BackgroundThe growing prevalence of cardiovascular diseases (CVDs) has created an increasing demand for alternative biologically functional vascular grafts. Among the explored tissue engineering strategies, decellularization provides a means to generate suitable acellular scaffolds from donor tissues with preserved complex extracellular matrix (ECM) architecture and vascular basement membrane (BM), both of which are critical for graft endothelialization and function. To assess decellularization efficiency and quality, proteomic profiling offers high-resolution characterization of tissue composition, enabling systematic analysis of extracellular matrix subgroups and residual proteins with the aid of open-access annotation tools, thus providing critical insights beyond conventional methods.MethodsHuman saphenous vein segments were decellularized using a CHAPS-based protocol, and decellularization efficiency was evaluated by DNA and glycosaminoglycan (GAG) quantification, together with histological evaluation. A subset of samples (n = 9) was further subjected to label-free quantitative proteomic profiling using LC-MS/MS to assess ECM composition, BM integrity, and residual cellular content.ResultsDecellularization led to 99.96% DNA removal and 53.78% GAG retention, with well-preserved tissue morphology. Decellularization efficiency was further assessed by label-free quantitative proteomics, which showed well-preserved ECM composition. Collagens, proteoglycans, and BM proteins were largely well-maintained, whereas ECM glycoproteins and matrisome-affiliated proteins were moderately reduced. The strongest decrease in protein abundance was observed among cellular proteins, and Gene Ontology analysis confirmed the reduction of key cellular protein groups.DiscussionIn this study, we provide a comprehensive proteomic evaluation of decellularized human saphenous veins, demonstrating selective preservation and loss across ECM subgroups and cellular proteins. The findings highlight proteomics as a valuable complement to conventional assays for assessing decellularization efficiency and establishing a reference framework for optimizing protocols in vascular tissue engineering.

Recent clinical trial data show curative potential of cell therapy for diabetes, however the cells required are a bottleneck. Cell differentiation exhibits substantial variability, even among clones of stem cells generated from the same patient. Human experts struggle to see the difference between highly- and lowly-efficient cell clones early. We therefore propose an image-based deep learning model to guide the selection of the most efficient clones. We apply different deep learning models to learn the morphological differences between good and bad stem cell clones and classify them based on phase-contrast imaging. To gain insight into the learned features, we use layer-wise relevance propagation, and Fourier-based frequency analysis. Using an EfficientNet-V2-S model, we obtain a novel early prediction for the outcome of the differentiation process from patient-derived stem cells to [Formula: see text] -cells using imaging. Clone level accuracy is 96.7 % at 53 hours after start of differentiation. The analysis of learned features shows that the structure of the cell population is an important predictive feature. This study is a proof-of-concept that deep learning combined with label-free imaging can be highly predictive and guide selection of stem cell clones, thereby reducing cost of [Formula: see text] -cell production.

The growing gap between organ demand and clinical availability has renewed interest in immune-evasive graft strategies, yet rejection and lifelong immunosuppression remain major barriers to durable success. Advances in genome editing enable immune-evasive cell platforms designed to avoid immune recognition while replacing missing function in allogeneic settings. This review summarizes current strategies for engineering immune-evasive grafts that simultaneously suppress adaptive and innate immune responses. We discuss how coordinated modulation of antigen presentation and immune checkpoint pathways can protect transplanted allogeneic cells and tissues from T, NK, and macrophage-mediated rejection. We also present the emerging concept of integrating hypoimmune engineering with genetically modified porcine donors, where extensive genome editing has reduced, but not eliminated, xenogeneic immune barriers. Combining donor genome modification with immune-evasive graft design represents a promising conceptual advance toward xenograft survival, though whether full elimination of systemic immunosuppression is achievable remains to be established clinically. We further examine how the regulatory landscape for these products is evolving across major jurisdictions, and how differences in approval pathways, manufacturing standards, and long-term surveillance requirements shape the path to clinical translation. Finally, we outline the safety considerations and remaining limitations in immune evasion that must be addressed to enable clinical implementation.

The need to suppress a patient's immune system after the transplantation of allogeneic cells is associated with wide-ranging side effects. We report the outcomes of transplantation of genetically modified allogeneic donor islet cells into a man with long-standing type 1 diabetes. We used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12b (Cas12b) editing and lentiviral transduction to genetically edit the cells to avoid rejection; the cells were then transplanted into the participant's forearm muscle. He did not receive any immunosuppressive drugs and, at 12 weeks after transplantation, showed no immune response against the gene-edited cells. C-peptide measurements showed stable and glucose-responsive insulin secretion. A total of four adverse events occurred, none of which were serious or related to the study drug. (Funded by the Leona M. and Harry B. Helmsley Charitable Trust; EudraCT number, 2023-507988-19-00; ClinicalTrials.gov number, NCT06239636.).

