Multi-scale vascularization strategy for 3D-bioprinted tissue using coaxial core-shell pre-set extrusion bioprinting and biochemical factors.

Three-dimensional bioprinting is a key technology in bioartificial organ production. However, production of bioartificial organs has significant limitations because it is hard to build vascular structures, especially capillaries, in printed tissue owing to its low resolution. As the vascular structure plays a critical role in delivering oxygen and nutrients to cells and removing metabolic waste, building vascular channels in bioprinted tissue is essential for bioartificial organ production. In this study, we demonstrated an advanced strategy for fabricating multi-scale vascularized tissue using a pre-set extrusion bioprinting technique and endothelial sprouting. Using a coaxial precursor cartridge, mid-scale vasculature-embedded tissue was successfully fabricated. Furthermore, upon generating a biochemical gradient environment in the bioprinted tissue, capillaries were formed in this tissue. In conclusion, this strategy for multi-scale vascularization in bioprinted tissue is a promising technology for bioartificial organ production.

Genomic comparison between an in vitro three-dimensional culture model of melanoma and the original primary tumor.

Three-dimensional (3D) melanoma culture is a personalized in vitro model that can be used for high-fidelity pre-clinical testing and validation of novel therapies. However, whether the genomic landscape of 3D cultures faithfully reflects the original primary tumor which remains unknown. The purpose of our study was to compare the genomic landscapes of 3D culture models with those of the original tumors. Patient-derived xenograft (PDX) tumors were established by engrafting fresh melanoma tissue from each patient. Then, a 3D culture model was generated using cryopreserved PDX tumors embedded in pre-gelled porcine skin decellularized extracellular matrix with normal human dermal fibroblasts. Using whole-exome sequencing, the genomic landscapes of 3D cultures, PDX tumors, and the original tumor were compared. We found that 91.4% of single-nucleotide variants in the original tumor were detected in the 3D culture and PDX samples. Putative melanoma driver mutations (BRAF p.V600E, CDKN2A p.R7*, ADAMTS1 p.Q572*) were consistently identified in both the original tumor and 3D culture samples. Genome-wide copy number alteration profiles were almost identical between the original tumor and 3D culture samples, including the driver events of ARID1B loss, BRAF gain, and CCND1 gain. In conclusion, our study revealed that the genomic profiles of the original tumor and our 3D culture model showed high concordance, indicating the reliability of our 3D culture model in reflecting the original characteristics of the tumor.

Development of In Situ Microfluidic System for Preparation of Controlled Porous Microsphere for Tissue Engineering.

In this study, we present an in situ microfluidic system to precisely control highly porous polycaprolactone microspheres as tissue templates for tissue engineering. The porosity of the microspheres was controlled by adjusting the flow rates of the polymer phase and the pore-generating material phase in the dispersed phase. The microfluidic flow-focusing technique was adopted to manufacture porous microspheres using a relatively highly viscous polymer solution, and the device was fabricated by conventional photolithography and PDMS casting. The fabricated in situ microfluidic system was used to precisely control the pore size of monodispersed polycaprolactone microspheres. The porous microspheres with controlled pore sizes were evaluated by culturing HDF cells on the surface of porous microspheres and injection into the subcutaneous tissue of rats. We found that the increased pore size of the microspheres improved the initial proliferation rate of HDF cells after seeding and relieved the inflammatory response after the implantation of porous microspheres in the subcutaneous tissue of rats.

Transwell-Hypoxia Method Facilitates the Outgrowth of 3D-Printed Collagen Scaffolds Loaded with Cryopreserved Patient-Derived Melanoma Explants.

A previous study from our laboratory demonstrated the effects of in vitro three-dimensional (3D)-printed collagen scaffolds on the maintenance of cryopreserved patient-derived melanoma explants (PDMEs). However, it remains unknown whether 3D-printed collagen scaffolds (3D-PCSs) can be harmonized with any external culture conditions to increase the growth of cryopreserved PDMEs. In this study, 3D-PCSs were manufactured with a 3DX bioprinter. The 3D-printed collagen scaffold-on-frame construction was loaded with fragments of cryopreserved PDMEs (approximately 1-2 mm). 3D-PCSs loaded with patient-derived melanoma explants (3D-PCS-PDMEs) were incubated using two types of methods: (1) in transwells in the presence of a low concentration of oxygen (transwell-hypoxia method) and (2) using a traditional adherent attached to the bottom flat surface of a standard culture dish (traditional flat condition). In addition, we used six different types of media (DMEM high glucose, MEM α, DMEM/F12, RPMI1640, fibroblast basal medium (FBM), and SBM (stem cell basal medium)) for 7 days. The results reveal that the culture conditions of MEM α, DMEM/F12, and FBM using the transwell-hypoxia method show greater synergic effects on the outgrowth of the 3D-PCS-PDME compared to the traditional flat condition. In addition, the transwell-hypoxia method shows a higher expression of the MMP14 gene and the multidrug-resistant gene product 1 (MDR1) than in the typical culture method. Taken together, our findings suggest that the transwell-hypoxia method could serve as an improved, 3D alternative to animal-free testing that better mimics the skin's microenvironment using in vitro PDMEs.

