Growing efforts toward long-duration human space missions demand novel strategies for the treatment of acute injuries in extreme environments. Technologies for space applications must perform reliably even under environmental stressors rarely encountered on Earth, such as gravitational fluctuations. However, space-compatible personalized therapy approaches for treatment of high-risk injuries remain scarce. 3D bioprinting represents an advanced technology with promising potential to address this unmet medical need by enabling the on-demand fabrication of patient-specific tissue constructs for regenerative wound care. Here, the robust 3D printing of both acellular and cell-laden hydrogel constructs is demonstrated using photo- and bioinks under diverse environmental conditions, including microgravity and hypergravity phases encountered during parabolic flight (0-1.8 g). Despite dynamic accelerative conditions, the developed flight-compliant, closed stereolithographic (SLA) bioprinting system successfully printed 3D structures with maintained dimensional fidelity. Irrespective of gravitational forces, high cell viability was preserved in both fibroblast- and keratinocyte-laden constructs. High-resolution features are achieved with precision comparable to normal-gravity controls. Complex architectures, including gyroids, can be fabricated with smooth, continuous surfaces. These findings establish SLA bioprinting as a robust and gravity-tolerant platform for fabricating viable, cell-laden constructs-offering a promising pathway for advancing tissue engineering in space and in extreme conditions on Earth.

Reconstructive surgery following breast cancer ablation is a surgical gold standard, but current options comprising autologous fatty tissue transfer and artificial soft tissue implants are inferior. With the advent of powerful biofabrication technologies, researchers for the first time have the tools to engineer life-like tissues with the ultimate goal of clinical application. Here, we apply multi-material stereolithographic bioprinting together with a novel sacrificial biomaterial system to engineer complex fatty tissue constructs. Biomaterials, cellular composition and cultivation conditions of these constructs were designed to enablein vitrocreation of vascularised fatty tissue. Cells within the constructs showed an overall good survival (>93%), indicated by live-dead cell staining, over the entire cultivation period of 27 d. Adipose-derived stem cells were successfully differentiatedin situ, forming fat vesicles and expressing adipocyte markers PPARγ, FAPB4 and S100B. Additionally, secretion of adipokines leptin and adiponectin into culture supernatants increased significantly. Endothelial cells vascularised the constructs, creating macro- and microvascular structures within the printed channels and extending beyond with culture time. Moreover, cells invaded into the surrounding hydrogel. The engineered fatty tissue constructs could serve as a base to develop patient-specific tissue building blocks with the final goal to achieve an all-natural reconstruction of the breast.

IntroductionEffective CAR T cell infiltration into solid tumors remains a major barrier to therapy success. Despite their clinical potential, few studies have evaluated phenotypes of CAR T cells successfully invading the tumor mass following infusion. Phenotypic information would enrich our understanding of the mechanisms governing CAR T cell migration into solid tumors. Here we implemented an in vitro strategy to identify genes driving L1CAM-CAR T cell migration into a 3D tumor mass.MethodsL1CAM-CAR T cells were separated into 2 groups by their capability to infiltrate (or not) a 3D bioprinted neuroblastoma model. Single-cell and bulk RNA sequencing was performed, and infiltrating CAR T cells were compared to noninfiltrating cells to seek genetic drivers of CAR T cell migration. CRISPR/Cas9 technology was used to generate modified L1CAM-CAR T cells.ResultsTumor-infiltrating L1CAM-CAR T cells expressed lower levels of the selectin P ligand (SELPLG) glycoprotein and higher levels of the T cell-specific adaptor protein, SH2D2A. Functional characterization of L1CAM-CAR T cells genetically modified to enforce these characteristics demonstrated that neither trait negatively impacted L1CAM-CAR T cell cytotoxicity, activation and cytokine release upon coculture with neuroblastoma target cells. Transgenic SH2D2A expression did not improve CAR T cell migration in an endothelial transmembrane assay. SELPLG knockout benefited CAR T cell in vitro trans-endothelial migration, but did not enhance anti-tumor efficacy in an immunodeficient mouse model.DiscussionOur findings reveal a key limitation of murine xenograft models, which are widely used as the gold standard for preclinical CAR T cell testing. The lack of conservation between the human and murine SELPLG proteins likely accounts for the discrepancy between enhanced in vitro migration of SELPLG-deficient L1CAM-CAR T cells and their lack of improved efficacy in the mouse model. This underscores the need for more predictive human-relevant models to better preclinically evaluate CAR T cell function.

