and polymer.^90–92 When a dry hydrogel makes contact with water, the most hydrophilic functional groups interact with the water molecules, forming a primary-bound water layer. Hydrophobic functional groups also interact with water molecules, creating a secondary-bound water layer. Upon saturation of the functional groups, the remaining free or bulk water molecules distribute themselves in the spaces throughout the hydrogel networks, due to osmotic pressure.⁹³ Many researchers have investigated alginate hydrogel swelling and its impact on 3D cell culture.^94–97 The capacity of alginate swelling is mainly dependent on the amount and type of divalent cations crosslinking alginate hydrogels.⁹² For example, Ba²⁺-incorporated alginate hydrogels show significantly lower swelling ratios compared to Ca²⁺-alginate hydrogels because the affinity of Ba²⁺-ions towards the G-blocks in alginate polymer is higher than that of Ca²⁺ ions, resulting in increased stiffness of hydrogels, as discussed in Section 2.1.

Typically, the average pore size of alginate hydrogels is in the nanometer scale (5–200 nm).^98,99 This range of pore size allows molecules less than ~ 650 kDa to be transported in and out through alginate matrices. Examples of such molecules include, oxygen (~16 Da), carbon dioxide (~44 Da), urea (60 Da), glucose (180 Da), insulin (~5700 Da), and other macromolecules.¹⁰⁰ Moreover, both the stability and permeability of alginate hydrogels can be modulated with the use of polycations. Examples of such polycations include poly-L-lysine (PLL),^101,102 poly-L-ornithine (PLO),¹⁰³ and poly-methylene co-guanidine.¹⁰⁴ The polycations interact electrostatically with the negative charged alginate surface which results in decreased pore sizes.^105,106 For example, alginate hydrogels encapsulating insulin-secreting cells such as islets of Langerhans are coated with PLL to decrease the outer pore size, while maintaining a liquid-core.¹⁰⁷ Another example is the use of PLL to decrease pore sizes and reduce the diffusion rate of encapsulated hemoglobin.

The crosslinking methods, as described earlier in Section 2.1, have an effect on the pore sizes of the alginate hydrogels produced (Figure 2b). In internally-gelled alginate hydrogels that use insoluble gas-forming divalent cation salts such as carbonates and citrates,¹⁰⁸ exposure to acid induces ion exchanges to promote a homogeneous crosslinking profile with large and uniform pore sizes on the hydrogel surface.¹⁰⁹ In contrast, inhomogeneous hydrogel structures obtained from external crosslinking has smaller gel networks on the alginate surface with pore sizes of 12–16 nm.¹¹⁰ Note that the pore size also depends on the type of crosslinking cations, alginate M/G ratios, and the concentration of alginate.^111–113 For instance, Thu et al. demonstrated that Bovine serum albumin (BSA) diffused out of alginate hydrogels at a higher rate in hydrogels with lower concentration of alginate.¹¹⁴ It is known that the porosity decreases with increasing amounts of G-blocks, which interact with cations to form crosslinks. Kierstan investigated the relationship between the pore size and crosslinking divalent cations such as Ca²⁺ and Ba²⁺ ions. As expected, it was demonstrated that Ba²⁺ produced hydrogels with reduced pore size due to its higher affinity to G-blocks.¹¹⁵ Moreover, the effective pore size can also be altered by introducing other polymers, such as chitosan, which reduced the pore size to 1.74 nm.¹¹⁶ This ability to tune pore sizes makes alginate useful for modulating biomolecule transport into and out of alginate structures. For example, encapsulation of insulin protected it from denaturation in the gastric environment (pH 1.2), while releasing the encapsulated protein with zero-order kinetics under a neutral pH condition.¹¹⁷

The thickness of alginate hydrogels is also a critical consideration for regulating mass transportation.^118–120 In general, as the thickness of the alginate hydrogels increases, transport limitations of small molecules including nutrients and wastes will occur from outer shell to center core, causing cellular necrosis under hypoxic and nutrient-limited environments. The concentration of O₂ in alginate hydrogels has been evaluated to be relatively constant up to depths of ~100 μm in the hydrogel.¹²¹ Based on this O₂ limitation, an alginate microcapsule with a diameter of 0.9–1 mm showed βTC3 cells to form a ~0.2 mm thick layer at the periphery, whereas cells located at the center of the microcapsule died.¹²² This growth pattern implies that nutrient gradients form with a high level at the outer layer and less towards the center of alginate microcapsules.