Impact of microstructure on cell behavior and tissue mechanics in collagen and dermal decellularized extra-cellular matrices.

Skin models are used for many applications such as research and development or grafting. Unfortunately, most lack a proper microenvironment producing poor mechanical properties and inaccurate extra-cellular matrix composition and organization. In this report we focused on mechanical properties, extra-cellular matrix organization and cell interactions in human skin samples reconstructed with pure collagen or dermal decellularized extra-cellular matrices (S-dECM) and compared them to native human skin. We found that Full-thickness S-dECM samples presented stiffness two times higher than collagen gel and similar to ex vivo human skin, and proved for the first time that keratinocytes also impact dermal mechanical properties. This was correlated with larger fibers in S-dECM matrices compared to collagen samples and with a differential expression of F-actin, vinculin and tenascin C between S-dECM and collagen samples. This is clear proof of the microenvironment's impact on cell behaviors and mechanical properties. STATEMENT OF SIGNIFICANCE: In vitro skin models have been used for a long time for clinical applications or in vitro knowledge and evaluation studies. However, most lack a proper microenvironment producing a poor combination of mechanical properties and appropriate biological outcomes, partly due to inaccurate extra-cellular matrix (ECM) composition and organization. This can lead to limited predictivity and weakness of skin substitutes after grafting. This study shows, for the first time, the importance of a complex and rich microenvironment on cell behaviors, matrix macro- and micro-organization and mechanical properties. The increased composition and organization complexity of dermal skin decellularized extra-cellular matrix populated with differentiated cells produces in vitro skin models closer to native human skin physiology.

Therapeutic Efficacy of Artificial Skin Produced by 3D Bioprinting.

The skin protects the body from external barriers. Certain limitations exist in the development of technologies to rapidly prepare skin substitutes that are therapeutically effective in surgeries involving extensive burns and skin transplantation. Herein, we fabricated a structure similar to the skin layer by using skin-derived decellularized extracellular matrix (dECM) with bioink, keratinocytes, and fibroblasts using 3D-printing technology. The therapeutic effects of the produced skin were analyzed using a chimney model that mimicked the human wound-healing process. The 3D-printed skin substitutes exhibited rapid re-epithelialization and superior tissue regeneration effects compared to the control group. These results are expected to aid the development of technologies that can provide customized skin-replacement tissues produced easily and quickly via 3D-printing technology to patients.

Bone Fracture-Treatment Method: Fixing 3D-Printed Polycaprolactone Scaffolds with Hydrogel Type Bone-Derived Extracellular Matrix and ß-Tricalcium Phosphate as an Osteogenic Promoter.

Bone formation and growth are crucial for treating bone fractures. Improving bone-reconstruction methods using autologous bone and synthetic implants can reduce the recovery time. Here, we investigated three treatments using two different materials, a bone-derived decellularized extracellular matrix (bdECM) and ß-tricalcium phosphate (ß-TCP), individually and in combination, as osteogenic promoter between bone and 3D-printed polycaprolactone scaffold (6-mm diameter) in rat calvarial defects (8-mm critical diameter). The materials were tested with a human pre-osteoblast cell line (MG63) to determine the effects of the osteogenic promoter on bone formation in vitro. A polycaprolactone (PCL) scaffold with a porous structure was placed at the center of the in vivo rat calvarial defects. The gap between the defective bone and PCL scaffold was filled with each material. Animals were sacrificed four weeks post-implantation, and skull samples were preserved for analysis. The preserved samples were scanned by micro-computed tomography and analyzed histologically to examine the clinical benefits of the materials. The bdECM-ß-TCP mixture showed faster bone formation and a lower inflammatory response in the rats. Therefore, our results imply that a bdECM-ß-TCP mixture is an ideal osteogenic promoter for treating fractures.

