A. 3D bioprinting of esophageal cancer models

Esophageal cancer is one of the most aggressive tumors in the gastrointestinal tract. It is the seventh most commonly occurring cancer and the sixth prime cause of cancer-related mortality. The overall 5-year survival rate ranges from 15% to 25% worldwide; approximately 400 000 people die every year because of this cancer. Squamous cell carcinoma and adenocarcinoma are the two main types of cancer. A prognosis of the tumor remains poor despite improved outcomes compared to other types of solid tumors.2828. M. Davern, N. E. Donlon, R. Power, C. Hayes, R. King, M. R. Dunne, and J. V. Reynolds, Br. J. Cancer 125, 479 (2021). https://doi.org/10.1038/s41416-021-01331-yEsophageal cancer very often requires surgical resection of the damaged portion of the esophagus through esophagectomy. A major disadvantage of this procedure is the creation of a large defect area on the wall of the esophagus. Incidences of other esophageal diseases like congenital esophageal stenosis, trachea-esophageal fistula, and esophageal atresia are also on the rise. Most often, these congenital or acquired esophageal disorders require reconstruction of the defective site. However, esophageal tissue is difficult to regenerate owing to its weak regenerative ability. Similarly, surgical resection and ablation in these cases can cause postoperative complications such as anastomotic leaks, cardiopulmonary complications, and infection, leading to a median survival time ranging from 13 to 19 months.2929. J. D. Urschel, Am. J. Surg. 169, 634 (1995). https://doi.org/10.1016/S0002-9610(99)80238-4 Hence, tissue engineering is suggested as an encouraging substitute for reconstructing circumferential defects.3030. H. Nam et al., Sci. Rep. 10, 7255 (2020). https://doi.org/10.1038/s41598-020-64049-6 Tissue-engineered tubular grafts can be alternatives because these could replace the excised esophageal tissue and can, therefore, restore the integrity and continuity of the esophagus with reduced complications.

An ideal substitute for a damaged esophagus is one that does not cause immunological rejection. The implant must be able to support cell adhesion and proliferation, exhibit optimum porosity with an acceptable pore size for cell invasion, and replicate the structure and function of the esophageal layers. It must be soft and elastic to allow the passage of food. Furthermore, it has to propagate peristalsis, as well as possess lubricating capacity and endurance to acid reflux disease. This biodegradable substitute would preferably act as a temporary template for the regeneration of a biologically functional esophagus and would be slowly replaced by de nova esophageal tissue, thereby avoiding subsequent surgeries.

Radiotherapy is an important treatment method for esophageal cancer, and it involves radiation esophagitis, which is a critical complication. This is characterized by inflammation of the esophagitis and results in discomfort, pain, and even death. This is the most commonly occurring acute adverse effect of radiation therapy, which is extensively used for treating cancer symptoms.31,3231. Z. Nesheiwat, H. Akbar, A. Kahloon, and K. Mahajan, “Radiation esophagitis,” in StatPearls (StatPearls Publishing, Treasure Island, FL, 2022), see https://www.ncbi.nlm.nih.gov/books/NBK499920/.32. L. Ma, Y. Men, L. Feng, J. Kang, X. Sun, M. Yuan, W. Jiang, and Z. Hui, Radiol. Oncol. 53, 6 (2019). https://doi.org/10.2478/raon-2019-0006 Around 25% of patients with radiation esophagitis develop complications like eating disorders requiring intubation, obstruction, and fistula formation.66. L. Ventola, Pharm. Ther. 39, 704 (2014). Currently available treatments for these are palliative with intraesophageal delivery of therapeutic medicines, which can significantly attenuate esophageal inflammation.33,3433. H. G. Wu, S. Y. Song, Y. S. Kim, Y. T. Oh, C. G. Lee, K. C. Keum, Y. C. Ahn, and S.-W. Lee, Cancer 115, 3699 (2009). https://doi.org/10.1002/cncr.2441434. M. I. Koukourakis et al., Clin. Cancer 5, 3970 (1999). These treatment protocols usually require repeated delivery and often have limited efficacy due to esophageal peristalsis, which results in rapid flushing. Hence, designing fundamental treatment targeting the damaged tissue is extremely crucial. Tissue engineering based on 3D bioprinting can provide ideal solutions and an unlimited supply of substitutes for esophageal engineering, where it is possible to exquisitely tailor esophageal substitutes for patients according to tissue morphology and pathology.

Until date, research on 3D bioprinting in esophageal cancer treatment has focused on the development of the 3D bioprinted esophageal models that have therapeutic applications in esophageal cancer patients who have undergone esophagectomy or those in whom radiation esophagitis has led to esophageal dysfunction. Hence, in this part of the review, we describe the 3D bioprinting of esophageal models.