Porcine corneas were decellularized for future use in corneal regeneration by using various washing steps with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) detergent, Benzonase, and Ethylenediaminetetraacetic acid (EDTA). The quality of the decellularized corneas was assessed by quantitative and qualitative measurement of DNA content, glycosaminoglycans (GAGs), immunofluorescent staining for Collagen (Col) I, V, Keratocan, Fibronectin, Laminin, Lumican and Decorin, and Transmission Electron Microscopy (TEM) to observe the structure of the collagen fibrils was used. The decellularization process showed 99.5% DNA content reduction in the corneas and a similar pattern was observed in the preservation of GAGs. Hematoxylin & Eosin (H&E) and immunofluorescent staining showed no presence of cell nuclei, while Alcian blue staining confirmed the presence of GAGs. Col I, V, Keratocan, Fibronectin, Laminin, Lumican and Decorin were still present in the decellularized corneas and TEM microscopy further confirmed the similar patterns of the collagen fibrils in the decellularized, compared to the native corneas. This pilot study showed our method is effective in decellularizing porcine corneas, with a very high amount of DNA being removed, while the GAGs being preserved to an acceptable extent, and the structure and pattern of the collagen fibrils maintained.

The generation of insulin-producing cells from human-induced pluripotent stem cells holds great potential for diabetes modeling and treatment. However, existing protocols typically involve incubating cells with un-physiologically high concentrations of glucose, which often fail to generate fully functional IPCs. Here, we investigated the influence of high (20 mM) versus low (5.5 mM) glucose concentrations on IPCs differentiation in three hiPSC lines. In two hiPSC lines that were unable to differentiate to IPCs sufficiently, we found that high glucose during differentiation leads to a shortage of NKX6.1+ cells that have co-expression with PDX1 due to insufficient NKX6.1 gene activation, thus further reducing differentiation efficiency. Furthermore, high glucose during differentiation weakened mitochondrial respiration ability. In the third iPSC line, which is IPC differentiation amenable, glucose concentrations did not affect the PDX1/NKX6.1 expression and differentiation efficiency. In addition, glucose-stimulated insulin secretion was only seen in the differentiation under a high glucose condition. These IPCs have higher KATP channel activity and were linked to sufficient ABCC8 gene expression under a high glucose condition. These data suggest high glucose concentration during IPC differentiation is necessary to generate functional IPCs. However, in cell lines that were IPC differentiation unamenable, high glucose could worsen the situation.

Organoids are 3D cell cultures with microanatomies mimicking aspects of real organs, useful for e.g. animal-free studies of development, disease, and drug discovery. The cell medium of organoid models of Langerhans islets, regulating blood glucose levels by insulin secretion, can be analyzed by liquid chromatography-mass spectrometry (LC-MS). However, organoid medium complexity is a major challenge, as matrix interferences can reduce sensitivity and selectivity, even with optimized LC-MS conditions. By applying preparative agarose gel electrophoresis-electrodialysis (PGE-ED), we were able to decrease the cell medium background signal, allowing for reduced interferences affecting LC-MS analysis of human insulin.

Stem cell-derived islets (SC-islets) are not only an unlimited source for cell-based therapy of type 1 diabetes but have also emerged as an attractive material for modeling diabetes and conducting screening for treatment options. Prior to SC-islets becoming the established standard for disease modeling and drug development, it is essential to understand their response to various nutrient sources in vitro. This study demonstrates an enhanced efficiency of pancreatic endocrine cell differentiation through the incorporation of WNT signaling inhibition following the definitive endoderm stage. We have identified a tri-hormonal cell population within SC-islets, which undergoes reduction concurrent with the emergence of elevated numbers of glucagon-positive cells during extended in vitro culture. Over a 6-week period of in vitro culture, the SC-islets consistently demonstrated robust insulin secretion in response to glucose stimulation. Moreover, they manifested diverse reactivity patterns when exposed to distinct nutrient sources and exhibited deviant glycolytic metabolic characteristics in comparison to human primary islets. Although the SC-islets demonstrated an aberrant glucose metabolism trafficking, the evaluation of a potential antidiabetic drug, pyruvate kinase agonist known as TEPP46, significantly improved in vitro insulin secretion of SC-islets. Overall, this study provided cell identity dynamics investigation of SC-islets during prolonged culturing in vitro, and insights into insulin secretagogues. Associated advantages and limitations were discussed when employing SC-islets for disease modeling.

