Background:Abdominal wall closure after transplantation is crucial to reduce morbidity and to ensure graft and patient survival, but it often remains challenging. Donor nonvascularized rectus fascia (NVRF) allotransplantation offers a valuable solution when primary closure fails. Despite the growing clinical use of NVRF, standardized protocols for NVRF preservation are lacking. This study evaluates the impact of short-term cold storage at 4 °C (8 d) and long-term cryopreservation (1 mo and 6 mo) on human NVRF at the mechanical, histological, and microbiological levels. Methods:NVRF was obtained from 10 human donors (6 men; 6 donation after circulatory death III; 4 donation after brain death; age 56 y [26-68 y]; BMI 28 kg/m2 [23-31 kg/m2]). Fresh tissue was compared with 3 preservation methods: (1) cold storage in Institute Georges Lopez-1 solution at 4 °C for 8 d; (2) 1 mo cryopreservation; and (3) 6 mo cryopreservation at -80 °C. For each condition, the analysis included uniaxial tensile testing, histological evaluation based on hematoxylin and eosin and picrosirius red staining, and microbiological analysis for yeast and bacteria. Results:After 8 d at 4 °C, ultimate strength and rigidity were reduced, whereas histological structure appeared intact. One month of cryopreservation showed no indications for altered mechanical or histological characteristics compared with fresh tissue. After prolonged cryopreservation for 6 mo, extensibility was increased. Yeast contamination was absent in all samples, and commensal bacteria were detected in 17 of 40 fresh and preserved samples, without impact of the preservation method. Conclusions:In conclusion, cryopreservation at -80 °C maintains biomechanical characteristics and structural integrity of NVRF for up to 1 mo, offering a promising method for prolonged NVRF preservation in clinical applications.

Pulsed electromagnetic field (PEMF) stimulation has been widely investigated in musculoskeletal applications. This study investigates its application to promote bovine myoblast differentiation and maturation for cultured meat production, aiming to generate muscle constructs that better resemble conventional meat. PEMF stimulation duration and the effect of intermittent recovery were evaluated in terms of metabolic activity, differentiation markers, and structural characteristics in both 2D myoblast cultures and 3D bio-artificial muscle constructs. The best results were obtained with daily intermittent PEMF stimulation consisting of 90s stimulations separated by 30s breaks for 2h per day. This intermittent protocol increased the metabolic activity of myotubes, particularly those derived from low-fusion donors. Additionally, an increase in myoblast fusion capacity was observed under this condition. When applied to bio-artificial muscle constructs, both intermittent and 2h continuous PEMF stimulation enhanced metabolic activity, multinucleation, and striation, while maintaining similar fusion capacities to non-stimulated muscle constructs. Furthermore, PEMF-stimulated constructs exhibited significant enhancements in mechanical properties, with stiffness increasing similar to 2-fold and ultimate tensile strength rising by similar to 1.7-fold, approaching values observed in native adult muscle tissue. Concurrently, continuous stimulation led to an increase in myotube diameter. These findings suggest that low-intensity PEMF stimulation is a promising tool for muscle tissue engineering. For cultured meat applications, PEMF stimulation could improve the texture and structure of muscle constructs, thereby enhancing consumer acceptance of structured meat products.

Volumetric muscle loss is the significant loss of skeletal muscle volume beyond the innate regenerative capacity, resulting in functional impairment. The current standard of care combines muscle autografting with physical therapy but is often insufficient to reach full recovery. Decellularized skeletal muscle (DSM) provides an interesting alternative to repair volumetric muscle loss. The native structure and composition of the extracellular matrix in these acellular implants provide a blueprint for muscle regeneration. Moreover, DSM can be combined with cells to facilitate the regeneration of the skeletal muscle defect. This systematic review provides a complete and thorough overview of the state-of-the-art applications and efficacy of DSM matrices in skeletal muscle repair in vivo, selected according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Technical information on the different methods to create DSM implants and the implantation studies is provided. Moreover, details on the evaluation of the structural and functional regeneration of the muscle defect after implantation of the DSM are described. Results reveal a large heterogeneity in the analysis of regeneration upon DSM implantation. This heterogeneity makes it difficult to fully assess the efficiency of DSM to regenerate skeletal muscle, hampering further translation of this technique. Therefore, we suggest a multi-level evaluation method to assess (i) muscle regeneration, (ii) vascularization, (iii) innervation of the regenerated muscle, and (iv) functional regeneration in a quantitative way.