3D bioprinting is a very new approach in esophageal tissue engineering, and Park et al.3535. S. Y. Park, J. W. Choi, J.-K. Park, E. H. Song, S. A. Park, Y. S. Kim, Y. S. Shin, and C-.H. Kim, Interact. CardioVasc. Thorac. Surg. 22, 712 (2016). https://doi.org/10.1093/icvts/ivw048 published one of the earliest remarkable studies fabricating an artificial scaffold for reconstructing partial esophageal defects. In this study, an extrusion-based bioprinter was used to build a grid structure of polycaprolactone (PCL), forming 5-mm-diameter/10-mm-high acellular cylindrical scaffolds with a wall thickness of 2 mm. In this model, the fibrin/MSC-coated 3D bioprinted PCL scaffolds were implanted on a 5 × 10 mm artificial esophageal defect in three rabbits (3D bioprinted/MSC group), and scaffolds with only 3D bioprinted PCL were implanted in three more rabbits (control group). Three weeks post-procedure, the implanted sites were assessed radiologically and histologically with no evidence of any infection or stenosis observed during computed tomography. In the 3D bioprinted/MSC group, the replaced scaffolds were entirely covered with regenerating mucosal epithelium and smooth muscle cells, as determined by hematoxylin and eosin and desmin staining; this was absent in the 3D bioprinted-only group. The authors concluded that the use of the 3D printed scaffolds coated with MSCs seeded in fibrin led to a successful restoration of the shape and histology of the cervical esophagus without causing any graft rejection, thus attesting their role as a promising material for use as an artificial esophagus. However, the long-term effects, translation to clinical practice, and subsequent immunologic reactions were not assessed in this study.Tan et al.3636. Y. J. Tan, X. Tan, W. Y. Yeong, and S. B. Tor, Sci. Rep. 6, 39140 (2016). https://doi.org/10.1038/srep39140 reported another 3D bioprinting technique, where instead of conventional scaffold-free cell-laden hydrogels and tissue spheroids/strands, cell-laden microscaffolds are used as building blocks in bioprinting. This could possibly solve the problem of cell source shortage. In this study, distinct types of cells were printed successfully. The cells were viable after printing, and they continued to proliferate with culture time. The bioprinted construct showed excessive biocompatibility with a cell viability of more than 90% following culturing for 2, 7, and 14 days. In addition, the mechanical strength of the construct was improved significantly by more than 100 times compared to that of the agarose–collagen composite hydrogel. Besides combining the advantage of solid scaffolds with the new 3D bioprinting concept, this work permitted the fabrication of multiscale and multicellular 3D tissue constructs, thus giving promising substitutes for bioprinted clinically relevant tissue replacements and functional in vitro biological models3636. Y. J. Tan, X. Tan, W. Y. Yeong, and S. B. Tor, Sci. Rep. 6, 39140 (2016). https://doi.org/10.1038/srep39140Another 3D bioprinting technique was introduced by Chung et al.3737. E. J. Chung et al., Nanomed. Biotechnol. 46, 885 (2018). using the 3D melt-extrusion method. In this model, the authors demonstrated vascularization of tubular scaffolds measuring approximately 5 mm in length and 1.6 mm in diameter, which was not reported previously. Structural analysis showed that the average pore size of scaffolds was approximately 5.1 μm and their ultimate tensile stress and yield strength were more than those of the native rat esophagus, although the elastic modulus appeared to be similar. The in vitro PCL-based scaffolds were able to support the survival and proliferation of fibroblasts. The tubular scaffolds were orthotopically implanted into surgically generated circumferential defects following the implantation of tubular scaffolds into the omentum of rats. Histological analysis via hematoxylin and eosin staining indicating the neoformation of blood vessels on the exteriors of scaffolds has been reported. The study revealed that the host cells could infiltrate the construct and spread throughout its inner and outer membranes, providing evidence supporting cell migration and survival, as well as tissue vascularization.3737. E. J. Chung et al., Nanomed. Biotechnol. 46, 885 (2018).A method combining electronanospinning and 3D bioprinting was developed by Kim et al.3838. I. G. Kim, Y. Wu, S. A. Park, H. Cho, J. J. Choi, S. K. Kwon, J.-W. Shin, and E.