3D Printed Tissue Models - American Chemical Society

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3D Printed Tissue Models: Present and Future Jinah Jang, Hee-Gyeong Yi, and Dong-Woo Cho ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00129 • Publication Date (Web): 30 Apr 2016 Downloaded from http://pubs.acs.org on May 5, 2016

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ACS Biomaterials Science & Engineering

3D Printed Tissue Models: Present and Future Jinah Jang‡, Hee-Gyeong Yi‡ and Dong-Woo Cho* Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-gu, Pohang, Kyungbuk, 37673, Korea ‡

These authors contributed equally to this work.

*E-mail address: [email protected] Keywords: 3D printing; in vitro tissue model; 3D cell culture; Bioinks; Organ-on-a-chip

ABSTRACT Three-dimensional (3D) tissue model is an emerging field of investigation for disease mechanism, drug testing, and therapeutic effect for human survival. Various methods have been developed to recapitulate tissue mimetic microenvironment; however, they could mimic only fragmentary phase of disease. Cells should be tested under two-dimensional (2D) substrate or encapsulated into hydrogels so that cannot mimic natural tissue behaviors or arrangements in the body. 3D printing technology allows us to create precisely controlled 3D tissue or organ model through localization of cells, biomolecules, and materials precisely similar to tissue specific microenvironment. In this article, we review the recent advances of 3D printed in vitro tissue models that can support normal or diseased tissue differentiation, integration, and spatiotemporal reaction by drug treatment or cancer metastasis. With development of fabrication methods, the 3D printed in vitro tissue model can be utilized to study the human complex physiology in tissue and organ contexts, which could potentially be used as substitute for animals applied in drug development and toxicology testing.

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INTRODUCTION Human tissues and organs are highly complex structures consist of various types of cells and their unique organizations. Different responses for each disease are usually observed in the same organ depending on the disease location.1 Although the developed humanized or transgenic animals edited through gene alteration techniques show a great potential for investigation of the fundamental human pathophysiology, recent studies demonstrated that the underlying mechanism is not much similar in an animal and human.2 Moreover, 2D cell culture validation method differentiating cell functions failed to mimic tissue or organ-level physiology, eventually led to biased results.3 In this regard, the new mode for development of 3D tissues or disease models is urgently required to gain a better understanding the involved issues. 3D cell culture study has been spotlighted as a great potential method to recapitulate physiological environment for studying in vitro human disease mechanism.4 The definition of 3D cell culture study in this review is a cell culture in its engineered construct. This approach can recreate more complex 3D organ-level structures and integrate mechanical as well as chemical cues that are crucial elements of the whole organ architecture. The use of 3D cell culture study results in a higher expression of tissue specific function that was not previously possible in 3D gel culture method. In this manner, various microfabrication techniques (e.g., photolithography5, soft-lithography6,7, microcontact printing8, and 3D printing9,10) are widely used to build the 3D cell culture system. In particular, 3D printing has recently been emerged as a key method. It can fabricate higher-order assembly of arbitrarily complex 3D living tissue constructs with multiple types of biocompatible materials and cells using 3D models (e.g., 3D scanned models or medical images).11,12 In addition, this technique capacitates to build 3D tissue or organ specific


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microenvironment through mimicking their highly dynamic and variable 3D structures, mechanical properties, and biochemical microenvironments. The most outstanding characteristic of 3D printing technique is to construct microscale structures consisting of physiologically relevant multiple cellular arrangements or basic unit of tissues in a single-step process.13 In addition, 3D printed in vitro tissues in conjunction with microfluidic system can provide an ideal test platform for the use in drug discovery; analysis of chemical, biological and toxicological agents; and basic research with offering design and system flexibilities.14 In this article, we review recent progress in the development of 3D printed in vitro models that are more likely to mimic the native physiology of normal or diseased tissues. We describe the 3D printing techniques to engineer in vitro tissue models and valuable printing materials to stimulate cells for tissue specific functions. Further, we discuss on recent advances in the construction of 3D in vitro tissue models and their associated characteristics and functions. Finally, we briefly suggest future perspectives for development of 3D printed in vitro tissue models with personalization approaches using stem cell technologies. Printing Techniques and Processes 3D cell-printing techniques were developed in three ways, depending on the principles of releasing the cells from the printing head: Microextrusion, inkjet, and laser-assisted printing methods. Microextrusion Printing Systems In microextrusion cell-printing systems, the cell-containing materials, usually called bioink, are extruded, using the forces driven by a pneumatic pressure15,16 or by a mechanical tool, such as piston,17,18 from the reservoir to the building platform through a nozzle (Figure 1A). The continuously extruded stream is rapidly deposited along the x, y, and z axes with robotic motion



