The utility of biomedical scaffolds laden with spheroids in various tissue engineering applications

A spheroid is a complex, spherical cellular aggregate supporting cell-cell and cell-matrix interactions in an environment that mimics the real-world situation. In terms of tissue engineering, spheroids are important building blocks that replace two-dimensional cell cultures. Spheroids replicate tissue physiological activities. The use of spheroids with/without scaffolds yields structures that engage in desired activities and replicate the complicated geometry of three-dimensional tissues. In this mini-review, we describe conventional and novel methods by which scaffold-free and scaffolded spheroids may be fabricated and discuss their applications in tissue regeneration and future perspectives.


Introduction
A spheroid is a three-dimensional (3D), self-assembling cell aggregate [1]. Before spheroids were introduced by Moscona and Moscona [1], conventional two-dimensional (2D) cell cultures created in Petri dishes or culture flasks were widely used; these were convenient, inexpensive, and very reproducible. All cells received identical nutrients. 2D cell cultures have found many applications in in vitro studies, disease modeling, analyses of cellular responses, drug screening, and regenerative medicine [2,3]. However, 2D cultures do not mimic the in vivo 3D microenvironment; cell-cell and cell-matrix interactions are limited [4]. 2D monocultures also do not engage in in vivo cellular behavior [5] and are much more drug-sensitive than are spheroids [6,7], reflecting differences in physical and physiological properties, the expression and spatial organization of surface receptors, cancer gene expression levels, cell stage, drug accessibility, and the local pH [8]. Spheroids thus find many applications in tissue engineering (TE) [9]. Spheroids feature extensive cellular interactions, mimicking the in vivo extracellular matrix (ECM) that mechanically supports cells and regulates most cellular functions, including migration, survival, proliferation, and differentiation. The ECM interacts with growth factors and facilitates contact between cells and ECM components [10]. The geometrical similarity between spheroids and cells of native tissue may allow spheroids to mimic cellular functions (including viability and differentiation) better than do monolayer cultures [11,12]. Furthermore, spheroids exhibit higher-level tissue-specific gene expression compared to monocultures, thus affecting the regulation of cell signaling and cytokine expression, because they more closely mimic in vivo microstructures [13]. Finally, as spheroids are cell aggregates, they protect cells from unwanted external stimuli or drug inflows by ensuring the maintenance of a dense ECM network. Spheroids more closely resemble the geometrical and physiological in vivo microenvironment than do monolayers, enhancing their clinical and regenerative utility.

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However, spheroids are associated with critical shortcomings attributable to their geometrical structure. Cells rely exclusively on diffusion to transport nutrients or eliminate waste. Therefore, the interiors of spheroids lack nutrients and oxygen and retain CO2, ultimately triggering necrosis, whereas the cells in the outer layer successfully transfer nutrients and remove waste [14,15]. Necrosis usually develops in spheroids of larger diameters, e.g., 400 to 600 µm, and reduces cell viability and proliferation [16]. To overcome these limitations, efforts have been made to artificially supply oxygen via fiber injection [17][18][19][20]. Moreover, spheroids scatter light and feature inhomogeneous dye diffusion [21], rendering imaging very challenging. In efforts to counter these limitations, cryoplastic sectioning, light-sheet fluorescence microscopy, and optical clearing methods have been employed [21,22].
Furthermore, it is difficult to accurately shape spheroids and obtain complex 3D cell constructs. Nonetheless, spheroids have been widely used for the regeneration of skin, blood vessels, nerves, and liver tissues. Some tissues (such as cardiac tissues) require extensive cell-ECM interactions to maximize tissue-specific functions [23]. Spheroids have thus been incorporated into various biomedical scaffolds (made of collagen, elastin, or ECM), yielding complex 3D structures. However, the scaffold materials may trigger immune responses and cause infections [24][25][26][27].
Several previous works have shown that spheroids can be used for various TE applications, alone or with a biomedical scaffold fabricated using conventional or novel processes such as electrospinning, 3D printing, and hydrogel contraction. In this mini-review, we explore the use of spheroids for the regeneration of bone, cartilage, liver tissues, nerves, and skin, dealing separately with scaffold-free spheroids and scaffolded spheroids. We describe their advantages and disadvantages and summarize future research directions.