-J. Chung, Tissue Eng., Part A 25, 1478 (2019). https://doi.org/10.1089/ten.tea.2018.0277 The major advantage of this hybrid method is the early implantation. The authors investigated the effects of the 3D printed esophageal grafts and bioreactor cultivation on muscle regeneration and re-epithelialization from circumferential esophageal defects in a rat model. For this model, the authors fabricated a novel two-layered tubular scaffold as an artificial esophagus comprising electrospun nanofibers (inner layer) and 3D printed strands (outer layer). Human mesenchymal stem cells (hMSCs) were seeded onto the inner layer of the scaffold to aid re-epithelialization. The inner nanofiber structure functioned as a template for the mucosal layer by bestowing topographical cues for cell migration, and the 3D printed strand presented good mechanical strength and flexibility as the framework of the esophagus. To attain full regeneration of the esophageal mucosa and muscle layer, additional bioactivity for esophageal reconstruction was conferred on this scaffold through bioreactor cultivation and convergence of the thyroid gland flap on the implanted scaffold.3838. I. G. Kim, Y. Wu, S. A. Park, H. Cho, J. J. Choi, S. K. Kwon, J.-W. Shin, and E.-J. Chung, Tissue Eng., Part A 25, 1478 (2019). https://doi.org/10.1089/ten.tea.2018.0277 One of the major challenges of 3D bioprinting of tissue constructs is the immunologic host response to the scaffold itself and its degradation. To overcome this challenge, Takeoka et al.3939. Y. Takeoka et al., PLoS ONE 14, e0211339 (2019). https://doi.org/10.1371/journal.pone.0211339 developed a scaffold-free structure with a mixture of cell types using 3D bioprinting technology and evaluated its characteristics in vitro and in vivo after transplantation into rats. The cell sources used were normal human dermal fibroblasts, human esophageal smooth muscle cells, human bone marrow-derived mesenchymal stem cells, and human umbilical vein endothelial cells. Following the preparation of multicellular spheroids, esophagus-like tube structures were prepared by 3D bioprinting. In a bioreactor, the structures were allowed to mature and later transplanted into male rats as esophageal implants with silicon stents, thereby providing a promising substitute for the repair of esophageal defects.3939. Y. Takeoka et al., PLoS ONE 14, e0211339 (2019). https://doi.org/10.1371/journal.pone.0211339Another advancement in the field of 3D bioprinted esophageal constructs is the development of an innovative artificial esophageal construct that can impersonate the native esophagus using a unique dragging 3D printing technique through an extrusion-based bioprinter. This is an advanced multilayered free-form porous tubular construct. Using mucosal and muscular layers of the esophagus, decellularized bioinks were developed to produce a structure that revealed the required cell specificity to accurately recreate the esophagus.55. H. Arun, Healthcare 3D Printing Market 2018 Prominent Players-Aprecia Pharmaceuticals, Aspect Biosystems, Bio 3D Technologies, Biobots, Cyfuse Biomedical, Digilab, 3 Dynamics Systems, Envision Tec, Luxexcel, Materialise NV, Nano 3D Biosciences, Oceanz, OrganovoHo. (2019), see http://www.openpr.com. The uniqueness of this model lies in the use of specific bioinks derived from different decellularized esophageal layers, which are extremely beneficial for inducing proliferation. To overcome the complications induced by radiation esophagitis, secondary to radiation therapy for esophageal cancer, Ha et al.4040. D. H. Ha et al., Biomaterials 266, 120477 (2021). https://doi.org/10.1016/j.biomaterials.2020.120477 developed 3D printed esophageal stents using a decellularized ECM (dECM)-based hydrogel with tissue-healing effects. An exclusive method using a rotating rod combined with a 3D printing system (2RPS) was employed for fabricating a rat model. The exclusivity of this stent was the novel design that included an outer open-groove reservoir structure effective for holding bioactive materials such as hydrogels and drugs. The study demonstrated the efficacy of the tissue-specific dECM hydrogel in retrieval from radiation through induced inflammation without the use of drugs or cells. As the hydrogel allows for cell encapsulation, the tissue-specific hydrogel functioned as a potential biomaterial for the carriers of various desired agents. This model can be expanded to the treatment of other inflammation-based local injuries like wounds, burns, and tissue necrosis. The tissue-specific dECM hydrogel-loaded stents could serve as promising platforms to deliver therapeutic cells. Moreover, this can be beneficial to develop drugs for complex and diverse disorders, including cancer.4040. D. H. Ha et al., Biomaterials 266, 120477 (2021). https://doi.org/10.1016/j.biomaterials.2020.120477This part of the review highlights the tremendous research being conducted in developing the 3D bioprinted constructs as implants in esophageal cancer and other diseases with esophageal defects, as shown in Table I. Until date, no cancer models have been reported for understanding cancer biology, and drug screening and discovery.

