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.

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.

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.

Retinal degenerative diseases are frequently caused by the loss of retinal neural cells such as photoreceptors. Cell replacement is regarded as one of the most promising therapies. Multiple types of stem and somatic cells have been tested for photoreceptor conversion. However, current induction efficiencies are still low and the molecular mechanisms underlying reprogramming remain to be clarified. In this work, by combining treatment with small molecules, we directly reprogrammed human fetal retinal pigment epithelial (RPE) cells into chemically induced photoreceptor-like cells (CiPCs) in vitro. Bulk and single-cell RNA sequencing, as well as methylation sequencing, were performed to understand the transcriptional and epigenetic changes during CiPCs conversion. A multi-omics analysis showed that the direct reprogramming process partly resembled events of early retina development. We also found that the efficiency of CiPCs conversion from RPE is much better than that from human dermal fibroblasts (HDF). The small molecules effectively induced RPE cells into CiPCs via suppression of the epithelial-to-mesenchymal transition (EMT). Among the signaling pathways involved in CiPCs conversion, glutamate receptor activation is prominent. In summary, RPE cells can be efficiently reprogrammed into photoreceptor-like cells through defined pharmacological modulations, providing a useful cell source for photoreceptor generation in cell replacement therapy for retinal degenerative diseases.

We aim to investigate a Layer-by-Layer (LBL) bioprinting process using stem cells for retinal tissue regeneration. The LBL nature of the bioprinting process matches nicely with the native, multilaminar anatomy of the retina. This research will bioprint retinal stem cells (RSCs) and appropriate growth factors (GF) encapsulated in a biomaterial (e.g. hyaluronic acid, HA). We will investigate the mechanical, chemical and biological properties of the hydrogel materials. We will study the cell viability and analyze the biological functions of the 3D printed retina tissue.