Engineered Biomaterials to Guide Spheroid Formation, Function, and Fabrication into 3D Tissue Constructs

Abstract

Cellular spheroids are aggregates of cells that are being explored to address fundamental biological questions and as building blocks for engineered tissues. Spheroids possess distinct advantages over cellular monolayers or cell encapsulation in 3D natural and synthetic hydrogels, including direct cell-cell interactions and high cell densities, which better mimic aspects of many tissues. Despite these advantages, spheroid cultures often exhibit uncontrollable growth and may be too simplistic to mimic complex tissue structures. To address this, biomaterials are being leveraged to further expand the use of cellular spheroids for biomedical applications. In this review, we provide an overview of recent studies that utilize engineered biomaterials to guide spheroid formation and function, as well as their fabrication into tissues for use as tissue models and for therapeutic applications. First, we describe biomaterial strategies that allow the high-throughput fabrication of homogeneously-sized spheroids. Next, we summarize how engineered biomaterials are introduced into spheroid cultures either internally as microparticles or externally as hydrogel microenvironments to influence spheroid behavior (e.g., differentiation, fusion). Lastly, we discuss a variety of biofabrication strategies (e.g., 3D bioprinting, melt electrowriting) that have been used to develop macroscale tissue models and implantable constructs through the guided assembly of spheroids. Overall, the goal of this review is to provide a summary of how biomaterials are currently being engineered and leveraged to support spheroids in biomedical applications, as well as to provide a future outlook of the field.

1. Introduction

Spheroids are dense cellular aggregates (~50 to 2000 μm in diameter) comprised of varied cell types, ranging from primary cells (e.g., chondrocytes) to mesenchymal stromal cells (MSCs) and pluripotent stem cells (PSCs) (e.g., induced-PSCs (iPSCs), embryonic stem cells (ESCs)). Spheroids, often termed “microtissues”, have become increasingly popular in the study of fundamental biology or for tissue engineering applications, particularly due to their advantages over the use of cells cultured on monolayers, encapsulated in natural and synthetic hydrogels, or seeded onto porous scaffolds. Specifically, the high cell density of spheroids results in increased cellular interactions, which is important in the fabrication of constructs that mimic high cell density tissues (e.g., cardiac) or developmental processes (e.g., N-cadherin cell-cell interactions during mesenchymal condensation).[1,2] Spheroids often possess marked benefits of improved cell survival, proliferation, and increased extracellular matrix (ECM) deposition when compared to other types of cultures, which can be important in tissue generation.[3–5] Further, spheroids (specifically MSC spheroids) possess a potent secretome of paracrine signals that are known to enhance cardiovascular and musculoskeletal tissue regeneration and protection.[6,7] Multiple cell types are also easily incorporated into spheroids to increase their complexity and application to model tissues.

There are numerous applications of spheroids in tissue engineering, including in regenerative medicine or as in vitro models for drug, disease, and tissue modeling. With these developments, biomaterials are playing a crucial role in the advancement of spheroid technologies. At the most basic level, biomaterials (e.g., hydrogels) have acted as substrates for the formation of spheroids, through the patterning of microwells that induce cell condensation.[8,9] This has enabled the large-scale production of uniform spheroids with methods that are adaptable across many laboratories. Beyond spheroid formation, biomaterials have been used to control spheroid function (e.g., cellular outgrowth, spheroid fusion, cell differentiation) through the engineering of controlled spheroid microenvironments, primarily with hydrogels. Additionally, the embedding of biomaterial microparticles into spheroids is useful to locally deliver growth factors or to modulate spheroid actomyosin dynamics.[10–12] Lastly, recent years have seen an increase in the development of methods to fabricate tissues from spheroids, where spheroids act as building blocks in tissue formation. Biofabrication techniques include the controlled seeding of spheroids into macroporous scaffolds, as well as aspiration-assisted and extrusion-based bioprinting to control spheroid deposition.[1,13,14]

Within these approaches, the fusion of multiple spheroids and maturation into functional macroscale tissue constructs is important to their use. Spheroid fusion is a biophysical process that occurs over a 24–48 hour period and is thought to be driven by the difference in interfacial surface tension between the surrounding culture media and cells, which can be modulated by ECM secretion, cell type, actin dynamics, and the formation of a condensation boundary (tenascin ECM deposition and peripheral localization) with time.[2,15,16] Spheroid fusion and the corresponding increase in inter-spheroid contact area results in reduced surface area and tension for individual spheroids.[17] Additionally, as thoroughly described by Bhumiratana et al., it is important that initial spheroid contact occurs before the setting of a condensation boundary (e.g., <7days for human MSCs)[2]; otherwise, spheroid fusion is limited and rather aggregates of spheroids loosely connected by peripherally deposited ECM are observed.[18] Even when adhering to these guidelines, the generation of macroscale tissue constructs that retain their shape, volume, and structure after spheroid fusion is quite challenging, as cells condense and continually reorganize their surroundings. Biomaterials provide an important opportunity to control these processes.

In this review, we summarize recent advances in the use of engineered biomaterials to guide spherical spheroid formation, function, and fabrication into tissue constructs (Fig. 1) and then comment on the future outlook for the use of biomaterials to guide spheroid behavior. We recognize that a substantial number of studies have recently been published regarding biomaterials and organoids, which are commonly defined as self-organizing structures sourced from stem cells that simplistically model organs. However, this work is outside the scope of this review.

Figure 1 |. Engineered Biomaterials to Guide Spheroid Behavior.

(A) Spheroid formation in biomaterial-based, confined geometries. (B) Spheroid function guided by embedding within biomaterials with controlled properties (top) or by incorporating microparticles within spheroids for molecule delivery or through biophysical cues (bottom). (C) Biofabrication methods used to organize spheroids, including spheroid seeding into macroporous sponge/foam and 3D lattice scaffolds (top), the bioassembly or bioprinting of spheroids into microchambers, encapsulated within bioinks, or embedded directly into biomaterials via extrusion (middle) or bioprinting of spheroids into support biomaterials through assisted aspiration methods (bottom).