TABLE I. Summary of the different methods used in the 3D bioprinting of the esophageal cancer models.

BioinksTechniqueApplicationReferencePCLExtrusion-based bioprinter3D printed PCL scaffold with allogenic MSCs can be potential materials for anatomical and functional reconstruction of the esophagus3535. S. Y. Park, J. W. Choi, J.-K. Park, E. H. Song, S. A. Park, Y. S. Kim, Y. S. Shin, and C-.H. Kim, Interact. CardioVasc. Thorac. Surg. 22, 712 (2016). https://doi.org/10.1093/icvts/ivw048Decellularized bioinksExtrusion-based 3D printing, namely, the dragging techniqueAdvanced multilayered construct for treatment of full-thickness circumferential esophageal defects3030. H. Nam et al., Sci. Rep. 10, 7255 (2020). https://doi.org/10.1038/s41598-020-64049-6MSC-seeded tubular scaffold with a bioreactor systemElectrospinning and 3D printingA novel approach for circumferential esophageal reconstruction3838. I. G. Kim, Y. Wu, S. A. Park, H. Cho, J. J. Choi, S. K. Kwon, J.-W. Shin, and E.-J. Chung, Tissue Eng., Part A 25, 1478 (2019). https://doi.org/10.1089/ten.tea.2018.0277Cell-laden poly(D,L-lactic-co-glycolic acid) (PLGA)Extrusion-based printing methodTissue replacements and biological models3636. Y. J. Tan, X. Tan, W. Y. Yeong, and S. B. Tor, Sci. Rep. 6, 39140 (2016). https://doi.org/10.1038/srep39140Human dermal fibroblasts, human esophageal smooth muscle cells, human bone marrow-derived mesenchymal stem cells, and human umbilical vein endothelial cells3D bioprintingRepair of esophageal defects3939. Y. Takeoka et al., PLoS ONE 14, e0211339 (2019). https://doi.org/10.1371/journal.pone.0211339dECM hydrogel-loaded stentRotating rod combined with 3D printing system (2RPS)Local treatment of radiation esophagitis4040. D. H. Ha et al., Biomaterials 266, 120477 (2021). https://doi.org/10.1016/j.biomaterials.2020.120477Poly(caprolactone) (PCL)Combining 3D printing and electrospinningRepair of circumferential esophageal defects3737. E. J. Chung et al., Nanomed. Biotechnol. 46, 885 (2018).

B. 3D bioprinting of liver cancer models

The liver has a pivotal role in a variety of critical bodily functions, such as detoxification of the systemic and portal blood, and secretion of multiple proteins and bile components. Cirrhosis of the liver is a major risk factor for hepatocellular carcinoma (HCC) and has emerged as an important public health problem worldwide. Many patients with end-stage liver disease die waiting for an organ because donors are scarce, leading to alternative strategies for liver replacement. Despite the increased morbidity and mortality associated with it, effective therapies are still scarce.4141. S. L. Friedman, D. Sheppard, J. S. Duffield, and S. Violette, Sci. Transl. Med. 5, 167sr161 (2013). https://doi.org/10.1126/scitranslmed.3004700 The current therapies for cirrhosis are mostly palliative.4242. D. Schuppan and Y. O. Kim, J. Clin. Invest. 123, 1887 (2013). https://doi.org/10.1172/JCI66028 One of the major challenges for developing new and efficient therapies is the lack of robust and biomimetic in vitro models of cirrhosis for drug screening studies. Traditional animal models are still the ones mostly being used for preclinical testing. However, animal models fare poorly in predicting human physiological responses in terms of efficacy and toxicity.43,4443. R. Greek and A. Menache, Int. J. Med. Sci. 10, 206 (2013). https://doi.org/10.7150/ijms.552944. D. Schuster, C. Laggner, and T. Langer, Curr. Pharm. Des. 11, 3545 (2005). https://doi.org/10.2174/138161205774414510 Furthermore, ethical concerns and the need for more predictive and efficient models led to studies developing in vitro technologies for hepatobiliary cancer.HCC accounts for 90% of primary malignant tumors of the liver. It is the fifth most common malignant cancer worldwide.4545. R. L. Siegel, K. D. Miller, and A. Jemal, CA: Cancer J. Clin. 70, 7 (2020). https://doi.org/10.3322/caac.21590 The overall survival rate of HCC is approximately 18%.4545. R. L. Siegel, K. D. Miller, and A. Jemal, CA: Cancer J. Clin. 70, 7 (2020). https://doi.org/10.3322/caac.21590 The poor prognosis is attributed to the advanced stage of diagnosis and limited treatment options available. Rodent models fail to fully simulate the complex human cancer phenotypes owing to variations in the functional profile of liver tissue among different species, thus hindering the progress in drug development.46,4746. V. L. Tsang, A. A. Chen, L. M. Cho, K. D. Jadin, R. L. Sah, S. DeLong, J. L. West, and S. N. Bhatia, FASEB J. 21, 790 (2007). https://doi.org/10.1096/fj.06-7117com47. N. J. Hewitt et al., Drug Metab. Rev. 39, 159 (2007). https://doi.org/10.1080/03602530601093489 Additionally, the phenotypic and genotypic differences in HCC among patients are not represented in the existing models. To overcome this drawback, various 3D bioprinting techniques have been used to develop biomimetic liver tissue constructs that can effectively recapitulate the in vivo liver tissue microenvironment, including angiogenesis and accurate spatiotemporal signaling.48–5148. A. Faulkner-Jones, C. Fyfe, D.-J. Cornelissen, J. Gardner, J. King, A. Courtney, and W. Shu, Biofabrication 7, 044102 (2015). https://doi.org/10.1088/1758-5090/7/4/04410249. K. Kang et al., Tissue Eng., Part A 24, 576 (2018). https://doi.org/10.1089/ten.tea.2017.016150. S. Mao, Y. Pang, T. Liu, Y. Shao, J. He, H. Yang, Y. Mao, and W. Sun, Biofabrication 12, 042001 (2020). https://doi.org/10.1088/1758-5090/ab97c051. X. Ma, C. Yu, P. Want, W. Xu, X. Wan, C. S. E. Lai, J. Liu, A. Koroleva-Maharajh, and S. Chen, Biomaterials 185, 310 (2018). https://doi.org/10.1016/j.biomaterials.2018.09.026