Large-scale production of cultured meat requires muscle cell culture in bioreactors, where microcarriers (MCs) support cell attachment, growth, and differentiation. However, most MCs are composed of inedible materials, requiring a cell detachment step, and/or contain animal-derived components, which are undesirable for cultured meat production. Therefore, we developed animal-free edible microcarriers based on soy protein isolate (SPI) that support muscle cell growth. SPI MCs supported cell attachment and growth similar to commercial collagen-coated dextran MCs, as bovine myoblasts expanded 24-fold over 8 days in a bioreactor. Moreover, myoblasts could differentiate into myotubes on the SPI-MCs. Importantly, SPI supported cell attachment in serum-free medium, as opposed to methacrylated gelatin (GelMA). Proteomics analysis revealed that, during SPI processing, cell adhesion peptides become available on the biomaterial, which also partially leach into the cell culture medium and replace serum components. To conclude, our study demonstrates the feasibility of growing and differentiating bovine muscle cells on edible, fully plant-based MCs, providing a scalable system for the production of cultured meat.

Mesenchymal stromal cells (MSCs) hold significant potential for various applications in regenerative medicine and tissue engineering. Initially considered as a single cell type with defined characteristics, MSCs are now known as a heterogeneous cell population with remarkable differences in their properties. No consensus exists on how donor age affects MSC characteristics, like proliferation. Additionally, differences in differentiation capacities and immunophenotype could arise when MSCs are isolated from different animals breeds, which is relevant for experimental and preclinical studies of MSC-based treatments. In this study, we isolated bovine adipose tissue-derived MSCs from three age categories, i.e. fetal, calf, and adult, and of two different breeds, i.e. Holstein Friesian (HF) and Belgian Blue (BB). MSC characterization included tri-lineage differentiation, proliferation and senescence assays, and immunophenotyping using multi-color flow cytometry. Especially fetal and calf HF-MSCs showed a high proliferation capacity, where 4 and 6 out of 7 donors, respectively, could surpass 30 population doublings. Adipogenic differentiation potential was higher for fetal and adult HF-MSCs. Furthermore, breed, but not age, affected their osteogenic differentiation potential, with BB-MSCs performing better. Evaluation of cell surface marker expression revealed a breed effect, as calf HF-MSCs showed a higher percentage of Cluster of Differentiation (CD)34+ cells compared to calf BB-MSCs, which was correlated with both osteogenic differentiation and proliferation potential. Our findings clearly show the impact of donor characteristics such as age and breed on MSC proliferation, immunophenotype, and differentiation potential, illustrating the importance of selecting the appropriate MSC donor for MSC-based treatments when allogeneic MSCs are considered.

As the global population continues to grow, the demand for sustainable food production methods becomes increasingly critical. This study investigates the presence of chromosomal abnormalities in bovine embryonic stem cells (bESCs), which hold potential for innovative food sources such as cultured meat. We derived three bESC lines from day-eight post-insemination blastocysts using a whole blastocyst plating approach. These cells maintained core pluripotency markers (POU5F1, SOX2, SALL4, NANOG) and demonstrated the ability to differentiate into the three germ layers, indicating their potential for sustainable food applications. Through shallow whole-genome sequencing, we identified various chromosomal anomalies, including mono-, tri-, and tetrasomies, with specific gains in chromosomes 7, 12, 27, and 29, and losses in chromosome 9. Notably, these aneuploidies progressively accumulated over time, raising concerns about genomic stability in long-term cultures. The implications of these genomic variants are significant for the development of cultured meat, as they may affect the efficiency and safety of production processes. This research underscores the necessity for systematic monitoring and optimizing culture conditions to mitigate genomic instabilities, ensuring the safe application of bESCs in sustainable food systems. Our findings pave the way for future innovations in clean food processing and utilising new food ingredients.