Spheroid Fabrication Methods
Spheroid formation does not require a special culture medium and can be divided into three steps: loose cellular aggregation caused by interactions between integrin and the ECM, accumulation of cadherin, and the formation of compact spheroids via cadherin-cadherin interactions [28]. Cell adhesion molecules such as cadherins and integrins play significant roles in spheroid formation [29]. During the first stage, the ECM binds single cells via their membrane integrins, and the cells become loosely aggregated. E-cadherin, the principal glycoprotein of Ca 2+ -dependent systems, mediates homotypic cell-cell adhesion [30] and triggers robust spheroid compaction after cell accumulation. Spheroids can be fabricated by various methods, using the same culture media as those of 2D culture. We next briefly review conventional spheroid fabrication techniques such as the hanging drop, nonadherent surface, spinning flask, and rotating vessel methods, as well as the more recently developed microfluidic, acoustic, water-inwater emulsion, and 3D printing methods.
The hanging drop, nonadherent surface, spinning flask, rotating vessel, external force-assisted, and matrix-embedded methods have all been employed to induce cell aggregation. The hanging drop method is cost-effective; cells aggregate within suspended droplets under the influence of surface tension and gravitational forces ( Figure 1A) [31,32]. The spheroid size is determined by the droplet volume. However, the procedure is labor-intensive and thus unsuitable for high-throughput production. With the nonadherent surface method, spheroids are readily fabricated in an environment in which cells cannot adhere. Although no special equipment is required, the process is inefficient and spheroid size cannot be controlled ( Figure 1B) [33]. In the spinning flask method, cells collide within rotating bioreactors, triggering aggregation ( Figure 1C). The centripetal force promotes nutrient transfer and waste removal, but the high shear stress can damage or kill cells [34,35]. To minimize shear stress, the rotating vessel maintains the cells in continuous suspension ( Figure  1D) [36,37]. Recent fabrication methods have employed micro-patterned molds ( Figure 1E) [38]. Spheroids are fabricated by seeding cells onto a non-adhesive mold material. However, the spheroid size is variable, throughput is low, and handling is difficult [39]. To overcome these limitations, methods such as microfluidics, micromolded non-adhesive hydrogels, acoustic, and water-in-water emulsions methods have been implemented to fabricate spheroids.
In microfluidics, low fluid volumes are processed, allowing multiplexing, automation, and high-throughput screening. Emulsions, microwells, and U-shaped microstructures have been used to fabricate spheroids (Figure 2A) [40][41][42]. Spheroid fabrication features cell encapsulation, with two fluids forming droplets and cells encapsulated in a hydrogel [43,44]. Micromolded non-adhesive hydrogels are used in combination with the hanging drop, rotating vessel, and microfabrication techniques; spheroids are fabricated as desired by seeding cells into micromold chambers formed from non-cell-adhesive polymers such as a polyacrylamide hydrogel or an agarose gel ( Figure 2B) [45][46][47]. A microfluidic system can be supplemented with an acoustic wave generator, such that injected cells aggregate in the trough of a wave ( Figure 2C). This allows high-throughput production, but the equipment is expensive [48][49][50]. In the water-in-water emulsion method, cells are injected into two different phases of an aqueous solution; an osmotic effect then triggers aggregation ( Figure 2D). This method is cost-efficient and enables highthroughput production [51,52]. More recently, 3D printing technology has emerged as a novel fabrication strategy; this is reproducible, allows highthroughput production, and is precise. In particular, a drop-on-demand method has been employed to fabricate a non-adhesive hydrogel cup. In this process, a hydrogel bio-ink is deposited layer-by-layer and cell droplets are placed in the center of the hydrogel cup. Over time, the cells aggregate into spheroids [53]. The precise spheroid fabrication capacity of 3D printing has greatly aided spheroid research. Kang et al. reported precise positioning of HepG2 and NIH3T3 spheroids (at the micrometer scale) using a 3D bio-dot printing method [54]. Such spheroids can be used alone for TE or combined with biocompatible supporting materials that remedy spheroid shortcomings or provide additional functions. In this review, we named the former as "scaffold-free" and the latter as "scaffolded".

Spheroid applications in TE
TE seeks to develop implantable biological substitutes that maintain or improve tissue function [55]. Many researchers have shown that mimicking the physiological environment of native tissue yields superior results. Therefore, spheroids have been increasingly used for TE, as their 3D form mimics native tissue. Scaffold-free approaches use spheroids alone, whereas scaffolded approaches feature scaffolds. Both approaches have unique advantages and disadvantages, but researchers have been versatile, as shown below.

