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Cultivated meat provides a sustainable and ethical alternative to traditional livestock production, addressing global challenges in meat demand and environmental impact. Fat is recognized as an essential component of meat products, but strategies for culturing animal fat precursors still have major limitations, such as the need for a serum-containing medium and inefficient adipogenic cell maturation. In this study, a serum-free medium (SFM) formulation for the cultivation of bovine adipose-derived stem cells (bASC) was first validated by comparison with serum-containing media (FBS). After a 14-day induction period, the SFM increased bASC proliferation by 20% and triglyceride accumulation by 66%. When the SFM was applied to a three-dimensional (3D) spheroid culture system, it resulted in up to 34% higher triglyceride accumulation compared to 2D cultures. These results were accompanied by a significant increase in the expression of key adipogenic markers and lipid droplet formation, suggesting a synergistic potential of SFM and 3D spheroid strategies for bASC culture. Overall, the results of this study provide a scalable and sustainable platform for cultured fat production and are an important step towards overcoming some of the challenges in cultured meat production.

Cryopreservation enhances the availability of “off-the-shelf” cell therapies. However, the choice between tissue culture polystyrene (TCP) and hollow fiber system (HFB) system for adipose-derived stem cell (ASC) production remains a critical decision, with implications for scalability, reproducibility, and the clinical efficacy. Therefore, the characteristics of ASCs expanded in TCP and HFB and cryopreserved were compared. TCP and HFB cultures were established, and cells were cryopreserved. Surface markers were analyzed to identify immunophenotypic changes and subpopulations. Clonogenicity, differentiation capability, and proliferation potentials were determined along with surrogate tests on wound healing. The expressions of the most markers were consistent before and after thawing for both systems. However, CD105 expression of TCP cells was significantly decreased by the freeze-thawing procedure. Also, CD274 was significantly less expressed on HFB-expanded cells before freezing, however, post-thawing, the proportion of CD274 positive cells was comparable to TCP cells. Besides, two expansions supported different subpopulations, influencing the heterogeneity within ASC cultures. Despite this heterogeneity, no statistical differences were observed as for ASC functional characteristics and the effects on fibroblasts. This study highlighted freeze-thaw does not interfere with the production of fully functional ASCs in either system, although it drives some differential changes in the subpopulations between systems.

Myogenic precursors sense and dynamically respond to mechanical stimulation through complex integrin-mediated mechanotransduction, in which focal adhesion kinase (FAK) is a fundamental intracellular signaling mediator. When skeletal myoblasts are exposed to uniaxial cyclic tensile strain (UCTS), they display uniform alignment and an enhanced rate of differentiation. In this work, we explored the role of FAK activation by using C2C12 myoblasts that were grown on flexible culture plates and exposed to UCTS during the early differentiation phase. After 24 h, the cells oriented perpendicularly to the direction of strain and exhibited an enhanced differentiation profile. Next, the cells were exposed to a strain field that was either kept in the same direction or rotated 90°, in the presence or not of an FAK phosphorylation inhibitor. On reorientation of the strain field by 90°, the cells reassembled their focal adhesions and actin cytoskeleton to regain the perpendicular position with respect to the engaging stress. After blocking the FAK, however, the cells failed to respond to the reoriented strain field and their differentiation was abrogated. Interestingly, when the strain field remained in the same direction, the FAK inhibitor compromised the differentiation, even though there was no evident change in cell orientation. Our data indicate that during exposure to UCTS, the activation of FAK is necessary for the myoblasts to undergo alignment and enhanced differentiation.

In pre-clinical studies, human adipose-derived stem cells (hASCs) have shown great promise as a treatment modality for healing of cutaneous wounds. The advantages of hASCs are that they are relatively easy to obtain in large numbers from basic liposuctions, they maintain their characteristics after long-term in vitro culture, and they possess low immunogenicity, which enables the use of hASCs from random donors. It has been hypothesized that hASCs exert their wound healing properties by reducing inflammation, inducing angiogenesis, and promoting fibroblast and keratinocyte growth. Due to the inherent variability associated with the donor-dependent nature of ASC-based products, it appears necessary that the quality of the different products is prospectively certified using a set of most relevant potency assays. In this review, we present an overview of the available methodologies to assess the Mode and the Mechanism of Action of hASCs, specifically in the wound healing scenario. In conclusion, we propose a panel of potential potency assays to include in the future production of ASC-based medicinal products.