Cellular agriculture and regenerative medicine strive to replace animal-derived products in their processes as much as possible. In these efforts dissociation enzymes required for cell detachment from their substrate are often overlooked. Cell detachment is an essential part of subculturing and to date it is achieved using either animal pancreas-derived trypsin or expensive recombinant enzymes. We found that nattokinase, a fermentation product of Bacillus subtilis subsp. Natto, can be effectively applied for the detachment of bovine myoblasts, mesenchymal stromal cells (MSCs) and human embryonic cell line (H9). We described the suitable enzyme concentrations, incubation times and inhibition procedures for each cell type. Long-term subculture with nattokinase did not negatively affect proliferation nor differentiation of the tested cells and even led to significantly higher number of CD56+ cells in myoblast culture after six passages (88 ± 3 % vs 73 ± 5 %, p = 0.03, n = 6) compared to trypsin control. Efficacy, affordability, and current “generally recognized as safe” status of nattokinase make it an attractive product for cell-based food manufacturing.

When considering non-genetically modified primary bovine cells for large-scale cultured meat production, one must account for their biological limitations, such as proliferation and differentiation capacity. This study tests these limitations in primary cells from fetal and adult bovine donors. Bovine myoblasts are expanded past 30 doublings and characterized for CD56 expression, senescence, and myogenic differentiation capacity. Contrary to the Hayflick limit, several cell lines from adult cows surpass 60 population doublings, all with normal karyotypes. One line maintains high CD56 expression for the first 25 doublings. However, differentiation capacity declines, with fetal-derived cells showing high fusion indexes early on, but minimal fusion in both adult- and fetal-derived cells past 25 doublings. Differential transcriptomic and proteomic analyses of adult myoblasts with higher versus lower fusion indices identify many significantly affected genes and pathways. Genes related to myogenesis, DNA repair, and calcium signaling, among others, are downregulated in low fusing cells. This research shows the potential to expand unmodified primary bovine myoblasts for industrial cultured meat production, but further research is needed to address the lack of differentiation in expanded cells to replicate the fibrous texture and protein composition of meat.

Background. Failure to close the abdominal wall after intestinal transplantation (ITx) or multivisceral Tx remains a surgical challenge. An attractive method is the use of nonvascularized rectus fascia (NVRF) in which both layers of the donor abdominal rectus fascia are used as an inlay patch without vascular anastomosis. How this graft integrates over time remains unknown. The study aims to provide a multilevel analysis of the neovascularization and integration process of the NVRF. Methods. Three NVRF-Tx were performed after ITx. Clinical, radiological, histological, and immunological data were analyzed to get insights into the neovascularization and integration process of the NVRF. Moreover, cryogenic contrast-enhanced microfocus computed tomography (microCT) analysis was used for detailed reconstruction of the vasculature in and around the NVRF (3-dimensional histology). Results. Two men (31- and 51-y-old) and 1 woman (49-y-old) underwent 2 multivisceral Tx and 1 combined liver-ITx, respectively. A CT scan showed contrast enhancement around the fascia graft at 5 days post-Tx. At 6 weeks, newly formed blood vessels were visualized around the graft with Doppler ultrasound. Biopsies at 2 weeks post-Tx revealed inflammation around the NVRF and early fibrosis. At 6 months, classical 2-dimensional histological analysis of a biopsy confirmed integration of the fascia graft with strong fibrotic reaction without signs of rejection. A cryogenic contrast-enhanced microCT scan of the same biopsy revealed the presence of microvasculature, enveloping and penetrating the donor fascia. Conclusions. We showed clinical, histological, and microCT evidence of the neovascularization and integration process of the NVRF after Tx.

The fusion index is a key indicator for quantifying the differentiation of a myoblast population, which is often calculated manually. In addition to being time-consuming, manual quantification is also error prone and subjective. Several software tools have been proposed for addressing these limitations but suffer from various drawbacks, including unintuitive interfaces and limited performance. In this study, we describe MyoFInDer, a Python-based program for the automated computation of the fusion index of skeletal muscle. At the core of MyoFInDer is a powerful artificial intelligence-based image segmentation model. MyoFInDer also determines the total nuclei count and the percentage of stained area and allows for manual verification and correction. MyoFInDer can reliably determine the fusion index, with a high correlation to manual counting. Compared with other tools, MyoFInDer stands out as it minimizes the interoperator variability, minimizes process time and displays the best correlation to manual counting. Therefore, it is a suitable choice for calculating fusion index in an automated way, and gives researchers access to the high performance and flexibility of a modern artificial intelligence model. As a free and open-source project, MyoFInDer can be modified or extended to meet specific needs.