Scaffold-free applications
Cell sheets, spheroids, or tissue strands are implanted alone ( Table 1) [56]. The spheroids serve as building blocks, with spheroids fusing to form larger 3D structures. The high initial cell density enables spheroid self-assembly. Immune responses to scaffold by-products are obviously absent. However, the absence of supporting material can create significant mechanical problems in dynamic in vivo applications. Scaffold-free strategies are commonly used for tissue self-assembly and microchannel fabrication [57][58][59][60]. In the former approach, spheroids are placed in a mold/support to obtain the desired macrostructure. The latter approach is typically used for drug testing or to observe cellular activities such as vascularization.   Schematic illustration of water-in-water emulsion method using phase separation between PEO and dextran. Adapted with permission from [52], copyright 2020 Royal Society of Chemistry.

Bone
Bones support weight and protect organs; bones are subjected to continuous mechanical stresses that can trigger fractures or osteoporosis [61,62], particularly in the elderly, who thus require bone regeneration. The cell type, biomaterial, bone architecture, and mechanical properties must all be considered [63]. Calcium phosphate-based ceramics such as hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) are widely used as scaffolds because they are osteoconductive [64]. Ceramics are commonly combined with spheroids to enhance osteogenic differentiation. Suenaga et al. investigated the effects of β-TCP on mesenchymal stem cell (MSC) spheroids used for in vivo calvarial regeneration, with the test components comprising MSC spheroids, β-TCP, and an MSC-spheroid/β-TCP composite. Surprisingly, the MSC spheroids afforded better bone regeneration than did the composite. The persistence of β-TCP particles between newly formed bone tissues hindered uniform bone formation. The computed tomography (CT) image in Figure 3A displays the bone regeneration of calvarial defects implanted with MSC spheroids (left) versus that of untreated defects (right) [65]. A recent report found that the addition of nano-hydroxyapatite particles (nHAps) to spheroids enhanced bone regeneration. Hasani-Sadrabadi et al. prepared an adhesive hydrogel (AdhHG) with tunable mechanical properties by modifying methacrylated alginate with dopamine and adding an arginine-glycine-aspartate (RGD) motif ( Figure 3B). To induce osteogenesis, nHAps were incorporated into MSC spheroids encapsulated in AdhHG. Spheroids of MSC cells aggregated with HA and AdhHG to afford significantly improved tissue mineralization and bone-filling at 8 weeks after implantation, compared to the alginate hydrogel, which was attributable to the enhanced crosslinking capacity of the modified AdhHG. The resultant mechanical properties were better [66].
Bone TE requires adequate nutrient transfer through vascularized connective tissue [67]. Osteogenic expression improved when human adipose-derived stem cell (ASC) spheroids and poly(L-lactic acid) (PLLA) fibers were coated with platelet-derived growth factors (PDGFs) and biominerals ( Figure 3C) [68]. The synergistic actions of the angiogenic growth factors and biominerals enhanced vascularized bone regeneration. Heo et al. used MSC/human umbilical vein endothelial cell (HUVEC) spheroids as building blocks for bone TE. The MSC/HUVEC spheroids were encapsulated in a collagen/fibrin hydrogel. Cell viability, proliferation, and osteogenic differentiation were enhanced compared to the three control groups consisting of hydrogels encapsulated with an MSC suspension, an MSC/HUVEC suspension, and MSC spheroids ( Figure 3D) [69].

