
Cell-based therapy is a promising approach for tissue regeneration. To fill tissue loss and restore tissue functions of injured tissues, a major cell type of the tissue to be regenerated is often chosen as a cell source for transplantation. For example, chondrocytes are used for cartilage defect repair and cardiomyocytes are used for myocardial infarction repair. Among many cell sources, mesenchymal stem cells (MSCs) are the most clinically relevant and frequently used cell type for regenerative therapy due to their self-renewal ability, multipotency, low immunogenicity, and secretion of regenerative paracrine factors (1-3). MSCs-based regenerative therapy has been clinically demonstrated to have versatile therapeutic potential. However, its clinical outcome is not satisfying due to poor survival and engraftment of transplanted MSCs
In the body, cells are constantly interacting with their surrounding extracellular matrix (ECM) and adjuvant cells to maintain their homeostasis and functions according to physicochemical cues from the surroundings (12). Thus, 3D cell culture systems should provide a physicochemical micro-environment mimicking the
In this review, we mainly focus on spheroid-based tissue regenerations of bone defects, cartilage defects, critical limb ischemia, cardiac defects, pancreas and hair follicles in which spheroid formation/configuration is essential for cell transplantation. We also discuss specific strategies of spheroid engineering combined with advanced biomaterials for targeting each tissue.
Unlike a 2D monolayer culture system, 3D spheroid culture systems can create a more
There are various methods including microwells, hanging-drop, microencapsulation, centrifugation, magnetic levitation and spinning/rotating for spheroid formation (Fig. 2). In principle, the methods increase cell-to-cell cohesion by inducing spontaneous cellular assembly, applying physical forces (e.g. gravitational, centrifugal, or magnetic forces) or confinement (e.g. micro-structured surfaces or microcapsules) (Table 1). However, most of methods involve labor-intensive process, low yield, and difficulty in spheroid size control. Therefore, various spheroid formation techniques using functional biomaterials have been developed (Fig. 3). For example, porous scaffolds can induce in situ spheroid formation in the pores and subsequently be co-transplanted to target tissues. The scaffolds protect spheroids not only from shear stress generated during injection, but also from oxidative stress at transplanted sites. On the other hand, nano-/microparticles (or fibers) incorporated in spheroids are beneficial to deliver drugs to cells in the core region of spheroids and create gaps improving oxygen and nutrient transportation. Indeed, advanced biomaterials improve survival and engraftment of cell spheroids and increase the potential of cell spheroids in regenerative therapy by strategizing target tissue specific engineering. Table 2 summarizes the current spheroid-based approaches according to target tissues.
Bone is a mineralized connective tissue that constitutes body structure and enable mobility (25). Bone tissues are capable of sufficient self-healing for small sized damages or defects that are not over the critical size threshold (> 2 cm) (2). However, large defects, fractures, and degenerative or congenital diseases will not achieve complete healing if unaided (26, 27). Although autografts and allografts are gold standard treatments for bone repair, they have inevitable side effects such as morbidity, infection, and hemorrhage at the donor site (28). To overcome these limitations, engineered bone spheroids composed of stem cells (e.g., bone marrow-derived stem cells and adipose-derived stem cells) have been used as micro-bone tissues and explored extensively due to their great therapeutic potential for the repair of nonhealing bone defects (2, 26, 29). In comparison with dissociated MSCs, MSC spheroids can enhance survival in a harsh microenvironment and maintain their osteogenic potential (30). In addition, pre-osteogenic induction of MSC spheroids before transplantation can enhance the potential contribution of transplanted cells toward bone formation (31). Furthermore, biomaterials-assisted cell delivery therapies can significantly improve the regenerative capacity of stem cells by recapitulating the complex bone microenvironment.