Hydrogel inks used for 3D bioprinting are mainly based on radical polymerization of methacrylate groups. Inks based on the radical thiol-ene polymerization have raised attention in recent years, as they are not susceptible to oxygen inhibition and require lower light doses for polymerization, therefore, they can be more benign to living cells. Here, we modified hyaluronic acid inks with allyl ether or norbornene groups, which can form a crosslinked network in the presence of a dithiol crosslinker. We performed systematic studies to compare precursor stability, photocrosslinking and printability of the thiol-ene inks with methacrylated hyaluronic acid inks. Our results showed higher storage stability of the thiol-ene hydrogel precursors over 15 months. Photorheology experiments demonstrated faster photocrosslinking and higher temporal control over the network formation in thiol-ene inks. The suitability of thiol-ene inks was demonstrated using digital light processing-based printing with a minimum print time of 2 s per layer and a xy resolution of 100 µm.

Photocrosslinkable formulations based on the radical thiol-ene reaction are considered better alternatives than methacrylated counterparts for light-based fabrication processes. This study quantifies differences between thiol-ene and methacrylated crosslinked hydrogels in terms of precursors stability, the control of the crosslinking process, and the resolution of printed features particularized for hyaluronic acid (HA) inks at concentrations relevant for bioprinting. First, the synthesis of HA functionalized with norbornene, allyl ether, or methacrylate groups with the same molecular weight and comparable degrees of functionalization is presented. The thiol-ene hydrogel precursors show storage stability over 15 months, 3.8 times higher than the methacrylated derivative. Photorheology experiments demonstrate up to 4.7-times faster photocrosslinking. Network formation in photoinitiated thiol-ene HA crosslinking allows higher temporal control than in methacrylated HA, which shows long post-illumination hardening. Using digital light processing, 4% w/v HA hydrogels crosslinked with a dithiol allowed printing of 13.5 × 4 × 1 mm3 layers with holes of 100 µm resolution within 2 s. This is the smallest feature size demonstrated in DLP printing with HA-based thiol-ene hydrogels. The results are important to estimate the extent to which the synthetic effort of introducing -ene functions can pay off in the printing step.

In light-based 3D-bioprinting, gelatin methacrylate (GelMA) is one of the most widely used materials, as it supports cell attachment, and shows good biocompatibility and degradability in vivo. However, as an animal-derived material, it also causes safety concerns when used in medical applications. Gelatin is a partial hydrolysate of collagen, containing high amounts of hydroxyproline. This causes the material to form a thermally induced gel at ambient temperatures, a behavior also observed in GelMA. This temperature-dependent gelation requires precise temperature control during the bioprinting process to prevent the gelation of the material. To avoid safety concerns associated with animal-derived materials and reduce potential issues caused by thermal gelation, a recombinant human alpha-1 collagen I fragment was expressed in Komagataella phaffii without hydroxylation. The resulting protein was successfully modified with methacryloyl groups and underwent rapid photopolymerization upon ultraviolet light exposure. The developed material exhibited slightly slower polymerization and lower storage modulus compared to GelMA, while it showed higher stretchability. However, unlike the latter, the material did not undergo physical gelation at ambient temperatures, but only when cooled down to below 10°C, a characteristic that has not been described for comparable materials so far. This gelation was not caused by the formation of triple-helical structures, as shown by the absence of the characteristic peak at 220 nm in CD spectra. Moreover, the developed recombinant material facilitated cell adherence with high cell viability after crosslinking via light to a 3D structure. Furthermore, desired geometries could be easily printed on a stereolithographic bioprinter.