Certain applications such as tissue replacement and controlled molecule release require gel degradation. Alginate is not metabolized by mammals; however, the crosslinked alginate hydrogels can slowly dissolve through exchange reactions of divalent cations with monovalent cations such as sodium ions in the surrounding milieu.^123–125 Physical and chemical modifications can modulate degradation. Degradation can be manipulated by controlling the molecular weight and composition of the alginate polymers.¹²⁶ A clever approach to create polymers of varying molecular weights and structures uses gamma radiation.^127,128 Irradiation doses below 8 Mrad divide MG chains while keeping the G content and G-block length constant. Such gamma-irradiated alginate hydrogels degrade more rapidly, and hence are cleared more quickly in vivo, leading to a significant improvement in bone regeneration from transplanted cells.^129,130 Degradation rates can also be controlled by incorporating alginates with different lengths of G-blocks to induce size mismatches among the crosslinking blocks.¹³¹ Another chemical method is to perform partial oxidation of alginate with sodium periodate which leads to faster degradation of alginate hydrogels.^99,132 This method cleaves cis-diol groups in uronate residues at the carbon-carbon bond and creates a hydrolytically labile bond, enabling degradation of the alginate chains. The rate of degradation is dependent not only on the degree of oxidation, but also on physical conditions of the surrounding environments such as temperature and pH of the media.^123,133 This method has been used to produce alginate gels that serve as delivery vehicles for drugs and cells. These findings collectively show that degradation kinetics of alginate hydrogels can be precisely controlled for a wide range of applications in 3D cell culture research. The ability to adjust swelling, porosity, and crosslinking of alginate enables versatile regulation of permeability and degradability of alginate hydrogel microencapsulation for applications in cell transplantation and tissue engineering.

2.3. Ease of modification

The control of soluble factor distribution within 3D hydrogels is important because the response of cells and tissues to biomolecules depends on the bulk concentration in the medium, diffusion-limited gradients within the gel, and rates of change in these parameters.¹³⁴ Recent advances in targeted delivery systems with 3D hydrogels show promise for drug delivery with enhanced pharmacokinetics and pharmacodynamics.^135–137 Soluble biomolecules frequently show enhanced bioactivity when encapsulated and attached to hydrogel networks. Growth factors are proteins that control cell differentiation, proliferation, tissue growth, and vascularization.^138–140 Various growth factors have been encapsulated within alginate hydrogels. For example, Mierisch et al. demonstrated the targeted delivery of TGF-β using calcium alginate hydrogels to treat osteochondral defects.¹⁴¹ Additionally, the temporal and spatial availability of growth factors can affect stem cell differentiation. Ansari et al. developed a stem cell delivery system by using alginate-hyaluronic acid (HA) gels loaded with TGF-β1 ligand and encapsulating periodontal ligament stem cells (PDLSCs) for chondrogenic differentiation.¹⁴² They demonstrated that sustained release of TGF-β1 can be achieved by regulating the hydrogel composition of alginate/HA, and higher expression of chondrogenesisrelated genes was observed from hydrogels with a 2:1 alginate/HA composition.

