Cytotherapy最新文献

{"title":"A Multifunctional Microfluidic Device for Tailoring Analytical Assay Development for Multiproduct Cell Therapy Products (CTPs)","authors":"A. Nisar ,&nbsp;S. Singh ,&nbsp;P. Gururaj Joshi ,&nbsp;K.R. Pandit ,&nbsp;G. Narula ,&nbsp;U. Mohanty ,&nbsp;J. Krishnan","doi":"10.1016/j.jcyt.2025.03.022","DOIUrl":"10.1016/j.jcyt.2025.03.022","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>The rising demand for cell therapies necessitates innovative tools to streamline analytical development &amp; QC assays. We propose a microfluidic device to transform analytical workflows by handling small samples, reducing reagents, &amp; automating processes.</div></div><div><h3>Methodology</h3><div>This device minimizes reagent requirements while maintaining high throughput, enabling cost-efficient and scalable processes. Its microfluidic architecture precisely manipulates &amp; separates T cells from whole blood samples, reducing sample volume significantly compared to conventional methods. Beyond T cell isolation, the device integrates a multifunctional platform capable of performing analytical assays, including ELISA, effectively replacing traditional 96-well plates. This integration enhances its utility by enabling real-time cytokine profiling, immunophenotyping, high-sensitivity biomarker detection, single-cell analysis &amp; multiplexed assays on a single platform.</div></div><div><h3>Results</h3><div>The design of this microfluidic device was developed after thorough review of existing literature &amp; iterative modifications. Key considerations included device dimensions, operational sequences, particle size compatibility, reagent material compatibility, hydrophobicity, and detection sensitivity. The final configuration comprises two pairs of hexagonal channels connected via one inlet and two outlets, utilizing a herringbone structure to optimize cell capture &amp; retention. This structure disrupts flow, increasing turbulence &amp; enhancing cell interaction with channel surfaces. The device leverages microfluidic principles, manipulating fluid behavior using microliter to picoliter volumes with channel dimensions in the micrometer range. It adheres to the Hele-Shaw flow property, defined as Stokes Flow between two parallel plates separated by a narrow gap, ensuring laminar flows with low Reynolds numbers. Fabricated from advanced polymers like PDMS and PMMA, the device ensures durability, biocompatibility, and seamless integration with automated workflows. It supports parallel processing of multiple samples, reducing assay time while maintaining precision and reproducibility. The system is compatible with existing laboratory tools like flow cytometry &amp; imaging systems, enabling effortless adoption in clinical and industrial settings.</div></div><div><h3>Conclusion</h3><div>By combining versatility, scalability, and cost-efficiency, this microfluidic system represents a robust platform for CAR T analytical development, poised to advance the manufacturing and QC landscapes of CGT.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S18"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888149","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Acute respiratory distress vs healthy lung environments differently affect mesenchymal stromal cell extracellular vesicle miRNAs","authors":"Sara Rolandsson Enes ,&nbsp;Irakli Dzneladze ,&nbsp;Thomas H. Hampton ,&nbsp;Samuel L. Neff ,&nbsp;Lori Asarian ,&nbsp;Jayita Barua ,&nbsp;Tobias Tertel ,&nbsp;Bernd Giebel ,&nbsp;Nicolas Pereyra ,&nbsp;David H. McKenna ,&nbsp;Pingzhao Hu ,&nbsp;Erica Acton ,&nbsp;Alix Ashare ,&nbsp;Kathleen D. Liu ,&nbsp;Anna D. Krasnodembskaya ,&nbsp;Karen English ,&nbsp;Bruce A. Stanton ,&nbsp;Patricia R.M. Rocco ,&nbsp;Michael A. Matthay ,&nbsp;Claudia C. dos Santos ,&nbsp;Daniel J. Weiss","doi":"10.1016/j.jcyt.2025.01.006","DOIUrl":"10.1016/j.jcyt.2025.01.006","url":null,"abstract":"<div><div>The acute respiratory distress syndrome (ARDS) inflammatory environment alters mesenchymal stromal cell (MSC) gene and protein expression but effects on microRNA (miRNA) content of MSC-extracellular vesicle (EVs) remain unknown. To assess this, sequencing analysis of EV-miRNAs prepared from human bone marrow-derived MSCs (hMSCs) exposed <em>ex vivo</em> to bronchoalveolar lavage fluid (BALF) from ARDS patients or healthy volunteers (HV) identified a number of differentially expressed miRNAs. Discriminant, differential expression, and functional enrichment analyses identified 14 miRNAs significantly changed following ARDS versus HV BALF exposure. Network analysis showed 4 (miR-760, miR-3175, miR-885-3p, and miR-766-3p) of the 14 EV-miRNAs formed a regulatory “hub”, suggesting co-targeting of specific gene pathways. <em>In silico</em> prediction identified a number of pathways important in lung injury. Two miRNAs involved in regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), miRNA-145-5p and miRNA-138-5p, were also significantly increased in ARDS BALF-exposed hMSCs EVs. Functionally, EVs from hMSCs exposed to either ARDS or HV BALF had differential effects on CFTR Cl^- secretion by cultured primary human bronchial epithelial cells, an effect predicted to reduce mucociliary clearance. The potential clinical impact of these finding highlights the need for further studies assessing the role of hMSC-EV miRNAs in regulating lung inflammation and mucociliary clearance.