This part of the review summarizes recent progress in the development of 3D bioprinted cancer models with a strong emphasis on the processing, properties, and therapeutic implications of the developed models.

In 2015, Faulkner-Jones et al. developed 3D constructs of the liver tissue for the first time using human pluripotent stem cells (hPSCs) in hydrogel bioinks (e.g., cross-linked calcium alginate). They evaluated the influence of nozzle geometry and length on the postprinting viability of cells, revealing that longer nozzles were associated with decreased viability of cells in the postprinting phase. The authors also demonstrated that on the 21st day of differentiation, the cells reached the peak albumin secretion in the 40-layer HLC containing alginate in the 3D bioprinted constructs. The authors observed that the cells were positive for albumin, indicating their hepatic origin. When compared to the normal duration of 17–24 days for 2D differentiation of hPSC-HLCs, 3D printed cells took a longer time to reach the maximum albumin secretion. Interestingly, there was a proportionate increase in albumin secretion with the increase in the number of layers. This supports the notion that 3D differentiation and maturation of cells are influenced by the permeability of alginate hydrogel that allows nutrition and differentiation reagents to enter the structure, and these are unrelated to the height of the printed structure. This work shows that the valve-based printing process is useful in printing hPSCs (both human embryonic stem cells and human induced pluripotent stem cells) quite gently without losing the pluripotency, while simultaneously inducing differentiation into specific lineages.

However, the authors do not explain the molecular mechanism and transcriptional profiling of the 3D printed liver cancer model. Nevertheless, these initial findings form the basis for personalized in vitro human liver cancer models to better understand tumor biology and serve as a potential platform for screening drugs, thereby leading to improved clinical and therapeutic outcomes.4848. A. Faulkner-Jones, C. Fyfe, D.-J. Cornelissen, J. Gardner, J. King, A. Courtney, and W. Shu, Biofabrication 7, 044102 (2015). https://doi.org/10.1088/1758-5090/7/4/044102

Furthermore, research on the 3D bioprinted hepatobiliary cancer models has explored gene expression profiles and dose–effect responses of the relevant anticancer drugs.