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of the printing head; thereby obtaining a significantly faster printing than the single dropletbased printing techniques, such as inkjet and laser assisted printing systems. In this way, although the bioinks with a wide range of viscosities (0.008–22 Pa·s) can successfully be dispensed,19 the hydrogels with larger viscosity values are usually used to maintain the shape of the construct by itself after printing. For example, the silk fibroin-gelatin bioink showed a high shape-fidelity at a viscosity of 3.25 Pa·s, which was measured at the calculated shear rate (200 s– 1

) on the nozzle of the customized printing system.20 However, the printed constructs composed

of the bioinks and the cells often have a smaller elastic modulus than the desired stiffness, especially in the case of musculoskeletal tissues, such as bone and cardiac muscles. To make the printed construct achieve the larger modulus, a hybrid-type structure consisting of a hydrogel and a polymeric framework was proposed by Shim et al.13 The printing system with multiple printing heads, named as multi-head tissue/organ building system (MHDS), was developed for the fabrication of a hybrid-type structure to build an osteochondral tissue-like construct.21 Moreover, reinforcement of hydrogel helps to accomplish the desired modulus of the printed tissue construct. The heart decellularized extracellular matrix (hdECM) bioink for printing the cardiac muscle tissue construct was reinforced with vitamin B2-mediated photo-crosslinking in the postprinting process.22 Another approach using microextrusion printing is scaffold-free tissue construction. To produce naturally cell-derived extracellular matrix (ECM) within the printed structure, the cellular aggregates are extruded through the capillary micropipettes, which undergo the self-assembly among themselves in the post-printing stage.23,24 Inkjet Printing Systems Inkjet printers produce droplets from a cell-suspended liquid in a ‘drop-on-demand’ manner. The droplets are generated through electrically heating the nozzle to induce a vapor bubble25 or


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breaking the liquid into droplets using a piezoelectric actuator (Figure 1B).26,27 Although the main drawback of the inkjet printing is the limitation in selecting bioinks to only low viscosity materials (~ 0.1 Pa·s),19 this technique enables to control the small volume of the liquid and achieve higher resolutions up to 20 µm.28 While the microextrusion printing technique is advantageous to produce the biomimetic structures in a large scale with the rapid printing speed, the inkjet printers have a high potential for accomplishment of fabricating the integrated multiple organ tissues or array within a narrow area. For example, this technique has been successfully used to fabricate a heterogeneous tissue composed of three compartments,29 a 3D cell-laden zigzag tube mimicking the shape of vessel,27 and a 3D 13-layer stacked cell-laden structure of the university logo30 in a space of sub-millimeter size. Laser-Assisted Printing Systems Laser-assisted printing systems apply ‘aim-and-shoot’ procedure to produce cell-containing droplets via laser beam.31 The printing methods using laser beam provide the highest resolution to control the droplets. Laser-guided direct writing method traps the cells in the laser beam using the difference of refractive indices between cells and media.32 The trapped cells are then axially pushed through a layer of a low-viscous material to a hydrogel-coated surface. Laser-induced cell-printing systems can produce droplets of cells by focusing the laser pulse onto a cell-laden hydrogel coated transparent support, called ribbon (Figure 1C).33,34 Because of the formation of a laser-induced vapor pocket, the droplet is separated from the ribbon and transferred to the receiving substrate. Therefore, as explained, with the various methods of delivering cells, 3D cell-printing techniques embody the cells within the ECM-like materials to mimic the complex and heterogeneous tissue seen in vivo.



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Stereolithography (SLA) is an interesting additive manufacturing technique for producing fully designed microfluidic devices (Figure 1D).35,36 This is the oldest 3D printing technique, developed in the 1980s. SLA enables the solidification of liquid photopolymer by selective photo-polymerization using ultraviolet (UV), infrared (IR), or visible light. 2D patterns, made by slicing of the 3D models, were exposed to the polymer reservoir through irradiation of the light sources.37 The reservoir contains bioink and cells with photo-initiator. 3D construct is then built by stacking up of solidified 2D patterns via a layer-by-layer process.