2. Leveraging Biomaterials to Form Spheroids

The condensation of cells into spheroids has enabled the formation of biomimetic tissue models that recapitulate aspects of native tissues. Original strategies for in vitro spheroid formation relied on spontaneous self-assembly or aggregation on non-adhesive plates, liquid droplets (hanging droplet method), or rotary and spinner flask cultures, all of which depend on either gravity or fluid dynamics for cell aggregation.[19–21] These methods are technically challenging, low-throughput, or result in heterogeneous spheroid sizes, which motivates the use of biomaterials as substrates to produce high-throughput, homogeneous spheroids. The earliest strategies for implementing biomaterials as substrates for spheroid formation relied on hydrogels that were cast and stabilized in polydimethylsiloxane (PDMS) molds to create cylindrical microwells. A variety of hydrogels, such as poly(ethylene glycol) dimethacrylate, methacrylated hyaluronic acid (MeHA), and azidobenzene-modified chitosan were used to generate spheroids with this approach where spheroid shape, size, and number were modulated through mold design.[22–24] Limitations of this approach include material cost, time-consuming multistep processes, and resource intensive photolithographic equipment needed to fabricate the original molds. These studies also demonstrated that the combination of cylindrical well shape with cell-adhesive materials interferes with the condensation process and generates hemisphere-shaped spheroids.[23] Further, challenges with spheroid removal from adhesive substrates motivated the need for cheaper, less cell adhesive biomaterials to fabricate microwells.

Towards non-adhesive platforms, agarose is an inexpensive biomaterial that has become a common choice to develop microwells.[25] In one example, agarose was used to fabricate U-shaped wells in flat 96-well plates for the formation of spheroids; however, 96-well plates are insufficient to generate the quantities of spheroids needed for some applications.[26] 3D molds introducing greater numbers of microwells within 96-well plates have been developed to improve spheroid formation throughput but are technically challenging to implement without robotics.[27] There have also been several recent advances within the field to address this challenge, including negative molds and 3D printed stamps (Fig 2A). The first approach was demonstrated recently by Valdoz and coworkers, where low-melt agarose was used to fabricate an array of U-shaped microwells via silicone rubber negative molds, which enables easy removal from agarose (Fig 2B).[9] The addition of a media reservoir reduced the disruption and lifting of spheroids during culture. One disadvantage to this strategy is the lack of reproducible casts within plastic well-plates, which may result in spheroid seeding issues. Advances in stereolithography (SLA)-based 3D printing of polymers enabled the rapid prototyping of micro-scale designs towards 3D-printed stamps. For example, Nulty et al. developed a stamp containing 401 U-shaped microwells that could imprint this array onto agarose within a 6-well tissue culture plate (Fig 2C).[28] The addition of 3D printed holders that screwed the stamps tight allowed each agarose mold to be cast in a precise and controlled manner. Regardless of the stamp design, one major limitation of 3D printed stamps is the method of sterilization, as most resin plastics cannot withstand autoclaving, in contrast to PDMS molds. As cylindrical and U-shaped molds have enabled the high-throughput generation of spheroids in the 100s per well, alternatives are still needed to increase the scale of spheroid formation.

Figure 2 |. Formation of Spheroids.

(A) (i) Negative molds and (ii) 3D printed stamps are common fabrication methods for imprinting microwells into biomaterials to enable spheroid formation. (B) Negative silicone mold fabrication of agarose U-shaped microwells with media reservoir that mitigates spheroid disruption.[7] (C) 3D printed stamps and holders to imprint agarose into 6-well tissue culture plates for the generation of hypertrophic spheroids. [25] (D) Fabrication of fibronectin-modified Tetronic^® square microwells whose gap distance can be controlled to dictate spheroid distance for transfer into hydrogels. Scalebar: 200μm [27] Figures B-D are credited to original authors and with written permission from Elsevier.

Inverted pyramid and square microarrays have also been implemented and have increased throughput to 1000s of spheroids per well. Similar to Valdoz and colleagues, researchers have used the negative mold strategy to fabricate inverted pyramids using PDMS as their positive cast. As PDMS can adhere to cells, a proprietary anti-adhesion solution was applied before seeding.[29] Others, such as Kim et al., have taken a different approach where a microarray of square wells was imprinted into temperature-responsive fibronectin-crosslinked Tetronic^® (BASF) hydrogels that expand at 4°C to allow for the culture and delivery of spheroids into a 3D Matrigel^® system (Fig 2D).[30] The distances between square microwells were altered to pattern multicellular spheroids and showed that short distances (~200μm) between spheroids improved migration and fusion within a hydrogel. Interestingly, the addition of fibronectin, a cell-adhesive biomaterial, molded into flat square microwells did not inhibit spherical spheroid formation and the addition of cold saline expanded the Tetronic^®-modified hydrogel to improve spheroid release, which has been a challenge for other microwell based systems due to the deposition of ECM over time. To improve on 3D-printed stamps and issues with bubble entrapment, Gonzalez-Fernandez et. al. included an inlet and outlet for agarose deposition and bubble release.[8] Although not high-throughput, this method was used to develop 29 microwells that formed spheroids comparable to commercially available microwell plates (AggreWell^™, STEMCELL Technologies Inc.) and hanging droplet plates (Perfecta3D^®, 3D Biomatrix). Overall, the formation of spheroids through biomaterial microwells engineered through negative molds and 3D printed stamps has enabled the homogeneous, high-throughput generation of spheroids that can be easily adopted by others within the field. Alternatively, single cell suspensions encapsulated within hydrogels may also form spheroids de novo based on their cell density and the hydrogel formulation; however, this approach results in heterogeneously-sized spheroids that may be limiting when compared to encapsulating pre-formed homogeneously-sized spheroids.[31,32]

3. Guiding Spheroid Function with Biomaterials

3.1. Spheroid Embedded Hydrogels

Cell-ECM interactions and the actin cytoskeleton are known to play an essential role in spheroid function, including spheroid fusion, cellular differentiation, matrix production, and cellular outgrowth, which are all heavily regulated by their surrounding microenvironment.[17,33,34] The natural ECM is generally fibrous, viscoelastic, and cell-adhesive. Naturally derived biomaterials (e.g. fibrin, collagen, Matrigel^®, and transglutaminase-crosslinked gelatin) that retain aspects of these properties have been leveraged with MSC spheroids for wound healing and tissue engineering applications, partly due to the action of paracrine signaling via the MSC secretome over single cells.[30,35–40] For example, MSC spheroids encapsulated within fibrin hydrogels have improved cell viability, proliferation, anti-inflammatory, and angiogenic cytokine secretion (VEGF/PGE₂) when compared to dissociated, individual cells.[37] Further, VEGF/PGE₂ cytokine secretion can be modulated based on fibrin hydrogel stiffness, where an intermediate stiffness (25kPa) hydrogel enhanced cytokine secretion.[38] In parallel, human umbilical vascular endothelial cell (HUVEC) and stem cell spheroids have been embedded within fibrin/collagen or Matrigel^® hydrogels towards vascularized bone formation and spheroid migration and fusion.[30,38] Overall, these findings suggest that co-cultured spheroids in close proximity (~200μm) improve cell migration for the formation of pre-vascularized networks, as well as promote higher cell viability, proliferation and spreading while enhancing osteogenesis.[30,38]

As natural biomaterials lack fine-tuned control over biomechanical and biochemical properties, synthetic hydrogels have also been explored, including those fabricated from modified biopolymers.[41] It is now well understood that hydrogel properties such as viscoelasticity, incorporation of cell-adhesion ligands, degradation, and architecture influence encapsulated cell behavior and function; however, little is known about how spheroids interact and interpret the signals engineered into surrounding hydrogels, particularly as only the cells on the outer surface of spheroids directly interact with these hydrogels.[42–46] As spheroids are gaining increased use, there is a need to better understand how hydrogel biomechanical and biochemical properties influence spheroid function (Fig. 3A).