Accordingly, Sun et al. developed a 3D bioprinted model using HepG2 cells and compared it with the 2D cultured tumor cells.5252. L. Sun et al., Front. Oncol. 10, 878 (2020). https://doi.org/10.3389/fonc.2020.00878 They used gelatin and sodium alginate as bioinks owing to their stable structures, adequate biocompatibility, and low cost. Figure 2(a) shows the printed liver cancer cell model, designated as 3DP-HepG2. The authors reported marked improvement in the expression of tumor-related genes including ALB, AFP, CD133, IL-8, EpCAM, CD24, and TGF-β in the 3D bioprinted model compared to that observed in the 2D model. They also found significant differences in the gene expression profiles related to liver cell function and tumors as well as in drug resistance genes in response to antitumor drugs among the 3D bioprinted models and the 2D ones (Fig. 3).Patient-derived intrahepatic cholangiocarcinoma (ICC) cells printed with gelatin–alginate Matrigel showed excellent results in terms of colony-forming abilities, high proliferation, and survival in a 3D bioprinted tumor model.5353. S. Mao, J. He, Y. Zhao, T. Liu, F. Xie, H. Yang, Y. Mao, Y. Pang, and W. Sun, Biofabrication 12, 045014 (2020). https://doi.org/10.1088/1758-5090/aba0c3 The viability of cells exceeded 90% in these constructs. The 3D bioprinted model demonstrated a remarkable up-regulation of various aggressive tumor characteristics such as the degree of malignancy, stemness, degree of fibrosis, invasion, and metastatic propensity as opposed to those in 2D cultures. Another notable finding was the stem cell-like properties of the ICC cells in the 3D constructs, indicated by their resistance to anticancer drugs. This 3D bioprinted cancer model comprising ICC cells and enriched with CSC can potentially be used for understanding carcinogenesis and in precision medicine.Additionally, a 3D bioprinted liver cancer model was developed using HepG2 cells and sodium alginate/gelatin/fibrinogen hydrogel as the bioink. This construct pertinently reproduced a metastatic microenvironment, which was revealed by the gene expression studies demonstrating an increased expression of the cancer-/stemness-associated cellular markers, matrix metalloproteinase, and EMT regulatory proteins.5454. X. Zhou et al., World J. Pharm. Pharm. Sci. 5, 196 (2016). Furthermore, in response to various anticancer drugs, like 5-Fluorouracil (5-FU) and mitomycin, the 3D models showed HepG2 cell behaviors different from those of the 2D cell models. This provided evidence of a closer physiological relevance of the bioprinted liver cancer models for in vitro drug screening. Developing an HCC model that depicts the relationship among fibrosis, cirrhosis, and HCC is important for exploring the mechanisms of carcinogenesis. In this context, a 3D HCC model composed of the fibrotic stromal compartment and vasculature was developed. Resistance to chemotherapeutic drugs was noted in this model. This 3D tumor construct could serve as an excellent platform for investigating multifocal HCCs that contribute to the early stages of cancer metastasis.5555. C. Calitz, N. Pavlovic, J. Rosenquist, C. Zagami, A. Samanta, and F. Heindryckx, “A biomimetic liver model recapitulating bio-physical properties and tumour stroma interactions in hepatocellular carcinoma: Three-dimensional cell culture,” bioRxiv: 2020.04.30.069823 (2020). https://doi.org/10.1101/2020.04.30.069823 Moreover, to promote navigation surgery in hepatocellular carcinoma, a 3D printed model of the intrahepatic vessel was developed, paving the way for the further development of vessel-like structures in bioprinted liver cancer constructs.5656. S. Kuroda, T. Kobayashi, and K. Ohdan, Int. J. Surg. Case Rep. 41, 219 (2017). https://doi.org/10.1016/j.ijscr.2017.10.015An advanced high-throughput 3D bioprinter (HT-3DP) was developed to print constructs with varying spatial geometries having biomimetic properties and tunable mechanics with high scalability and reproducibility. Both HCC and heterogeneous HCC/human umbilical vein endothelial cell combinations were used to bioprint tissue constructs in multiwell plates. These constructs had the benefit of depicting in vivo tumor heterogeneity as well as enabling large-scale drug screening.5757. H. H. Hwang et al., Biofabrication 13, 025007 (2021). https://doi.org/10.1088/1758-5090/ab89ca A satisfactory response to doxorubicin was noted in the functional drug response assay. This work provides an excellent example of the advancement in 3D bioprinting, providing