As mentioned previously, alginate hydrogels have many desirable characteristics for 3D cell culture such as controllable mechanical strength, porous structures, permeability to essential substances, and degradation. However, alginate, like most unmodified polysaccharides, lacks binding sites for cell adhesion and attachment, and has low adsorption capacity for proteins. Because of this inherent characteristic, cells encapsulated within native alginate hydrogels do not undergo proliferation, migration or exhibit other cellular responses.^143,144 One approach to provide sites for cell adhesion is to form an interpenetrating polymer network (IPN), which is a 3D network structure of at least two polymers consisting of alginate plus one other material such as: collagen;^145,146 hyaluronic acid;^147,148 gelatin;^149,150 and chitosan.^151,152 IPN is a combination of more than two polymer chains each in network formation without any covalent bonds between them (Figure 2c). This network structure is classified into two categories, namely, a full IPN and a semi-IPN. The former is formed when two (or more) different polymers are crosslinked independently, whilst the latter refers to networks with only one polymer of the assembly crosslinked, leaving the other in a linear form.^153,154 Mahou et al. proposed IPN hydrogels from an alginate-collagen blend and evaluated its property in tissue engineering applications.¹⁵⁵ Ionotropic crosslinking of alginate was performed with collagen fibrillogenesis, and the resulting IPN hydrogels exhibited stiff mechanical property with great resistance towards enzymatic degradation. In addition, they showed that the viability of the embedded mesenchymal stromal cells (MSCs) was unaffected, and human umbilical vein endothelial cells (HUVECs) were able to attach themselves and proliferate on the collagen-coated IPN alginate hydrogels. More recently, Vorwald et al. developed fibrin-alginate IPN hydrogels to increase cell adhesion and mechanical strength simultaneously.¹⁵⁶ Cellular responses of co-cultures of MSCs and endothelial cells (ECs) was modulated by alginate crosslinking density and fibrinogen concentration. As IPN stiffness increased, total cell density decreased with increases in cell circularity. Challenges for these methods include the need to optimize polymer concentrations and crosslinking ratios to avoid negative effects of unreacted materials.¹⁵⁷ Various types of extracellular matrix proteins and synthetic peptides are employed to overcome the need for increasing cell adhesion. Commonly used biomolecules include gelatin, collagen, arginine-glycine-aspartic acid (RGD) peptide derived from fibronectin, Asp-Gly-Glu-Ala (DGEA), and tyrosine-isoleucine-glycine-serinearginine (YIGSR) derived from laminin, that are the functional adhesion ligands in the ECM (Figure 2d). Among them, gelatin has been extensively studied for increasing cell adhesion and differentiation in alginate hydrogels.^158,159 For instance, Balakrishnan et al. demonstrated that oxidized alginate having appropriate molecular weight and degree of oxidation rapidly crosslinks with gelatin.¹⁶⁰ The gelatin incorporated alginate hydrogels successfully encapsulated hepatocytes and increased their albumin secretion. Inclusion of RGD-peptidefunctionalized co-monomers in alginate hydrogels allows tailored biological recognition to promote cell adhesion, cellular remodeling, and migration.¹⁶¹ The RGD tripeptide motif has been widely used as ligands for cell adhesion, because they are recognized by transmembrane integrin receptor proteins (more specifically, α_vβ₃ and α₅β₁ for various cell types).^162,163 The conjugation of RGD peptides to alginate chains can be chemically performed by using water soluble carbodiimide chemistry.¹⁶⁴ RGD-modified alginates maintain viability and promote (1) proliferation of bone marrow MSCs;¹⁶⁵ (2) muscle differentiation of umbilical cord MSCs in alginate-fibrin-RGD hydrogels;¹⁶⁶ (3) osteogenic differentiation;¹⁶⁷ (4) vascular endothelial growth factor (VEGF) secretion from MSC spheroids.¹⁶⁸ Additional incorporation of a metalloproteinase cleavable peptide (proline-valine-glycine-leucine-isoleucine-glycine) further enhances adhesion and provides better elongation of MSCs compared to RGD-alginate hydrogels without the cleavable peptides.¹⁶⁹ Different peptide motifs including DGEA¹⁷⁰ and YIGSR¹⁷¹ sequences have also been investigated for modification of alginate hydrogels and found to improve the adhesion of various cells.¹⁷² The majority of the studies to date, however, has focused on RGD-alginate hydrogels because of its higher solubility in aqueous media, short and easy manufacturing process, and well-defined characteristics.¹⁷³

3. Alginate encapsulation for microscale 3D cell culture

This section is divided into three sub-sections depending on the structures and compositions of the microscale alginate constructs used. The first sub-section will focus on examples where the material prepared is homogeneous throughout. The second sub-section will focus on use of two different types of alginate material combined in different microstructures and patterns. The third sub-section will describe structures where an alginate shell is used to encapsulate another material in its core.