Figure 3 |. Spheroids Encapsulated in 3D Hydrogels.

(A) Schematic representation portraying how biomaterial properties can enhance (left) or limit (right) spheroid functions (e.g., spheroid growth, cellular proliferation, cellular outgrowth). (B) GelMA hydrogel stiffness (due to crosslink density) influences spheroid proliferation, fusion, and cellular outgrowth. [44] (C) Viscoelastic alginate hydrogels induce an osteogenic phenotype through adhesion-ligands and actomyosin contractility when compared to elastic hydrogels. Scalebar: 200μm [46] (D) MSC spheroid sequestered growth factors enhance myoblast infiltration in sulfated alginate hydrogels (HSO₃Cl amount). Scalebar: 100μm [49] Figures B-D are credited to original authors and with written permission from Elsevier and Wiley and Sons.

Biomechanical properties, such as stiffness, viscoelasticity, and architecture can be engineered into hydrogels to guide cell behavior. Spheroids, unlike cell suspensions, sense these biophysical cues primarily from their boundary cells and transduce these signals inwards via the actin cytoskeleton and cell-cell interactions. This differential mechanosensing may result in heterogeneous differentiation throughout spheroids.[47] Hydrogel stiffness has been heavily investigated in cell cultures and is known to drive cellular processes such as differentiation, proliferation, and migration.[45,48] In a similar manner, MSC spheroids encapsulated within gelatin methacryloyl (GelMA) hydrogels of varying stiffnesses (0.5–3kPa) showed altered proliferation, outgrowth, and differentiation based on hydrogel properties (Fig 3B).[49] At lower stiffnesses, MSCs within spheroids were able to easily remodel their surroundings, proliferate, and migrate out to fuse with neighboring spheroids, while high stiffnesses elicited an increased hypoxic environment and drove osteo/pro-angiogenic gene expression. Other studies exploring higher stiffness ranges of GelMA hydrogels demonstrated similar results with lower stiffnesses (<3.5kPa) allowing cells within spheroids to remodel, migrate out, and differentiate towards a chondro/osteogenic lineage even without differentiation medium.[50] Interestingly, stiff hydrogels (>8kPa) supported adipogenic differentiation, as spheroids remained spherical and static within the hydrogel, suggesting a reduction of cytoskeletal tension. Hydrogel stiffness has also been tailored through crosslink irradiation time or polymer concentration to investigate the formation of pre-vascularized spheroid constructs. Stiffer GelMA hydrogels reduced vessel sprouting number and length; however, MSCs and endothelial co-cultures can synergize to improve pre-vascularization as demonstrated in a 3D collagen/fibrin hydrogel.[51–53] It should be noted that with stiffness changes in these studies, other features such as diffusivity, adhesion ligand concentration, and degradability are likely also changed.

Beyond stiffness, the viscoelasticity or plasticity of engineered hydrogels can also impart different spheroid responses by allowing cells to deform and remodel their surrounding environment. For example, Whitehead et. al. demonstrated that spheroids encapsulated within viscoelastic alginate hydrogels elicited an increased osteogenic phenotype mediated through adhesive ligands and actomyosin contractility when compared to elastic hydrogels with reduced stress relaxation (Fig 3C).[54] The inclusion of bone morphogenetic protein-2 (BMP-2) coated hydroxyapatite microparticles within these spheroids further enhanced osteogenic differentiation and bone formation in both in vitro and in vivo settings. In vivo, varied biological responses due to stiffness and viscoelasticity are convoluted by other biomechanical factors (e.g., fiber density). The decoupling of stiffness and fiber density with GelMA/Collagen I interpenetrating networks (IPNs) demonstrated that breast cancer spheroid outgrowth is enhanced with the addition of fibers in a density dependent manner, regardless of bulk stiffness, likely through topographical cues.[55] While this model demonstrates the fundamental aspects of fiber density on cancer spheroid outgrowth, the introduction of the secondary crosslinked GelMA network may impact fibrillogenesis and alignment of the collagen network, resulting in differences in spheroid outgrowth compared to metastatic breast tissue. Altogether, hydrogel biomechanical properties play an important role in regulating spheroid function; however, these are often tied to hydrogel biochemical properties.

Biochemical properties, such as adhesion ligand density and sulfated glycosaminoglycan (sGAG) presentation can influence how spheroids sense their surrounding environment and locally sequester growth factors. Adhesion ligands, such as RGD motifs, are naturally found on ECM molecules (e.g., fibronectin, collagen) and can also be tethered to hydrogels. Ho et. al. demonstrated that the inclusion of RGD moieties on alginate hydrogels improves spheroid-encapsulated cell survival, outgrowth, and pro-angiogenic factor secretion.[3] Further, RGD ligand density was controlled independent of stiffness and spheroids encapsulated within a high RGD density hydrogel exhibited enhanced osteogenic potential, pro-angiogenic factor secretion, and cellular outgrowth when compared to lower RGD densities.[4] As biomechanical and biochemical properties are intrinsically coupled in many systems, Hung. et. al. utilized a design-of-experiments approach where hydrogel stiffness and RGD ligand density, as well as cell type and spheroid size were altered to determine their influence on MSC phenotypes within spheroids. [56] This study demonstrated that, in general, a smaller spheroid encapsulated within a soft hydrogel with higher adhesion ligands will more easily remodel its surrounding environment and invoke a chondro/osteogenic phenotype, while stiffer matrices support an adipogenic phenotype. These behaviors were also maintained in vivo; however, chemical induction of MSCs towards a specific cell lineage had a stronger influence on spheroid behavior than biomechanical cues.