Cartilage
Cartilage is a protective, blood vessel-free connective tissue located between bones or joints that softens impacts and supports bony motion. The self-repair capacity is limited but cartilage is not easily damaged [70,71]. However, constant loading or stress may cause wear, and a decrease in glucosamine synthesis with aging may reduce the levels of proteoglycans, the principal components of articular cartilage, triggering degenerative disease [72]. Several studies have shown that spheroids better enhance cartilage regeneration than do 2D cultured cells. Ishihara et al. used bone-marrow-derived MSC (BM-MSC) spheroids to regenerate osteochondral defects in rabbit knee joints. The spheroids were loaded into tube-shaped Teflon molds to form cylinders. In vivo, the spheroids differentiated into both bone and cartilage ( Figure 4A) [73]. Human nasal septum-derived chondrocyte (hNC) spheroids repaired osteochondral defects both in vitro and in vivo, resulting in improved cell viability, proliferation, and gene expression, as well as generated more ECM components, compared to a 2D monoculture of hNCs ( Figure 4B) [74].
The cartilaginous trachea sends air to the lungs; thus, tracheal defects are serious [75,76]. Tracheal TE is considered ideal in terms of tracheal replacement [77]. Machino et al. constructed a trachea-like tube composed of various cell types [78]. Artificial tracheae featuring two types of hybrid spheroids -one consisting of fibroblasts, HUVECs, and MSCs, and the other consisting of chondrocytes, HUVECs, and MSCs -were transplanted into rats. Immunohistological staining revealed the development of an epithelium and capillary network similar to those of the native trachea ( Figure 4C). Complete tubular grafting of human cells may become possible.  [66]. C. Schematic illustration and osteogenic differentiation of a spheroid building block including biominerals and PDGF-coated PLLA fibers [68]. D. Schematic illustration of spheroid encapsulation within a collagen/fibrin matrix [69].

Liver
The liver performs vital regulatory functions; produces bile acids; engages in hormone excretion, fat and protein metabolism, and vitamin and mineral storage; and exerts other physiological functions [79]. Liver cell spheroids are commonly used for drug testing. C3A hepatocarcinoma cell spheroids were fabricated using an overlay method [80] and exhibited in vivo-like features such as 3D cell-cell interactions, zonation, and structural and functional polarization. Moreover, the spheroids expressed and secreted significantly higher levels of albumin and urea compared to a 2D monoculture (Figure 5A).
Liver fibrogenesis is caused by injury and characterized by the excessive deposition of ECM [81]. The differentiation of hepatic stellate cells (HSCs) into collagen-secreting myofibroblasts activates fibrosis, as does hepatocyte (HEP) damage. HSC/HEP cell spheroids are used to test drugs that may counter fibrosis, as the spheroids exhibit in vivo-like HSC behavior [82].

Nerves
The brain is composed of neurons, glial cells such as astrocytes, microglia, oligodendrocytes, pericytes, and endothelial cells. Brain disease often develops after a brain or spinal cord injury, or may be neurodegenerative in nature (Parkinson's, Alzheimer's, or Huntington's disease) [83,84]. Spheroids may be used to effectively deal with these conditions. For example, as shown in Figure 5B, human gingiva-derived MSC (GMSC) spheroids stacked in a needle array and immunostained for axonal SMI-31/32 (neurofilament H) markers produced signals indicative of possible peripheral nerve regeneration [58]. An autologous, 3D nerve conduit graft consisting of human normal dermal fibroblast (hNDFB) spheroids regenerated peripheral nerve defects [85]. The histological analysis results shown in Figure 5C show that the myelinated axon diameter and myelin thickness were significantly greater than those of the control (with a silicon graft).

Skin
The skin is the largest organ of the human body and is responsible for thermoregulation and protection against solar radiation. However, the skin is susceptible to damage, being the first line of defense against external insult. Natural healing features sequential hemostasis, inflammation, cell proliferation, and cell maturation [86,87]. Umbilical cord tissue-derived MSC spheroids have been used to accelerate this process [88].The immunostaining results of extracellular components (laminin, collagen, and fibronectin) indicated that ECM components were synthesized. The levels of hepatocyte growth factor (HGF), transforming growth factor-β1, granulocyte-colony stimulating factor, vascular endothelial growth factor (VEGF)-A, fibroblast growth factor (FGF)-2, and interleukin-6 were significantly higher than those of a 2D monoculture. Low-level light irradiation induced secretion of angiogenic growth factors (VEGF and FGF) by adipose-derived stromal cell spheroids ( Figure 5D) [89,90].

Vessels
Angiogenic growth factors including VEGF and stromal cell-derived factors activate various signaling pathways [91,92]. Hypoxia (tissue oxygen deprivation) may develop in the spheroid core; the cells then secrete angiogenic growth factors. Figure 6A shows immunofluorescence images of MSC spheroids and a 2D monoculture after hypoxia induction. Proliferating cell nuclear antigen (PCNA) and human nuclear antigen (HNA) markers (cell proliferation markers) were more highly expressed by the spheroids [93].
Many attempts have been made to fabricate tubular structures using spheroids. A 3D bioprinter and skewered spheroids were employed to fabricate 3D tubular structures consisting of HUVECs, human aortic smooth muscle cells, and hNDFBs [94]. As shown in Figure 6B, the fabricated tubular grafts were then cultured in a dynamic environment featuring perfusion by an endothelial cell medium, a smooth muscle cell medium, and a fibroblast medium, which enlarged the lumen. Norotte et al. used an agarose mold to fabricate a microtubular vascular graft consisting of hybrid spheroids (smooth muscle cells and fibroblasts) (Figure 6C) [95]. A conventional 3D printer facilitated rapid and reproducible construction of vessels similar to native vessels in terms of both composition and architecture.

