3D Printing for In vitro and In vivo Disease Models

CHAPTER 7

3D Printing for In vitro and In vivo Disease Models ABHAY SACHDEV, PHD  •  ROCKY RAJ, M.TECH  •  ISHITA MATAI, PHD

INTRODUCTION Biomedical research mainly focuses on the development of new and more effective tools and technologies such as biosensors, biomaterials, image processing, and artificial intelligence for research, disease diagnosis and prevention, and therapeutic interventions.1 It is of utmost importance for a medical researcher to understand all the biological mechanisms, including physiologic, cellular, molecular, and genetic mechanisms, associated with a disease and to predict the response of the treatment before moving to human clinical trials. This requires proficient modeling systems that can faithfully reciprocate the actual human microenvironment.2 Both in vitro and in vivo disease models are extensively utilized in scientific research to enhance our understanding of health issues and to make progress in the biomedical field. Conventional disease models (Fig. 7.1) have contributed a lot toward our understanding of numerous diseases and have played a crucial role in disease diagnostics, therapeutics, surgical research, toxicologic testing, and screening of potential drugs.3 In vivo disease models have played a critical role in the exploration and characterization of disease pathophysiology, in target identification, and in the evaluation of novel therapeutic agents and treatments.4,5 The use of lower invertebrates such as zebrafish,6,7 roundworm,8 or fruit fly9,10 and animals such as humanized mice,11 rabbits,12 guinea pigs,13 or monkeys14 is well established but the transferability of the results obtained from these models to humans is questionable. Current preclinical animal models do not fully recapitulate the physiologic and/or pathologic mechanisms underlying human diseases.15 The efficacy and toxicity of the drugs tested using animal models do not always predict their exact response in human patients because of the differences in their respective microenvironments. Animal models also exhibit limitation in controlling variables, which are crucial for understanding certain cellular or molecular parameters associated with progression of

a disease or drug response. High expenses and ethical concerns associated with animal use as disease models further alleviate the need for the development of less expensive and high-throughput alternatives.16 In vitro disease models, such as anatomic models, two-dimensional (2D) cell culture, and stem cell culture, are used worldwide for initial testing of drug efficacy and toxicity as well as to study disease pathology. Although 2D cell cultures serve as a feasible alternative, they exhibit problems in maintaining stability and viability. Due to the lack of “integrity of an organism as a whole,” 2D cell cultures cannot explain much about the mechanisms beyond a particular cell type. Recent advances in the tissue engineering and microfabrication techniques for the development of three-dimensional (3D) tissue cultures,17 engineered in vitro tissue constructs,16 and stem cell–derived organoid cultures18 have subdued some of the limitations of conventional disease models.19–21 The 3D in vitro models replicate in vivo conditions better than both 2D cell cultures and animal models. However, the 3D disease models are produced in a low-throughput manner and often lack the nativelike structure.16 The emergence of 3D printing in the field of medicine has increased the possibilities of patient-specific treatments and development of more faithful disease models with a great potential of lowering the manufacturing costs and time.22–24 3D printing is an innovative manufacturing technology that was first developed and employed by Hull in the 1980s for the fabrication of a plastic device.25 Since then, it has been considered revolutionary in the field of engineering, product design, and manufacturing. It has gained immense interest by medical researchers for its application in the health sector.26,27 Today, 3D printing has advanced to the stage where it can efficiently print conventional biocompatible materials and even viable cells into complicated 3D functional tissue constructs.22,28,29 It has shown tremendous potential to develop desired tissue and organ models that are

3D Printing Technology in Nanomedicine. https://doi.org/10.1016/B978-0-12-815890-6.00007-4 Copyright © 2019 Elsevier Inc. All rights reserved.

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FIG. 7.1  Study models of human diseases.

suitable for disease modeling, drug screening, organ transplantation, prosthetics, implantation, and surgical planning and training.16,30,31 As discussed in the succeeding sections, 3D printing has been used to generate models of various diseases including cardiovascular diseases, hepatic disorders, neural diseases, musculoskeletal diseases, and even cancer. 