Due to the abundance of paracrine signals from the MSC secretome during spheroid cultures, negatively charged sulfate groups have been tethered to alginate hydrogels to mimic natural ECM properties and enhance secreted growth factor sequestering via electrostatic charge.[7] Sulfated alginate gels readily sequestered secreted growth factors and elicited myoblast infiltration without hindering MSC spheroid viability and metabolic activity (Fig 3D). Interestingly, endothelial cells treated with conditioned medium from spheroid cultures in non-sulfated hydrogels exhibited enhanced tubule formation, suggesting that growth factor sequestration and release can be controlled via sulfate modification. Overall, it is apparent that biomechanical and biochemical biomaterial properties influence spheroid functions, such as cellular differentiation and outgrowth; yet, more studies are needed to better understand how other hydrogel properties, such as degradation, influence spheroid behavior and if biomaterial properties function similarly across varying spheroid sizes and densities. Spheroid size has been shown to dictate trophic factor secretion, proliferation, metabolic activity, and formation of a hypoxic core; however, few studies have shown how spheroid size can dictate their interactions with surrounding biomaterials and subsequent downstream cellular functions.[57,58]

3.2. Incorporating Microparticles into Spheroids

Challenges of nutrient and growth factor diffusion and heterogeneous differentiation have been major obstacles to the use of spheroids and has motivated the use of incorporated biomaterial microparticles to internally guide and improve spheroid function (Fig 4A). For scale reference, biomaterial microparticles (typically <50μm) are being embedded within spheroids (typically >200um). Early reports include the incorporation of natural biomaterial microparticles (e.g. collagen) into hepatocyte spheroids to upregulate hepatocyte-specific genes and functions.[59] Unfortunately, natural microparticles suffer poor physical stability and often require unsuitable processing conditions that can hinder spheroid viability. In comparison, cell-derived ECM has become an attractive alternative for altering spheroid function when incorporated within spheroids.[60,61] For example, the Leach group recently demonstrated that increasing amounts of cell-derived ECM incorporated within MSC spheroids during condensation improves mechanosensing (e.g., via α₂β₁ integrin signaling and YAP/TAZ translocation).[61] Interestingly, cell-derived ECM incorporated MSC spheroids alone or those encapsulated within high RGD ligand alginate hydrogels improved chondrogenic and osteogenic differentiation, further supporting the importance of cell-ECM integrin signaling. While natural biomaterial microparticles and ECM recapitulate aspects of native ECM, tuning their properties remains a challenge and motivated the use of modified biopolymer or synthetic biomaterial microparticles.[62] These microparticles allow for tailored properties to leverage both biophysical and biochemical cues, such as modulating spheroid actomyosin dynamics or locally delivering growth factors to regulate function, respectively. Overall, the inclusion of microparticles to guide spheroid function has been primarily demonstrated through changes in microparticle properties, growth factor delivery, or gene delivery.

Figure 4 |. Microparticle Incorporated Spheroids.

(A) Microparticles incorporated within spheroids to release cargo over time. (B) Incorporation of varying genipin-crosslinked gelatin microparticles (GMA) within PSC spheroids drives epithelial to mesenchymal transition. Scalebar: 200μm [10] (C) BMP-2 loaded mineral-coated HA (hydroxyapatite) microparticle incorporation within MSC spheroids (blue box) drives osteogenic differentiation without the presence of soluble osteogenic factors. Scalebar: 500μm [58] (D) Dex-HEMA microparticles incorporating siRNA for gene silencing within MSC spheroids.[65] Figures B-D are credited to original authors and with written permission from Elsevier and ACS Publications.

Microparticle properties have been tailored within spheroids to introduce homogeneous biophysical cues throughout to guide cell proliferation and differentiation. For example, PDMS or polyacrylamide microparticles with varying stiffnesses were incorporated within MSC spheroids to investigate cell differentiation and spheroid growth.[10,63] In these studies, stiffer microparticles (~.01–1 MPa) primed spheroids to exhibit reduced growth and increased f-actin filaments, which induced an osteogenic phenotype while inhibiting adipogenesis, without the addition of soluble factors. Interestingly, the addition of cell-adhesive coatings to the microparticles, such as Type I collagen or gelatin, was needed to prevent spheroid dissociation. As these biomaterials are intrinsically inert, gelatin-based microparticles have been used to control microparticle degradation. For instance, Nguyen et. al. demonstrated that GelMA microparticles induced the production of metalloproteinases within PSC spheroids, which guided their epithelial to mesenchymal transition (Fig 4B).[12] Additionally, previous reports observed that glutaraldehyde-crosslinked gelatin microparticles alone primed PSCs toward an endodermal lineage through biophysical cues, even without added soluble factors.[64] Finally, gelatin microparticles incorporated within MSC spheroids induced spheroid stiffening, likely due to increased local remodeling within the spheroid; however, MSC spheroid differentiation was not observed and cells remained in an undifferentiated phenotype.[65] Beyond biophysical cues, gelatin also contains native cell-binding motifs (e.g. RGD) that exhibit a slight negative surface charge that is favorable for cell adhesion and proliferation. Kim et. al. modified the surface charge and surface area of glutaraldehyde-crosslinked gelatin microparticles incorporated within MSC spheroids by modifying gelatin with a variety of positively or negatively charged proteins and sieving microparticles based on size.[66] Surprisingly, surface charge modification of any kind resulted in reduced cell proliferation, while increased surface area improved proliferation.

Local growth factor delivery via microparticles has also been advantageous to present biochemical cues within spheroids, overcoming diffusion restrictions of growth factors presented only from culture media. This method of delivery has been heavily adopted for applications in bone and cartilage tissue engineering, particularly with the delivery of bone morphogenetic proteins (BMPs) and transforming growth factor betas (TGFβs). Early work by the Alsberg group used PLGA microparticles incorporated with TGF-β1 to locally deliver the factor to MSC spheroids to induce chondrogenesis. [67] Since then, they have also reported TGF-β1 incorporated genipin-crosslinked gelatin microparticles and BMP-2 loaded mineral-coated hydroxyapatite microparticles to control the single or dual delivery of soluble factors within MSC spheroids to drive endochondral ossification or chondrogenesis (Fig 4C).[68,69] Other groups have also used calcium phosphate-based biomaterials, such as hydroxyapatite, β-tricalcium phosphate, or β-glycerophosphate, to fabricate micro/nanoparticles, as their intrinsic osteoconductive properties alone may promote bone repair.[70–72] Whitehead et. al. found that absorbing small doses of BMP-2 onto hydroxyapatite nanoparticles initiated and sustained osteogenic differentiation when incorporated within MSC spheroids.[72] Engineered heparin microparticles, which exhibit a negative charge, were incorporated within ATDC5 cell spheroids at varying particle ratios to improve the sequestration of cell-secreted proteins and induce chondrogenic differentiation.[73] However, chondrogenesis was reduced compared to poly(ethylene glycol) microparticles, as the sequestered growth factors (i.e. insulin growth factor) likely had limited interactions with surrounding cells.