Failure to close the abdomen after intestinal or multivisceral transplantation (Tx) remains a frequently occurring problem. Two attractive reconstruction methods, especially in large abdominal wall defects, are full-thickness abdominal wall vascularized composite allograft (AW-VCA) and nonvascularized rectus fascia (NVRF) Tx. This review compares surgical technique, immunology, integration, clinical experience, and indications of both techniques. In AW-VCA Tx, vascular anastomosis is required and the graft undergoes hypotrophy post-Tx. Furthermore, it has immunologic benefits and good clinical outcome. NVRF Tx is an easy technique without the need for vascular anastomosis. Moreover, a rapid integration and neovascularization occurs with excellent clinical outcome.

With the current environmental impact of large-scale animal production and societal concerns about the welfare of farm animals, researchers are questioning whether we can cultivate animal cells for the purpose of food production. This review focuses on a pivotal aspect of the cellular agriculture domain: cells. We summarised information on the various cell types from farm animals currently used for the development of cultured meat, including mesenchymal stromal cells, myoblasts, and pluripotent stem cells. The review delves into the advantages and limitations of each cell type and considers factors like the selection of the appropriate cell source, as well as cell culture conditions that influence cell performance. As current research in cultured meat seeks to create muscle fibers to mimic the texture and nutritional profile of meat, we focused on the myogenic differentiation capacity of the cells. The most commonly used cell type for this purpose are myoblasts or satellite cells, but given their limited proliferation capacity, efforts are underway to formulate myogenic differentiation protocols for mesenchymal stromal cells and pluripotent stem cells. The multipotent character of the latter cell types might enable the creation of other tissues found in meat, such as adipose and connective tissues. This review can help guiding the selection of a cell type or culture conditions in the context of cultured meat development.

Decellularized organs and tissues are emerging within the field of regenerative medicine to meet the growing demand for organ and tissue transplantation. Quality control of these acellular matrices prior to transplantation is of paramount importance to ensure the absence of an adverse reaction. In particular, thorough evaluation of the DNA content is essential but also poses technical challenges. Therefore, in this study, we compared different methods for quantitative and qualitative evaluation of DNA content in native and decellularized skeletal muscle tissue to identify strengths and weaknesses for each. Histological analysis revealed that Feulgen staining is more sensitive and robust than the commonly used hematoxylin-eosin and 4',6-diamidino-2-phenylindole staining for detection of remaining nuclear material. Furthermore, gel electrophoresis allowed to identify the quality and length of remaining DNA fragments. The results of the quantitative analysis indicated that direct measurement of DNA content in tissue lysates is preferred over silica-based extraction methods, since the latter resulted in the loss of small DNA fragments during extraction. Moreover, a weight loss correction factor should be implemented to take into account the impact of the decellularization on the extracellular matrix. With regard to the detection method, the results revealed that a fluorescence-based approach is more accurate than the use of UV/VIS absorbance. Through combination of the proposed methods, it should be possible to achieve a more standardized evaluation of novel acellular matrices in terms of DNA content and to enhance the predictability of clinical success.

Decellularized matrices are an attractive choice of scaffold in regenerative medicine as they can provide the necessary extracellular matrix (ECM) components, signals and mechanical properties. Various detergent-based protocols have already been proposed for decellularization of skeletal muscle tissue. However, a proper comparison is difficult due to differences in species, muscle origin and sample sizes. Moreover, a thorough evaluation of the remaining acellular matrix is often lacking. We compared an in-house developed decellularization protocol to four previously published methods in a standardized manner. Porcine skeletal muscle samples with uniform thickness were subjected to in-depth histological, ultrastructural, biochemical and biomechanical analysis. In addition, 2D and three-dimensional cytocompatibility experiments were performed. We found that the decellularization methods had a differential effect on the properties of the resulting acellular matrices. Sodium deoxycholate combined with deoxyribonuclease I was not an effective method for decellularizing thick skeletal muscle tissue. Triton X-100 in combination with trypsin, on the other hand, removed nuclear material but not cytoplasmic proteins at low concentrations. Moreover, it led to significant alterations in the biomechanical properties. Finally, sodium dodecyl sulphate (SDS) seemed most promising, resulting in a drastic decrease in DNA content without major effects on the ECM composition and biomechanical properties. Moreover, cell attachment and metabolic activity were also found to be the highest on samples decellularized with SDS. Through a newly proposed standardized analysis, we provide a comprehensive understanding of the impact of different decellularizing agents on the structure and composition of skeletal muscle. Evaluation of nuclear content as well as ECM composition, biomechanical properties and cell growth are important parameters to assess. SDS comes forward as a detergent with the best balance between all measured parameters and holds the most promise for decellularization of skeletal muscle tissue.

