Hybrid cell constructs consisting of bioprinted cell‐spheroids

Abstract Bioprinted cell constructs have been investigated for regeneration of various tissues. However, poor cell–cell interactions have limited their utility. Although cell‐spheroids offer an alternative for efficient cell–cell interactions, they complicate bioprinting. Here, we introduce a new cell‐printing process, fabricating cell‐spheroids and cell‐loaded constructs together without preparation of cell‐spheroids in advance. Cells in mineral oil droplets self‐assembled to form cell‐spheroids due to the oil‐aqueous interaction, exhibiting similar biological functions to the conventionally prepared cell‐spheroids. By controlling printing parameters, spheroid diameter and location could be manipulated. To demonstrate the feasibility of this process, we fabricated hybrid cell constructs, consisting of endothelial cell‐spheroids and stem cells loaded decellularized extracellular matrix/β‐tricalcium phosphate struts for regenerating vascularized bone. The hybrid cell constructs exhibited strong angiogenic/osteogenic activities as a result of increased secretion of signaling molecules and synergistic crosstalk between the cells.


| INTRODUCTION
Recently, cell printing has been used to fabricate a variety of complex tissue constructs, such as biomedical scaffolds and in-vitro models used to evaluate therapeutic biocomponents, among others. [1][2][3] This fabrication technique can provide biochemical/biophysical cues and even place cells in a desired region to achieve native tissue-mimetic structures or patterns of multiple types of cells. [3][4][5] Because cell printing allows encapsulation of cells into microscale hydrogel struts, such cell-loaded structures have been widely used as tissue engineering substitutes for bone, skin, muscle, cardiac, and other tissues. [3][4][5][6][7][8] However, the homogeneously distributed cells in bioprinted threedimensional (3D) constructs often require extremely long times in culture to form the strong cell-cell attachments needed for a natural cellular microenvironment. For this reason, several researchers have been pursuing new innovative methods to induce strong cell-cell interactions within cell-loaded constructs. [9][10][11] Cell-spheroids, which are 3D spherical aggregates of cells, have been used in tissue engineering applications because of their strong cell-cell/cell-ECM interactions that mimic those formed in cellular microenvironments in vivo. [12][13][14] Cells in 3D spheroids have been reported to exhibit improved organ-specific activities as a result of increased cell-cell communication via signaling factors, such as growth factors, chemokines, and cytokines, when compared with single cells in 2D culture. [14][15][16] Due to the outstanding in-vivo-like 3D microenvironment, cell-aggregates can be used not only as biomimetic tissue models, but also as models for various diseases for screening, diagnosis, treatment, and drug tests. [17][18][19] Therefore, combining cellspheroids with bioprinting constitutes a synergistic approach that can overcome weak cell-cell interactions in cell-loaded constructs fabricated by cell printing. 14 To obtain spheroid-based constructs, cellspheroids fabricated by conventional methods (e.g., hanging drop, microcavity, non-adhesive surface) have been seeded into predesigned scaffolds by bioprinting a bioink that contains spheroids, as shown in Figure 1. 14,20-23 However, a two-step procedure can cause cell-spheroid aggregation in the bioink, spheroid breakage resulting from high shear stresses near walls during extrusion through microscale printing nozzles, loss of spheroids, and low homogeneity of spheroids seeded into scaffolds ( Figure 1). In this respect, efficient fabrication methods are needed to overcome these previous limitations of spheroid printing. 24,25 To address the issues, various methods have been proposed to hybridize the cell-spheroids with hydrogels. [26][27][28][29] Williams et al. 26 printed adipose stromal fraction cells encapsulated in alginate hydrogel into the CaCl 2 solution. They successfully printed sphereshaped cell-loaded alginate structures. Wu et al. 27 also fabricated bone marrow stem cells-loaded microspheres using gelatin methacryloyl (GeMA) hydrogel and a microfluidic system. By loading cells into the porous GelMA microspheres produced in advance, they obtained porous hydrogel-based cell-beads. While the cell-beads have been produced with the hydrogels, several limitations for hybridizing the cell-spheroids with 3D cell-constructs need to be overcome.
In this study, a new cell-printing system incorporating in-situ spheroid formation has been developed to fabricate 3D bioengineered constructs containing multiple types of cells as well as precise patterns of the printed cell-spheroids. A one-step hybrid printing process was developed to simultaneously fabricate both cell-spheroids with similar biological functions to the conventional cell-spheroids, and cell-printed constructs without preparation of cell-spheroids in advance. Cell-spheroids were obtained by printing cells in droplets of mineral oil, and the spheroids were designed to influence biofunctional characteristics of surrounding cells loaded into supporting struts. Cell-spheroid size and location were easily manipulated by controlling various printing parameters.
To show the effect of these hybrid constructs, endothelial cells were used for cell-spheroids, and human adipose stem cells (hASCs)loaded bioink was used to fabricate the cell-laden struts for regenerating bone tissue. In vitro cellular activities of these constructs were significantly improved over those of conventional cell constructs printed using a mixture of the endothelial cells and hASCs. These differences were a result of reinforced synergistic crosstalk between the endothelial spheroids and hASCs, which accelerated angiogenesis and osteogenesis in vitro.