For bone repair, various cell spheroids in combination with engineered 3D scaffolds such as hydrogels, microparticles, and electrospun fibers have been attempted (32, 33). Entrapment of MSC spheroids within engineered 3D scaffolds can localize cells at the implantation site, regulate cell migration from spheroids to surrounding tissues, and modulate spheroid function. The function of MSCs can be influenced by many properties of engineered 3D scaffolds including bulk mechanical properties, degradation profiles, and densities of adhesive ligands (34, 35). For example, Ho
Due to compact cell-cell interactions of spheroids, penetration of soluble osteogenic/angiogenic cues (e.g., BMP-2, adenosine, and platelet-derived growth factor [PDGF]) into spheroids is insufficient to stimulate inner cells of spheroids. For example, spheroids cultured in media containing soluble BMP-2 exhibit differentiation only at the periphery of spheroids. On the other hand, MSC spheroids incorporating BMP-2-loaded hydroxyapatite (HA) nanoparticles exhibit greater alkaline phosphatase activity and more uniform spatial expression of osteocalcin than spheroids with uncoated HA nanoparticles (37). Similarly, incorporation of adenosine-modified microfibers to MSC spheroids can stimulate adenosine 2b receptor signaling of MSCs thus significantly upregulating osteogenic markers (i.e., Runt-related transcription factor 2 (
3D scaffolds (e.g., microparticles and microfibers) have been incorporated within cell alone spheroids not only to deliver bioactive molecules, but also to improve viability of inner cells in spheroids (39, 40). In general, inadequate mass transport and development of a hypoxic core within spheroids limit their long-term culture and practical application. Incorporation of microparticles or microfibers generates relatively less compact spheroids of cells, thus improving cell viability. Microfibers are especially beneficial for bone tissue engineering due to their hierarchically organized architecture mimicking bone microenvironment. Ahmad
Cartilage healing is extremely slow due to its avascular properties (43). Once cartilage is damaged, it is often healed with fibrocartilage scar tissue which remains a challenge (44). Hyaline cartilage consists of specialized ECM including collagen type II (COL II) and proteoglycans, while fibrocartilage produces more collagen type I (COL I) but less COL II and exhibits inferior capacity to support high dynamic compressive loads (45-47). Thus, it is important to engineer cell spheroids to produce hyaline cartilage-specific ECM for successful cartilage repair.
Similarly, in situ formation of MSC spheroids inside a 3D porous scaffold is another strategy for engineering cartilage tissue substitute. Cell repulsive scaffolds such as chitosan film can generate spontaneous cellular aggregation due to their low adhesive force between cells and scaffolds (55). The highly hydrophilic layer of scaffolds can tightly bind to water via hydrogen bonds or electrostatic interaction, thus achieving a non-fouling ability. For example, a porous scaffold made of zwitterionic poly (L-glutamic acid)-chitosan co-polymer (PLGA-CS) can generate MSC spheroids with diameter of 80-100 μm in situ (55). MSC spheroids in PLGA-CS scaffold exhibited significantly upregulated chondrogenic genes (COL II and glycosaminoglycans (GAG) expression and decreased expression of a marker for fibrocartilage (
Transforming growth factor beta (TGF-β) superfamily plays essential roles in all phases of chondrogenesis, mesenchymal condensation, chondrocyte proliferation, extracellular matrix deposition, and finally terminal differentiation (56). TGF-β is a key initiator of chondrogenesis. Cellular condensation is strongly stimulated by TGF-β-induced elevation of N-cadherin expression (49). In addition, TGF-β can stimulate the proliferation of chondroblasts and deposition of cartilage-specific ECMs (e.g., aggrecan and collagen type II). It has been demonstrated that exogenous TGF-β treatment could enhance chondrogenic differentiation of MSCs (57). However, as MSCs form spheroids, it is difficult to achieve efficient TGF-β delivery to cells inside spheroids due to diffusional limitation of TGF-β. To overcome this limitation, substrate-mediated TGF-β delivery systems have been developed. For example, Yoon
Cell-based therapies are promising for reconstructing blood vessels and restoring blood perfusion of various injuries or diseases. MSCs have been extensively used for critical limb ischemia (CLI) repair due to their proangiogenic and immunoregulatory functions (60, 61). However, low retention and poor viability of transplanted cells at the implantation site limit their therapeutic effects. It has been known that MSC spheroids exhibit higher cell viability
Recently, macrophages have been emerged as a promising cell source for CLI repair. Macrophages are primary effector cells of the immune system that dominantly provide cytokines to regulate angiogenesis and matrix remodelling during tissue repair (66, 67). Ran