Background: Reliable in vitro cellular models are needed to study the phenotypic modulation of smooth muscle cells (SMCs) in health and disease. The aim of this study was to optimize gelatin methacrylate (GelMA)/alginate hydrogels for bioprinting three-dimensional (3D) SMC constructs. Methods: Four different hydrogel groups were prepared by mixing different concentrations (% w/v) of GelMA and alginate: G1 (5/1.5), G2 (5/3), G3 (7.5/1.5), and G4 (7.5/3). GelMA 10% was used as control (G5). A circular structure containing human bladder SMCs was fabricated by using an extrusion-based bioprinter. The effects of the mixing ratios on printability, viability, proliferation, and differentiation of the cells were investigated. Results: Rheological analysis showed that the addition of alginate significantly stabilized the change in mechanical properties with temperature variations. The group with the highest GelMA and alginate concentrations (G4) exhibited the highest viscosity, resulting in better stability of the 3D construct after crosslinking. Compared to other hydrogel compositions, cells in G4 maintained high viability (> 80%), exhibited spindle-shaped morphology, and showed a significantly higher proliferation rate within an 8-day period. More importantly, G4 provided an optimal environment for the induction of a SMC contractile phenotype, as evidenced by significant changes in the expression of marker proteins and morphological parameters. Conclusion: Adjusting the composition of GelMA/alginate hydrogels is an effective means of controlling the SMC phenotype. These hydrogels support bioprinting of 3D models to study phenotypic smooth muscle adaptation, with the prospect of using the constructs in the study of therapies for the treatment of urethral strictures.

The corneal homeostasis is maintained by limbal epithelial stem cells (LESCs), which reside in the limbal niche. This microenvironment comprises the cells, the extracellular matrix (ECM), and their interactions that balance the quiescent and proliferative states of LESCs. The stress caused by removing the cells from their niche triggers the quiescent stem cells to enter the proliferative state, which is beneficial for expansion, but reduces their self-renewal capability, making them less suitable for transplantation. Fibronectin (FN), a key ECM component, widely used in tissue engineering and scaffold structure, has been shown to preserve the self-renewal ability of LESCs . In parallel, paracrine growth factors are crucial for maintaining limbal niche homeostasis and promoting corneal epithelial regeneration. Limbal-niche-cells-conditioned media is a potential reservoir of limbal niche paracrine growth factors. However, whether utilizing fibronectin and limbal-niche-cells-conditioned media can sustain or enhance the stemness and proliferation ability of LESCs has not yet been investigated. Primary cultures of limbal niche cells, including LESCs, limbal mesenchymal stromal cells (LMSCs), and limbal melanocytes (LM), were established from remnant human corneal transplant specimens, and human epidermal melanocytes (HEMn) were included as a negative control. The proliferation ability (doubling time) and self-renewal potential (as assessed by and gene expressions) of LESCs were evaluated after culture in LM-, LMSC-, and HEMn-conditioned media, as well as coating with 3, 5, and 8 µg/cm concentrations of FN. Compared to the control group, the LMSC- and LM-conditioned media showed a clear trend towards upregulated and gene expressions. FN coating generally upregulated the expression of and genes, with this effect being most prominent at 3 µg/cm . These findings illustrate the potential of utilizing niche-cell-conditioned media and direct contact with FN on the self-renewal of LESCs . Further research is required to provide a more comprehensive understanding of these effects and to elucidate the underlying mechanisms of action.

Ex vivo engineering of skeletal muscle represents an exciting new area of biotechnology. Although the ability of skeletal muscle cells to sense and respond to mechanical forces is well known, strategies based on the use of mechanical stimuli to optimize myogenesis in vitro remain limited. In this work, we describe a simple but powerful method based on uniaxial cyclic tensile strain (CTS) to induce assembly and differentiation of skeletal myocytes in vitro. Confluent mouse myoblastic precursors cultured on flexible-bottomed culture plates were subjected to either uniaxial or equibiaxial CTS. The uniaxial CTS protocol resulted in a highly aligned array of cross-striated fibers, with the major axis of most cells aligned perpendicularly to the axis of strain. In addition, a short period of myogenin activation and significant increase in the myotube/myoblast ratio and percentage of myosin-positive myotubes was found, indicating an enhanced cell differentiation. In contrast, cells under equibiaxial strain regimen had no clear orientation and displayed signs of membrane damage and impaired differentiation. These results, thus, demonstrate that the selection of a proper paradigm is a key element when discussing the relevance of mechanical stimulation for myogenesis in vitro. This study provides a rational framework to optimize engineering of functional skeletal muscle.