The field of organ transplantation is experiencing a transformative shift with the rise of Advanced Therapy Medicinal Products (ATMPs), which include gene therapies, somatic cell therapies, and tissue-engineered products. These therapies offer new, potentially curative treatments for longstanding medical challenges, impacting numerous patients. However, their adoption is hindered by complex regulatory frameworks, high production costs, and inconsistent access across Europe. The ESOT ATMP Task Force’s position paper analyzes these challenges from research to clinical application, advocating for a coordinated strategy to position Europe as a leader in ATMP development. It proposes specific actions such as streamlining regulatory pathways to accelerate approvals, boosting funding for ATMP research, and creating specialized facilities for development and implementation. The paper also highlights the critical roles of patient engagement and real-world evidence in optimizing clinical and regulatory practices.

Background: Tailoring surgical treatment is mandatory to optimize outcomes in chronic pancreatitis. Total pancreatectomy (TP) offers pain relief in a subset of patients. TP with islet autotransplantation (IAT) has the potential to reduce the burden of postsurgical diabetes. We present the first Scandinavian prospective study assessing outcomes following total pancreatectomy and islet autotransplantation (TPIAT) in chronic pancreatitis. Our aim was to assess short- and long-term outcomes following implementation of a nationwide program of TPIAT at a tertiary reference center for pancreatic surgery in Norway.Methods: A prospective, observational single-center study enrolling consecutive patients undergoing TPIAT for chronic pancreatitis at Oslo University Hospital. The selection of potential candidates for TPIAT was based on discussions at multidisciplinary team (MDT) meetings, focusing on tailored surgery in chronic pancreatitis. Patients were finally evaluated in a dedicated TPIAT team. The outcome measures included pain relief, quality of life (QoL) assessed by EORTC QLQ-C30, complications, and glycemic control.Results: Between August 2017 and November 2022, 15 patients underwent TPIAT. The follow-up rate was 87% with a median follow-up of 26 months (range = 14-65). Pain relief was achieved in 92%. EORTC QLQ-C30 analysis revealed clinically significant improvements in 28 of 30 domains, particularly in pain and role- and social-functioning. The Clavien-Dindo >= IIIa complications occurred in one patient. There was no 90 days mortality. All patients maintained C-peptide positivity, although none of the patients reached insulin independence.Conclusion: TPIAT was as a safe and effective treatment for a selected group of patients with chronic pancreatitis, providing substantial pain relief and enhanced QoL. Islet autotransplantation prevented complete insulin deficiency, reducing diabetes severity postpancreatectomy. Dedicated chronic pancreatitis MDT meetings were key factor in the success of the program.

Purpose: To effectively decellularize human and porcine corneas for future use in corneal regeneration by using an already established decellularization technique being efficient in decellularizing blood vessels. Methods: One human and 4 porcine corneas were decellularized by using various washing steps with 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propanesulfonate (CHAPS) detergent, Benzonase, Ethylenediaminetetraacetic acid (EDTA), 5% Dextran or 5% D‐Mannitol. The quality of the decellularization process was assessed by quantitative and qualitative measurement of DNA content, glycosaminoglycans (GAGs), immunofluorescent staining for Collagen I, V and keratocan, and Transmission Electron Microscopy (TEM) to observe the structure of the collagen fibrils was used. Results: The decellularization process showed 99,94% and 99,52% DNA content reduction in the human and porcine corneas when using Dextran, respectively, while more DNA remained in the porcine corneas when D‐Mannitol was used. Similar pattern was observed in the preservation of GAGs, with the highest amount being found in the human cornea, and less in the porcine corneas. H&E and Immunofluorescent staining showed no presence of cell nuclei when compared to the control native corneas, while Alcian blue staining qualitatively confirmed the presence of GAGs. Collagen I, V and keratocan were still present in the decellularized corneas when compared to the native ones, while TEM microscopy further confirmed the similar patterns of the collagen fibrils in the decellularized‐, compared to the native‐ corneas. Conclusions: This pilot study showed that our decellularization method is effective in decellularizing human and porcine corneas, with very high amount of DNA being removed from the corneas, while the GAGs were preserved to an acceptable extent, and the structure and pattern of the collagen fibrils maintained.