Production of Multiple Cell-Laden Microtissue Spheroids with a Biomimetic Hepatic-Lobule-Like Structure.

The construction of an in vitro 3D cellular model to mimic the human liver is highly desired for drug discovery and clinical applications, such as patient-specific treatment and cell-based therapy in regenerative medicine. However, current bioprinting strategies are limited in their ability to generate multiple cell-laden microtissues with biomimetic structures. This study presents a method for producing hepatic-lobule-like microtissue spheroids using a bioprinting system incorporating a precursor cartridge and microfluidic emulsification system. The multiple cell-laden microtissue spheroids can be successfully generated at a speed of approximately 45 spheroids min-1 and with a uniform diameter. Hepatic and endothelial cells are patterned in a microtissue spheroid with the biomimetic structure of a liver lobule. The spheroids allow long-term culture with high cell viability, and the structural integrity is maintained longer than that of non-structured spheroids. Furthermore, structured spheroids show high MRP2, albumin, and CD31 expression levels. In addition, the in vivo study reveals that structured microtissue spheroids are stably engrafted. These results demonstrate that the method provides a valuable 3D structured microtissue spheroid model with lobule-like constructs and liver functions.

NecroX-5 Can Suppress Melanoma Metastasis by Reducing the Expression of Rho-Family GTPases.

NecroX-5 (NX-5) is a cell-permeable necrosis inhibitor with cytoprotective effects. Although it has been reported to inhibit lung and breast cancer metastasis by modulating migration, its therapeutic effect on melanoma metastasis is still unknown. In this study, we examined the anti-metastatic effect of NX-5 on melanoma cell lines and its related therapeutic mechanism. The anti-metastatic effect of NX-5 on melanoma cell lines was determined using a transwell migration assay. We performed a quantitative real-time polymerase chain reaction and western blot analysis to measure changes in the expression of mRNA and protein, respectively, for major mediators of Rho-family GTPases after NX-5 treatment in melanoma cells. In addition, after constructing the 3D melanoma model, the expression of Rho-family GTPases was measured by immunohistochemistry. NX-5 (10 µM and 20 µM) treatment significantly reduced melanoma cell migration (p < 0.01). Additionally, NX-5 (20 µM) treatment significantly decreased the mRNA and protein expression levels of Cdc42, Rac1, and RhoA in melanoma cells compared with the untreated group (p < 0.001 and p < 0.05, respectively). Immunohistochemistry for our 3D melanoma model showed that Cdc42, Rac1, and RhoA were constitutively expressed in the nuclei of melanoma cells of the untreated group, and NX-5 treatment decreased their expression. These results demonstrate that NX-5 can suppress melanoma metastasis by reducing the expression of Rho-family GTPases.

Study of the process-induced cell damage in forced extrusion bioprinting.

With remarkable developments in technologies, the possibility of replacing injured tissue or organs with artificial ones via three-dimensional bioprinting is being improved. The basic prerequisite for successful application of bioprinting is high cell survival following printing. In this study, numerical calculations and experiments were performed to understand cell damage process incurred by forced extrusion bioprinters. Compressible and shear stresses were presumed to play a pivotal role within the syringe and needle, respectively, based on numerical calculation. To verify the numerical results, two experiments-pressurization in a clogged syringe and extrusion through syringe-needle-were conducted, and the damaged cell ratio (DCR) were measured by live/dead assays. Shear stress of needle flow had a great influence on DCR of discharged bioink, whereas effect of compressible stress in clogged syringe was relatively small. Cell damage in the needle flow is affected by moving distance under load as well as magnitude of shear stress. Applying this concept the differential equation of DCR growing was established, similar to the historied logistic equation for population dynamics, and the mathematical formula to predict DCR was explicitly represented splendidly as a function of only one independent variable, pressure work. The proposed formula was able to effectively predict DCR measurements for 43 bioprinting conditions, and the exactness confirmed the hypothesis for the theory. The presence of safe core zone, which may be related to the critical shear stress and stressed duration on cells, was theoretically conjectured from the DCR measurements, and further studies are necessary for an extensive and profound understanding. Fast printing is required for efficiency of a bio-structure fabrication; however, the higher shear stress accompanying increased operating pressure to speed up bioink discharge rate causes more cell damage. Employing the accurate formula presented, the optimal bioprinting conditions can be designed with ensuring targeted cell viability.