3D PRINTING The captivating and inventive technique of 3D printing allows the conversion of digitalized information into 3D physical objects with accuracy and in much shorter duration as compared with the conventional techniques of product design.32 In the medical field, 3D printing has been used for the rapid and accurate production of pharmaceuticals,27 biomodels,31 scaffolds,24 prosthetics, and implants.33 The basic components of 3D printing (Fig. 7.2) can be divided as follows: a. Hardware: The printer itself is used to print a 3D physical object, such as inkjet, microextrusion, or laser-assisted 3D printer. b. Software: The software used for communicating between the computer and the printer and the software

 

FIG. 7.2 Basic components of 3D printing.

used for converting collected images or computationa­ lly designed images to the printer-recognizable format. c. The printing material: The material used as ink for printing the 3D physical object, including polymers, metals, ceramics, nanomaterials, or biological materials (bioink).

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FIG. 7.3  Process for generating patient specific 3D printed human tissue models. (Reproduced from

­Murphy and Atala, 2014 with permission from Springer Nature. Copyright 2014).

The collaborative advancement in the fields of tissue engineering, materials science, and cell biology has led to the advent of bioprinting. Bioprinting is concerned with the application of 3D printing technology to biomedical field. 3D bioprinted tissues have the capability to better mimic in vivo conditions because of the precise control offered by the bioprinters for spatial positioning of the bioink.34 Design approaches for reproduction of functional cells, tissue, or organs using 3D printing include biomimicry (manufacturing physiologically accurate biomaterial types and gradients), independent self-assembly (using embryonic development as guide), or fabrication of miniaturetissue blocks.35 

3D PRINTING FOR DISEASE MODELS 3D bioprinting approaches are capable of generating diseased or physiologically normal microenvironments with tunable biophysical and biochemical properties to better mimic in vivo conditions. The process of printing human disease models (Fig. 7.3) involves accumulation of the 3D image of the targeted body part, organ, or tissue; conversion of images into a printer-readable format; and followed by printing the model using a suitable bioink and appropriate bioprinters.34 The first step for production of precise 3D printed disease models is to collect images of the targeted tissue or organ for gaining a comprehensive understanding about the composition and organization, which

is crucial for reproducing the complex heterogeneous structure. Various medical imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) are being used for imaging purposes. In a typical CT scan, X-rays are employed to take a series of 2D images of the patient’s whole body or the targeted body part virtually from all the angles. In the MRI technique, magnetic and radio waves are used to scan and produce cross-sectional images of the soft tissues. The obtained 2D images from CT or MRI scan are then processed and stacked together with the help of computer-aided design (CAD) software to reconstruct an informational 3D image. However, both CT and MRI scans give very limited information about the cellular composition and distribution in the targeted tissue. In such cases, computer simulation, in combination with mathematic modeling and various imaging modalities, could be opted for generating a precise 3D image of the target. Once the comprehensive 3D image of the target is generated, it is converted to a printer-readable stereolithographic format. Software such as FreeCAD, Slic3r, Blender, Onshape, and MeshLab can be used for this purpose. Depending on the composition of the target and approach of designing disease model, appropriate printing material is selected. Various organic materials including hydrogels (such as alginate, collagen, agarose, or gelatin), decellularized matrix components, nanocellulose, and hyaluronic acid have been used in combination with specific cell types for bioprinting

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applications.36,37 The choice of bioink depends on various factors such as the supporting bioprinting modalities, bioprintability, cell viability, affordability, scalability, practicality, mechanical and structural integrity, bioprinting and postbioprinting maturation times, tissue fusion and formation after implantation, degradation characteristics, commercial availability, immune compatibility, and application areas.36 Next step in the process is to print the 3D physical model using an appropriate bioprinter. Bioprinting processes combine controlled 3D spatial movement to deposit printing material using modules such as inkjet, microextrusion, or laser-assisted method.34,38

