Multifaceted cancer alleviation by cowpea mosaic virus in a bioprinted ovarian cancer peritoneal spheroid model.

Ovarian cancer (OvCa) is a leading cause of mortality among gynecological malignancies and usually manifests as intraperitoneal spheroids that generate metastases, ascites, and an immunosuppressive tumor microenvironment. In this study, we explore the immunomodulatory properties of cowpea mosaic virus (CPMV) as an adjuvant immunotherapeutic agent using an in vitro model of OvCa peritoneal spheroids. Previous findings highlighted the potent efficacy of intratumoral CPMV against OvCa in mouse tumor models. Leveraging the precision control over material deposition and cell patterning afforded by digital-light-processing (DLP) based bioprinting, we constructed OvCa-macrophage spheroids to mimic peritoneal spheroids using gelatin methacrylate (GelMA), a collagen-derived photopolymerizable biomaterial to mimic the extracellular matrix. Following CPMV treatment, bioprinted spheroids exhibited inhibited OvCa progression mediated by macrophage activation. Our analysis indicates that CPMV regulates and activates macrophage to both induce OvCa cell killing and restore normal cell-cell junctions. This study deepened our understanding of the mechanism of CPMV intratumoral immunotherapy in the setting of OvCa. This study also highlights the potential of studying immunotherapies using high throughput tissue models via DLP bioprinting.

Heat-inducible CAR-T overcomes adverse mechanical tumor microenvironment in a 3D bioprinted glioblastoma model.

Glioblastoma (GBM) presents a significant therapeutic challenge due to the limited efficacy of existing treatments. Chimeric antigen receptor (CAR) T-cell therapy offers promise, but its potential in solid tumors like GBM is undermined by the physical barrier posed by the extracellular matrix (ECM). To address the inadequacies of traditional 2D cell culture, animal models, and Matrigel-based 3D culture in mimicking the mechanical characteristics of tumor tissues, we employed biomaterials and digital light processing-based 3D bioprinting to fabricate biomimetic tumor models with finely tunable ECM stiffness independent of ECM composition. Our results demonstrated that increased material stiffness markedly impeded CAR-T cell penetration and tumor cell cytotoxicity in GBM models. The 3D bioprinted models enabled us to examine the influence of ECM stiffness on CAR-T cell therapy effectiveness, providing a clinically pertinent evaluation tool for CAR-T cell development in stiff solid tumors. Furthermore, we developed an innovative heat-inducible CAR-T cell therapy, effectively overcoming the challenges posed by the stiff tumor microenvironment.

Integration of 3D bioprinting and multi-algorithm machine learning identified glioma susceptibilities and microenvironment characteristics.

Glioma, with its heterogeneous microenvironments and genetic subtypes, presents substantial challenges for treatment prediction and development. We integrated 3D bioprinting and multi-algorithm machine learning as a novel approach to enhance the assessment and understanding of glioma treatment responses and microenvironment characteristics. The bioprinted patient-derived glioma tissues successfully recapitulated molecular properties and drug responses of native tumors. We then developed GlioML, a machine learning workflow incorporating nine distinct algorithms and a weighted ensemble model that generated robust gene expression-based predictors, each reflecting the diverse action mechanisms of various compounds and drugs. The ensemble model superseded the performance of all individual algorithms across diverse in vitro systems, including sphere cultures, complex 3D bioprinted multicellular models, and 3D patient-derived tissues. By integrating bioprinting, the evaluative scope of the treatment expanded to T cell-related therapy and anti-angiogenesis targeted therapy. We identified promising compounds and drugs for glioma treatment and revealed distinct immunosuppressive or angiogenic myeloid-infiltrated tumor microenvironments. These insights pave the way for enhanced therapeutic development for glioma and potentially for other cancers, highlighting the broad application potential of this integrative and translational approach.

High cell density and high-resolution 3D bioprinting for fabricating vascularized tissues.

Three-dimensional (3D) bioprinting techniques have emerged as the most popular methods to fabricate 3D-engineered tissues; however, there are challenges in simultaneously satisfying the requirements of high cell density (HCD), high cell viability, and fine fabrication resolution. In particular, bioprinting resolution of digital light processing-based 3D bioprinting suffers with increasing bioink cell density due to light scattering. We developed a novel approach to mitigate this scattering-induced deterioration of bioprinting resolution. The inclusion of iodixanol in the bioink enables a 10-fold reduction in light scattering and a substantial improvement in fabrication resolution for bioinks with an HCD. Fifty-micrometer fabrication resolution was achieved for a bioink with 0.1 billion per milliliter cell density. To showcase the potential application in tissue/organ 3D bioprinting, HCD thick tissues with fine vascular networks were fabricated. The tissues were viable in a perfusion culture system, with endothelialization and angiogenesis observed after 14 days of culture.