| Cells and bioinks
In the present study, human adipose-derived stem cells (hASCs; Lonza, USA) and human umbilical vein endothelial cells (HUVECs; Lonza, USA) were used to formulate bioinks. Before preparing the F I G U R E 1 Application of conventionally prepared cell-spheroids. Schematic drawings illustrating use and limitations of cell-spheroids placed on cell-loaded 3D scaffolds using conventional methods. 3D, three-dimensional bioinks, the cells were cultured in cell culture plates with different culture media at 37 C and 5% CO 2 . Dulbecco's Modified Eagle's Medium-low-glucose (DMEM-L; Sigma-Aldrich, USA)-based GM, containing 10% fetal bovine serum (BioWest, USA) and 1% penicillinstreptomycin (PS; Thermo-Fisher Scientific, USA), were used for hASCs. In the case of HUVECs, an EGM™-2 endothelial SingleQuots™ Before preparing bone-specific bioinks, bone tissues were isolated from the lower limbs of Yorkshire pigs (females, 10-15 months old) and demineralized/decellularized in accordance with protocols described in previous paper. 39 The

| One-step spheroid printing
To fabricate the spheroid-based cell-printed construct, a 3D bio-

| Deposition of cell-loaded mineral oil droplets into collagen hydrogels
To observe formation of cell-spheroids in mineral oil, HUVEC-loaded mineral oil and collagen hydrogel were used. Briefly, mineral oil, alginate (4 wt%), or collagen (5 wt%) droplets (0.1 μl) loaded with HUVECs were added to collagen hydrogel (5 wt%) in 96-well plates using a micro pipette (Gilson Inc., France). The collagen hydrogel containing cell-loaded droplets was cultured in GM at 37 C and 5% CO 2 .
The GM was changed every 2 days.

| Preparation of conventional HUVECspheroids
Conventional HUVEC-spheroids (~200 μm diameter) were prepared using non-adherent agarose molds in accordance with the manufacturer's protocols to confirm the efficacy of generated one-step spheroid printing (One-SP)-spheroids. 43 Briefly, the agarose molds were prepared by casting 2% agarose (in DPBS; Invitrogen, USA) into a 3D Petri dish (Sigma-Aldrich, USA). Then, the HUVEC suspension (190 μl; 1.4 Â 10 6 cells/ml) was seeded into the agarose mold and cultured with GM.

| Cellular responses of hASCs to HUVEC-spheroids
To evaluate the osteogenic responses of stem cells to endothelial cells and spheroids, hASCs (1 Â 10 5 cells/cm 2 ) were seeded onto 6-well tissue culture plates. The One-SP-spheroids-based dECM construct (containing 56 spheroids) was placed onto the top level of transwell inserts (SPL Life Science, USA). As controls, 2D cultured isolated HUVECs and conventionally prepared HUVEC-spheroids (C-Spheroids) were positioned on the top level in a dECM construct. The GM was changed every 2 days, and the cells were cultured at 37 C and 5% CO 2 .

| Characterization of cell constructs
The 3D printed constructs were visualized using an optical microscope (BX FM-32; Olympus, Japan) with a digital camera and a scanning electron microscope (SEM) (SNE-3000M; SEC Inc., South Korea).
The mechanical properties of the bioprinted constructs To measure the content of bioceramics in the fabricated constructs, thermogravimetric analysis was performed under a nitrogen atmosphere using a thermogravimetric analyzer (TGA-2050; TA-Instruments, USA). The freeze-dried scaffolds were heated from 30 C to 800 C (typical sample mass: 10 mg; ramp rate: 20 C/min).