Inkjet Bioprinters Inkjet printers are the most widely used 3D printers for bioprinting applications.34 Robert J. Klebe, for the first time, printed biological material (cell adhesion proteins and monoclonal antibodies) onto a flexible substrate by using commercially available 2D inkjet printers.39 Since then, inkjet printers were modified by incorporating an elevating platform (for vertical movement), replacing the ink by biological components including cells, proteins, growth factors (GFs), extracellular matrix components, and other materials of biological origin.40 Presently, the inkjet bioprinters can be used to print cell-containing droplets with a very good resolution (20–100  μm) at an exceptionally higher speed of about 10,000 droplets per second. These printers deliver controlled volumes of the printing material to the predefined locations using thermal or piezoelectric actuators.38 In thermal inkjet printers, the printer head is heated electrically to generate pressure pulses for forced ejection of droplets from the nozzle.41 These printers offer the advantages of high print speed, cost-effectiveness, and wide availability but they pose the risk of causing damage to the cells and the material because of their exposure to heat and pressure. Nonuniformity in the droplet size, low droplet directionality, and high frequency of nozzle clogging further limit their application for bioprinting.34 Acoustic inkjet printers contain a piezoelectric crystal that regularly generates acoustic waves on application of voltage for breaking the printing liquid material into droplets. These printers are able to generate uniform droplets with appropriate directionality. Open-pool nozzle-less ejection systems can be used for reducing imposed sheer stress on the cells and to avoid nozzle clogging. Inkjet bioprinters, however, have limitations on material viscosity and require excessive force to eject droplets of solutions with viscosity above 10 cP (mPa-s).38 

Microextrusion Bioprinters Microextrusion printers employ pneumatic, piston, or screw pumps for controlled extrusion of the printing material onto the substrate. Unlike liquid droplets in the inkjet printers, small beads of the printing material are deposited with the help of a microextrusion head. The deposition occurs in two dimensions and the stage is moved vertically, as directed by the CAD-CAM (computer-aided manufacturing) software.42 A variety of materials including hydrogels, copolymers, and cell spheroids are compatible for microextrusion printing. It can even be employed for printing using highly viscous materials (30–6 × 107 mPa-s or even above) and sheer thinning materials.38 Also, a number of curing methods such as ionic cross-linking, photo-cross-linking, and thermal solidification can be employed for the fabrication of functional, complex, and composite tissues.43 However, use of viscous ink and encapsulation of cells reduces the viability. In comparison to both inkjet and laser-assisted printing, the printing speed of microextrusion bioprinters is very low.34,38 

Laser-Assisted Bioprinters Laser-assisted bioprinters are composed of a laser source, focusing lens, ribbon, absorbent layer, biomaterial layer, and the substrate.44 The laser beam generates a high-pressure bubble in the energy-absorbing layer that results in the ejection of a bioink droplet toward the substrate. This method is used to deposit a very small amount of bioink in both the liquid and solid phases, with microscale resolution. Nozzle-free deposition and low mechanical stress offer the advantages of high cell viability.45 Materials with wider viscosity range (1–300 mPa-s) can be printed. These printers are capable of capturing a single cell in one droplet and thus can be used for the deposition of multiple types of cells. However, preparing ribbons in such cases requires excessive efforts.34 

3D PRINTED DISEASE MODELS 3D bioprinting is still in its infancy but recent investigations for the development of 3D printed tissues, such as cardiovascular tissues, hepatic tissues, and neural tissues, have captivated medical researchers to utilize its potential in disease modeling.