Cardiac muscle allows the heart to pump blood through circulatory system. Its dysfunction can lead to heart failure. Myocardial infarction (MI) can cause loss of gap junction-expression cardiac cells and cardiac fibrosis, thus increasing the risk of arrhythmia (69). For functional recovery of infarcted hearts, cardiac spheroids composed of MSCs or cardiomyocytes have been implanted into the ischemic myocardium (70-72). Although cardiac spheroids can restore cardiac functions of infarcted hearts, they often yield unsynchronized contraction, leading to a potential risk of arrhythmia. To overcome this limitation, electrically conductive materials can be incorporated into cardiac spheroids. For example, incorporating silicon nanowires into cardiac spheroids can improve the formation of an electrically conductive network in spheroids, leading to significantly synchronized and enhanced contraction as compared to non-incorporated cardiac spheroids (Fig. 4) (71). Furthermore, in combination with external electrical stimulation (recapitulating natural pacemaker-initiated excitation of cardiomyocyte contraction), silicon nanowire-incorporated cardiac spheroids can further improve cell-cell junction formation and the development of a contractile machinery and decrease the spontaneous beat rate of spheroids, thus reducing arrhythmogenic potential (70). Park
Type 1 diabetes is a chronic autoimmune disease. Due to T-cell mediated attack, b cells in pancreatic islets cannot produce adequate insulin, resulting in hyperglycemia (73). Although insulin uptake through intensive insulin injection or pumps can effectively decrease blood glucose levels, exogenous insulin therapy has a risk of complications such as hypoglycemia and ketoacidosis (73, 74). In addition, exogenous insulin uptake cannot ameliorate symptoms of patients with type 1 diabetes who exhibit severe hypoglycemia complicated by impaired hypoglycemia awareness or excessive glycemic lability (74). Although pancreas transplantation is the best option, it is an invasive surgery that needs lifelong immunosuppression to relieve massive immune attack to the graft. More importantly, the scarcity of donor pancreas limits its extensive application. Transplantation of allogenic islet or stem cell-derived islets is a promising alternative that can avoid an invasive surgery by directly injecting islets to the hepatic portal vein (75). However, islet anoikis can be triggered during an ex vivo isolation. In addition,
In the pancreas, islets of Langerhans are surrounded by a layer of ECM defined as the peri-insular basement membrane composed of collagen type IV, laminin, and fibronectin (84). During an enzymatic isolation of islets from pancreas tissues, the interaction between the islet and the ECM is disrupted. Subsequent reduction of cell adhesion can trigger b cell apoptosis and decrease insulin secretion (77). To overcome this limitation, pancreatic decellularized ECM (dECM) hydrogels have been used because they can mimic physicochemical cues of
Encapsulation islets in biomaterials can protect islets from a harsh microenvironment (e.g., immune attack, inflammatory conditions, etc.). Alginate microencapsulation of islets has been extensively used due to its ease of use. Alginate can be rapidly crosslinked in the presence of divalent cations (e.g. Ca2+) (86). However, the size of alginate microspheres (0.5-1.5 mm) often exceeds the physiological oxygen diffusion range, resulting in reduced islet survival and function (78). Yang
Islet transplantation is limited not only by host immune responses, but also by toxic effects of chronic immunosuppression required to control immune rejection (87). Recently, biomaterials-assisted strategies have focused on providing a local immunosuppression microenvironment. Biomaterials presenting checkpoint proteins (e.g., programmed cell death-1 ligand [PD-L1] and Fas ligand [FasL]) can modulate local immune responses and avoid the need for systemic chronic immunosuppression. For example, Headen
Hair loss is a significant concern of human disorder regardless of age and gender. This can be attributed to aging, environmental reasons, stress, or the lack of hair follicle recovery due to poor tissue regeneration of injured tissues (88). Hair follicle is an ectodermal organ composed of two main parts, including the epithelium of keratinocytes and the mesenchyme of dermal papilla (DP) cells (89). DP cells are known as a master regulator of HF cycle. They give instructive signals to epithelial bulge to initiate follicle formation, growth, and proliferation during HF regeneration (90). Currently, DP spheroids implantation is the best option for HF regeneration. To construct efficient and functional DP spheroids, non-adhesive scaffolds made of hydrophilic polymers have been used. After cells are seeded onto non-adhesive/non-fouling scaffolds, they rather spontaneously aggregate to each other and form a spheroid (91). Huang
Wang
A spheroid formation is indeed a promising approach to mimic
This research was supported by the research fund of Dankook University in 2020.
The authors have no conflicting interests.
Methods for spheroid formation
Method | How to | Driving force | Advantages | Disadvantages |
---|---|---|---|---|
Hanging drop | Make droplets of cell suspensions on a lid of tissue culture plate, the lid is flipped upside-down, and culture in a humid condition | Gravitational forces and physical confinement | Easy to control spheroid size | A limited volume of droplets (<50 μl) Difficulty in changing culture medium |
Microwells | Confine cells in physical compartments at a micrometer scale | Gravitational forces and physical confinement | A difficulty in harvest | |
Centrifugation | Force cells aggregate at the bottom of a centrifuge tube | Centrifugal forces | Cellular damages by excessive external forces | |
Magnetic levitation | Force magnetized cells form into spheroids | Magnetic force | Cytotoxicity of magnetic materials | |