Photopolymerizable formulations based on the thiol-ene crosslinking are considered better alternatives than methacrylated counterparts for light-based fabrication processes. The thiol-ene photoreaction requires lower light doses for polymerization and leads to networks with lower molecular heterogeneity. For hydrogels at low polymer concentrations as used in bioinks (<8 wt%), faster polymerization rates and higher conversion of the crosslinking reaction have been reported in thiol-ene systems. Here we quantify further differences between thiol-ene and methacrylated crosslinked hydrogels for HA functionalized with norbornene, allyl ether or methacrylate groups. We show storage stability of the thiol-ene hydrogel precursors over 15 months, 3.8 times higher than the methacrylated derivative. Photorheology experiments demonstrated up to 4.7-times faster photocrosslinking. Network formation in photoinitiated thiol-ene HA crosslinking allows higher temporal control than in methacrylated HA, which shows long post-illumination hardening. Using digital light processing, 4% w/v HA hydrogels polymerized with a dithiol crosslinker allowed printing of 13.5x4x1 mm3 layers with holes of 100 µm resolution within 2 s. Considering that ene-derivatized hydrogel precursors are less commercially available than methacrylated counterparts, our results are important to estimate the extent to which the synthetic effort of introducing –ene functions can pay off in the printing step.

To date, islet transplantation to treat type 1 diabetes mellitus remains unsuccessful in long-term follow-up, mainly due to failed engraftment and reconstruction of the islet niche. Alternative approaches, such as islet embedding structures (IESs) based on 3D printing have been developed. However, most of them have been implanted subcutaneously and only a few are intended for direct integration into the vascular system through anastomosis. In this study, we 3D printed a proof-of-concept IES using gelatin methacrylate biocompatible ink. This structure consisted of a branched vascular system surrounding both sides of a central cavity dedicated to islets of Langerhans. Furthermore, we designed a bioreactor optimized for these biological structures. This bioreactor allows seeding and perfusion experiments under sterile and physiological conditions. Preliminary experiments aimed to analyze if the vascular channel could successfully be seeded with mature endothelial cells and the central cavity with rat islets. Subsequently, the structures were used for a humanized model seeding human endothelial progenitor cells (huEPC) within the vascular architecture and human islets co-cultured with huEPC within the central cavity. The constructs were tested for hemocompatibility, suture strength, and anastomosability. The 3D printed IES appeared to be hemocompatible and anastomosable using an alternative cuff anastomosis in a simple ex vivo perfusion model. While rat islets alone could not successfully be embedded within the 3D printed structure for 3 days, human islets co-cultivated with huEPC successfully engrafted within the same time. This result emphasizes the importance of co-culture, nursing cells, and islet niche. In conclusion, we constructed a proof-of-concept 3D printed islet embedding device consisting of a vascular channel that is hemocompatible and perspectively anastomosable to clinical scale blood vessels. However, there are numerous limitations in this model that need to be overcome to transfer this technology to the bedside. Impact statementExtensive research exists on 3D bioprinting of islet containing tissues, each tackling islet engraftment and protection against immune reaction. Rarely has the focus been put on bioprinting a construct consisting of at least one perfusable vascular channel that can withstand anastomosis using sutures and perfusion with physiological flow rates, which has close physical contact to all cell-containing, that is permeable to important components of cell medium and that provides a bidirectional exchange of substances. In this study, we depict first technical notes and preliminary results established in the process of constructing and testing a perfusable, anastomosable, and biocompatible 3D printed islet embedding structure.