Loss of prolyl endopeptidase-like ( PREPL ) encoding a serine hydrolase with (thio)esterase activity leads to the recessive metabolic disorder Congenital Myasthenic Syndrome -22 (CMS22). It is characterized by severe neonatal hypotonia, feeding problems, growth retardation, and hyperphagia leading to rapid weight gain later in childhood. The phenotypic similarities with Prader-Willi syndrome (PWS) are striking, suggesting that similar pathways are affected. The aim of this study was to identify changes in the hypothalamic-pituitary axis in mouse models for both disorders and to examine mitochondrial function in skin fibroblasts of patients and knockout cell lines. We have demonstrated that Prepl is downregulated in the brains of neonatal PWS-IC -p/+m mice. In addition, the hypothalamic-pituitary axis is similarly affected in both Prepl -/- and PWS-IC -p/+m mice resulting in defective orexigenic signaling and growth retardation. Furthermore, we demonstrated that mitochondrial function is altered in PREPL knockout HEK293T cells and can be rescued with the supplementation of coenzyme Q10. Finally, PREPL-deficient and PWS patient skin fibroblasts display defective mitochondrial bioenergetics. The mitochondrial dysfunction in PWS fibroblasts can be rescued by overexpression of PREPL. In conclusion, we provide the first molecular parallels between CMS22 and PWS, raising the possibility that PREPL substrates might become therapeutic targets for treating both disorders.

Complex abdominal wall repair remains a major surgical challenge. In transplant patients, non-vascularized rectus fascia (NVRF) is successfully used to bridge the defect. To extrapolate this to non-transplant patients, we developed a rabbit model of NVRF-transplantation without immunosuppression comparing syngeneic versus allogeneic transplants. Short-term outcome (4 weeks) was evaluated macroscopically (ingrowth, seroma/hematoma, herniation, and infection), histologically at the graft interface and center (inflammation, neovascularization, and collagen deposition) and by mechanical testing. In both groups a similar macroscopic ingrowth of the NVRF was observed. In the syn-group, one seroma and one hematoma was seen. Two small herniations were detected at the suture line in the allo-group. No surgical site infections were observed. Histologically, graft neovascularization was observed in all animals. Infiltration of T-lymphocytes was seen at the graft interface in both groups, but more in the allo-group (p < 0.0001). Deposition of collagen was not different between groups. Macrophages were present in both groups around sutures and in the center more abundantly in the allo-group (p = 0.0001). Graft stiffness and strength were similar for both groups. With this model, we showed that allogeneic transplantation without immunosuppression results in favorable short-term inflammatory and mechanical outcomes. Long-term experiments are needed to further evaluate the effect on graft integration and hernia development.

Failure to close the abdomen after intestinal or multivisceral transplantation (Tx) remains a frequently occurring problem. Two attractive reconstruction methods, especially in large abdominal wall defects, are full-thickness abdominal wall vascularized composite allograft (AW-VCA) and nonvascularized rectus fascia (NVRF) Tx. This review compares surgical technique, immunology, integration, clinical experience, and indications of both techniques. In AW-VCA Tx, vascular anastomosis is required and the graft undergoes hypotrophy post-Tx. Furthermore, it has immunologic benefits and good clinical outcome. NVRF Tx is an easy technique without the need for vascular anastomosis. Moreover, a rapid integration and neovascularization occurs with excellent clinical outcome.