Smooth muscle differentiated adipose tissue-derived stem cells are a valuable resource for regeneration of gastrointestinal tissues, such as the gut and sphincters. Hypoxia has been shown to promote adipose tissue-derived stem cells proliferation and maintenance of pluripotency, but the influence of hypoxia on their smooth myogenic differentiation remains unexplored. This study investigated the phenotype and contractility of adipose-derived stem cells differentiated toward the smooth myogenic lineage under hypoxic conditions. Oxygen concentrations of 2%, 5%, 10%, and 20% were used during differentiation of adipose tissue-derived stem cells. Real time reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of smooth muscle cells-specific markers, including early marker smooth muscle alpha actin, middle markers calponin, caldesmon, and late marker smooth muscle myosin heavy chain. The specific contractile properties of cells were verified with both a single cell contraction assay and a gel contraction assay. Five percent oxygen concentration significantly increased the expression levels of α-smooth muscle actin, calponin, and myosin heavy chain in adipose-derived stem cell cultures after 2 weeks of induction (p < 0.01). Cells differentiated in 5% oxygen conditions showed greater contraction effect (p < 0.01). Hypoxia influences differentiation of smooth muscle cells from adipose stem cells and 5% oxygen was the optimal condition to generate smooth muscle cells that contract from adipose stem cells.

Corneal epithelium is maintained throughout life by well-orchestrated proliferation of limbal epithelial stem cells (LESCs), followed by migration and maturation centripetally towards the ocular surface. Disturbance of LESCs can potentially lead to a blinding condition, which can be reversed by reconstitution of a functional LESC pool. The current clinical procedures are effective to some degree, however, deeper knowledge of the molecular interplay within the limbal niche is necessary to achieve a fully satisfactory patient outcome. The present study was thus undertaken to carry out a comprehensive transcriptome analysis of four distinct human limbal compartments, including basal limbal crypts (BLCs), superficial limbal crypts (SLCs), cornea, and the supporting stroma, with the aid of laser capture microdissection and deep RNA sequencing. The tissue harvest pipeline was rigorously optimized so that the exposure to cold ischemia would be less than five minutes. The global gene ontology analysis confirmed existence of primitive cells in BLCs, migratory and activated cells in SLCs, and differentiated cells in cornea. Interestingly, many significantly upregulated genes in SLCs mapped to processes involved in regulation of vasculature, such as sFLT1. In contrast, BLCs exhibited many genes mapping to neurogenic processes and processes related to cell development. The primitive nature of BLCs was, furthermore, confirmed by the KEGG pathway analysis, and some potential regulators of LESCs were revealed, such as Lrig1 and SOX9. The analysis also yielded comprehensive lists of uniquely expressed genes in both BLCs and cornea, which may be useful to identify possible biomarkers. In conclusion, the current investigation provides new insight into the relationship between distinct cell populations within the limbal niche, identifies candidates to be verified for novel biological functions, and yields a wealth of information for prospective data mining.

Treatment of severe burn wounds presents a daunting medical challenge, and novel approaches promoting healing and reducing scarring are highly desirable. The application of mesenchymal stem/stromal cells (MSCs) has been suggested as a novel treatment. In this paper, we present systematic reviews of pre-clinical and clinical studies of MSC therapy for second- or third-degree thermal burn wounds. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, the PubMed and Embase databases were searched, and interventional studies of MSC therapy using rodent models (21 studies) or human burn patients (three studies) were included in the pre-clinical and clinical reviews, respectively, where both overall outcome and wound-healing-phase-specific methodologies and effects were assessed. The pre-clinical studies demonstrated a promising effect of the application of MSCs on several wound healing phases. The clinical studies also suggested that the MSC treatment was beneficial, particularly in the remodeling phase. However, the limited number of studies, their lack of homogeneity in study design, relatively high risk of bias, lack of reporting on mode of action (MOA), and discontinuity of evidence restrict the strength of these findings. This comprehensive review presents an overview of available methodologies to assess the MOA of MSC treatment for distinct wound healing phases. Furthermore, it includes a set of recommendations for the design of high-quality clinical studies that can determine the efficacy of MSCs as a therapy for burn wounds.