| In-vitro cellular responses
To estimate numbers and proliferation of cells, the MTT assay was All values are expressed as means ± SDs (n = 4).
To separately visualize the printed hASCs and HUVECs, the cells were prestained using Cell-Tracker™ (Molecular probes, USA) according to the manufacturer's protocol before formulating the bioinks.
After cells were harvested, they were incubated in prewarmed stain- To evaluate osteogenic differentiation of hASCs cultured on 2D plates, cells were stained for alkaline phosphatase (ALP) and calcium.
Alizarin red S (ARS) was used to stain for calcium, and nitro blue tetra-

| Immunofluorescence
To evaluate differentiation of stem cells and endothelial cells,

| Combined in-situ cell-spheroid formation and cell printing
To achieve in-situ cell-spheroid formation using the cell-loaded mineral oil droplets, a cell-printing system was used for effective spheroid formation within a 3D meshwork of printed cylindrical struts, as shown in Figure 3a. We call this process one-step spheroid printing.
As shown schematically in Figure 3a

F I G U R E 2 Fabrication of cell-spheroids using cell-loaded mineral oil. (a) Schematic drawings illustrating the deposition of a cell-loaded mineral oil droplet into a collagen-based hydrogel leading to the formation of a cell-spheroid. (b) Optical and live (green)/dead (red) and nuclei (blue)/F-actin (red) images of cells loaded into a droplet of mineral oil at 1 and 3 days. (c) Schematic drawings and fluorescence images (live/dead and nuclei/F-actin) at 3 days of cells loaded into droplets of alginate and collagen hydrogels within a collagen hydrogel. (d) Expression of
Ve-cadherin, Tgfb1, and Bmp-2 genes at 7 days in cells loaded into mineral oil, collagen, and alginate droplets (n = 4). *p < 0.050, ***p < 0.001, one-way ANOVA with Tukey's HSD post-hoc test. ANOVA, analysis of variance; Tukey's HSD, Tukey's Honest Significant Difference

| Procedure for stable in-situ spheroid formation in printed constructs
In the One-SP method, stable cell-spheroids with controllable diame-