3.1. Homogeneous alginate hydrogels

Cell microencapsulation is a strategy for 3D cell culture in which cells are isolated by a semipermeable material that permits diffusion of nutrients, gases, and metabolites.⁶⁵ Alginate solutions can be mixed with a cell suspension, then the mixtures rapidly crosslinked by divalent cations (i.e., Ca²⁺, Sr²⁺, Fe²⁺, and Ba²⁺) under various physical and chemical conditions.¹⁷⁴ This is an advantage for the encapsulation of living cells to enable cell culture in a 3D environment where soluble factors can diffuse into the cell encapsulating gel. When chemically modified alginate is used, adhesive cues can also be presented to cells from all directions.

Some examples of homogeneous alginate microencapsulation are shown in Figure 3. Microenvironmental factors influence overall cell fate and is mediated in part through specific cell morphology induced. Utech et al. prepared RGD-peptide-functionalized alginate hydrogels using carbodiimide chemistry and encapsulated MSCs into alginate microgels using PDMS a microfluidic device that produce monodisperse alginate droplets with size adjustability.¹⁷⁵ The crosslinking of cell containing alginate-Ca-EDTA mixture takes place by introduction of acetic acid which triggers internal gelation. The encapsulated cells show growth and proliferation in alginate hydrogels with a high viability of 70% after two weeks of seeding (Figure 3a). The mechanical properties of alginates can also alter cellular responses. The growth of the hepatocarcinoma cell line (HepG2) differed significantly in alginate hydrogels with a liquefied core versus a solidified core.¹⁷⁶ Alginate-poly-lysine-alginate (APA) hydrogels fabricated with an electrostatic droplet generator are microcapsules (300–400 μm) with high uniformity. APA hydrogels are obtained by external crosslinking and each microcapsule contained 150–180 mouse embryonic stem (ES) cells. As shown in Figure 3b, ES cells aggregate into a clump and proliferate to form a large spheroid in the liquefied core, whereas the cells just proliferate and grow into small multi-spheroids in an alginate hydrogel filled core. This result clearly suggests that the liquefied and soft capsules, rather than rigid gel core beads might provide a more suitable 3D culture environment for the growth of certain types of cells. In another example, Richardson et al. elucidated that the proliferation of human embryonic stem cells (hESCs) is diminished in mechanically strong alginate hydrogels, in which alginate G-blocks were crosslinked with a high affinity cation.¹⁷⁷ Homogeneous barium alginates (BAlg) have shown significantly higher proliferation of hESCs compared to inhomogeneous counterparts observed through 10 days post-encapsulation (Figure 3c). In addition, a reduction in proliferation was also observed with alginate hydrogels composed of alginate with high concentration of Ba²⁺ ions which increase stiffness. The microencapsulation of cancer cells in alginate hydrogels has been used as tumor models.¹⁷⁸ In this study, serotonin (5-HT) producing small intestinal neuroendocrine tumor spheroids (KRJ-1) were encapsulated in alginate hydrogels of diverse capsule sizes, M/G ratio, and crosslinking ions. Spheroids encapsulated in alginate with increased G-blocks experienced increased physical stress, which gradually induced cell death (Figure 3d). According to this result, they found that the KRJ-1 cells are sensitive to matrix resistance and require cell-cell signaling for growth. Alginate microencapsulation offers an important gastrointestinal neuroendocrine neoplasms (GI-NEN) models to develop, test and validate new drugs.¹⁷⁹ Physical confinement without any basement membrane ligands has been shown to cause malignant transformation.^180,181 Chaudhuri et al. demonstrated in vitro that increased ECM stiffness is associated with malignant phenotype transformation of normal human mammary epithelium (MCF10A).¹⁸² The growth and proliferation of MCF10A encapsulated within porous IPNs of alginate and Matrigel matrix with stiffness ranging from 30 to 310 Pa was created by modulating the concentration of Ca²⁺ ions. As shown in Figure 3e, the development into growth-arrested acinar structures or invasive clusters is more apparent with increases in the hydrogel stiffness, independent of ligand density and polymer concentration. Enhanced expression of ESR1, a gene whose function is to initiate an estrogen receptor α (ER α) of breast cancers, was observed within the MCF10A cells in stiff IPNs.¹⁸² Moreover, Mao et al. proposed a microfluidic-based single cell encapsulation that increases the proportion of encapsulation efficiencies over 90% (Figure 2f).¹⁸³ In this study, they created in vitro microenvironments of alginate hydrogel by controlling CaCO₃ nanoparticle concentrations, acetic acid concentrations, and alginate molecular weight to achieve higher stiffness (~16 kPa) cell-encapsulating microgels. With their system, murine mesenchymal stem cells (mMSCs) and murine pre-adipocyte cells (OP9s) were exposed to a calcium carbonate (CaCO₃) solution, allowing adsorption of CaCO₃ to the cell surface. Addition of these nanoparticles to an alginate solution then initiated cross-linking. A cross junction microfluidic device was used to generate the alginate emulsion by applying an acetic acid-dissolved oil phase which induces internal gelation of the alginate hydrogels. Genomic analysis of alkaline phosphatase and osteogenic transcription factor Runs2 show that the stiffer microgels induced an increased osteogenesis of the mMSCs. Furthermore, they demonstrated therapeutic efficacy of MSC therapy in NOD/SCID/IL2γ^−/− (NSG) mice by measuring pharmacokinetics and pharmacodynamics of the hydrogel drug. Therefore, recent studies of integration between microfluidics and microencapsulation of cells using alginate hydrogels show several key advancements such as control over mechanical property, narrow size distribution, cell encapsulation with high efficiency, and drug delivery with enhanced efficacy.¹⁸⁴ In short, the encapsulation of cells in homogeneous alginate hydrogels is important for regulating cellular behaviors as well as for studying mechanotransduction and malignant phenotypes.