Although multi-modal and ablative treatment strategies for breast cancer are becoming more popular, partial or full mastectomy is still a most common intervention. While proving to be effective in curing the cancer, it leaves the patient disfigured, which can pose a considerable psychological burden. Hence, reconstructive breast surgery is continuing to increase in importance as it offers the possibility to at least partially restore the original look of the breast. Autologous adipose tissue transfer techniques, amongst them the surgical gold standard flap surgery and liposuction-based fat grafting, present natural solutions with the patient’s own tissue, but either produce a considerable secondary wound or are insufficient to address larger defects. Synthetic solutions like silicone implants on the other hand can also be used to fill large defects and only require minimal surgical intervention, but suffer from foreign body-associated complications like fibrosis. Tissue engineering biological breast implants of arbitrary size with no or minor insult to the patient’s body and excellent biocompatibility, exclusively using biomaterials and patient-specific adipose cells, could open a new chapter for breast reconstruction. Such an approach has become an intriguing possibility through the rise of powerful biofabrication technologies in the last two decades, namely three-dimensional bioprinting. Bioprinting marries additive manufacturing with biomaterials and living cells with the aim to create life-mimicking tissues and organs, and while it is mainly focused on research today, current endeavors push for application in regenerative medicine. In our work we sought to fabricate adipose tissue constructs using light-based bioprinting to investigate the potential application of such technology for future reconstructive breast surgery. The engineered tissue blocks were designed to mimic native human adipose tissue by comprising fat, connective tissue and endothelial cells. Being specifically placed by the bioprinter within extracellular matrix-like hydrogels, the cells were cultured in vitro and differentiated within the cultivation time of up to 4 weeks to form an adipose-like tissue. Live imaging showed high viability of bioprinted tissue blocks over the whole cultivation period and pronounced cellular reorganization. Confocal and histological analyses revealed a dynamic tissue maturation process with increasing proportions of differentiated adipocytes, ECM remodeling and formation of microvascular structures. In conclusion, the obtained results gave proof to the concept of engineering adipose tissue through bioprinting and could lay the groundwork for the development of all-natural biological breast implants. Citation Format: Nina Hedemann, Alexander Thomas, Nils Tribian, Lutz Kloke, Dirk Bauerschlag, Marion van Mackelenbergh. Light-based bioprinting of vascularized fatty tissue for the development of biological breast implants [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-15-06.

Abstract Although multi-modal and ablative treatment strategies for breast cancer are becoming more popular, partial or full mastectomy is still a most common intervention. While proving to be effective in curing the cancer, it leaves the patient disfigured, which can pose a considerable psychological burden. Hence, reconstructive breast surgery is continuing to increase in importance as it offers the possibility to at least partially restore the original look of the breast. Autologous adipose tissue transfer techniques, amongst them the surgical gold standard flap surgery and liposuction-based fat grafting, present natural solutions with the patient’s own tissue, but either produce a considerable secondary wound or are insufficient to address larger defects. Synthetic solutions like silicone implants on the other hand can also be used to fill large defects and only require minimal surgical intervention, but suffer from foreign body-associated complications like fibrosis. Tissue engineering biological breast implants of arbitrary size with no or minor insult to the patient’s body and excellent biocompatibility, exclusively using biomaterials and patient-specific adipose cells, could open a new chapter for breast reconstruction. Such an approach has become an intriguing possibility through the rise of powerful biofabrication technologies in the last two decades, namely three-dimensional bioprinting. Bioprinting marries additive manufacturing with biomaterials and living cells with the aim to create life-mimicking tissues and organs, and while it is mainly focused on research today, current endeavors push for application in regenerative medicine. In our work we sought to fabricate adipose tissue constructs using light-based bioprinting to investigate the potential application of such technology for future reconstructive breast surgery. The engineered tissue blocks were designed to mimic native human adipose tissue by comprising fat, connective tissue and endothelial cells. Being specifically placed by the bioprinter within extracellular matrix-like hydrogels, the cells were cultured in vitro and differentiated within the cultivation time of up to 4 weeks to form an adipose-like tissue. Live imaging showed high viability of bioprinted tissue blocks over the whole cultivation period and pronounced cellular reorganization. Confocal and histological analyses revealed a dynamic tissue maturation process with increasing proportions of differentiated adipocytes, ECM remodeling and formation of microvascular structures. In conclusion, the obtained results gave proof to the concept of engineering adipose tissue through bioprinting and could lay the groundwork for the development of all-natural biological breast implants. Citation Format: Nina Hedemann, Alexander Thomas, Nils Tribian, Lutz Kloke, Dirk Bauerschlag, Marion van Mackelenbergh. Light-based bioprinting of vascularized fatty tissue for the development of biological breast implants [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-15-06.