Figure 1. Schematic illustrations of (A) microextrusion, (B) inkjet, (C) laser-assisted printing, and (D) stereolithography techniques. Materials for Printing Tissue Models


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Biocompatible materials play various roles in printing tissue models. During the printing process, cells are protected from the external forces through encapsulation in hydrogel-form materials, usually called bioink.38 These bioinks should meet several physicochemical characteristics, such as proper viscosity and sufficient mechanical strength after crosslinking, to sustain their 3D shapes.39 The biocompatible materials are also used for fabrication of polymeric frameworks or sacrificial parts to generate complex 3D structures maintaining high pattern fidelity.13,21,40 There are two primary kinds of printing materials that are mostly used in 3D cell printing techniques: Synthetic and natural polymers with their pros and cons. For example, synthetic materials are used for fabrication of mechanically robust constructs with lower biological affinity, whereas natural materials provide significantly greater biological responses and similarity to the native tissue with weaker mechanical properties. In this regard, the development and selection of appropriate printing materials are highly crucial for successful printing tissue construct. Synthetic Polymers Most synthetic polymers have advantages on the modification of physicochemical and mechanical properties for various biomedical applications. These materials can be produced at low cost and reduce the risk of being pathogens in a printed construct due to their biologically inert characteristics.10 Moreover, they also can be used as either bioink or supporting materials.41 Blends of synthetic and natural polymers can also lead to get significant effects in an attempt to enhance cellular responses.42 There are various synthetic polymers that are extensively used in 3D printing, including commercialized polymers for printing, polycaprolacton (PCL), poly(lactic-co-glycolic acid) (PLGA), Poly(ethylene glycol) (PEG), and Poloxamer 407 (Pluronic F127).



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Among various synthetic polymers, PCL and PLGA are the most widely used thermoplastic and biocompatible polymers. PLGA is a Food and Drug Administration (FDA)-approved material that has controllable degradation characteristic depending on the ratio between the poly(lactic acid) (PLA) and poly(glycolic acid) (PGA) in the copolymer. PCL and PLGA cannot be used for encapsulating cells due to their high glass transition temperature. PCL is extensively used as a polymeric framework providing high load-bearing mechanical properties for the hydrogel-based 3D printed constructs.21,40,43 PLGA is used for fabrication of biopaper to provide the binding capacity between the cell-printed layers.44 PEG-based materials are also largely used in printing tissue constructs through conjugation with other functional groups. PEG, also an FDA-approved biopolymer, is safe for clinical applications. It shows great hydrophilicity and water-soluble characters that make it suitable as a sacrificial bioink.45 Rutz et al explored PEG-based bioinks through modification of functional group in PEG.46 They developed multiple types of modified PEGs, each material was mixed together to achieve gel formation that occurred by click chemistries or Michael-type additions mechanism.46 The mixtures of PEG-based materials can help to control and customize the degree of crosslinking so that the final mechanical properties can be tailored. Another method to achieve gel formation property from PEG is acrylation reaction in which the chemically modified PEGbased material can be gelled by photo-crosslinking under UV exposure.47,48 Pluronic 127 has a thermoreversible gelation behavior from an insoluble to a soluble state when the concentration of Pluronic polymer in aqueous solution is over 15 wt %.49 Printed Pluronic can be easily removed below 4–5 °C and turning it into a gel above 16 °C, which is a very useful character in printing sacrificial parts.50 For example, Wu et al. fabricated biomimetic


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omnidirectional microvascular networks in 3D large volume hydrogel block using Pluronic as a sacrificial bioink.49 Natural Polymers For the use of natural polymers as bioinks, the form of hydrogel that is typically used to print living cells is made through encapsulation in that material.51 Natural materials show better cell affinity and structural similarity to the native tissue environment than those of the synthetic material; thus, they can stimulate cellular behaviors, including migration, proliferation, differentiation, and maturation.12,52 Collagen hydrogel contains a large quantity of glycine, proline, and hydroxyproline residues so that it can facilitate thermoresponsible gelation under physiological conditions, which is a primary advantage for 3D printing application.13,53 Gelatin, also derived from collagen, is widely used as gelling agent for foods, pharmaceuticals, and cosmetics.54 However, it shows different crosslinking mechanism from that of collagen due to the denaturation process during gelatin production. It is usually liquid above 40 °C and reversibly forms an alpha helix structure below 30 °C by coiling its molecular structures; thus, it requires additional agent for crosslinking, such as glutaraldehyde and genipin.55 Gelatin methacrylate (GelMA) bioink, widely used in biomedical applications, is a semi-synthetic or semi-natural bioink modified with photopolymerizable methacrylate (MA) groups, allowing the matrix to be covalently crosslinked with exposure to UV radiation.56–58 This bioink also provides ECM-like microenvironment to the encapsulated cells because the gelatin in the bioink forms the assembly of intermolecular triple helices using physical gelation process. In addition, it provides versatility of materials properties, including shear yield stress and modulus of liquid GelMA bioink.