Cardiovascular Disease Models Cardiovascular diseases encompass a group of disorders that affect the heart and blood vessels. 3D printed disease models are a great alternative to understand the physiologic and biochemical mechanisms behind these

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B FIG. 7.4  (A) Confocal images of a 3D printed cardiac tissue model. (i) condensed cardiac tissue with

cardiomyocytes and fibroblasts. (ii) cardiomyocetes aligned on one fiber showing the sarcomere structure ensuring the electrical integration within the cardiac tissue. (iii & iv) LQT3 iPS-CMs growing on the middle layer of filamentous matrices. (Reproduced from Ma et al., with permission from Elsevier Ltd. Copyright 2014). (B) Fluorescent images of a 3D printed cardiac tissue construct i), ii), & iii) with uniform channel width and iv), v) & vi) with gradient channel widths. HUVECs (red) are encapsulated in the intended channels and HepG2 (green) are encapsulated in the surrounding area. Scale bars, 250 mm. (Reproduced from Zhu et al., with permission from Elsevier Ltd. Copyright 2017).

disorders. Mosadegh and group46 reported the development of a paper-based 3D culture model to mimic cardiac ischemia. Cardiac cells printed onto paper sheets were stacked together in a hydrogel to form a layered 3D model of cardiac tissue. To replicate cardiac ischemia, stress was generated by modulating mass transport of oxygen and glucose. As a result of the induced stress, the cardiac cells secreted chemokines that triggered

the migration of cells toward the ischemic region in the tissue. Ma and group developed a patient-specific 3D cardiac model to replicate condensed and aligned human ventricular myocardium (Fig. 7.4A). Synthetic filamentous matrices using a laser-based writing technique were fabricated and populated with cardiac cells from a healthy volunteer and induced pluripotent stem cells from a patient with long QT syndrome type 3.

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The developed model was then used to study the associated contractility malfunctions and response of the model to various drugs. The generated tissue model exhibited different levels of contractile abnormality and susceptibility to drug-induced cardiotoxicity on varying matrix stiffnesses.47 Bioprinting has also been employed for the fabric­ ation of vascular tissues. A complex 3D vascular mic­ rostructure was printed using cells encapsulated in hydrogels. The printed endothelial cells spontaneously produced a lumenlike structure in vitro (Fig. 7.4B) and formed endothelial network in the prevascularized tissue when implanted under the dorsal skin of an immunodeficient mice.48 In a study, calcific aortic valve disease was modeled using a 3D bioprinted construct. In this study, valvular interstitial cells were encapsulated in methacrylated gelatin/methacrylated hyaluronic acid hydrogels to recapitulate the leaflet layer biomechanics of the human aortic valve. Microcalcification in the models was induced by exposure to osteoge­nic media and pathogenesis assessment was conducted by near-infrared or immunofluorescence microscopy.49 

Neural Disease Models Neurologic diseases affect more than 1 billion people globally. Lack of suitable disease models limits the therapeutic interventions for coping neurologic disorders because of the complexity of the nervous system. 3D bioprinting has been used in the recent years to generate controllable neural disease models that can replicate the complex microscale features while accounting for the diversity of cells and tissuelevel responses. Complex 3D neural structures were printed using rat embryonic hippocampal and cortical neurons by a thermal inkjet bioprinter. The printed neurons maintained their functionality including cellular properties, healthy neuronal phenotype, and electrophysiologic characteristics.50 In addition, 3D nanosheets were also fabricated using NT2 cells (clonally derived, pluripotent human embryonal carcinoma cell line). An artificial 3D printed neural tissue model indicated that time-released delivery of soluble GFs promotes the migration and proliferation of neural cells. Murine neural stem cells were fabricated in collagen hydrogel with engineered fibrin hydrogel (containing vascular endothelial GF). The cells in the collagen hydrogel immigrated toward the fibrin gel and showed GF-induced morphologic changes.51 Moreover, 3D bioprinted neural constructs were shown to be capable of rescuing neural injuries in animal models. Neural stem cell–laden thermoresponsive biodegradable