As an alternative to growth factor delivery, gene delivery within spheroids can also improve cell fate and function. Traditional gene delivery through addition to culture media is limited, as peripheral cells act as a barrier for transfection to the core cells of the spheroids; however, recent advances using biomaterial micro/nanoparticles embedded within spheroids have improved plasmid DNA (pDNA) or silencing RNA (siRNA) delivery. Khalil et. al. demonstrated this technique by incorporating pDNA lipoplexes encoding luciferase, EGFP, or BMP-2 within hydroxyapatite microparticles and introducing them to MSC spheroids.[74] Over 7 days, luciferase activity (indicative of successful transfection) was sustained with pDNA incorporated within particles when compared to exogenous delivery and BMP-2 encoded pDNA was sufficient to enhance calcium deposition during osteogenic differentiation. Another approach to guide cell fate and function is through siRNA silencing of specific genes of interest. McMillan et. al. used a biomimetic and tunable microparticle biomaterial, dextran modified hydroxyethyl methacrylate (Dex-HEMA), to deliver siRNA micelles and silence GFP-expression within MSC spheroids over 28 days (Fig 4D).[75] Unfortunately, no functional assessment of siRNA besides GFP was demonstrated, leaving open the question of whether such approaches are sufficient to drive cell fate and function. Regardless, gene delivery through microparticles within spheroids holds great promise in manipulating cellular behavior.

4. Biofabrication Approaches with Spheroids

4.1. Macroporous Scaffolds with Spheroids

Scaffold-free biofabrication approaches with spheroids have enabled the production of macroscale tissue constructs for tissue engineering and regenerative applications.[76] Unfortunately, these approaches suffer drawbacks of mechanical instability and volume maintenance due to spheroid fusion and contraction. For this reason, macroporous biomaterial scaffolds have been used, including foam/sponge and 3D lattice structures to mechanically support and provide a defined geometry for culturing spheroids (Fig 5A). Unlike hydrogels described above, these scaffolds are engineered to exhibit macroporous features with a high interconnectivity that enhances cell migration, nutrient diffusion, and structural stability while also maintaining a relatively low volume fraction of material. Macroporous scaffolds have previously been developed through porogen-leaching, cryo-processing, or 3D manufacturing techniques to introduce an interconnected pore network and then subsequently seeded with cell suspensions or spheroids to elicit tissue growth, spheroid formation and fusion.[77–79] Natural biomaterial (e.g. HA/collagen) macroporous scaffolds fabricated via cryo-processing have been combined with spheroids for a variety of tissue engineering and tumor modeling applications.[80,81] Generally, these macroprous scaffolds leverage native cell-adhesive sites to improve the predictive capabilities of cancer drug screening by altering the cancer cell niche microenvironment and priming tumour signalling pathways.[81]

Figure 5 |. Macroporous Scaffolds with Spheroids.

(A) Representation of the use of two macroporous scaffolds for spheroid culture: foam/sponge (top) and 3D lattices (bottom). (B) MeHA cryogels seeded with CD44+ breast cancer spheroids to provide structural support as well as adhesion and HA biochemical cues on spheroids. Scalebar: 200μm [72] (C) Dissolvable PEGNB-Dopa microgels create macroporous PEGNB hydrogels to elicit controllable MSC spheroid formation and size. [74] (D) PCL MEW scaffolds seeded with MSC spheroids at day 0 (left) and day 14 (right). [78] Figures B-D are credited to original authors and with written permission from Elsevier and Wiley and Sons.

Unfortunately, naturally-derived microporous scaffolds can be limited by the difficulty in tuning their biochemical and structural properties, motivating the use of synthetic biomaterial or modified biopolymer scaffolds. As an example, Rezaeeyazdi et. al. recently implemented methacrylated hyaluronic acid (MeHA) cryogel scaffolds with CD44+ breast tumor spheroids to combine structural support and inherent HA biochemical cues to spheroids (Fig 5B).[82] Interestingly, the reduction of RGD bound to MeHA cyrogels allowed spheroids to interact solely through CD44 interactions and elicited a highly aggressive and drug-resistant tumor phenotype with increased cellular invasion. Macroporous scaffolds have also been engineered with granular hydrogels, where the void space between particles can be easily controlled to dictate spheroid size (~20–100μm diameter) and interconnectivity.[83–85] Morley et al. seeded CHO cells in varying volume fractions within jammed polyacrylamide granular hydrogels to understand cell aggregation dynamics and cohesive forces. As expected, increasing the cell volume fraction improved interconnectivity; however, enhanced cell-cell interactions resulted in more irregular-shaped aggregates (Fig 5C). [83] These interconnected structures form initially from cells aggregating into spheroids that then interconnect over time. An alternative approach by Jiang et al. used hydrolyzable PEG-norbornene-dopamine (PEGNB-Dopa) within a PEGNB bulk matrix to produce controllable macropores. MSCs were incorporated within PEGNB-Dopa microgels to facilitate controllable spheroid formation and size (~20–80μm diameter) within the macroporous gel. (Fig 5C).[84] Interestingly, when gelatin norbornene hydrogels were used as the bulk matrix, MSCs were allowed to further interact with their surrounding matrix to form hollow spheres rather than spheroids. Unfortunately, seeding spheroids or cells uniformly into macroporous scaffolds still remains a challenge and can often result in a heterogeneous spheroid distribution, which motivates the use of additive manufacturing to produce more controlled, engineered scaffolds that allow for uniform spheroid deposition.