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Fibrin can be rapidly gelled through enzymatic interaction between fibrinogen and thrombin, which is a widely known mechanism for blood coagulation.59 This rapid crosslinking character provides a key feature of fibrin gel as a bioink. The mixture of fibrin with other hydrogels, for instance with collagen60,61, alginate62,63, and gelatin62,64, have been extensively used in 3D printing applications. Matrigel® is a gelatinous protein mixture produced using Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It contains laminin, collagen IV, entactin, heparin sulfate proteoglycan, and numerous growth factors that are naturally observed in EHS tumor.65 It forms a gel under physiological conditions. This complex mixture is widely used to mimic in vivo environments for 2D and 3D gel culture applications from cancer and stem cell research to metabolism and toxicology studies.23,44,66,67 Recent advances in decelluarized extracellular matrix (dECM) have been recognized as an ideal bioink to recreate the natural microenvironment that cells experience in their native tissue.18,22,43 In dECM hydrogel, a variety of proteins, proteoglycans, glycoproteins, and cytokines exist that can promote tissue-specific stem cell differentiation. Pati et al. were the first research group to use dECM hydrogel for printing constructs, which was printed below 15 °C and transformed into a hydrogel above 37 °C.18 The fabricated constructs using dECM bioink revealed better differentiation of the printed cells into the tissue-specific lineages in comparison with the use of pure collagen and alginate bioinks.18 Applications of 3D Printed Tissue Models Significant characteristics of human tissues, such as biological, structural, and physiological complexity with fluid flow conditions, have been comprehensively characterized using 2D, 3D gel, and 3D cell culture models.3,4 These traditional models offer significant advantages to gain a biomimetic pattern in the culture environment made by nanofabrication/microfabrication


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techniques or a faster processing using a microfabricated high throughput device.5–8 However, there have been numerous limitations for complete production of the predominant features and the functions of the target tissues due to the inherent drawbacks in the traditional models for reflecting the structural complexity of human tissue/organs. The use of traditional models for studying human physiology has also been hampered by a variety of restrictions, such as fabrication of multiple layers or complex and composite construct designs that require distinct multi-materials to be stacked or aligned because these processes involve a manual layer-by-layer assembly process. Moreover, these processes are limited in terms of recapitulation of physiological organization in the body that is composed of very complex and non-planar pattern. In this regard, a 3D printing technique can be used as an alternative fabrication method that could potentially provide significant advantages for the development of tissue mimetic microenvironment. Here, we present the current state of the art of the normal or diseased tissue modeling fabricated by 3D printing technology (Table 1). Table 1. Various 3D Printed in vitro Tissue Models Examples 3D printing method LaserAssisted Printing

Ref 44

68

Target tissue or disease

Cell types

Bioinks

Embryonic carcinoma

Murine embryonal carcinoma cell line (P19)

Ribbon: cell-encapsulated Matrigel

Colon cancer

Human colon cancer cell line (HT-29)

Ribbon: cell-suspended medium

Receiving substrate: Matrigel

Receiving substrate: culture medium 33

Skin tissue

Murine fibroblast (NIH-3T3), human keratinocytes (HaCaT)

Ribbon: cell-suspended media Receiving substrate: Matriderm™

Ejection printing

69-70

Ovarian cancer

Human ovarian (OVCAR-5),

cancer

cell

line

Ejectors: cell-suspended media Stage: Matrigel

human ling fibroblast cell line (MRC-5) Inkjet printing

71

72

Breast cancer

Liver cancer

Murine mammary cancer cell line (4T07), murine mesenchymal stem cells (D1)

Cartridges: cell-suspended media

Hep G2, human umbilical endothelial cells (HUVEC)

Cartridges for printing the cells: cellsuspended media

vein


Stage: Matrigel


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Cartridge for printing the adhesive film: fibronectin-gelatin solution Stage: N/A Extrusion printing

73

Breast cancer

Breast cancer cell, adipocytes, mammary fibroblasts, and endothelial cells

N/A (scaffold free method)