polyurethane hydrogels were bioprinted and tuned for mechanical properties. The bioprinted hydrogels were then injected into zebrafish embryo neural injury model to evaluate their repairing potential. The results showed that the developed cell-laden hydrogels had the potential to rescue traumatic brain injury in the zebrafish model.52 Another research group constructed a brainlike 3D structure using peptide-modified biopolymer and gellan gum-RGD, combined with primary cortical neurons.53 The peptide modification of the hydrogel enhanced cellular proliferation and networking of the neural cells. 3D printed artificial axons were engineered for studying the myelination process, which is associated with various neural diseases such as multiple sclerosis. The bioprinted axons resembled the geometric, mechanical, and surface chemistry components of the biological axons. The study demonstrated that myelination and proliferation of oligodendrocytes is highly dependent on the stiffness, surface chemistry, and size (diameter) of the axons (Fig. 7.5).54 

Hepatic Disease Models Bioprinting has also been used to print hepatic tissue models. A 3D model of hepatic tissue consisting of 30 layers of the printing material was constructed (hepatocytes in gelatin hydrogel).55 The printed cells remained viable and functional even after 2 months of printing. Zhong and group56 also fabricated hepatic cells in hydrogel scaffolds and tested their biocompatibility in nude mice. The responses between mice engrafted with control, hydrogel alone, hydrogel with hepatocytes, and hydrogel with hepatocytes and GF were compared (Fig. 7.6). The mice treated with 3D hydrogel scaffold containing hepatocytes had longer survival time than other groups. A functional 3D printed mini-liver was also constructed for studying in vitro drug metabolism. An automated syringe-based direct cell writing bioprinting process with micropatterning techniques was employed for the fabrication of a microscale device encapsulating a hydrogel-based liver tissue construct. The fabricated model recapitulated the dynamic perfusion for the collective drug metabolic function.57 A patient-specific hepatic model that replicates the native cellular architecture and microenvironment has also been demonstrated. Hepatic cells derived from humaninduced pluripotent stem cells and supporting cells (originated from endothelial and mesenchymal cells) were encapsulated in hydrogel to mimic hepatic lobule structure, using photopolymerization-based hydrogel activation. The researchers were able to create hepatic models with improved morphologic organization,

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B

C FIG. 7.5  Bioprinted neural constructs. (A) Confocal microscopy images of printed rat embryonic hip-

pocampal neurons stained with neuronal markers after 15 days of culture highlighting. (i) The cell bodies and dendrites (in green) and (ii) The cell bodies and dendrites (in green), and the axons (in red). (Reproduced from Xu et al., with permission from Elsevier Ltd. Copyright 2006). (B) Artificial 3D printed axons as examined by phase contrast microscopy (left) and confocal microscopy (right). (Reproduced from Espinosa et al., with permission from Nature Scientific Reports. Copyright 2018). (C) 3D printed collagen scaffold as evaluated by bright-field microscopy (left column), Calcein AM live (middle column) and ethidium-homodimer (EthD-1) dead fluorescence (right column) tests after 3 days of culture in serum-containing media. (Reproduced from Lee et al., with permission from Elsevier Inc. Copyright 2010).

higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction.58 

Musculoskeletal Disease Models 3D bioprinters are also being employed to print stable and clinically relevant musculoskeletal tissue con­ structs. Spatially organized bone tissue using cell-laden hydrogel by a microextrusion bioprinter was fabricated.59 The viability of the printed cells was found to be similar to that of the cells that were cultured

using conventional in vitro assays. The study also suggested the feasibility of printing two different types of cells within a single scaffold simply by exchanging the microsyringe for depositing different cell types. A 3D printed model of bladder tissue using droplet printing method was also reported.60 The bioprinted blocks of smooth muscle cells were found to be capable of consistent proliferation. Also, these blocks were assembled together and cultured for 51 days to create a smooth muscle construct patch, which resembled the native structure of the rat bladder.