As additive manufacturing techniques, such as melt electrowriting (MEW) and electrospinning, have advanced within the field, spheroid seeded constructs have been developed to engineer high-resolution scaffolds with enhanced structural stability and shape fidelity. MEW combines 3D printing with electric fields to allow the precise, high-resolution deposition of polymer melts (primarily poly-ε-caprolactone, PCL) in 3D lattice structures by pneumatically driving a polymer melt through a charged nozzle head.[86] Previous studies have utilized cell suspensions alone or with hydrogel carriers to seed within these lattices to form composites that exhibit exceptional biomechanical properties when compared to hydrogels alone.[87,88] Alternatively, the Dalton and Blunk groups have developed multimodal and multiphasic MEW scaffolds by modulating the flow rate and collector speed to fabricate a range of PCL scaffolds with varying filament and pore diameters that can be subsequently seeded with MSC spheroids and cultured for several weeks.[89] Due to the control over fiber deposition, “catching fibers” could be deposited at the bottom of the scaffold to improve spheroid seeding efficiency. This technique was further explored to induce MSC spheroids into an adipogenic lineage with PCL scaffolds (Fig 5D).[90] Recently, MEW has also been integrated with inkjet bioprinting to form spheroids in situ within MEW scaffolds and to generate cartilage tissue with near native biomechanics.[91] Similar to MEW, electrospinning uses a high-voltage charged nozzle head and a collector to fabricate nanoscale fibers that can be engineered to elicit varying filament dimensions and biomechanical properties.[92] Extrusion-based printing of alginate lattices has been recently combined with electrospun alginate to entrap MSC spheroids and harness the angiogenic factors secreted within the medium.[93] Conditioned medium from these biofabricated scaffolds was found to enhance tubule formation of HUVECs embedded in Matrigel^® when compared to scaffold-free spheroids. Overall, macroporous scaffolds to induce spheroid assembly or to modulate construct properties will greatly impact the field.

4.2. Bioassembly and Extrusion-Based 3D Bioprinting

Bioassembly and 3D bioprinting are emerging parallel technologies within the biofabrication field that enable the precise assembly or deposition of biological material (i.e., cells, spheroids, biomaterials) into pre-defined 3D structures that can mature into functional tissue constructs for in-vitro modeling or implantable tissue engineering applications. As both technologies rapidly advance within the field and facilitate the same goal of fabricating biological tissue constructs, their definitions often become convoluted and are used interchangeably. Consistent with Groll et al., we define “bioassembly” in this review as the fabrication of pre-defined 3D biological structures through the automated assembly of preformed cellular building blocks and a 3D biomaterial structure.[94] “3D bioprinting” can be defined as the fabrication of pre-defined 3D biological structures through the automated spatial deposition of cells or spheroids encapsulated within or printed into a structurally supporting biomaterial.[95] With time, an array of bioassembly and extrusion-based bioprinting strategies have been developed whereby spheroids can be deposited in a controlled manner with biomaterials that then allow spheroid fusion and growth into scalable tissues. There are three main strategies for bioassembly and extrusion-based 3D bioprinting with spheroids, which include microchamber bioassembly, encapsulated bioprinting, and embedded bioprinting (Fig 6A).

Figure 6 |. Extrusion-Based 3D Bioprinting.

(A) Microchamber, encapsulated, and embedded extrusion-based bioprinting strategies. (B) Proof-of-concept osteochondral microchamber bioassembly printed with PCL filaments, as well as chondrogenic and osteogenic spheroids. Scalebar: 2mm [99] (C) MSC spheroids encapsulated in GelMA bioinks printed into 3D lattices that differentiate into cartilage tissue. Scalebars: (c) 800μm, (d,e) 500μm, (f) 200μm [16] (D) The SWIFT embedded method (top) and printed perfusable cardiac tissue models formed through sacrificial writing (bottom). Scalebars: (a) 2mm, (b) 500μm, (c) 10μm [26] Figures B-D are credited to original authors.

The microchamber assembly strategy combines traditional extrusion 3D printing of thermoplastic polymers (e.g., PCL, polylactic acid (PLA)) with automated bioassembly robotics that allow for the precise deposition of individual spheroids within thermoplastic printed microchambers. In contrast to the previously described use of macroporous scaffolds for spheroid culture, this approach provides controlled placement of spheroids. Initial attempts used a ‘wood-pile’ microstructure consisting of printed PCL filaments, which were then manually seeded with spheroids to develop vascularized adipose, bone, and cartilage tissues.[96–98] Over time, automated bioassembly robotics were developed to enable the precise positioning of individual spheroids or microtissues into predetermined locations within the microchambers to enable the biofabrication of heterogeneous tissue constructs.[99] Recently, Mekhileri et. al. used this strategy to first print a PCL scaffold and subsequently pattern microtissues in a layer-by-layer manner to assemble a proof-of-concept osteochondral tissue construct (Fig 6B).[100] On a smaller scale, this microchamber scaffold has also been used to investigate co-cultures of spheroids, such as with chondrocyte and MSC spheroids.[101] Combining bioassembly and 3D bioprinting strategies, the Ozbolat and Kelly groups have used inkjet bioprinting to dispense droplets of cell suspensions within pre-formed hydrophobic microchambers whose surface tension drives spheroid condensation.[102,103] This approach has been used to develop spatially organized implantable osteochondral constructs and joint anchoring pin devices that self-organize to drive tissue growth.[103,104] One limitation to the microchamber approach is that PCL filaments can block spheroid fusion and restrict them to their respected microchamber.

As an alternative, the encapsulated bioprinting strategy relies on biomaterial-based bioinks that act as carriers for spheroids during the printing process to stabilize them into printed constructs. Engineered bioinks are commonly stabilized through temperature or light-initiated crosslinking either in situ or post-printing. For example, the Declercq group recently reported the use of GelMA bioinks with MSC, HUVEC and human foreskin fibroblast (HFF) spheroids to engineer vascular, adipose and cartilage tissue structures.[18,51,105] MSC spheroids were incorporated within a GelMA bioink, printed into 3D lattice structures, post-crosslinked with light, and differentiated down a chondrogenic lineage (Fig 6C).[18] Spheroids maintained high cell viability throughout the entire printing process and demonstrated comparable deposition of cartilage-specific ECM to single cell suspensions; however, the low spheroid density within the bioink limited spheroid fusion over time and produced a construct with only isolated spheroids. Other biomaterial options, such as alginate/gelatin composite bioinks, that are not covalently crosslinked have been explored to address this and have enabled spheroid fusion.[106] Thus, increasing spheroid density or introducing a fast degrading bioink could increase spheroid fusion during the printing process. Unfortunately, another limitation to this approach is the settling of spheroids during printing, which has been addressed by Horder et. al. with the incorporation of high-MW HA within their bioink to increase viscosity.[107]

The embedded bioprinting strategy, which involves the printing of cells and spheroids into support baths either alone or with biomaterials overcomes some of the challenges described above.[108] Originally, embedded bioprinting was performed with natural biomaterials such as type I collagen precursor solution or bio-paper, to investigate spheroid fusion kinetics and assembly into topologically defined structures.[16,109] As collagen gels quickly and introduces errors during the printing process, this biomaterial system was replaced by support baths. Support baths are yield-stress hydrogels composed of microparticles or self-assembled structures that elicit shear-thinning and self-healing properties to allow needle movement within the material without permanently damaging its structure. As an example, SWIFT was recently developed by the Lewis group, where sacrificial gelatin is printed into a support bath of collagen, Matrigel^®, and spheroids. The support bath is stabilized through collagen polymerization and spheroid fusion while the sacrificial hydrogel is evacuated to retain a channel that could be subsequently perfused with media to support long-term vascular and cardiac tissue culture (Fig 6D).[29] After 8 days in culture, cardiac tissue was observed to function through calcium imaging and electrical stimulation. Overall, it becomes apparent that embedded bioprinting has many advantages over traditional extrusion-based printing and has enabled the engineering of more complex, functional cell-based structures.

