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

Abstract

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.

PBP‐based bioprinting of hydrogels for biomedical applications

“…Different materials with different properties can affect cells differently. Zhong et al 67 prepared GelMA and glycidyl methacrylated hyaluronic acid (HAGM) microscale hydrogel scaffolds using PBP technology. Encapsulated limbal stem/progenitor cells (LSCs) remain active in GelMA but static in HAGM, providing an innovative “yin‐yang” model that offers new insights into stem cell therapy and corneal reconstruction as well as novel engineering approaches for new disease models.…”

3D bioprinting of complex tissues in vitro: state-of-the-art and future perspectives

“…GelMA, a gelatin-based biomaterial in which the primary amines in the lysine backbone of gelatin are replaced with methacrylate groups to facilitate photo-initiated free-radical polymerization, is one of the most common (van Hoorick et al 2019 ). For example, GelMA-based micro-constructs with encapsulated conjunctival stem cells were created using DLP-based bioprinting (Zhong et al 2021a , b ), where the GelMA provided a nurturing 3D environment that maintained stem cell phenotype and differentiation potency while maintaining the vitality. Click chemistry methods have been emerging as a way to modify gelatin because of its high efficiency, high selectivity, and minimal side reactions at mild reaction conditions.…”

“…In another example, HAGM was used to print the scaffold to support the quiescence state of the limbal stem/progenitor cells (LSC), while the cells remained active when encapsulated in GelMA. The distinct states of the cells in different biomaterials enabled the fabrication of dual-state cells in a single construct, addressing a better mimicry of the native LSC niche (Zhong et al 2021a , b ). For further applications, HA can be modified for click chemistry crosslinking via a variety of mechanisms.…”

Digital light processing (DLP)‐based (bio)printing strategies for tissue modeling and regeneration

“…[30,55,56] Moreover, multiple ECM components can be considered as an ideal bioink to recapitulate the complex native microenvironment to simulate different targeted tissues for cell proliferation and differentiation. [57][58][59] Figure 1B exhibits the number of papers published on the DLP printing tissue model since 2016, reflecting the numbers of research article have been increasing sharply, and covered various fields (Figure 1C).…”

“…[12][13][14] Currently, 3D bioprinting improved the application of tissue structure in drug screening, [15,16] disease modeling, [17,18] and tissue repair and regenerative medicine. [19,20] Most of importantly, heart, [21,22] blood vessels, [23,24] bone, [25,26] cartilage, [27,28] liver, [1,29,30] lung, [1,31] eye, [32,33] neuronal tissue, [34,35] and pancreatic tissue [36] have been successfully designed and developed through 3D extrusion-based bioprinting and DLPbased bioprinting strategies (Figure 1A). However, the resolution of extrusion-based 3D bioprinting structure is relatively limited, and the resolution of conventional extrusion printing is usually less than 200 μm, and the obtained structure is also easy to cause stress relaxation and permanent deformation due to the different crosslinking rate during the printing process.…”