Figure 3.

Homogeneous alginate microencapsulation with various physicochemical properties, a) Micrographs of RGD modified alginate hydrogels encapsulating mesenchymal stem cells (MSCs) at day 0 and day 15.¹⁷⁵ Reproduced with permission from ref 175. Copyright 2015 John Wiley and Sons, b) Growth profile difference of mouse embryonic stem (ES) cells enclosed in liquefied and unliquefied alginate microcapsules.¹⁷⁶ Reproduced with permission from ref 176. Copyright 2006 John Wiley and Sons, c) Crosslinking density control by using 10 mM Ba²⁺ and 50 mM Ba²⁺ alginate hydrogel with human embryonic stem cells (hESCs) at day 10.¹⁷⁷ Reproduced with permission from ref 177. Copyright 2016 Elsevier, d) Small intestinal neuroendocrine tumor (KRJ-1) spheroids encapsulating alginate microspheres using high-G alginate.¹⁷⁸ Reproduced with permission from ref 178. Copyright 2012 John Wiley and Sons, e) Different stiffness of alginate hydrogels lead to the malignant transformation of MCF10A spheroids.¹⁸² f) Schematic illustration showing the single cell encapsulation process.¹⁸³ Scale bars: a, 25 μm; b, 100 μm; c, 450 μm; d, 100 μm.

3.2. Patterned alginate hydrogels

In vivo microenvironments are composed of anisotropic and hierarchical structures with different types of cells and ECM.¹⁸⁵ Different cell types require distinct and particular environments, as cells differ in differentiation profile, proliferation rates, spatial organization, and nutrient metabolism during growth.