4.3. Aspiration-Assisted 3D Bioprinting

The assembly of spheroids into macroscopic tissues has advanced within the field due to the interest in assembling spheroids of uniform or mixed compositions into 3D structures. This technology enables spheroids to be deposited and stabilized into complex 3D geometries, either through spheroid fusion or with biomaterial supports, to allow for the generation of cell-dense engineered tissues and tissue models. Traditional 3D bioprinting (e.g., extrusion-based) can mechanically perturb spheroids when deposited through a capillary or syringe and typically allows only the continuous deposition of spheroids, which can limit the printing of complex 3D heterogeneous structures. As an alternative, spheroids can be organized through the placement of individual spheroids with some order that then fuse together into tissues, particularly with aspiration-assisted 3D bioprinting (AAB). This technology uses a 3D printer or micromanipulator with an aspiration-controlled needle to individually pick and place spheroids into 3D heterogeneous patterns or geometries. For this reason, AAB can be mistaken as a “bioassembly” technology; however, spheroids are placed on a needle or within a biomaterial support bath that does not have a pre-formed 3D structure. One example is the Kenzan printing method (now sold by Cyfuse Biomedical) where spheroids are placed onto needle arrays.[110–113] In recognition of the damage to spheroids through these needles, hydrogels have also been used to support spheroid deposition with AAB, particularly by the Ozbolat and Burdick groups.[1,114] Two main AAB strategies with biomaterials include layer-by-layer and embedded printing, both of which enable the precise patterning of spheroids (Fig 7A). This technology has improved the utility of bioprinting approaches to produce promising bone, cartilage, vascular tissues, and tissue models.[1,13,114–116]

Figure 7 |. Aspiration-Assisted 3D Bioprinting of Spheroids.

(A) (i) layer-by-layer and (ii) embedded aspiration-assisted bioprinting (AAB) strategies. (B) Heterogeneous pyramid and diamond geometries printed layer-by-layer in sodium alginate with HUVEC and MSC spheroids and (C) bone formation of osteogenically differentiated HUVEC/MSC spheroids printed in a triangular pattern with the layer-by-layer technique. [86] (D) MSC spheroids printed into an agarose support bath and patterned in a ring geometry either before (strategy I) or after (strategy II) chondrogenic differentiation.[11] (E) Healthy and scarred cardiac tissue rings developed through iPSC cardiomyocyte and fibroblast spheroids printed into GH hydrogels via an aspiration-assisted micromanipulator. Scalebars: 100μm, insets 50μm [1] Figures B-E are credited to original authors.

In the layer-by-layer approach, developed by the Ozbolat group, a variety of spheroid types, as well as functional and sacrificial hydrogels were printed into complex 3D heterogeneous structures.[114] This AAB system patterns spheroids in a layered approach within hydrogels of varying crosslink chemistries such as: collagen I (enzymatic), GelMA (photocrosslinking), and alginate (ionic, CaCl₂ aerosol), where each layer is crosslinked in sequence (Fig 7B). The technology was applied to several biomedical applications, including investigation of cellular crosstalk (e.g., HUVECs and MSCs (or MSC/human dermal fibroblast) spheroids patterned at varying distances to examine angiogenic sprouting within fibrin hydrogels or to fabricate tissue constructs (e.g., MSC spheroids printed into bone tissue) (Fig 7C). Interestingly, spheroid culture times before scaffold-free printing were important - the printing of spheroids 2 days after condensation resulted in construct contraction, whereas spheroids maintained for 2 weeks before printing produced partially fused constructs that maintained their geometry. The construct contraction from this study demonstrates and motivates the need for a biomaterial that structurally supports spheroid constructs during the fusion process. With this layer-by-layer AAB technique, subsequent reports developed osteochondral and HUVEC containing bone tissue constructs.[115,117] While the layer-by-layer method is effective, this strategy is very time-consuming, introduces stresses to spheroids when passing through the air-liquid interface, and requires external stimuli to initiate hydrogel crosslinking.

As an alternate AAB strategy, the embedding of spheroids within yield-stress hydrogel supports allows their continuous printing and then stabilization through hydrogel yielding and healing. Yield-stress hydrogels, such as microparticle-based support baths, maintain a stable solid state but then transition to a fluid state as the needle moves through them. The Ozbolat group implemented this approach using Carbopol^® or agarose supports, deemed aspiration-assisted freeform bioprinting (AAfB), to print and pattern 3D complex spheroid geometries.[13] This study demonstrated that the properties (e.g., polymer concentration) of the support bath influences print construct stability, as well as print accuracy and cytotoxicity. Demonstrating the functionality of their printed constructs, circular rings and triangles were printed within agarose support baths and differentiated into cartilage and bone tissue, respectively (Fig 7D). Pre-differentiating MSCs in monolayer culture and longer spheroid cultures improved printed construct differentiation capacity and shape fidelity; however, pre-culture for too long limited spheroid fusion. Further, the use of agarose microparticles limited visualization and print precision, which was improved upon in a subsequent paper.[116] As an alternative to particle-based support baths for spheroid printing, the Burdick lab explored yield-stress guest-host (GH) hydrogels synthesized through hyaluronic acid modified with adamantane or β-cyclodextrin.[118] This GH hydrogel has previously demonstrated its utility as injectable hydrogels, as well as support baths for biomaterial printing.[119–121] An AAB strategy was explored with the GH hyaluronic acid hydrogels for the printing of spheroids and fusion into heterogenous tissue models, including with iPSC-derived cardiomyocytes.[1] Specifically, a ring-shaped cardiac model was developed for disease modeling where spheroids of mixed ratios of cardiomyocytes and cardiac fibroblasts were printed to model the heterogeneity of cardiac tissue after myocardial infarction (Fig 6E). The introduction of spheroids with high fibroblast content to model scarred regions of myocardium disrupted uniform tissue contraction and electroconductivity via calcium signaling and was used to evaluate miRNA therapeutics (i.e., miRNA-302b/c induction of cardiomyocyte proliferation). Overall, these AAB/AAfB strategies have enabled complex patterning of heterogenous spheroid structures that would not be feasible with traditional extrusion-based bioprinting. The AAB field is rapidly growing and the Angelini group recently introduced an AAB technique that enables patterning on a single-cell scale.[122]

5. Final Remarks and Outlook

The use of engineered biomaterials with spheroids is an emerging field that holds great promise in applications of regenerative medicine and in vitro tissue models. Advancements in biomaterial chemistry and biotechnologies (e.g., 3D bioprinting) have enabled studies that range from the understanding of spheroid functions to the biofabrication of macroscale tissues. In this review, we have covered recent approaches in the application of engineered biomaterials to guide spheroid formation, function, and fabrication into tissue models and constructs. Unlike scaffold-free approaches, engineered biomaterials can provide structural stability and instructive cues to support and direct tissue function and growth.