Other tissues
Scaffold-free spheroids enhance the cell-ECM interactions of cardiac tissues. Scaffold-free cardiac patches consisting of multiple cellular spheroids (human esophageal microvascular endothelial cells, rat neonatal ventricular cardiomyocytes, and hNDFBs) have yielded promising results [96]. Cardiac patches grafted into the hearts of nude rats formed vascular networks. However, the contractions were not synchronized to those of native tissue, and some spheroids were inappropriately positioned. To ensure precise positioning, 3D printers have become widely used. A recent vacuum system featuring a bioprinting pipette was used to relocate spheroids of induced, pluripotent stem cell-derived cardiomyocytes in a self-healing support hydrogel to desired locations [97]. After removal of the 3D microtissues from the hydrogel, the structure was maintained. Thus, cardiac regeneration may be possible; this is a novel approach toward accurate positioning of individual spheroids. Cardiac spheroids may be used to treat myocardial infarction. Injection of human cardiospheres into the infarct border zones of mice enhanced angiogenesis, tissue protection, and cardiomyocyte proliferation compared to a control group [98].
Hypoparathyroidism is associated with reduced production of parathyroid hormone (PTH), resulting in an imbalance between calcium and phosphorus. Park et al. developed scaffold-free spheroids of differentiated tonsil-derived MSCs (dTMSCs) [99] and implanted them in parathyroidectomized rats expressing a parathyroid-specific protein marker. Spheroid cell viability was reasonable (more than 50%), and high levels of intact PTH were synthesized. Such scaffold-free dTMSCs spheroids are novel and may find clinical application. Oral ulcers caused by cancer therapies are becoming an increasing concern and may even trigger treatment discontinuation. Ulcer treatment using a 3D ASC spheroid sheet has been investigated [100]. An in vivo study using white rabbits showed that such a sheet formed a 3D multilayered epithelium that exhibited immunofluorescent staining for CK5 and CK13 (markers of basal and suprabasal cells, respectively), affording better epithelialization and faster wound closure compared to a 2D culture. Thus, the ASC spheroid sheet may effectively treat oral ulcers.
As populations age, periodontitis causing tooth loss has increased [101]. Spheroids consisting of a mixture of human periodontal ligament MSCs and HUVECs may reduce periodontitis [102]. Spheroid transplantation into rat periodontal tissue defects shows increased bone and cementum formation to a greater extent than did spheroids with only one cell type. Periodontal regeneration may thus be possible.
According to the World Health Organization, lung disease is one of the top five causes of death. Autologous tissue implants may enhance lung regeneration. Recently, Cheng et al. showed that lung spheroids composed of a mixture of progenitor cells and supporting stromal cells from healthy lungs improved pulmonary fibrosis in rats [103]. Compared to MSC spheroids, the lung spheroids better reduced fibrotic thickening and tissue infiltration. The same team treated idiopathic pulmonary fibrosis via inhalation of a lung spheroid cell secretome (LSC-Sec) and exosomes. LSC-Sec inhalation reversed the fibrosis caused by bleomycin or silica particles. Currently, idiopathic pulmonary fibrosis remains incurable; hence, the novel inhalation strategy may be very valuable [104].

Scaffolded Spheroid Applications
A fundamental advantage of scaffolding is the mechanical reinforcement thus afforded to the cells. A scaffold may be formed using tissue-specific ECM and then biomimetically functionalized [56]. As shown in Table 2, spheroids may be directly seeded into scaffolds or embedded between scaffold layers.