74

Cervical cancer

Human cervical cancer cell line (Hela)

Syringe: cell-encapsulated gelatin/alginate/ fibrinogen hydrogel

75

Skin tissue

keratinocytes and fibroblasts

Cartridges for printing collagen layer: 0.3 wt % collagen Cartridges for cells: cell-suspended media

Stereolithography

76

77

Liver analog

lobule

Liver tissue

hiPSC-HPCs,

Bioink: 5% GelMa (hiPSC-HPCs)

supporting cells (HUVEC, Adipose derived stem cells (ADSCs))

2.5% GelMA with (supporting cells)

Human hepatocellular carcinoma cell line (Hep G2)

N/A (pipetting of cell-gel mixture)

2%

GMHA

Skin Tissue Models Skin consists of multiple layers of dermal tissues that protects the underlying muscles, bones, and internal organs. It also plays a vital role in maintaining body homeostasis and provides a physical barrier to the invasion of external pathogens while regulating the exchange of water and metabolites from the outside of the body. Engineered skin tissue can serve as an extremely valuable for tissue regeneration and in vitro platform for testing topical drug or cosmetic agent discovery.78 In particular, the ban on testing of cosmetic products on animals by European Union is accelerating the development of in vitro skin tissue models as a replacement.79 Although conventional co-culture of keratinocytes and fibroblasts using air-liquid interface (ALI) culture method has facilitated the development of the first generation of in vitro skin model,80–82 this method did not take into account the precise positioning of each cell for the fabrication of highly complex, hierarchical and stratified structure of skin tissue. Thus, this model failed for development of fully differentiated or functional skin tissue models. 3D printing approach has


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significant advantages to mimic hierarchical structures of native human skin in terms of a wide range of sizes, a high throughput and reproducible characteristics. In the late 2000s, skin tissue models produced via microextrusion and laser-assisted printing methods have demonstrated good strengths and other advantages compared with the manually developed skin tissue model.83,84 In 2013, Lee et al. fabricated human skin tissue model using keratinocytes and fibroblasts representing the epidermis and dermis layers of skin, respectively.75 They localized 0.3 wt % collagen I hydrogel between the cell layers, representing as the dermal matrix of skin. The printing conditions (e.g. number of cells and printing parameters) were optimized for high cell viability after printing process. After 7–14 days ALI culture, epithelialization was observed and maturation and stratification were promoted. This model also offers significant advantages in terms of shape and form retention. On the other hand, manually deposited structures shrink and form concave shapes under culture conditions after 7 days. Koch et al. fabricated 3D arrangement of vital cells by Laser-assisted BioPinting (LaBP) as multicellular tissue mimicking 3D in vitro models (Figure 2A).33 They have printed the 20 layers of fibroblasts and 20 layers of keratinocytes embedded in collagen, and observed skin mimicking bi-layered construct 10 days after printing (Figure 2B).



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Figure 2. 3D printed skin tissue model. (A) Schematic of the laser printing setup for construction of skin tissue model. A patterned structure of fibroblasts (green) and keratinocytes (red) demonstrates micropatterning capabilities of the laser-assisted printing technique. (B) Skin mimicking bi-layered construct containing 20 layers of fibroblasts (NIH-3T3) and 20 layeres of human keratinocytes (HaCaT) in collagen. Reproduction with permission.33 Liver Tissue Models Liver is a critical organ in the human body that has an important role for the synthesis of vital proteins and metabolism of xenobiotics.85 The development and fabrication of in vitro liver tissue model is difficult because the functional maturation should be achieved through mimicking the sophisticated liver microenvironment.86–88 The complex microstructure and cell combinations are significantly related to disease development, and the hexagonal liver lobule unit construct is widely known as an essential factor to regulate the liver functions.89 Ma et al. created this 3D assembly of construct consisting of hepatocytes with the supporting cells into a hexagonal patterns using projection-based 3D printing method (Figure 3A).76 They used human induced pluripotent stem cells-derived hepatic progenitor cells (hiPSCs-HPCs) for depicting the personalized in vitro drug screening and disease study models. These cells were mixed with 5 w/v % GelMA bioink with similar mechanical stiffness to the healthy liver tissues. Human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem cells (ADSCs) were also chosen as the supporting cells representing the potentials in forming vasculatures nearby the natural liver lobule in the human body (Figure 3B). The supporting cells were encapsulated into the mixture of 2 w/v % glycidal methacrylate-hyaluronic acid (GMHA) and 5 w/v % GelMA at a 1:1 ratio for promoting neovascularization. All these liquid bioinks were then polymerized using UV exposure generated from projection-based 3D printer. This 3D hepatic triculture model