| Cellular activities of the printed HUVEC-spheroids
The microenvironment within cell-spheroids resembles that in vivo in terms of robust cell-cell and cell-matrix interactions. 20,21 In particular, cells exhibit greatly upregulated tissue-specific gene expression and secrete various intercellular signaling molecules, including cytokines, chemokines, and growth factors, at higher levels than do cells in 2D monoculture. [14][15][16]46,47 In this section, HUVEC-spheroids fabricated by the One-SP method were assessed by comparing them with single cells in 2D culture and cell-spheroids prepared using a conventional microwell process, as shown in Figure S4. Conventional cell-spheroids (C-spheroids) were fabricated using an agarose mold (seeding cell density, and Opn at 28 days was significantly elevated in both spheroid groups compared with the single-cell group (Figure 5g). These results are consistent with signaling between the HUVEC-spheroids and hASCs having promoted robust hASC osteodifferentiation due to angiogenic and osteogenic growth factors secreted by each cell type. 40,[48][49][50][51] 3.5 | Use of One-SP for regenerating bone tissue As we developed the cell-spheroid printing process, which can produce simultaneously cell-spheroids and cell-loaded constructs together, various applications of the system can be further extended in the several tissue engineering field. We carefully expect that the hybrid cell constructs, such as stem cell-spheroid-carrier, which can secret bioactive molecules (exosome, growth factor, cytokine, etc.), 18 vascularized cell constructs for bone, muscle, liver, skin, etc., 22,40,[52][53][54] and neuromuscular structure, 55 can be obtained using the One-SP process efficiently. In this study, we applied the One-SP system to fabricate a vascularized cell construct for regenerating bone tissue.
Several biofabrication methods, such as encapsulation of soluble factors and coculturing of multiple cells, have been employed to obtain efficient regeneration of bone tissues supported by effective blood vessel formation in vitro and in vivo. 56 In particular, coculture of endothelial and stem cells has been widely used to promote formation of vascularized bone tissues in response to secretion of various signaling molecules that promote several signaling pathways involved in angiogenesis and osteogenesis of each cell type (Figure 6a). In this work, we identified synergistic effects between the endothelial and the stem cells that upregulate vessel and bone formation in vitro and compared them with pure hASC-loaded bone constructs.
To improve upon previous results that took advantage of the synergism between HUVECs and hASCs, we used One-SP to obtain a cell-loaded construct that can more efficiently regenerate bone tissues. Figure 6b shows schematic diagrams of the One-SP process.
As shown in the SEM and nuclei/F-actin images in Figure 6c To assess the proliferation rates of the cells at 7 days, MTT assays were performed on the experimental and two control groups ( Figure 6g). Although cell proliferation of the constructs increased F I G U R E 7 Expression of genes in hASCs and HUVEC-spheroids in the cocultured hybrid constructs. (a) Schematic diagram illustrating the expected biological responses and crosstalk between cocultured hASCs and HUVEC-spheroids in the EXP cell constructs. Expression of (b, c) signaling factors (Et1, Fgf2, Vegf, Bmp-2, Cxcl2, Cxcr4, Tgf-β1, Tnfa), (d) NOTCH (Notch1, Notch2, Jag1, Hes1, and Heyl), (e) Wnt/β-catenin (Wnt and Ctnnb), (f) MAPK (Mapk1, Mapk8, and MAPK14), (g) PI3K (Pi3k and Akt), and (h) SMAD (Smad1, Smad4, Smad5, and Smad8) signaling pathway-related genes in the CON-1, CON-2, and EXP constructs (n = 4). *p < 0.050, **p < 0.010, ***p < 0.001, one-way ANOVA with Tukey's HSD post-hoc test. ANOVA, analysis of variance; hASCs, human adipose stem cells; HUVECs, human umbilical vein endothelial cells; Tukey's HSD, Tukey's Honest Significant Difference over the culture period, that for the CON-2 (227.3%) and EXP (251.5%) groups was significantly greater than for the CON-1 group (192.7%). This difference was the result of synergistic crosstalk between the endothelial and stem cells, which enhanced the intracellular activities of each, 48,57 and bioactive growth factors secreted from the HUVEC-spheroids that accelerated cell. 51,58 To estimate osteogenic and angiogenic potential of the con- Expression of several signaling pathway genes was compared between the cell-loaded constructs (CON-1, CON-2, and EXP). To confirm activation of signaling cascades and cellular responses in the two cell types, expression of representative genes from each pathway was estimated after 7, 14, and 21 days in culture ( Figure S8).
Activation of the NOTCH signaling pathway, which promotes cellular proliferation, was observed at early times, but was reduced after about 14 days in culture ( Figure S8A). 59 The Wnt/β-catenin signaling pathway was significantly activated after 14 days in culture and affected early stages of osteogenesis and angiogenesis in hASCs and HUVEC-spheroids ( Figure S8B). 59,60 In addition, MAPK, PI-3K, and SMAD signaling pathways were gradually activated over time in culture, inducing late stages of osteogenic differentiation and formation of vascular networks (Figure 7c-e). 59,[61][62][63] Based on the gene expression analyses, we confirmed that the cellular activities of the spheroid-based (EXP) construct could significantly promote osteogenic and angiogenic activities in these cells.
While the spheroid-based cell constructs showed efficient osteogenic activities and vascular network formation in vitro, the mechanical properties of the construct were still low compared to those of natural bone. Considering the meaningful bioactive properties of the cell-constructs, the structure could be applied into the non-load bearing region of defected bones. However, the low mechanical properties should be handled in future to applicate the hybrid cell construct in various translational medicine fields. 64 To improve the mechanical strength of the constructs, our previous strategies, such as printing core (high concentrated hydrogel)/shell (cell-laden bioink) struts, 65 poly(ε-caprolactone)-alginate interdigitated struts, [66][67][68] and cell-laden bioink coating on collagen/calcium deficient hydroxy apatite struts, 69 can be applied.
Although the in vitro cellular activities demonstrated that the multiple-cell-constructs supported with the spheroids can provide a highly efficient platform to crosstalk between laden cells, the use of the mineral oil to fabricate the spheroids has several limitations to apply directly in clinical applications because the remnant mineral oil may cause inflammation and harmful effects in various tissues. 70,71 For this reason, the effect of the mineral oil that was used in the formation of the spheroids on in vivo results and complex biological functions will be studied in our future work. Additionally, more biocompatible and safer hydrophobic oils 72-74 also could be considered for the One-SP.

| CONCLUSION
Here, we developed a novel 3D cell-printing process in which cells aggregated into spheroids, and their location could be precisely manipulated to generate hybrid constructs containing multiple interacting cell types for use in regeneration of vascularized tissues.
Toward that goal, a cell-spheroid fabrication method was developed that used mineral oil loaded with cells, and spheroid formation was optimally selected by adjusting various 3D printing parameters. To demonstrate the feasibility of the in-situ cell-spheroid printing process, we used two cell types, endothelial cells and hASCs, and a cell- of Bioabsorbable Hydroxyapatite that is less than micrometer in size).