Alginate hydrogels can act as a physical or chemical “barrier” to provide cellular microenvironments for artificial tissue models. Different configurations, such as Janus, coreshell, multicompartment structure, have been achieved by advanced techniques.^186–189 These systems mimic physical and chemical characteristics in terms of microenvironment heterogeneity and provide a means of functional regulation of mechanical and biochemical properties. In addition, researchers have demonstrated in vitro systems that mimic anisotropic ECM structures to confirm the adaptation of specific types of cells. For example, Yoshida et al. developed Janus alginate microgels as an anisotropic cellular scaffold for 3D cell culture.¹⁹⁰ To include different types of ECM in the alginate microgel hemispheres, they developed a centrifuge based microfluidic device that produces monodispersed alginate microgels as the alginate solution is ejected out of a theta-shaped capillary. As shown in Figure 4a, two different mixtures such as collagen and Matrigel incorporating alginate were separately ejected by centrifugal force (~2000 G) and immersed into a calcium chloride solution for external crosslinking, and the resulting anisotropic gels used to encapsulate mouse embryonic fibroblast cells (NIH/3T3). The viability of the encapsulated cells was more than 79% and the cells proliferated into fibrillar networks. In short, it was found that alginate microgels with different ECM compositions could be utilized for providing spatially anisotropic microenvironments. Cell-cell communication is important for maintaining normal cellular morphology and functionality of tissues.¹⁹¹ Spatially controlled alginate microgels provide useful 3D scaffolds for micropattemed cell co-culture. Figure 4b shows that three different type of alginate microgels (core-shell, side-by-side, and triple-layer) can be fabricated by a multi-fluidic electrostatic spraying technique.¹⁹² In this study using model cells including MDA-MB-231, normal human lung fibroblasts (NHLFs), and MCF-10A, the authors demonstrated that different types of cells can be co-encapsulated in different compartments. Recently, Zhang et al. demonstrated a one-step microfluidic method to generate multi-compartment alginate hydrogels, where the fabricated compartments can be independently controlled.¹⁹³ They used RGD-modified alginate hydrogels for encapsulating MSCs and HUVECs to create complex stem cell niche microenvironments to study single cell-cell interaction. Thus, the alginate microgels offer flexibility in terms of controllable structures and can also reduce spatial distance among co-cultured cells.

Figure 4.

Patterned alginate systems for 3D cell culture, a) Confocal image of heterogeneous material-based alginate hydrogels composed of hemispheres with different volume ratio of collagen and Matrigel.¹⁹⁰ Reproduced with permission from ref 190. Copyright 2017 John Wiley and Sons, b) Alginate hydrogel designs for cell encapsulation. Double layer, side-byside, and triple-layer alginate hydrogels encapsulating different types of cells (green cells: MDA-MB-231 expressing GFP; red cells: normal human lung fibroblasts expressing RFP; blue cells: MCF-10A stained with Hoechst).¹⁹² Reproduced with permission from ref 192. Copyright 2015 The Royal Society, c) Cell-laden alginate microfibers with Janus-compartment and twoshell layer hollow structure (red cells: NIH 3T3 cells stained with a red fluorescent dye; green cells: HepG2 cells stained with a green fluorescent dye).¹⁹⁶ Reproduced with permission from ref 196. Copyright 2014 John Wiley and Sons. Scale bars: a, 50 μm; c, 200 μm.

Fibrous alginates can induce the growth, alignment, and migration of cells and promote cell-cell interactions.¹⁹⁴ Cell encapsulating microfibers can be used to generate tubular structures for vascular tissue engineering.¹⁹⁵ By using a glass capillary device that modulates alginate crosslinking chemistry, Cheng et al. developed a scalable, one-step, and continuous process for formation of hollow microfibers.¹⁹⁶ This procedure produces microfibers by crosslinking multiple, parallel, laminar flows of alginate solutions arranged in different orientations to control overall fiber cross-sectional morphology. Alginate suspensions of HepG2 and NIH/3T3 cells were used to fabricate Janus compartments and layered-shell structures (Figure 4c). Cells in the layered-shell microfibers show higher viability (-85%) than the Janus microfibers (~79%). This result suggests that the layered-shell arrangements enhance cell-cell interactions via a larger interaction area. Other microfluidic approaches for alginate fiber formation include use of a coaxial triple cylinder to form 3D micro-vascularized structures¹⁹⁷ and use of microfabricated nozzles¹⁹⁸ to produce microfibers for the encapsulation and co-culture of cells.