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A

B FIG. 7.6  3D printed liver tissue construct. (A) 3D printed porous scaffold with hepatocytes. (B) The

engrafted tissue morphology of 3D printed hepatic cells in hydrogel scaffold in different experimental mice groups. The “liver” indicates the mice liver, “hy” represent hydrogel, and “3D” represents the 3D scaffold loaded with hepatic cells. cKit and CK18 were the specific antigens in hepatocytes. (Reproduced from Zhong et al., with permission from Elsevier Pte Ltd. Copyright 2016).

Similarly, cartilage tissue was bioprinted using chondrocytes in nanocellulose–alginate bioink.61 The printability and shape fidelity of the printed construct was evaluated and anatomic models for human ear and sheep meniscus were printed using MRI and CT images. Notably, the use of integrated tissue–organ printer to

fabricate the mandible and calvarial bone (Fig. 7.7B), cartilage (Fig. 7.7C), and skeletal muscle (Fig. 7.7D) using cell-laden composite hydrogels, supporting poly(ε-caprolactone) polymer and a sacrificial Pluronic hydrogel, was also demonstrated.30 A novel microplate 3D bioprinting platform with automated production

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B

C

D

E FIG. 7.7  (A) 2D patterning of ‘WFIRM’ characters written by cell-laden hydrogels through the integrated

organ printing. Microscopic (left) and Fluorescent (Right). (B) Two different patterns of printing. (C) Calvarial bone reconstruction. (D) 3D printing of ear construct. 3D printed muscle cell-laden hydrogel; bioprinted muscle construct after 2 weeks of implantation, and immunostaining images confirming the presence of organized muscle fibers and innervating capability within the implanted construct. (Reproduced from Kang et al., with permission from Springer Nature. Copyright 2016).

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A

B FIG. 7.8  3D printed cancer model showing. (A) Different printing patterns and (B) colocalization of het-

erotypic co-cultures of macrophages (in green) and tumor cells (in red) initially (left) and after 4 days (right). Dotted gray lines represent the hollow inner channel. Scale bar is 400 μm. (Reproduced from Grolman et al, with permission from John Wiley and Sons. Copyright 2015).

of muscle and tendon tissue was developed for drug screening.62 Monocultures and cocultures of human primary skeletal muscle cells and rat tenocytes were printed in a standard 24-well plate screening format for addressing musculoskeletal diseases. 

Cancer Disease Models Despite many advances in treatment over the past decades, cancer is still the second leading cause of death after cardiac diseases, globally. Yet, very little is known about the exact mechanism behind cancer progression and metastasis. Application of 3D printing for fabricating cancer models can serve as robust platforms to study mechanisms of cancer progression and high-throughput screening of drugs and will aid in the development of new therapies. The key characteristics of cancerous cells’ uncontrolled proliferation and metastasis have been studied using 3D printed models. A cancer model was fabricated using 3D polyethylene glycol (PEG) scaffolds to compare the 3D migration properties of normal breast epithelial cells and twisttransformed cells.63 The results of the study indicated that there is substantial difference in the migration of cells in the 2D and 3D models, which included differences in cell displacement, velocity, and direction.

A 3D microchip having varying channel width to mimic blood vessels was printed using hydrogel and analyzed for the differences between cancerous and noncancerous cell models. It was found that the migration of cancerous cells in the printed 3D model increased with decrease in the size of the channel. A customized bioprinting system was used to demonstrate the programmed fabrication of cellular spheroid on a chip.64 Breast cancer cell–laden sacrificial gelatin arrays were printed and used as templates for the development of concave wells with PEG-DMA (dimethacrylate) hydrogel. Reversing gelatin hydrogel into the sol phase facilitated the in situ formation of tumor spheroids with high cell viability. Moreover, scaffold-free 3D neural tissue culture for examining the invasion of glioma cells has also developed.49 Such 3D spheroid systems are highly beneficial for testing and screening of drugs and therapeutics. A cervical tumor model was constructed by printing cervical cancer cells encapsulated in gelatin/alginate/fibrinogen hydrogels.65 The study highlighted that the 3D printed spheroid had higher (90%) cell viability, higher matrix metalloproteinase protein expression, and higher resistance to paclitaxel than the 2D cultured cells. The glioma stem cell 3D model was bioprinted using gelatin/alginate/fibrinogen hydrogel,