Bone
A high scaffold macroporosity (similar to that of native bone) facilitates cellular infiltration and migration and thus, bone regeneration [105]. Foaming and freeze-drying techniques are regularly used to ensure macroporosity. A gelatin/carboxymethyl chitosan/nHAp matrix was vigorously mixed to form bubbles and then lyophilized (the SGC scaffold) (Figure 7A) [106]. The macropore diameter was 500-800 µm, aiding spheroid penetration. Alizarin red S staining indicated enhanced calcium deposition inside the scaffold. Efficient bone regeneration requires topological scaffold cues. For example, a negatively charged polydimethylsiloxane (PDMS) template was used to fabricate patterned nanofibrous poly(L-glutamic acid) (PLGA)/collagen/nHAp fibers that enhanced osteogenic differentiation (Figure 7B) [107]. Alkaline phosphatase staining of MSC spheroids seeded into the scaffold was significantly stronger than that of a 2D culture and spheroids cultured on an unpatterned scaffold. The synergistic effects of the patterned scaffold and 3D spheroids, and the fact that the biophysical environment closely resembled that of native tissue, were beneficial.

Cartilage
Subchondral bone regeneration is challenging given the limited self-repair capacity of articular cartilage. To overcome this issue, bi-compartmented (multilayered) therapeutics have been used, with bone morphogenetic protein-2 and MSC spheroids compartmentalized and inserted into sheep osteochondral defects [108]. The volume of articular cartilage thus created in the therapeutic group was three-fold that of the untreated group. Maintenance of spheroid morphology is important in terms of effective tissue regeneration [109]. Huang et al. thus developed chitosan (CS)-coated, plasma-treated 3D PLGA scaffolds (Figure 8A) [110]. The scaffolds were seeded with placenta-derived MSC spheroids and transplanted into chondral defects in rabbit knees. Safranin O and collagen II staining ( Figure 8A) indicated significantly enhanced glycosaminoglycan and type II collagen production in the spheroid group compared to the single-cell group. The scaffold macroporosity preserved the spheroid morphology. PLGA and CS form non-fouling fibrous networks because they are oppositely charged proteinrepulsing polyelectrolytes [111]. The results shows that the lyophilized scaffold did not re-assume the original hydrogel structure when soaked, thus retaining the porous network. At 14 days after ASC seeding, cells aggregated inside the pores, forming a spheroid. In vivo tests (using cartilage defects in rabbit knees) revealed hyaline cartilage of appropriate structure and function, suggestive of cartilage regeneration.   Articular cartilage is a smooth white tissue that forms joints at the ends of bones. To mimic the transition from cartilage to bone, bilayered scaffolds consisting of PLGA, PLGA-grafted nonohydroxyapatite (nHA-g-PLGA), and CS have been employed ( Figure 8B). Polylactic acid (PLA) rods 200-300 µm in length served as porogens in molds filled with the PLGA/CS hydrogel. Lyophilized nHA-g-PLGA/CS constructs were placed in bone. The seeded ASCs aggregated near cartilage, and the PLA rods were removed. The chondrogenic cell spheroids were distributed throughout the bony scaffold and played roles in both cartilage and bone regeneration [112].

Liver
Decellularized tissues are becoming the gold standard for liver TE given the biochemical similarities to native tissues. Decellularized liver tissues are routinely used to induce maturation of hepatocyte-like cells from mouse BM-MSCs [113]. Spheroids cultured with decellularized liver scaffolds exhibited significantly enhanced albumin and urea secretion. The principal drawback of conventional spheroid fabrication is the inability to precisely control the spheroid diameter. The 3D bio-dot printing method, in which cell-laden spheroids are ejected directly into a sacrificial bio-ink (an alginate hydrogel) and then into a pre-printed polycaprolactone (PCL) mold, shows promise in terms of diameter control (Figure 9A) [54]. The novel spheroids exhibited significantly higher albumin secretion compared to conventional spheroids.
As results from animal models may not adequately represent drug toxicity in humans, liver-on-a-chip models are regularly used to this end. The incorporation of spheroids into liver-on-a-chip models creates an in vivo-like environment, improving model reliability [114]. HepG2/C3A spheroids encapsulated in 3D hepatic constructs have been used to study acetaminophen toxicity (Figure 9B). A 3D hepatic construct was fabricated using a 3D bioprinter and photo-crosslinkable gelatin methacrylate (GelMA) and HepG2/C3A spheroids. The printed structure was packaged in a PDMS/poly(methyl methacrylate) (PMMA) bioreactor.
The recent introduction of 3D spheroid bioprinting suggests that 3D-printed autologous liver grafts may find clinical applications. 3D bioprinting of toroidal, induced pluripotent stem cell (iPSC)-derived hepatic spheroids has been used to explore hepatic functions. The bioprinted, hepatic spheroid structure exhibited significantly higher expression of liver genes and proteins compared to singly dispersed hepatocytes [115].