3D-Printed Collagen Scaffolds Promote Maintenance of Cryopreserved Patients-Derived Melanoma Explants.

The development of an in vitro three-dimensional (3D) culture system with cryopreserved biospecimens could accelerate experimental research screening anticancer drugs, potentially reducing costs and time bench-to-beside. However, minimal research has explored the application of 3D bioprinting-based in vitro cancer models to cryopreserved biospecimens derived from patients with advanced melanoma. We investigated whether 3D-printed collagen scaffolds enable the propagation and maintenance of patient-derived melanoma explants (PDMEs). 3D-printed collagen scaffolds were fabricated with a 3DX bioprinter. After thawing, fragments from cryopreserved PDMEs (approximately 1-2 mm) were seeded onto the 3D-printed collagen scaffolds, and incubated for 7 to 21 days. The survival rate was determined with MTT and live and dead assays. Western blot analysis and immunohistochemistry staining was used to express the function of cryopreserved PDMEs. The results show that 3D-printed collagen scaffolds could improve the maintenance and survival rate of cryopreserved PDME more than 2D culture. MITF, Mel A, and S100 are well-known melanoma biomarkers. In agreement with these observations, 3D-printed collagen scaffolds retained the expression of melanoma biomarkers in cryopreserved PDME for 21 days. Our findings provide insight into the application of 3D-printed collagen scaffolds for closely mimicking the 3D architecture of melanoma and its microenvironment using cryopreserved biospecimens.

Continuous pressure measurement and serial micro-computed tomography analysis during injection laryngoplasty: A preliminary canine cadaveric study.

Injection laryngoplasty (IL) has been used to treat various types of glottal insufficiency. The precise volume and location of the injected materials impact the outcomes. However, exactly how increasing volumes of material are distributed is unknown. In fact, the amount of IL material required to medialize a vocal cord tends to be determined empirically. Thus, the goal of this study was to investigate the pattern of IL material distribution by checking serial micro-computed tomography (MCT) and pressure changes during ILs. This experimental study used 10 excised canine larynges. Experimental devices included the IL syringe, pressure sensor, infusion pump, fixed frame, and monitoring system. We injected calcium hydroxyapatite in the thyroarytenoid muscle; whenever 0.1 mL of material was injected, we obtained an MCT scan while simultaneously measuring the pressure. After the experiments, we performed histologic analyses. MCT analyses showed that materials initially expanded centrifugally and then expanded in all directions within the muscle. The pressure initially increased rapidly but then remained relatively constant until the point at which the materials expanded in multiple directions. Histologic analyses showed that the IL material tended to expand within the epimysium of the thyroarytenoid muscle. However, in some cases, the MCT revealed that there were leakages to the surrounding space with a corresponding pressure drop. If the IL material passes through the epimysium, leakage can occur in the surrounding space, which can account for the reduction in resistance during ILs.

Bioprinting of Multiscaled Hepatic Lobules within a Highly Vascularized Construct.

Highly vascularized complex liver tissue is generally divided into lobes, lobules, hepatocytes, and sinusoids, which can be viewed under different types of lens from the micro- to macro-scale. To engineer multiscaled heterogeneous tissues, a sophisticated and rapid tissue engineering approach is required, such as advanced 3D bioprinting. In this study, a preset extrusion bioprinting technique, which can create heterogeneous, multicellular, and multimaterial structures simultaneously, is utilized for creating a hepatic lobule (≈1 mm) array. The fabricated hepatic lobules include hepatic cells, endothelial cells, and a lumen. The endothelial cells surround the hepatic cells, the exterior of the lobules, the lumen, and finally, become interconnected with each other. Compared to hepatic cell/endothelial cell mixtures, the fabricated hepatic lobule shows higher albumin secretion, urea production, and albumin, MRP2, and CD31 protein levels, as well as, cytochrome P450 enzyme activity. It is found that each cell type with spatial cell patterning in bioink accelerates cellular organization, which could preserve structural integrity and improve cellular functions. In conclusion, preset extruded hepatic lobules within a highly vascularized construct are successfully constructed, enabling both micro- and macro-scale tissue fabrication, which can support the creation of large 3D tissue constructs for multiscale tissue engineering.

Decellularized extracellular matrix-based bio-ink with enhanced 3D printability and mechanical properties.