Advances in cellular engineering, as well as gene, and cell therapy, may be used to produce human tissues with programmable genetically enhanced functions designed to model and/or treat specific diseases. Fabrication of synthetic human liver tissue with these programmable functions has not been described. By generating human iPSCs with target gene expression controlled by a guide RNA-directed CRISPR-Cas9 synergistic-activation-mediator, we produced synthetic human liver tissues with programmable functions. Such iPSCs were guide-RNA-treated to enhance expression of the clinically relevant CYP3A4 and UGT1A1 genes, and after hepatocyte-directed differentiation, cells demonstrated enhanced functions compared to those found in primary human hepatocytes. We then generated human liver tissue with these synthetic human iPSC-derived hepatocytes (iHeps) and other non-parenchymal cells demonstrating advanced programmable functions. Fabrication of synthetic human liver tissue with modifiable functional genetic programs may be a useful tool for drug discovery, investigating biology, and potentially creating bioengineered organs with specialized functions.

Stereolithographic bioprinting holds great promise in the quest for creating artificial, biomimetic cartilage-like tissue. To introduce a more biomimetic approach, we examined blending and stratifying methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA) bioinks to mimic the zonal structure of articular cartilage. Bioinks were suspended with porcine chondrocytes before being printed in a digital light processing approach. Homogenous constructs made from hybrid bioinks of varying polymer ratios as well as stratified constructs combining different bioink blends were cultivated over 14 days and analyzed by histochemical staining for proteoglycans/collagen type II, cartilage marker expression analysis, and for cellular viability. The stiffness of blended bioinks increased gradually with HAMA content, from 2.41 ± 0.58 kPa (5% GelMA, 0% HAMA) to 8.84 ± 0.11 kPa (0% GelMA, 2% HAMA). Cell-laden constructs maintained vital chondrocytes and supported the formation of proteoglycans and collagen type II. Higher concentrations of GelMA resulted in increased formation of cartilaginous matrix proteins and a more premature phenotype. However, decreased matrix production in central areas of constructs was observed in higher GelMA content constructs. Biomimetically stratified constructs retained their gradient-like structure even after ECM formation, and exclusively exhibited a significant increase in COL2A1 gene expression (+178%). Concluding, we showed the feasibility of blending and stratifying photopolymerizable, natural biopolymers by SLA bioprinting to modulate chondrocyte attributes and to create zonally segmented ECM structures, contributing to improved modeling of cartilaginous tissue for regenerative therapies or in vitro models.

Nutrient supply via a functional vasculature is essential during regenerative processes, tissue growth, and homeostasis. 3D bioprinting offers the opportunity to engineer vascularized constructs by combining cells and biocompatible materials in specifically designed fashions. However, the complexity of microvascular dynamic networks can hardly be recapitulated yet, even by sophisticated 3D manufacturing. Ideally, the natural organizational competences of endothelial cells will be harnessed such that engineered vascular networks self-assemble to form complex, controllable microvascular patterns. Here, a bioengineering approach is presented to control microvascular structure formation and to steer cellular self-assembly of endothelial and supporting cells within a multi-material stereolithographic 3D bioprinting concept. Bioengineered vascularized constructs are generated by controlled cell deposition in an enzymatically degradable or a non-degradable material. In vitro, the microvascular structures are regulated in distribution, network orientation, vessel length and branching behavior and developed lumen, signs of vascular stabilization and an interconnected vascular network including anastomosis. This novel biofabrication approach demonstrates the capability to control microvascular network formation by using cellular and spatial cues allowing the generation of distinctly yet precisely vascularized constructs. Such novel approach of controlled microvascular formation may play a fundamental role in the development of vascularized implants or in vitro screening models.