3.3. Composite alginate hydrogels

Microencapsulation of complementary cell types can recapitulate some in vivo microenvironment to provide more physiologically-relevant in vitro models.¹⁹⁹ Multiple cell types have been encapsulated within the same alginate microcapsule to enhance paracrine signaling between cells.²⁰⁰ Alginate microcapsules with hollow cores have been investigated to minimize interactions between the encapsulated cells and the surrounding network. These hollow core-shell structures are comprised of liquid core and/or ECM materials that are surrounded by a semipermeable alginate shell.^200–202

One study showed that microencapsulation promotes cellular reorganization within the liquid core to form spheroids. As shown in Figure 5a, Alessandri et al. succeeded in forming spheroids from HeLa cells, which normally have a low propensity to form 3D aggregates.²⁰³ They also demonstrated that murine sarcoma S180 cells expressing low levels of E-cadherin can also be induced to form spheroids. The spheroids formed in confined core-shell environments formed stronger cell-cell interactions, and hence were more compact, compared to unconfined multicellular spheroids. In another study, Chen et al. described controlled 3D assembly of liver hepatocellular carcinoma cells (HepG2) in microcapsules.²⁰⁴ Microcapsules with a media core and an alginate shell were generated by using two connected microfluidic flow-focusing PDMS channels. With slight alteration in encapsulating procedure, different cells (HepG2 and NIH/3T3) can be loaded into different regions within microcapsules (Figure 5b). A microstructure with hepatocytes in the core and fibroblasts around an alginate hydrogel shell formed co-culture spheroids with improved heterotopic cell-cell paracrine signaling. In addition, the highly permeable alginate shell allowed long-term culture (> 14 days) of spheroids to develop microtissues in the microcapsules. In some applications, having both a protective shell and an interior space for development is critical. For example, microcapsules with a soft core mimicking the medulla and stiff shell to resemble the cortex have guided ovarian follicle development.²⁰⁵ Early secondary preantral follicles were encapsulated within a soft collagen core inside of a stiff alginate microcapsule, so that they can locate themselves at the boundary between core and shell where they experience mechanical heterogeneity, similar as in the native microenvironment (Figure 5c). A total of ~28% of the preantral follicles in the appropriate biomimetic microcapsule formed antra. In contrast, when embedded in oxidized, soft, alginate material alone, no follicles proceeded to the antral phase because degradation of this alginate occurred rapidly in 5–7 days exposing the follicle prematurely. The authors further demonstrated in vitro ovulation in the microcapsules without addition of any additional growth factors.

Figure 5.

Composite alginate hydrogels, a) Time sequential phase contrast micrographs shows HeLa and murine sarcoma SI80 spheroids encapsulated in media-core and alginate shell structure.²⁰³ Reproduced with permission from ref 203. Copyright 2013 National Academy of Sciences, b) Spatial assembly of core-shell structure. HepG2 cells confined in the media core and NIH/3T3 fibroblasts immobilized by the alginate shell forming an artificial liver in a drop.²⁰⁴ Reproduced with permission from ref 204. Copyright 2016 The Royal Society, c) Schematic illustration and micrographs of mouse ovary that consists of two mechanically distinct tissue layers consisting of a rigid cortex and a softer medulla.²⁰⁵ Reproduced with permission from ref 205. Copyright 2014 Elsevier, d) Alginate capsules with a Matrigel inner lining for human neural stem cells (hNSC) differentiation. Inset shows confocal image of the Matrigel forming a basement membrane-mimetic lining (red) on the inner surface of an alginate shell (green) microcapsule.²⁰⁹ Reproduced with permission from ref 209. Copyright 2016 The Royal Society. Scale bar: a, 100 μm; c, 100 μm; d, 10 μm.