Perinatal derivatives have been proposed as adjunct therapeutic strategies or innovative treatments. Undoubtedly, perinatal derivatives can offer the opportunity and source material to isolate multipotent stem cells, but both maternal- and fetal-derived tissues can be processed and transformed into engineered tissues or advanced biomedical devices, whose potential remains to be fully elucidated. Promising preclinical and clinical results collected so far clearly foresee an escalation of such novel treatments. Market forecasts predict exponential growth in such advanced medicinal products during the next decade, with a pragmatic innovation for medicine into a more advanced biomedical version, enlarging the portfolio for treating a wide range of congenital and acute conditions. However, all these promising and fascinating therapeutic possibilities cannot gain a solid and recognized role in established medical practice without rigid and harmonized manufacturing strategies. The implementation of strategies according to guidelines and directives compiled by Regulatory Agencies, in conformity to (European) Pharmacopoeia and for Good Manufacturing Practice -conforming production of such products, represent critical steps required to translate perinatal technologies into effective therapeutic approaches. During the past 5 years, a panel of European experts and developers, gathered under the umbrella of the COST Sprint Action, supported by the European Cooperation in Science and Technology action, had the opportunity to revise and summarize experience and recommendations for a fruitful and proficient generation of perinatal biomedical products. In order to facilitate the creation and potential commercialization of perinatal bioengineered and advanced pharmaceutical products and technologies, such a collection of data and recommendations is described and discussed here.

Aims/Purpose: To assess whether 3D bioprinted, scaffold‐immobilized antimicrobial peptides (AMPs) derivatives, BMAP27(1–18) and hLF(1–11) can inhibit microbial colonization to obtain infection‐resistant 3D bioprinted hemi‐corneas. Methods: The research followed the tenets of the Declaration of Helsinki. 3D bioprinting was used to produce transparent nanocellulose‐ and alginate‐based 3D constructs. Functionalized 3D bioprinted scaffolds with AMPs were characterized by contact angle and transparency measurements. The antimicrobial and antifungal activity of the peptides in solution were investigated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods against Staphylococcus epidermidis ATCC 35984, S. aureus ATCC 25923, S. epidermidis ATCC 12228, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and Candida albicans ATCC 90028. The antimicrobial activity of the AMP‐modified scaffolds was studied against P. aeruginosa and C. albicans reference strains by colony forming unit (CFU) counts, and their biocompatibility was investigated by measuring the viability of primary human/cadaver‐isolated corneal stromal‐mesenchymal stromal cells (CS‐MSCs). Results: The original and modified peptides showed MIC (MBC) values of 1–4 (2–8) μM and 16 – >128 (32 – >128) μM, respectively, against S. epidermidis ATCC 35984, S. aureus , S. epidermidis ATCC 12228, P. aeruginosa , E. coli and C. albicans , while ncBP‐BMAP27(1–18) and ncBP‐hLF(1–11) values were 1–16 (2–32) μM 4–128 (4 – >128) μM, respectively, against all the pathogens. The modified scaffolds containing the AMPs attached to the scaffolds, demonstrated the ability to kill 20–96% of P. aeruginosa , and between 60–70% of C. albicans . All the 3D bioprinted scaffolds showed no cytotoxicity towards CS‐MSCs. Conclusions: We could successfully combine 3D bioprinting with immobilization of AMPs that show promising antimicrobial activity against two of the most dangerous pathogens to the cornea, while a good cytocompatibility for the CS‐MSCs could be achieved. Optimal bioink composition and cellularization of tissue equivalents are essential in fine‐tuning a method to promote the current 3D bioprinting technique as a future treatment modality for corneal regeneration.

Type 2 diabetes mellitus (T2DM), obesity, and metabolic dysfunction-associated steatotic liver disease (MASLD) are epidemiologically correlated disorders with a worldwide growing prevalence. While the mechanisms leading to the onset and development of these conditions are not fully understood, predictive tissue representations for studying the coordinated interactions between central organs that regulate energy metabolism, particularly the liver and pancreatic islets, are needed. Here, a dual pump-less recirculating organ-on-chip platform that combines human pluripotent stem cell (sc)-derived sc-liver and sc-islet organoids is presented. The platform reproduces key aspects of the metabolic cross-talk between both organs, including glucose levels and selected hormones, and supports the viability and functionality of both sc-islet and sc-liver organoids while preserving a reduced release of pro-inflammatory cytokines. In a model of metabolic disruption in response to treatment with high lipids and fructose, sc-liver organoids exhibit hallmarks of steatosis and insulin resistance, while sc-islets produce pro-inflammatory cytokines on-chip. Finally, the platform reproduces known effects of anti-diabetic drugs on-chip. Taken together, the platform provides a basis for functional studies of obesity, T2DM, and MASLD on-chip, as well as for testing potential therapeutic interventions.