Microencapsulation | Confine cells into microcapsules | Physical confinement | Batch variations | |
Non-adherent plates | Interrupt cell adhesion to the plates, make cells rather aggregate to each other | Spontaneous aggregation | One step spheroid formation and suspension culture Less labor intensive Suitable for large scale spheroid formation |
Low yield and various spheroid size |
Rotating wall vessels | Create a microgravity environment | Shear force | ||
Spinner flasks | Generate dynamic fluid shear force | Shear force |
Summary of biomaterials and strategies of spheroid engineering according to the target applications
Target | Cell type | Functional biomaterials | A method for spheroid formation | Strategy |
---|---|---|---|---|
Bone defect repair | hBMSC | RGD-modified alginate gels (31) | Microwells | Controlling MSC migration from spheroids to enhance spheroid osteogenic potential |
hBMSC | Alginate hydrogel (36) | Microwells | Applying dynamic mechanical stimulation to spheroids for enhancing osteogenic potential of MSC | |
hADSC | Adenosine and polydopamine coated PLLA fragmented fibers (38) | Centrifugation | Scaffolds-mediated adenosine delivery to improve osteogenic differentiation of MSCs | |
rbBMSC | Silk fibroin microfiber (39) | Centrifugation | Creating gaps in spheroids, leading to enhanced transportation of oxygen and nutrients to the core region | |
hADSC | Biomineral-coated PLLA fragmented fibers (41) | Centrifugation | Accelerating osteogenic differentiation by providing bone-like mineralized environments | |
hADSC | PDGF/biomineral-coated PLLA fragmented fibers (42) | Centrifugation | Providing bone-mimicking multiple factors for vascularized bone regeneration | |
Cartilage defect repair | rBMSC | Magnetic nanoparticles (54) | Magnetic condensation using magnetic devices | Controlling sizes and patterns of spheroids at the millimetric scale by using magnetic devices |
rbADSC | PLGA/chitosan porous scaffold (55) | In situ aggregation in pores | Forming denser mass of spheroids in the scaffold, leading to enhanced chondrogenic differentiation capacity of stem cells | |
hADSC | TGF-β3 and FN adsorbed graphene oxide sheet (58) | Hanging-drop | Providing a cell-adhesion substrate and simultaneously delivering chondrogenic growth factors for improving chondrogenic differentiation of stem cells | |
UCB-MSC | hFDM and TGF-β1-coated PLGA/PLLA microfiber (59) | Non-adherent plates | ||
Critical limb ischemia repair | UCB-MSC | Hyaluronic acid/alginate core-shell microcapsules (64) | Microencapsulation | Encapsulating spheroids to protect and retain the cells from harsh environments after transplantation |
hADSC | Poly(L-glutamic acid)/PEG-based porous hydrogel (65) | In situ aggregation in pores | In situ spheroid formation via gel-sol transition |
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RAW 264.7 | Chrysin-encapsulated fiber fragments (68) | Electrosprayed microcapsulation | Promoting vascular anastomosis via chronological shifting from M1 to M2 phenotypes, regulated by chrysin delivery | |
Cardiac repair | hiPSC-CM | Silicon nanowires (70, 71) | Microwells | Incorporating electrically conductive biomaterials to achieve synchronized and enhanced contraction of cardiac spheroids |
Used both exogenous and endogenous electrical stimuli for advanced structural and functional development of cardiac spheroids | ||||
hBMSC | Reduced graphene oxide flake (72) | Hanging-drop | Incorporating electroconductive biomaterials to spheroids for enhancing paracrine factors and connexin 43 expression | |
Islet transplantation | Human pancreatic islets | ECM hydrogels made of porcine decellularized tissues (83) | Encapsulation | Recapitulating the |
Mouse pancreatic islets | Chondroitin sulfate incorporated starPEG (80) | Nanocoating | Nanocoating of islets to reduce blood coagulation, improve islet cells survival, and protect against disruption | |
Fas ligand-conjugated PEG microgel (81) | Microencapsulation | Local immunomodulation to avoid acute rejection of islet allografts, avoiding the need for systemic chronic immunosuppression | ||
Programmed cell death-1-conjugated PEG microgel (79) | Microencapsulation | |||
TGF-β1-loaded PLG microporous scaffold (82) | In situ aggregation in pores | Localized TGF-β1 delivery to modulate the immunological environment of transplanted sites | ||
Hair follicle regeneration | hDPC | Polyvinyl alcohol (PVA) (91) | PVA-coated plates | Developed a controllable spheroid formation technique |
mDPC | Chitosan/PVA nanofiber sponge (92) | In situ aggregation in pores | Developed a technique for controllable and scalable spheroids formation | |
Gelatin and alginate (93) | Layer-by-layer nanoencapsulation | Developed a tunable and scalable spheroid formation model by inducing aggregation of nanoencapsulated cells |
MSC, mesenchymal stem cell; hBMSC, human bone marrow-derived MSC; hADSC, human adipose-derived stem cell; rbBMSC, rabbit bone marrow-derived MSC; rbADSC, rabbit adipose-derived stem cell; UCB-MSC, human umbilical cord blood-derived MSC; hiPSC-CM, human induced pluripotent stem cell-derived cardiomyocytes; hDPC, human dermal papilla cells; mDPC, mouse DPC; RGD, Arg-Gly-Asp; PLLA, poly (L-lactic acid); PDGF, platelet-derived growth factor; PLGA, poly(lactic-co-glycolic acid); TGF, transforming growth factor; FN, fibronectin; hFDM, human lung fibroblast decellularized ECM; PEG, polyethylene glycol; PLG, poly(lactide-co-glycolide).
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