Recently, decellularized extracellular matrix-based bio-ink (dECM bio-ink) derived from animal organs is attracting attention because of its excellent biocompatibility. However, its poor 3D printability and weak mechanical properties remain a challenge. Here, we developed a new dECM bio-ink with enhanced 3D printability and mechanical properties. dECM micro-particles of about 13.4 µm in size were prepared by decellularizing a porcine liver followed by freeze-milling. The new bio-ink, named as dECM powder-based bio-ink (dECM pBio-ink), was prepared by loading the dECM micro-particles into a gelatin mixture. The usefulness of the dECM pBio-ink was evaluated by assessing its mechanical properties, printability, and cytocompatibility. The results showed that its mechanical properties and 3D printability were greatly improved. Its elastic modulus increased by up to 9.17 times that of the conventional dECM bio-ink. Micro-patterns with living cells were successfully achieved with 93% cell viability. Above all, the new bio-ink showed superior performance in stacking of layers for 3D printing, whereas the conventional bio-ink could not maintain its shape. Finally, we demonstrated that the dECM pBio-ink possessed comparable cytocompatibility with the conventional dECM bio-ink through in vitro tests with endothelial cells and primary mouse hepatocytes.

A Microfluidic Chip Embracing a Nanofiber Scaffold for 3D Cell Culture and Real-Time Monitoring.

Recently, three-dimensional (3D) cell culture and tissue-on-a-chip application have attracted attention because of increasing demand from the industries and their potential to replace conventional two-dimensional culture and animal tests. As a result, numerous studies on 3D in-vitro cell culture and microfluidic chip have been conducted. In this study, a microfluidic chip embracing a nanofiber scaffold is presented. A electrospun nanofiber scaffold can provide 3D cell culture conditions to a microfluidic chip environment, and its perfusion method in the chip can allow real-time monitoring of cell status based on the conditioned culture medium. To justify the applicability of the developed chip to 3D cell culture and real-time monitoring, HepG2 cells were cultured in the chip for 14 days. Results demonstrated that the cells were successfully cultured with 3D culture-specific-morphology in the chip, and their albumin and alpha-fetoprotein production was monitored in real-time for 14 days.

A potential dermal substitute using decellularized dermis extracellular matrix derived bio-ink.

Upon bioprinting, cells are mixed with a biomaterial to fabricate a living tissue, thus emphasizing the importance of biomaterials. The biomaterial used in this study was a bio-ink prepared using skin decellularized extracellular matrix (dECM). Skin dECM was extracted by treating the dermis with chemicals and enzymes; the basic structural and functional proteins of the ECM, including collagen, glycosaminoglycans (GAGs), bioreactive materials and growth factors, were preserved, whereas the resident cells that might cause immune rejection or inflammatory responses were removed. The bio-ink based on dECM powder, together with human dermal fibroblasts (HDFs), was loaded into the nozzle of the 3D bioprinter to create the 3D construct. This construct underwent gelation with changing temperature while its shape was maintained for 7 days. The cells showed over 90% viability and proliferation. By analysing the gene expression pattern in the cells of the construct, the skin regenerative mechanism of the bio-ink was verified. Microarray results confirmed that the gene expression related to skin morphology and development had been enhanced because the bioreactive molecules and growth factors, in addition to residual ECM in dECM, provided an optimal condition for the HDFs.

Pre-set extrusion bioprinting for multiscale heterogeneous tissue structure fabrication.

Recent advances in three-dimensional bioprinting technology have led to various attempts in fabricating human tissue-like structures. However, current bioprinting technologies have limitations for creating native tissue-like structures. To resolve these issues, we developed a new pre-set extrusion bioprinting technique that can create heterogeneous, multicellular, and multimaterial structures simultaneously. The key to this ability lies in the use of a precursor cartridge that can stably preserve a multimaterial with a pre-defined configuration that can be simply embedded in a syringe-based printer head. The multimaterial can be printed and miniaturized through a micro-nozzle without conspicuous deformation according to the pre-defined configuration of the precursor cartridge. Using this system, we fabricated heterogeneous tissue-like structures such as spinal cords, hepatic lobule, blood vessels, and capillaries. We further obtained a heterogeneous patterned model that embeds HepG2 cells with endothelial cells in a hepatic lobule-like structure. In comparison with homogeneous and heterogeneous cell printing, the heterogeneous patterned model showed a well-organized hepatic lobule structure and higher enzyme activity of CYP3A4. Therefore, this pre-set extrusion bioprinting method could be widely used in the fabrication of a variety of artificial and functional tissues or organs.