Radiotherapy of head and neck squamous cell carcinoma can lead to long-term complications like osteoradionecrosis, resulting in severe impairment of the jawbone. Current standard procedures require a 6-month wait after irradiation before dental reconstruction can begin. A comprehensive characterization of the irradiation-induced molecular and functional changes in bone cells could allow the development of novel strategies for an earlier successful dental reconstruction in patients treated by radiotherapy. The impact of ionizing radiation on the bone-forming alveolar osteoblasts remains however elusive, as previous studies have relied on animal-based models and fetal or animal-derived cell lines. This study presents the first in vitro data obtained from primary human alveolar osteoblasts. Primary human alveolar osteoblasts were isolated from healthy donors and expanded. After X-ray irradiation with 2, 6 and 10 Gy, cells were cultivated under osteogenic conditions and analyzed regarding their proliferation, mineralization, and expression of marker genes and proteins. Proliferation of osteoblasts decreased in a dose-dependent manner. While cells recovered from irradiation with 2 Gy, application of 6 and 10 Gy doses not only led to a permanent impairment of proliferation, but also resulted in altered cell morphology and a disturbed structure of the extracellular matrix as demonstrated by immunostaining of collagen I and fibronectin. Following irradiation with any of the examined doses, a decrease of marker gene expression levels was observed for most of the investigated genes, revealing interindividual differences. Primary human alveolar osteoblasts presented a considerably changed phenotype after irradiation, depending on the dose administered. Mechanisms for these findings need to be further investigated. This could facilitate improved patient care by re-evaluating current standard procedures and investigating faster and safer reconstruction concepts, thus improving quality of life and social integrity.

Reconstruction of segmental bone defects by autologous bone grafting is still the standard of care but presents challenges including anatomical availability and potential donor site morbidity. The process of 3D bioprinting, the application of 3D printing for direct fabrication of living tissue, opens new possibilities for highly personalized tissue implants, making it an appealing alternative to autologous bone grafts. One of the most crucial hurdles for the clinical application of 3D bioprinting is the choice of a suitable cell source, which should be minimally invasive, with high osteogenic potential, with fast, easy expansion. In this study, mesenchymal progenitor cells were isolated from clinically relevant human bone biopsy sites (explant cultures from alveolar bone, iliac crest and fibula; bone marrow aspirates; and periosteal bone shaving from the mastoid) and 3D bioprinted using projection-based stereolithography. Printed constructs were cultivated for 28 days and analyzed regarding their osteogenic potential by assessing viability, mineralization, and gene expression. While viability levels of all cell sources were comparable over the course of the cultivation, cells obtained by periosteal bone shaving showed higher mineralization of the print matrix, with gene expression data suggesting advanced osteogenic differentiation. These results indicate that periosteum-derived cells represent a highly promising cell source for translational bioprinting of bone tissue given their superior osteogenic potential as well as their minimally invasive obtainability.

Chimeric antigen receptor (CAR) T cell performance against solid tumors in mouse models and clinical trials is often less effective than predicted by CAR construct selection in two-dimensional (2D) cocultures. Three-dimensional (3D) solid tumor architecture is likely to be crucial for CAR T cell efficacy. We used a three-dimensional (3D) bioprinting approach for large-scale generation of highly reproducible 3D human tumor models for the test case, neuroblastoma, and compared these to 2D cocultures for evaluation of CAR T cells targeting the L1 cell adhesion molecule, L1CAM. CAR T cells infiltrated the model, and both CAR T and tumor cells were viable for long-term experiments and could be isolated as single-cell suspensions for whole-cell assays quantifying CAR T cell activation, effector function and tumor cell cytotoxicity. L1CAM-specific CAR T cell activation by neuroblastoma cells was stronger in the 3D model than in 2D cocultures, but neuroblastoma cell lysis was lower. The bioprinted 3D neuroblastoma model is highly reproducible and allows detection and quantification of CAR T cell tumor infiltration, representing a superior in vitro analysis tool for preclinical CAR T cell characterization likely to better select CAR T cells for in vivo performance than 2D cocultures.

Jawbone differs from other bones in many aspects, including its developmental origin and the occurrence of jawbone-specific diseases like MRONJ (medication-related osteonecrosis of the jaw). Although there is a strong need, adequate in vitro models of this unique environment are sparse to date. While previous approaches are reliant e.g. on scaffolds or spheroid culture, 3D bioprinting enables free-form fabrication of complex living tissue structures. In the present work, production of human jawbone models was realised via projection-based stereolithography. Constructs were bioprinted containing primary jawbone-derived osteoblasts and vasculature-like channel structures optionally harbouring primary endothelial cells. After 28 days of cultivation in growth medium or osteogenic medium, expression of cell type-specific markers was confirmed on both the RNA and protein level, while prints maintained their overall structure. Survival of endothelial cells in the printed channels, co-cultured with osteoblasts in medium without supplementation of endothelial growth factors, was demonstrated. Constructs showed not only mineralisation, being one of the characteristics of osteoblasts, but also hinted at differentiation to an osteocyte phenotype. These results indicate the successful biofabrication of an in vitro model of the human jawbone, which presents key features of this special bone entity and hence appears promising for application in jawbone-specific research.

