Search

Recent advances in scaffold-free three-dimensional (3D) culture methods have significantly enhanced the potential of stem cell-based therapies in regenerative medicine. This cutting-edge technology circumvents the use of exogenous biomaterial and prevents its associated complications. The 3D culture system preserves crucial intercellular interactions and extracellular matrix support, closely mimicking natural biological niches. Therefore, stem cells cultured in 3D formats exhibit distinct characteristics, showcasing their capabilities in promoting angiogenesis and immunomodulation. This review aims to elucidate foundational technologies and recent breakthroughs in 3D scaffold-free stem cell engineering, offering comprehensive guidance for researchers to advance this technology across various clinical applications. We first introduce the various sources of stem cells and provide a comparative analysis of two-dimensional (2D) and 3D culture systems. Given the advantages of 3D culture systems, we delve into the specific fabrication and harvesting techniques for cell sheets and spheroids. Furthermore, we explore their applications in pre-clinical studies, particularly in large animal models and clinical trials. We also discuss multidisciplinary strategies to overcome existing limitations such as insufficient efficacy, hostile microenvironments, and the need for scalability and standardization of stem cell-based products.

The management of diabetic wounds presents a considerable challenge within the realm of clinical practice. Cellular-derived nanoparticles, or extracellular vesicles (EV), generated by human adipose-derived stem cells (hASCs) have been investigated as promising candidates for the treatment of diabetic wounds. Nevertheless, limitations on the yield, as well as the qualitative angiogenic properties of the EV produced, have been a persistent issue. In this study, a novel approach involving the use of various cell culture morphologies, such as cell spheroids, on hASC was used to promote both EV yield and qualitative angiogenic properties for clinical use, with an emphasis on the in vivo angiogenic properties exhibited by the EV. Moreover, an increase in the secretion of the EV was confirmed after cell spheroid culture. Furthermore, microRNA(miRNA) analysis of the produced EVs indicated an increase in the presence of wound healing-associated miRNAs on the cell spheroid EV. Analysis of the effectiveness of the treated EVs in vitro indicated a significant promotion of the biological function of fibroblast and endothelial cells, cell migration, and cell proliferation post-cell spheroid EV application. Meanwhile, in vivo experiments on diabetic rats indicated a significant increase in collagen production, re-epithelization, and angiogenesis of the diabetic wound after EV administration. In this investigation, we posit that the use of cell spheroids for the culture of hASC represents a novel approach to enhance the substantial secretion of extracellular vesicles while increasing the angiogenic wound healing properties. This innovation holds promise for augmenting the therapeutic potential of EVs in diabetic wound healing, aligning with the exigencies of clinical applications for these nanoparticles.

Abstract Background Application of autologous adipose-derived stem cells (ASC) for diabetic chronic wounds has become an emerging treatment option. However, ASCs from diabetic individuals showed impaired cell function and suboptimal wound healing effects. We proposed that adopting a low-glucose level in the culture medium for diabetic ASCs may restore their pro-healing capabilities. Methods ASCs from diabetic humans and mice were retrieved and cultured in high-glucose (HG, 4.5 g/L) or low-glucose (LG, 1.0 g/L) conditions. Cell characteristics and functions were investigated in vitro. Moreover, we applied diabetic murine ASCs cultured in HG or LG condition to a wound healing model in diabetic mice to compare their healing capabilities in vivo. Results Human ASCs exhibited decreased cell proliferation and migration with enhanced senescence when cultured in HG condition in vitro. Similar findings were noted in ASCs derived from diabetic mice. The inferior cellular functions could be partially recovered when they were cultured in LG condition. In the animal study, wounds healed faster when treated with HG- or LG-cultured diabetic ASCs relative to the control group. Moreover, higher collagen density, more angiogenesis and cellular retention of applied ASCs were found in wound tissues treated with diabetic ASCs cultured in LG condition. Conclusions In line with the literature, our study showed that a diabetic milieu exerts an adverse effect on ASCs. Adopting LG culture condition is a simple and effective approach to enhance the wound healing capabilities of diabetic ASCs, which is valuable for the clinical application of autologous ASCs from diabetic patients.

Sophisticated axolotl limb regeneration is a highly orchestrated process that requires highly regulated gene expression and epigenetic modification patterns at precise positions and timings. We previously demonstrated two waves of post-amputation expression of a nerve-mediated repressive epigenetic modulator, histone deacetylase 1 (HDAC1), at the wound healing (3 days post-amputation; 3 dpa) and blastema formation (8 dpa onward) stages in juvenile axolotls. Limb regeneration was profoundly inhibited by local injection of an HDAC inhibitor, MS-275, at the amputation sites. To explore the transcriptional response of post-amputation axolotl limb regeneration in a tissue-specific and time course-dependent manner after MS-275 treatment, we performed transcriptome sequencing of the epidermis and soft tissue (ST) at 0, 3, and 8 dpa with and without MS-275 treatment. Gene Ontology (GO) enrichment analysis of each coregulated gene cluster revealed a complex array of functional pathways in both the epidermis and ST. In particular, HDAC activities were required to inhibit the premature elevation of genes related to tissue development, differentiation, and morphogenesis. Further validation by Q-PCR in independent animals demonstrated that the expression of 5 out of 6 development- and regeneration-relevant genes that should only be elevated at the blastema stage was indeed prematurely upregulated at the wound healing stage when HDAC1 activity was inhibited. WNT pathway-associated genes were also prematurely activated under HDAC1 inhibition. Applying a WNT inhibitor to MS-275-treated amputated limbs partially rescued HDAC1 inhibition, resulting in blastema formation defects. We propose that post-amputation HDAC1 expression is at least partially responsible for pacing the expression timing of morphogenic genes to facilitate proper limb regeneration.

Axolotls have amazing ability to regenerate their lost limbs. Our previous works showed that after amputation the remnant muscle ends remained at their original location whilst sending satellite cells into the regenerating parts to develop into early muscle fibers in the late differentiation stage. The parental and the newly formed muscle fibers were not connected until very late stage. The present study used non-invasive diffusion tensor imaging (DTI) to monitor weekly axolotl upper arm muscles after amputation of their upper arms. DTI tractography showed that the regenerating muscle fibers became visible at 9-wpa (weeks post amputation), but a gap was observed between the regenerating and parental muscles. The gap was filled at 10-wpa, indicating reconnection of the fibers of both muscles. This was confirmed by histology. The DTI results indicate that 23% of the muscle fibers were reconnected at 10-wpa. In conclusion, DTI can be used to visualize axolotls' skeletal muscles and the results of muscle reconnection were in accordance with our previous findings. This non-invasive technique will allow researchers to identify the timeframe in which muscle fiber reconnection takes place and thus enable the study of the mechanisms underlying this reconnection.