Skin
Lee et al. described a spheroid-entrapping alginate hybrid obtained by seeding ASC spheroids onto a 3D-printed alginate-based mesh, followed by electrospinning of alginate/polyethylene oxide fibers directly onto the spheroids (Figure 10A) [116]. The experimental group (with the 3D-printed/electrospun alginate scaffold and ASC spheroids) evidenced greater angiogenic gene expression (of PECAM-1, VEGF-A, and HGF) and angiogenesis-related growth factor secretion compared to two control groups (pure spheroids lacking a scaffold and single cells seeded onto an alginate scaffold), suggesting potential applications in wound healing.  Other research teams have confirmed the skin regenerative capacity of ASC spheroids in the rat skin repair model. As shown in Figure 10B, ASCs/ spheroids were seeded into wounds in rat dorsal skin and covered with a hyaluronan gel/CS membrane to maintain a moist environment. The experiments featured a blank, the hyaluronan gel, single ASCs covered by the gel, ASC spheroids covered by the gel, the gel covered by a CS membrane, single ASCs covered by the hyaluronan gel and CS membrane, and ASC spheroids covered by the hyaluronan gel and CS membrane. Wounds treated with spheroids exhibited faster closure and significantly greater angiogenesis. Tracking of fluorescently labeled ASCs showed that the ASC spheroids were localized near micro-vessels, suggesting that angiogenesis was enhanced in a paracrine manner. Thus, based on both the in vitro and in vivo results, self-assembled ASC spheroids show promise in treatments facilitating skin TE and wound regeneration [117].

Vessels
To mimic the layered structure of the native vasculature, Shimazu et al. developed a doublelayered vascular structure with a HUVEC core and a shell composed of human aortic SMCs [118]. A gold needle coated with HUVECs was placed in the center of the chamber, and the rest of the chamber was filled with SMC spheroids and photo-crosslinkable GelMA. The samples were incubated for 4 days, and the gold needle was then removed, yielding a vascular-like layered structure with a HUVEC inner layer and an SMC outer layer.
The RGD-modification technique was used to mimic the ECM structure. Centrifugation of RGDmodified PLLA nanofibers (NFs) and hMSCs yielded spheroids [119]. Alternate stacking of the spheroids and a pre-printed, two-layered, PCL-meshed scaffold yielded cylinders (Figure 10C). Given the biomimetic environment imparted by the RGD modification, enhanced neovascularization and in vivo vascularized adipose tissue formation were observed.

Other tissues
Spheroids can be used to regenerate hair follicles. An open-cell NF sponge comprising electrospun CS and polyvinyl alcohol (PVA) fibers generated dermal papilla (DP) cell spheroids [120]. As shown in Figure 10D, DP cells were seeded into the pores of the sponge and aggregated into spheroids over 3 days. A chamber patch assay of a combination of DP spheroids and epidermal cells revealed significantly elevated hair follicle formation, as revealed by hematoxylin-and-eosin (H&E) staining. Additionally, immunofluorescent staining for keratin (a common hair protein) was positive, indicating successful regeneration of hair follicles.

Conclusion
Spheroids engage in intense cell-cell and cell-matrix interactions, which elevate growth factor secretion, thus improving cellular activity, organspecific gene expression, and cytokine production compared to 2D monocultured cells. Spheroids are robust to external stimuli, including drug entrance. As such, spheroids are attractive building blocks for TE. We reviewed some key examples of spheroid applications in TE and bioreactors, dividing the applications into approaches based on "scaffold-free spheroids" and "scaffolded spheroids." Current spheroid applications seek to enhance tissue regeneration and 3D structure formation using Kenzan needles or 3D printers. However, spheroids do not adequately mimic multiplex cellular tissues that make up native organs. It is essential to identify the specific biochemical/mechanical factors responsible for spheroid formation. Additionally, the large diffusion gradients and low cell proliferation of spheroids must be improved. Despite these issues, spheroids are projected to play critical roles in tissue regeneration. We strongly believe that future developments will facilitate the personalized treatment of various diseases and improve disease prognoses.