Introduction of cavities and channels into 3D bioprinted constructs is a prerequisite for recreating physiological tissue architectures and integrating vasculature. Projection-based stereolithography inherently offers high printing speed with high spatial resolution, but so far has been incapable of fabricating complex native tissue architectures with cellular and biomaterial diversity. The use of sacrificial photoinks, i.e. photopolymerisable biomaterials that can be removed after printing, theoretically allows for the creation of any construct geometry via a multi-material printing process. However, the realisation of this strategy has been challenging because of difficult technical implementation and a lack of performant biomaterials. In this work, we use our projection-based, multi-material stereolithographic bioprinter and an enzymatically degradable sacrificial photoink to overcome the current hurdles. Multiple, hyaluronic acid-based photoinks were screened for printability, mechanical properties and digestibility through hyaluronidase. A formulation meeting all major requirements, i.e. desirable printing properties, generation of sufficiently strong hydrogels, as well as fast digestion rate, was identified. Biocompatibility of the material system was confirmed by embedding of human umbilical vein endothelial cells with followed enzymatic release. As a proof-of-concept, we bioprinted vascular models containing perfusable, endothelial cell-lined channels that remained stable for 28 days in culture. Our work establishes digestible sacrificial biomaterials as a new material strategy for 3D bioprinting of complex tissue models.

Studies of virus–host interactions in vitro may be hindered by biological characteristics of conventional monolayer cell cultures that differ from in vivo infection. Three-dimensional (3D) cell cultures show more in vivo-like characteristics and may represent a promising alternative for characterisation of infections. In this study, we established easy-to-handle cell culture platforms based on bioprinted 3D matrices for virus detection and characterisation. Different cell types were cultivated on these matrices and characterised for tissue-like growth characteristics regarding cell morphology and polarisation. Cells developed an in vivo-like morphology and long-term cultivation was possible on the matrices. Cell cultures were infected with viruses which differed in host range, tissue tropism, cytopathogenicity, and genomic organisation and virus morphology. Infections were characterised on molecular and imaging level. The transparent matrix substance allowed easy optical monitoring of cells and infection even via live-cell microscopy. In conclusion, we established an enhanced, standardised, easy-to-handle bioprinted 3D-cell culture system. The infection models are suitable for sensitive monitoring and characterisation of virus–host interactions and replication of different viruses under physiologically relevant conditions. Individual cell culture models can further be combined to a multicellular array. This generates a potent diagnostic tool for propagation and characterisation of viruses from diagnostic samples.

Barrier organ models need a scaffold structure to create a two compartment culture. Technical filter membranes used most often as scaffolds may impact cell behaviour and present a barrier themselves, ultimately limiting transferability of test results. In this work we present an alternative for technical filter membrane systems: a 3D bioprinted biological membrane in 24 well format. The biological membrane, based on extracellular matrix (ECM), is highly permeable and presents a natural 3D environment for cell culture. Inspired by the human placenta we established a coculture of a trophoblast-derived cell line (BeWo b30), together with primary placental fibroblasts within the biological membrane (simulating villous stroma) and primary human placental endothelial cells-representing three cellular components of the human placental villus. All cell types maintained their cell type specific marker expression after two weeks of coculture on the biological membrane. In permeability assays the trophoblast layer developed a barrier on the biological membrane, which was even more pronounced when cocultured with fibroblasts. In this work we present a filter membrane free scaffold, we characterize its properties and assess its suitability for cell culture and barrier models. Further we show a novel placenta inspired model in a complex bioprinted coculture. In the absence of an artificial filter membrane, we demonstrate barrier architecture and functionality.