Co-targeting of Tiam1/Rac1 and Notch ameliorates chemoresistance against doxorubicin in a biomimetic 3D lymphoma model.

Lymphoma is a heterogeneous disease with a highly variable clinical course and prognosis. Improving the prognosis for patients with relapsed and treatment-resistant lymphoma remains challenging. Current in vitro drug testing models based on 2D cell culture lack natural tissue-like structural organization and result in disappointing clinical outcomes. The development of efficient drug testing models using 3D cell culture that more accurately reflects in vivo behaviors is vital. Our aim was to establish an in vitro 3D lymphoma model that can imitate the in vivo 3D lymphoma microenvironment. Using this model, we explored strategies to enhance chemosensitivity to doxorubicin, an important chemotherapeutic drug widely used for the treatment of hematological malignancies. Lymphoma cells grown in this model exhibited excellent biomimetic properties compared to conventional 2D culture including (1) enhanced chemotherapy resistance, (2) suppressed rate of apoptosis, (3) upregulated expression of drug resistance genes (MDR1, MRP1, BCRP and HIF-1α), (4) elevated levels of tumor aggressiveness factors including Notch (Notch-1, -2, -3, and -4) and its downstream molecules (Hes-1 and Hey-1), VEGF and MMPs (MMP-2 and MMP-9), and (5) enrichment of a lymphoma stem cell population. Tiam1, a potential biomarker of tumor progression, metastasis, and chemoresistance, was activated in our 3D lymphoma model. Remarkably, we identified two synergistic therapeutic oncotargets, Tiam1 and Notch, as a strategy to combat resistance against doxorubicin in EL4 T and A20 B lymphoma. Therefore, our data suggest that our 3D lymphoma model is a promising in vitro research platform for studying lymphoma biology and therapeutic approaches.

Three-Dimensional Hepatocellular Carcinoma/Fibroblast Model on a Nanofibrous Membrane Mimics Tumor Cell Phenotypic Changes and Anticancer Drug Resistance.

Three-dimensional (3D) in vitro tissue or organ models can effectively mimic the complex microenvironment of many types of human tissues for medical applications. Unfortunately, development of 3D cancer models, which involve cancer/stromal cells in a 3D environment, has remained elusive due to the extreme complexity of the tumor microenvironment (TME) and the stepwise progression of human cancer. Here, we developed hepatocellular carcinoma (HCC) models, which consist of fibroblasts as stromal cells, HCC cells, and a nanofibrous membrane to mimic the complex TME. The 3D HCC models were fabricated using three distinct culture methods: cancer cells grown directly on the nanofibrous membrane (mono model), fibroblasts covering the nanofibrous membrane (layer model), and both cancer cells and fibroblasts grown on the nanofibrous membrane (mixed model). Interestingly, the mono model and layer model showed similar tissue structures, whereas the mixed model resulted in phenotypic changes to the cancer cells. Further analysis demonstrated that the mixed models promoted the expression of fibronectin and vimentin, and showed higher resistance to anticancer drugs compared with the other models. Thus, our 3D HCC model could be utilized for testing efficient anticancer therapies at various stages of cancer, with potential application to different tumor types.

Fabrication of In Vitro Cancer Microtissue Array on Fibroblast-Layered Nanofibrous Membrane by Inkjet Printing.

In general, a drug candidate is evaluated using 2D-cultured cancer cells followed by an animal model. Despite successful preclinical testing, however, most drugs that enter human clinical trials fail. The high failure rates are mainly caused by incompatibility between the responses of the current models and humans. Here, we fabricated a cancer microtissue array in a multi-well format that exhibits heterogeneous and batch-to-batch structure by continuous deposition of collagen-suspended Hela cells on a fibroblast-layered nanofibrous membrane via inkjet printing. Expression of both Matrix Metalloproteinase 2 (MMP2) and Matrix Metalloproteinase 9 (MMP9) was higher in cancer microtissues than in fibroblast-free microtissues. The fabricated microtissues were treated with an anticancer drug, and high drug resistance to doxorubicin occurred in cancer microtissues but not in fibroblast-free microtissues. These results introduce an inkjet printing fabrication method for cancer microtissue arrays, which can be used for various applications such as early drug screening and gradual 3D cancer studies.