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maintained intrinsic cell reorganization and promoted liver specific gene expression level as well as metabolic product secretion (Figure 3C). Another approach to develop 3D liver construct for parallel toxicology examinations have been explored by Skardal et al.77 It is not a direct tissue printing method but they applied 3D printing technique for fabrication of microfluidic devices. They initially printed replica mold of 3D fluid device with inverted channel design and then PDMS were casted repeatedly into that mold, producing high throughput formats. HepG2 cells, a human liver cancer cell line, were encapsulated into hyaluronic acid (HA)/gelatin bioink mixed with PEGDA photocrosslinker. The solution was then filled into the fabricated PDMS device and crosslinked using UV radiation. For in situ toxicology examinations, alcohol was exposed into the microfluidic liver tissue model using a peristaltic pump system.

Figure 3. 3D printing of hydrogel-based hepatic construct. (A) Structural characterization of hepatic model. (B) 3D reconstruction of the construct showing patterns of hepatic cells (green) and supporting cells (red). (C) Gene expression profiles comparing the level of HPCs in 2D



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monolayer culture, 3D HPC-only culture model, and 3D printed model on day 7 following printing process. Reprints with permission.76 Cancer Models As cancer is one of the most common diseases causing death, clinically relevant cancer model is highly required to find an advanced anti-cancer treatment. Cancer development and progression are significantly influenced by complex microenvironments, including multiple cell types, various kinds of ECM molecules, and interactions between them.22 In addition, the toxicity of anti-cancer drug have been extensively reported; thus, the necessity of testing platform, such as in vitro cancer model, is rapidly growing in the field of pharmaceutical development. In particular, in vitro cancer models have been studied for mimicking the 3D microenvironments by putting the cancer cells within scaffolds fabricated from various kinds of materials; for example, Matrigel,23 hyaluronic acid,24 and decellularized matrices,25,26 porous PLGA,27 and electrospun PCL28. The scaffolds have been demonstrated to promote the cell-ECM interactions, resulting in increase of potential for malignancy of cancer cells. In general, the cellular and biological components are simply mixed in the bulk hydrogels or directly seeded onto prefabricated porous structures; however, these conventional methods have faced difficulties for production of complex structures. Therefore, advanced techniques are required to fabricate a more reliable research platform and recreate a physiologically relevant cancerous tissue with a high structural and functional similarity to the native cancer cells. 3D cell-printing techniques have advantage of placing the viable cells at a defined location via the robotic motions of the printing head. The printed embryonic carcinoma cells29 and the printed colon cancer cells30 have demonstrated that the laser-assisted printing system, named as matrix-assisted pulsed laser evaporation direct write (MAPLE DW), could accurately transfer a


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single droplet containing the cells on the Matrigel coated surfaces with maintaining the high cell viability greater than 95%. Furthermore, the 3D printing of multiple types of cells enables to study the effects of variations of spatial geometry on the modeling of tumors. Heterogeneous ovarian cancer models have been generated using dual-ejection systems to investigate the effects of the distances between ovarian cancer cells and fibroblasts (Figure 4A).31,32 The dual-ejection systems have

automated stage equipped with two ejectors that are filled with the media

suspended with OVCAR-5 cells, human ovarian cancer cell line, and another media suspended with MRC-5 cells, human fibroblast cell line. Each droplet containing OVCAR-5 or MRC-5 cells was deposited apart from each other by a certain distance on a Matrigel coated surface. The system was able to control the distance between the printed two droplets and number of cells per droplet via control of the dispensing valve. The printed cells maintained greater than 90% viability over 2 weeks and formed acini under the influence of the 3D microenvironment. Notably, the spatially controlled ovarian cancer models have demonstrated that the nodular formation of the cancer cells enlarge with the increasing proximity of the fibroblasts. In addition to the precise control of the cell-containing droplets, a high-throughput assay can be performed due to the high productivity of 3D cell-printing techniques.33 Co-culture models of breast cancer with repetitive patterns were fabricated using a thermal inkjet printing system equipped with two cartridges.34 The individual HP26 series cartridge was loaded with 4T07 cells, murine mammary cancer cell line, or D1 cells, murine mesenchymal stem cells, and alternately expelled the cells on a collagen coated surface. The 4T07 and D1 cells were pre-labeled as red and green, respectively, and printed in the circular and checked patterns that were repeated three to five times within a space of 2.4 mm length (Figure 4B). A liver cancer model was generated through a layer-by-layer assembly of HepG2 cells, human liver cancer cell line and human