a high production rate of tissue scaffolds with controllable spatial architectures and mechanical properties. This enables rapid generation of in vitro 3D tissue models within the regular multiwell cell culture plates, which can be scaled up for high-throughput preclinical drug screening and deciphering disease biology.Recently, a co-culture system based on the principle of 3D extrusion bioprinting was developed to explore the influence of the microenvironment on cellular responses.5858. R. Taymour, D. Kilian, T. Ahlfeld, M. Gelinsky, and A. Lode, Sci. Rep. 11, 5130 (2021). https://doi.org/10.1038/s41598-021-84384-6 Initially, a bioink based on alginate and methylcellulose (algMC) was used, which was found to be suitable for the bioprinting of hepatocytes. Later, the addition of Matrigel to algMC led to the increased proliferation in monophasic scaffolds. Furthermore, to study cellular interactions, core–shell bioprinting was developed to tailor the 3D co-culture models for hepatocytes. The bioinks were specifically functionalized using natural matrix components (based on human plasma, fibrin, or Matrigel). Fibroblasts and hepatocytes were co-printed in a spatially defined, coaxial manner. Fibroblasts, which are stromal cells, supported hepatocytes and induced the expression of biomarkers of hepatocytes like albumin. Moreover, matrix functionalization promotes properties such as adhesion, viability, proliferation, and function for both cell types in their respective compartments (Fig. 4). Finally, the authors demonstrated a functional co-culture model with independently tunable compartments for different cell types using core–shell bioprinting. This study provides a proof-of-principle concept to develop more complex in vitro models wherein the co-cultivation of hepatocytes with other liver-specific cell types is possible, which can closely resemble the liver microenvironment.Until date, most studies have utilized cell lines for 3D bioprinting cancer models in hepatobiliary systems. However, for such models to closely mimic the in vivo environment and be useful in precision medicine, it is necessary to develop patient-specific cancer models using patient-derived tissue. In this milieu, Xie et al. established the first individualized model for hepatocellular carcinomic specimens from six patients after surgery. Isolated primary HCC cells from the patient samples were mixed with gelatin and sodium alginate to form the bioink. The 3D constructs developed demonstrated satisfactory growth during long-term culture. As anticipated, the genetic alterations and expression profiles, including biomarker expressions, were similar to those noted in the patient samples.5959. F. Xie et al., Biomaterials 265, 120416 (2021). https://doi.org/10.1016/j.biomaterials.2020.120416 To assess the suitability of the 3DP-HCC model for precision medicine, the efficacies of four commonly used targeted drugs were tested on the patient-derived 3DP-HCC models of the six patients. Each 3DP-HCC model was treated with a series of dilutions of each drug for six consecutive days. Each construct was monitored for cell viability using a Cell Counting Kit 8 (CCK8) assay. The results indicated insensitivity in most of the models derived from the six patients to the four targeted drugs (Fig. 5). The authors found that the bioprinting-HCC models of patients no. 2 and no. 5 were resistant to all four drugs, with a half-maximal inhibitory concentration (IC50) greater than the maximum screening concentration. However, one or more drugs proved effective for the 3DP-HCC models of the other four patients, namely, no. 1, no. 2, no. 4, and no. 6. In most of these positive cases, a dose-dependent manner was observed. For patients no. 1 and no. 3, sorafenib and lenvatinib showed better antitumor effects than the other drugs, respectively. In addition, sorafenib inhibited cell survival effectively even at a very low concentration in patient no. 1.Whole exon sequencing was performed to elucidate the mutational profiles in the six patients (Table II). There was a general positive correlation between drug sensitivities and mutated targets. Patients no. 1, no. 3, no. 4, and no. 6 who showed mutations in the PDGFR and VEGFR family were sensitive to either sorafenib or lenvatinib, or both; however, the same was not seen in patients no. 2 and no. 5. These findings indicated that the 3DP-HCC models can serve as realistic in vitro models that are consistent and can be employed as predictive models in precision medicine.