Microencapsulation with ECM components such as collagen and Matrigel can modulate cellular differentiation of stem cells and induced pluripotent stem cells (iPSCs).^206,208 Human neural stem cells (hNSC) encapsulated in alginate-Matrigel core-shell microcapsules differentiate into neurons with 97.8% viability (Figure 5d).²⁰⁹ Using a 3Dprinted microfluidic device, the authors produced hollow cell-laden spheres where the Matrigel is anchored to the inner surface of the alginate capsule to mimic a basement membrane. Under differentiating conditions the neural stem cells differentiate to form a 3D network of mature neurites that criss-cross each other within a defined size sphere. In another example, Perez at al. demonstrated osteogenic programming of MSCs when encapsulated in collagen or poly(lactic-co-glycolic acid) (PLGA) core-alginate shell microcapsules.^210,211 Other studies have shown hepatocyte differentiation²¹² and differentiation into pancreatic lineages,²¹³ both from encapsulation of human embryonic stem cells. Composite alginate microcapsules with a core-shell structure provide a controlled and protected microenvironment for a variety of core materials, including physiological ECM gels and basement membrane mimetics, to facilitate cell growth, cell-cell interaction, cell transplantation, and stem cell differentiation.

4. Conclusions and Future directions

In the past few decades, alginate-based microscale 3D cell culture platforms have emerged as promising physiological 3D model systems for basic biology, tissue engineering, and stem cell applications. In this review, we summarize the use of alginate hydrogels for microscale 3D cell culture as an alternative to other microscale cell culture systems, including PDMS microdevices. The characteristics of the alginate hydrogels, such as controllable mechanical properties, semipermeable and degradable encapsulation, and ease of modification, are particularly useful for 3D cell culture systems. In particular, advanced techniques with enhanced alginate crosslinking have paved the way for alginate hydrogels to be fabricated with improved control of their structures and functions for applications in 3D cell culture.

Despite these major advances, many challenges remain. The alginate substrates on their own are insufficient to instruct cells to form functional tissue structures. ECM-enriched alginate hydrogels can provide a promising approach to alleviate such problems. It is, however, critically important to carefully consider unknown side effects such as abnormal vascularization,²¹⁴ diffusion limitations of some amino acid,²¹⁵ and many other possible complications. In addition, current systems often lack vascularization and therefore fail to fully model adult tissue or organ biology. Nevertheless, maturation of 3D cellular structure has been demonstrated, for example, by using tubular alginate hydrogel networks, fabricated with advanced fabrication techniques such as microfluidics^216–218 and bioprinting.^219–221 Experimental protocols such as cell seeding, medium change, and analytic methods should all be further assessed to enhance reproducibility. This is critical because variations in, for instance, cell counts and media change cycle disturb systematic comparison of batch-to-batch experimental results. Development of real-time 3D imaging-based readout systems can enable monitoring of cellular behaviors and microenvironmental parameters, which can further accelerate research. Moreover, real-time biochemical readouts may provide quantitative methods tailored for analysis of hydrogel-based 3D cell culture models. For example, optical coherence tomography (OCT)²²² can provide a non-destructive and non-invasive way of imaging encapsulated 3D cell structures without cell fixation and staining procedures. This optical system has an advantage over conventional 3D imaging techniques such as confocal laser scanning microscopy (CLSM),²²³ and multiphoton imaging,²²⁴ as OCT-based imaging systems use near-infrared radiation that can enable imaging depth to be in the order of microns up to centimeters in intact biological 3D structures.

Importantly, the ready accessibility and ease of use of alginate hydrogels for microscale 3D cell culture systems provide an attractive materials platform for interdisciplinary collaborations involving bioengineering, materials science, medicine, biology, and chemistry, to better understand and mimic the complicated in vivo microenvironments for tissue engineering, personalized medicine, and high throughput drug screening.