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umbilical vein endothelial cells (HUVECs) using a piezoelectric inkjet printing system (Figure 4C).35 The printing system dispensed the fibronectin-gelatin solution onto each printed cells to solidify the cell-laden layers for stacking. The multiple simplified liver cancerous tissues were generated on the array of micro-wells to have three different hierarchical structures: A monolayer of HepG2 cells, a HepG2 cells laying on a HUVECs-layer, and a HepG2 cells-layer sandwiched by two-layers of HUVECs (Figure 4C). The high-throughput assay using the array of the printed liver cancers revealed the largest secretion of the paracrine factors, such as albumin from the cancer cells of the sandwiched structure. 3D cell-printing technologies offer great potentials for production of a highly biomimetic cancer models through construction of heterogeneous compositions and arrangements of both cells and ECM components. A heterogeneous breast cancer model was produced using Organovo’s printing system—called NovoGen BioprintingTM Platform—to mimic the original structure of breast tissues, where the cancer arises.36 Clumps of human breast cancer cells were deposited within the printed stromal milieu, including human adipocytes, mammary fibroblasts, and endothelial cells. The neo-tissue of breast cancer was fabricated via self-assembly of the printed clumps of the cells, and displayed an in vivo-like structure of the cancer cells surrounded by the breast stroma. Additionally, the biomimetic breast cancer tissues were directly printed onto a multi-well plate to conduct high-throughput therapeutic tests against the anticancer agents and exhibited an increase in chemoresistance to the tamoxifen treatment. A cervical cancer model was fabricated through printing a blend of gelatin/alginate/fibrinogen hydrogel containing Hela cells (human cervical cancer cell line) in the grid shape to allow the diffusion of oxygen and nutrients (Figure 4D).37 The cervical cancer cells in the printed construct showed a cell viability of greater than 90% and the formation of spheroid (Figure 4D). Compared with the


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conventional 2D monolayer cultures, the printed cervical cancer cells within the ECM-like materials displayed a higher synthesis of matrix metalloproteinase (MMP) proteins and a lower sensitivity against paclitaxel, one of the anti-metabolite drugs. The printed biomimetic biological environments have provided the platforms to investigate the cancer initiation, propagation, and therapeutic responses to the treated anti-cancer agents. In this way, 3D cell-printing technique would enable to produce various kinds of tissue-specific cancer models due to its unique versatility.



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Figure 4. 3D cell-printed cancer models. (A) Schematic diagram of dual-ejection system and precise control of the cell-containing droplets. The acini were formed by the printed OVCAR-5 cells. (B) Fluorescent image showing the inkjet-printed circular and checked patterns composed of D1 cells (green) and 4T07 cells (red). (C) Illustration of layer-by-layer assembly process in inkjet-printing and three different assemblies of cell-laden layers. (D) Drawing of microextrusion printing process to construct cervical cancer model in a grid shape. Hela cells in the construct exhibited morphology of spheroids at day 8. Reproduced with permission.70–72,74 Conclusions and Perspectives The development of 3D printed in vitro tissue models, which enables accurately mimicking the diversity of in vivo microenvironment and the complexity of human physiological or pathophysiological conditions, provides significant potential for a better understanding of treatments. The 3D printing-based in vitro tissue model platforms are still at their initial stages of development requiring significant levels of additional researches. The future of 3D printed tissue models should be highly advanced in terms of both technical and biological aspects to accomplish these goals. High precision can be potentially achieved through modification of the nozzles sizes, but adjustment of proper shear stress is necessary to prevent cell death during the printing process.90 Moreover, the speed of printing is also a critical factor to improve cell viability that could be overcome through application of multiple nozzles for simultaneous printing of complex biological structures.91 Development of tissue specific bioink is another issue because proper bioink can improve the design flexibility of the system.12 This attribute is a great potential for using dECM materials as bioink to recreate a healthy or diseased tissue microenvironment into a 3D printed tissue model.18 As for the cell source, earlier studies focused on animal derived or immortalized cell lines due to their easy accessibility. The paradigm is now