TABLE II. Targets and the mutational profiles of six patients [reproduced with permission from Xie et al., Biomaterials 265, 120416 (2021). Copyright 2020, Elsevier Ltd].

TargetsDrugsaP. no. 1P. no. 2P. no. 3P. no. 4P. no. 5P. no. 6VEGFR2SLRA−++++↓−PDGFRβ+−−++↑−B-RafR−+−+−↑−FLT3+++++↑+Raf-1R+++++↓+B-Raf(V599E)−−−−−−KitLR−−−−−−PDGFRα+++++↑+FGFR1+−−−−↑−RETR+++++↑+VEGFR1L+++−+↓+B-Raf (V600E)−−−−−−An overview of the literature on the current 3D bioprinted liver cancer models is given in Table III. These studies highlight the significant potential liver-related biomedical applications of 3D bioprinting technology, such as drug screening and toxicology along with other preclinical applications.

TABLE III. Current 3D bioprinted liver cancer models.

Bioink/techniqueCharacteristicsDrug screeningReferenceSodium alginate solution/extrusion bioprintingThe 3DP-HepG2 model demonstrated significantly higher levels of various liver function-related proteins and genes. Similarly, increased expression of proteins and genes involved in proliferation, metastasis, drug resistance, antitumor immunosuppression, and epithelial-to-mesenchymal transition of tumor cells were also observed.IC50 values of the screened anticancer drugs were significantly higher in the 3DP-HepG2 model than in the 2D-HepG2 model.5252. L. Sun et al., Front. Oncol. 10, 878 (2020). https://doi.org/10.3389/fonc.2020.00878Gelatin and alginate with MatrigelThe patient-derived ICC cells demonstrated marked cellular proliferation, progression, and migration.Stem-like properties of ICC cells were indicated by the resistance of the ICC cells in the 3D construct to anticancer drugs.5353. S. Mao, J. He, Y. Zhao, T. Liu, F. Xie, H. Yang, Y. Mao, Y. Pang, and W. Sun, Biofabrication 12, 045014 (2020). https://doi.org/10.1088/1758-5090/aba0c3Sodium alginate, gelatin, and fibrinogen hydrogel/extrusion bioprintingThe biological characterization results indicated that HepG2 cells in the printed 3D liver tumor model grew very well with excellent livability (100%). The shapes of the HepG2 cells loaded in the 3D liver tumor model changed from separated spheroids to small clusters, and gradually to large hepatocellular carcinoma tissues after two weeks of in vitro culturing.Treatment effects of different anticancer drugs, such as 5-Fluorouracil, mitomycin, and their combination, on HepG2 cells were satisfactory.5454. X. Zhou et al., World J. Pharm. Pharm. Sci. 5, 196 (2016).Poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacryloyl (GelMA)/extrusion bioprintingViability of the cells was seen at the end of one week by live/dead cell quantification, which revealed viability in majority of the cell population (>85%).The dose- and time-dependent doxorubicin toxicities of 3D-printed HepG2 scaffolds were effective.5757. H. H. Hwang et al., Biofabrication 13, 025007 (2021). https://doi.org/10.1088/1758-5090/ab89ca5% gelatin (Sigma) and 1% sodium alginate/extrusion bioprintingThe biological and genetic features of the original tumor were retained, and they were independent of the intrinsic proliferative capacity of the original tumor.Personalized drug screening is possible, with obvious antitumor effects and reproducible experiments.5959. F. Xie et al., Biomaterials 265, 120416 (2021). https://doi.org/10.1016/j.biomaterials.2020.120416

C. 3D bioprinting of colorectal cancer models

Colorectal cancer is one of the most common cancers worldwide, next only to lung and prostate cancer in men and breast cancer in women. According to the GLOBOCAN 2018 report, colorectal cancer is the fourth most common cause of death due to cancer throughout the world.6060. P. Rawla, T. Sunkara, and A. Barsouk, Gastroenterol. Rev. 14, 89 (2019). https://doi.org/10.5114/pg.2018.81072 In 2020, colorectal cancer accounted for the highest number of cancer-related deaths (9.4%), next only to lung cancer (18%) globally. By 2030, the incidence of colorectal cancer is projected to increase by 60%, with 2.2 × 106 new cases and 1.1 × 106 new deaths, particularly in developed countries.6161. M. Arnold, M. S. Sierra, M. Laversanne, I. Soerjomataram, A. Jemal, and F. Bray, Gut 66, 683 (2017). https://doi.org/10.1136/gutjnl-2015-310912 The increase in the incidence of colorectal cancer, a strong marker of epidemiologic and nutritional transition, has been linked to the western lifestyle. A high intake of processed and red meat, an increase in body fat, a sedentary lifestyle, an increase in tobacco and alcohol intake, and a decrease in the consumption of high-fiber foods, essential minerals, and vitamins are considered highly influential factors for the development of colorectal cancer.62,6362. M. M. Center, A. Jemal, R. A. Smith, and E. Ward, CA: Cancer J. Clin. 59, 366 (2009). https://doi.org/10.3322/caac.2003863. F. Bray, A. Jemal, N. Grey, J. Ferlay, and D. Forman, Lancet Oncol. 13, 790 (2012). https://doi.org/10.1016/S1470-2045(12)70211-5 Though the mortality and incidence trends are stabilizing or decreasing in developed countries, Arnold et al. have mentioned the increasing trend in middle- and low-income countries, attributed to economic and social developments.6262. M. M. Center, A. Jemal, R. A. Smith, and E. Ward, CA: Cancer J. Clin. 59, 366 (2009). https://doi.org/10.3322/caac.20038 Colorectal cancer originates from the glandular epithelium lining the colon. Most colorectal cancers are adenocarcinomas. Most adenocarcinomas arise from a pre-existing adenoma, which is amenable to surgical resection. The adenoma–carcinoma transformation occurs over a duration spanning decades. Stepwise accumulation of myriad genetics and epigenetic mutations in the epithelium is responsible for the development of cancer, with the two most important pathways being the APC/β-catenin pathway and th