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FIG. 7.9  3D printed model for conjuctivital defect in rabbits. (A) The final defect (B) 3D printed Gelatin-

based membrane, (C) AM cut into an 8-mm round piece, (D) Representative eye implanted with Gelatinbased membrane, (E) Representative eye grafted with AM. (Reproduced from Dehghani et al., with permission from Elsevier Ltd. Copyright 2018.

which showed the characteristics of cancer stem cells, including vascularization. This 3D model showed enhanced resistance to the drug temozolomide in comparison to the 2D cell culture studies.66 3D printed models have also been developed to study interactions of cancerous cells with other cells. A 3D bioprinted model of ovarian cancer cells and healthy fibroblasts was created to study the cellular interactions. Cancerous cells were patterned at a distance of 1 and 10 mm from fibroblasts in two different models. It was found that in the model where cells were patterned close to each other, larger 3D micronodules were produced.67 3D cancer models were also constructed using alginate fiber to study the colocalization of adenocarcinoma cells and macrophages.68 The cells were printed in different structures having macrophages in the core and cancer cells in the sheath (Fig. 7.8A) and were analyzed with CellTracker on the day of culture and after 4 days (Fig. 7.8B). Depending on the architecture of the model, outward migration of macrophages occurred, which altered the macrophage/tumor cell ratios. 

Other Disease Models A 3D printed membrane was fabricated using gelatin, elastin and sodium hyaluronate blend for application in conjunctival reconstruction.69 The printed membrane was characterized for the mechanical and physical parameters to compare with amniotic membrane and tested in vitro (for viability, proliferation, and adhesion of epithelial cells) and in vivo (in rabbits) for analyzing the healing potential (Fig. 7.9). A study demonstrated

the development of a 3D printed model for mimicking the intestinal villi epithelium.70 Cell-laden collagen bioink cross-linked with tannic acid was used to obtain a 3D collagen villus structure with appropriate geometry. In vitro analysis of the model suggested that the viability and differentiation of the cells in the tested model was higher than that of the control. In the work by Rong Lu and group, 3D printing was employed to develop models to study host–pathogen interactions in communicable diseases.71 A human intestinal epithelial model with high cell viability and proper morphology was printed and the inner side of the printed model was infected with the bacteria Salmonella. The results of western blot analysis and cytokine analysis showed enhanced gene expression of the inflammatory regulators (p65 and interleukins) and decreased concentration of the protein occludin, which are similar to observations made under in vivo conditions. 

CONCLUSION AND FUTURE SCOPE Recent advancements in the field of 3D printing have enabled the scientists to build models with a physiologically relevant cell composition, appropriate material properties, and a complex architecture with proper vascularization. However, bioprinting is still in its infancy and to thrive on, further research and innovations are needed to possibly eliminate the need for animal or human clinical trials in the future. Some of the innovations that should be considered may involve the development of technologies for integration of printed

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constructs for in vivo safety and efficacy ­studies, development of postprinting culture platforms, enhancement of the capability to print and handle large volumes of printing materials, incorporation with other technologies such as microfluidics, and advancements of the imaging systems and analysis tools.

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FURTHER READING 1. Costa PF, Albers HJ, Linssen JE, et al. Mimicking arterial thrombosis in a 3D-printed microfluidic in vitro vascular model based on computed tomography angiography data. Lab Chip. 2017;17(16):2785–2792.

2. Roopavath UK, Kalaskar DM. Introduction to 3D printing in medicine. In: 3D Printing in Medicine. 2017:1–20. 3. Zhang YS, Davoudi F, Walch P, et al. Bioprinted thrombosis-on-a-chip. Lab Chip. 2016;16(21):4097–4105.