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shifting toward the use of human origin, particularly patient-derived iPSCs, which have a great potential to create personalized tissue models for drug discovery studies.92,93 In particular, patient-derived cells can reflect patient-specific fundamentals of diseases, including geneassociated congenital disorder, malignancy or tumor invasion so that it can be also applied for development of patient-specific disease models to a personalized treatment plan.94,95 In addition to the so far presented 3D printed tissue, it is important to recapitulate organ-specific mechanical microenvironments such as microfluidic perfusion system. In a related study, Au et al. have shown the capability of developing the optically cleared 3D microfluidic devices using SLA technique, which have higher order design modularity compared to the soft lithography.36 They also have developed fluidic valves and pump systems integrated within microfluidic devices by SLA technique.96 As an application, Lee et al. successfully utilized this 3D printed microfluidic device with helical microchannel designs for detecting pathogenic bacteria based on the size difference between the particles.97 These examples shows the possibility to develop the 3D printed tissue model in conjunction with the microfluidic system for using in various biomedical applications. Furthermore, it will be important to ensure that appropriate culture and investigation methods should be developed to validate the extrapolation of in vitro tissue model results to the human situation. Organ-to-organ connection and its culture method are important to reflect the in vitro physiological conditions; however, these subjects are still under development. Nevertheless, these 3D printed in vitro tissue models could be powerful alternatives to use as a substitute for animals applied in drug development and toxicology testing. Research works should be focused on these challenges to realize the potentials of 3D printing technique to be transformed into the organ-on-a-chip level. AUTHOR CONTRIBUTIONS



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‡

J.J. and H.–G.Y. contributed equally to this work. The paper was written through contributions

of all authors (J.J., H-G.Y. and D-W.C.). All authors have given approval to the final version of the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by the National Research Foundation of Korea (NRF), grant funded by the Korea government (MEST) (No. 2010-0018294), and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2015R1A6A3A04059015). REFERENCES 1. Benam, K. H.; Dauth, S.; Hassell, B.; Herland, A.; Jain, A.; Jang, K.-J.; Karalis, K.; Kim, H. J.; MacQueen, L.; Mahmoodian, R. Engineered in vitro disease models. Annu. Rev. Pathol. Mech. Dis. 2015, 10, 195–262. 2. Seok, J.; Warren, H. S.; Cuenca, A. G.; Mindrinos, M. N.; Baker, H. V.; Xu, W.; Richards, D. R.; McDonald-Smith, G. P.; Gao, H.; Hennessy, L. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (9), 3507–3512. 3. Huh, D.; Torisawa, Y.-s.; Hamilton, G. A.; Kim, H. J.; Ingber, D. E. Microengineered physiological biomimicry: organs-on-chips. Lab Chip 2012, 12 (12), 2156–2164. 4. Ryu, H.; Oh, S.; Lee, H. J.; Lee, J. Y.; Lee, H. K.; Jeon, N. L. Engineering a blood vessel network module for body-on-a-chip applications. J. Lab. Autom. 2015, 20 (3), 296–301. 5. Qi, H.; Du, Y.; Wang, L.; Kaji, H.; Bae, H.; Khademhosseini, A. Patterned differentiation of individual embryoid bodies in spatially organized 3D hybrid microgels. Adv. Mater. 2010, 22 (46), 5276–5281. 6. Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X.; Ingber, D. E. Soft lithography in biology and biochemistry. Annu. Rev. Biomed. Eng. 2001, 3 (1), 335–373. 7. Khademhosseini, A.; Ferreira, L.; Blumling, J.; Yeh, J.; Karp, J. M.; Fukuda, J.; Langer, R. Co-culture of human embryonic stem cells with murine embryonic fibroblasts on microwellpatterned substrates. Biomaterials 2006, 27 (36), 5968–5977. 8. Qin, D.; Xia, Y.; Whitesides, G. M. Soft lithography for micro-and nanoscale patterning. Nat. Protoc. 2010, 5 (3), 491–502. 9. Mironov, V.; Visconti, R. P.; Kasyanov, V.; Forgacs, G.; Drake, C. J.; Markwald, R. R. Organ printing: tissue spheroids as building blocks. Biomaterials 2009, 30 (12), 2164–2174.


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3D Printed Tissue Models: Present and Future Jinah Jang*, Hee-Gyeong Yi*, and Dong-Woo Cho 3D printed in vitro tissue models can be utilized to study the human complex physiology in tissue and organ contexts.


3D Printed Tissue Models - American Chemical Society

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