The use of engineered biomaterials for the formation of spheroids has been rapidly developing over the past decade and has allowed for the high-throughput generation of homogeneous spheroids. Non-adhesive biomaterials (e.g., agarose) and engineered biomaterials have primarily been used to condense spheroids into a variety of geometrically shaped microwells. Unfortunately, long-term culture and removal of spheroids from microwells for post-processing remains a challenge. Non-adhesive agarose microwells tend to release and aggregate spheroids, while adhesive engineered biomaterials tend to have problems removing spheroids from their microwells. One solution is to make the wells deeper to prevent aggregation; however, 3D printing templates and casting these structures introduces additional technical challenges. Kim et. al. proposed an innovative solution using the thermo-expanding Tetronic^® polymer that releases bound spheroids when cold saline is applied.[30] As the field is expanding, other shape-memory or temperature-regulated polymers may be implemented to provide additional control over the release of spheroids. Another avenue could be to engineer photodegradable polymer molds that can release spheroids when exposed to light.[123]

Beyond just the formation of spheroids, approaches could be used to further guide spheroid behavior with biomaterials during the formation process, including spheroid shape and differentiation. These approaches are being used for the formation of organoids[124,125] and the ability to control microwell geometry and engineered cues has guided organoid patterning and folding.[126] A final emerging approach for spheroid fabrication is the use of macroporous scaffolds or granular hydrogels that allow seeded cell suspensions to condense within their structures. [83,84,127]

In the ‘function’ section of this review, we summarized how biophysical and biochemical cues can be engineered to guide spheroid function and fusion either externally through encapsulated hydrogels or internally through embedded microparticles. It becomes apparent that like single cells, spheroids respond to biomechanical and biochemical cues presented within their surrounding environment. As many biomechanical and biochemical properties have been decoupled, the role of degradability and viscoelastic dynamics remains unclear with spheroid cultures and needs to be further investigated. Additionally, little is known about how biomaterial properties influence spheroid fusion, particularly outside the scope of naturally derived biomaterials. Regardless, biomaterials have been recently implemented with organoids to guide differentiation into intestinal or hepatocyte-like organoids.[128,129] As Matrigel^® is still the most common ECM for organoid culture, engineered biomaterials will become more common and be advantageous to decouple specific biomaterial properties that drive different spheroid and organoid functions. Overall, the field is continually learning the roles of biomaterial properties on spheroid function, and this will likely grow further in upcoming years.

Macroporous scaffolds and 3D bioprinting approaches have found great utility with spheroids by providing a supported structure that enables tissue growth while also allowing for complex geometries to be fabricated to recapitulate complex native tissues. As this review summarizes, there have been a variety of strategies (MEW, electrospinning, extrusion-based 3D bioprinting, etc.) used. One major issue within the field is that there is no standard consensus on the timing between spheroid formation, fabrication, and removal from support structures. Spheroid formation and fusion are highly time-dependent cellular processes and if spheroids are condensed too long, fusion is negatively impacted; whereas, too early of use and the biofabricated structures may collapse and condense into large cellular aggregates. Different AAB culture strategies helped demonstrate this phenomenon, but the challenge of maintaining proper fusion and construct shape fidelity still remains. With the recent advances in support baths, embedded 3D bioprinting holds great promise in allowing printed structures to be cultured long-term while also constantly providing structural support to mitigate contraction. As of recent, only cell-only or high-cell density bioinks that condense once printed into support baths have been investigated; however, printing of pre-condensed spheroids can be advantageous over this method by having additional control over spheroid size and modularity. The Lutolf group recently used this approach, deemed the BATE method, but within the application of bioprinting complex organoid structures. With this BATE method, MSC aggregates, intestinal and vascular organoids were able to be printed and fabricated into vascular beds or macroscopic intestinal tubes.[130] As the field grows, support baths will undoubtedly find utility within biofabrication approaches.

In summary, this review has covered how engineered biomaterials can be leveraged to guide spheroid formation, function, and fabrication. The field is rapidly expanding, and the future will certainly provide new and innovative approaches to combine biomaterials with spheroids.

Table 1.

Abbreviated Terminology Legend

Abbreviation
MSCs	Mesenchymal Stromal Cells
PSCs	Pluripotent Stem Cells
iPSCs	induced-Pluripotent Stem Cells
ESCs	Embryonic Stem Cells
ECM	Extracellular Matrix
PDMS	Poly (dimethyl siloxane)
MeHA	Methacrylated Hyaluronic Acid
GelMA	Gelatin Methacrylate
sGAG	sulfated Glycosaminoglycans
IPN	Interpenetration Networks
PLGA	Poly (lactic-co-glycosidic) acid
BMP-2	Bone Morphogenic Protein-2
TGFβ	Transforming Growth Factor β
pDNA	plasmid Deoxyribonucleic acid
SiRNA	silencing Ribonucleic acid
Dex-HEMA	Dextran Hydroxyethyl Methacrylate
MEW	Melt Electrowriting
PCL	Poly-ε-Caprolactone
AAB	Aspiration Assisted Bioprinting
AAfB	Aspiration Assisted freeform Bioprinting
HUVEC	Human Umbilical Vascular Endothelial Cells
GH	Guest-Host
PLA	Poly(lactic) acid
FRESH	Freeform Reversible Embedding of Suspended Hydrogels
SWIFT	Sacrificial Writing into Functional Tissues

Statement of Significance:

Cellular spheroids are becoming increasingly used as in vitro tissue models or as ‘building blocks’ for tissue engineering and repair strategies. Engineered biomaterials and their processing through biofabrication approaches are being leveraged to structurally support and guide spheroid processes. This review summarizes current approaches where such biomaterials are being used to guide spheroid formation, function, and fabrication into tissue constructs. As the field is rapidly expanding, we also provide an outlook on future directions and how new engineered biomaterials can be implemented to further the development of biofabricated spheroid-based tissue constructs.