Rapid 3D bioprinting of a multicellular model recapitulating pterygium microenvironment.

Pterygium is an ocular surface disorder with high prevalence that can lead to vision impairment. As a pathological outgrowth of conjunctiva, pterygium involves neovascularization and chronic inflammation. Here, we developed a 3D multicellular in vitro pterygium model using a digital light processing (DLP)-based 3D bioprinting platform with human conjunctival stem cells (hCjSCs). A novel feeder-free culture system was adopted and efficiently expanded the primary hCjSCs with homogeneity, stemness and differentiation potency. The DLP-based 3D bioprinting method was able to fabricate hydrogel scaffolds that support the viability and biological integrity of the encapsulated hCjSCs. The bioprinted 3D pterygium model consisted of hCjSCs, immune cells, and vascular cells to recapitulate the disease microenvironment. Transcriptomic analysis using RNA sequencing (RNA-seq) identified a distinct profile correlated to inflammation response, angiogenesis, and epithelial mesenchymal transition in the bioprinted 3D pterygium model. In addition, the pterygium signatures and disease relevance of the bioprinted model were validated with the public RNA-seq data from patient-derived pterygium tissues. By integrating the stem cell technology with 3D bioprinting, this is the first reported 3D in vitro disease model for pterygium that can be utilized for future studies towards personalized medicine and drug screening.

Bioprinting of dual ECM scaffolds encapsulating limbal stem/progenitor cells in active and quiescent statuses.

Limbal stem cell deficiency and corneal disorders are among the top global threats for human vision. Emerging therapies that integrate stem cell transplantation with engineered hydrogel scaffolds for biological and mechanical support are becoming a rising trend in the field. However, methods for high-throughput fabrication of hydrogel scaffolds, as well as knowledge of the interaction between limbal stem/progenitor cells (LSCs) and the surrounding extracellular matrix (ECM) are still much needed. Here, we employed digital light processing (DLP)-based bioprinting to fabricate hydrogel scaffolds encapsulating primary LSCs and studied the ECM-dependent LSC phenotypes. The DLP-based bioprinting with gelatin methacrylate (GelMA) or hyaluronic acid glycidyl methacrylate (HAGM) generated microscale hydrogel scaffolds that could support the viability of the encapsulated primary rabbit LSCs (rbLSCs) in culture. Immunocytochemistry and transcriptional analysis showed that the encapsulated rbLSCs remained active in GelMA-based scaffolds while exhibited quiescence in the HAGM-based scaffolds. The primary human LSCs encapsulated within bioprinted scaffolds showed consistent ECM-dependent active/quiescent statuses. Based on these results, we have developed a novel bioprinted dual ECM 'Yin-Yang' model encapsulating LSCs to support both active and quiescent statues. Our findings provide valuable insights towards stem cell therapies and regenerative medicine for corneal reconstruction.

Rapid bioprinting of conjunctival stem cell micro-constructs for subconjunctival ocular injection.

Ocular surface diseases including conjunctival disorders are multifactorial progressive conditions that can severely affect vision and quality of life. In recent years, stem cell therapies based on conjunctival stem cells (CjSCs) have become a potential solution for treating ocular surface diseases. However, neither an efficient culture of CjSCs nor the development of a minimally invasive ocular surface CjSC transplantation therapy has been reported. Here, we developed a robust in vitro expansion method for primary rabbit-derived CjSCs and applied digital light processing (DLP)-based bioprinting to produce CjSC-loaded hydrogel micro-constructs for injectable delivery. Expansion medium containing small molecule cocktail generated fast dividing and highly homogenous CjSCs for more than 10 passages in feeder-free culture. Bioprinted hydrogel micro-constructs with tunable mechanical properties enabled the 3D culture of CjSCs while supporting viability, stem cell phenotype, and differentiation potency into conjunctival goblet cells. These hydrogel micro-constructs were well-suited for scalable dynamic suspension culture of CjSCs and were successfully delivered to the bulbar conjunctival epithelium via minimally invasive subconjunctival injection. This work integrates novel cell culture strategies with bioprinting to develop a clinically relevant injectable-delivery approach for CjSCs towards the stem cell therapies for the treatment of ocular surface diseases.