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages 581-596"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143411368","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of CD141+vasculogenic precursor cells from human bone marrow and their endothelial engagement in the arteriogenesis by co-transplantation with mesenchymal stem cells","authors":"Y. Son ,&nbsp;G. Park ,&nbsp;D. Hwang ,&nbsp;S. Yim ,&nbsp;H. Hong","doi":"10.1016/j.jcyt.2025.03.025","DOIUrl":"10.1016/j.jcyt.2025.03.025","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Critical limb ischemia (CLI) is a condition characterized by insufficient blood flow to the lower limbs, resulting in severe ischemia and potentially leading to amputation. This study aims to identify novel vasculogenic precursor cells (VPCs) in human bone marrow and evaluate their efficacy in combination with bone marrow-derived mesenchymal stem cells (BM-MSCs) for the treatment of CLI</div></div><div><h3>Methodology</h3><div>VPCs and BM-MSCs from bone marrow were cultured <em>ex vivo</em> and comparatively characterized, whose therapeutic effects on neovascularization and long-term tissue regeneration were investigated after singular or a combination transplantation in a mouse CLI model</div></div><div><h3>Results</h3><div>VPCs, expressing high levels of hepatocyte growth factor and c-MET, were identified from human bone marrow aspirates. These cells exhibited strong vasculogenic capacity <em>in vitro</em> but possessed a cellular phenotype distinct from those of previously reported endothelial precursor cells in circulation or cord blood. They also expressed most surface markers of BM-MSCs and demonstrated multipotent differentiation ability. Screening of 376 surface markers revealed that VPCs uniquely display CD141 (thrombomodulin). CD141⁺VPCs are present in BM aspirates as a rare population and can be expanded <em>ex vivo</em> with a population doubling time of approximately 20 h, generating an elaborate vascular network even under angiogenic factor-deficient conditions and recruiting BM-MSCs to the network as pericyte-like cells. Intramuscular transplantation of a combination of human CD141⁺VPCs and BM-MSCs at a ratio of 2:1 resulted in limb salvage, blood flow recovery, and regeneration of large vessels in the femoral artery-removed CLI model, with an efficacy superior to that of singular transplantation. Importantly, large arteries and arterioles in dual cell transplantation expressed human CD31 in the intima and human α-smooth muscle actin in media layer at 4 and 12 weeks, indicating their lineage commitment to endothelial cells and vascular smooth muscle, respectively, <em>in vivo</em>.</div></div><div><h3>Conclusion</h3><div>Dual cell therapeutics comprising human CD141+VPCs and BM-MSCs could be developed for clinical trial to cure human PAD.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages S19-S20"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888157","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advancing Allogenic Cell Therapy with Automated iPSC Processing and Engineering: Benefits of a Closed Modular Approach.","authors":"R.R. Somasagara ,&nbsp;O. Cohen ,&nbsp;Y. Kim ,&nbsp;P. Chaluvappa ,&nbsp;L. Bailey Steinitz ,&nbsp;V. Chandra ,&nbsp;C.T. Dargitz ,&nbsp;A. Chandrasekaran ,&nbsp;N. Ravinder","doi":"10.1016/j.jcyt.2025.03.077","DOIUrl":"10.1016/j.jcyt.2025.03.077","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells have emerged as a promising platform for next-generation immunotherapy, offering a homogeneous, scalable and versatile approach for consistent large-scale manufacturing of off the shelf allogeneic therapies. This approach involves multiple steps including iPSC culturing and banking followed by gene editing and differentiation to tumor specific iNK cells to enhance cytotoxicity and tumor-targeting specificity, while minimizing risks of graft-versus-host disease. Building closed modular automated workflows will help minimize risks associated with manual processes associated with these methods. Through this work we built closed automated cell harvest, gene delivery and editing protocols that can enable iNK-based cell therapy manufacturing.</div></div><div><h3>Methodology</h3><div>In our current workflow, we cultured and expanded iPSCs up to a billion cells in a 10-layer cell factory system for master cell bank preparation. iPSC manual process and harvest in 10-layer cell factory system is very labor intensive and prone to contamination. Utilizing CTS Rotea counterflow centrifugation system minimized human intervention at multiple stages, including removal of media, washing of cells, addition of cell detachment media, collecting, concentrating iPSCs and delivering cells to collecting bags. Using this protocol, we processed the harvesting of entire iPSC culture in 10-layer cell factory system in a single batch to create a master bank for cell therapy development. We then successfully used these iPSC banks and carried out CRISPR-based non-viral gene editing using Neon NxT or CTS Xenon closed automated electroporation system for gene delivery, GMP grade CTS™ StemFlex media and high fidelity CRISPR-Cas9 system.</div></div><div><h3>Results</h3><div>The iPSCs processed using this method retain pluripotency characteristics with good viability and expansion rate. The CRISPR-based non-viral gene editing results shows successful generation of engineered CAR-iPSC with reproducible KI efficiency of up to 15%. We established the optimal target gene and promoter combination to stabilize transgene expression during the differentiation of iPSCs to iNK cells.</div></div><div><h3>Conclusion</h3><div>With methods developed through this work, we successfully generated potent CAR iNK cells. Together the workflows described here utilizing the clean room compliant closed automated cell processing and gene delivery platforms and the GMP compatibility iPSC and NK media systems enable clinical scale iNK cell therapy manufacturing.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages S47-S48"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888221","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Scalable Production of Hematopoietic Stem Cells and Microglia from iPSCs Using Stirred Tank Bioreactors for Consistent Cell Therapy Manufacturing","authors":"J. Cotovio ,&nbsp;P. van Loenen ,&nbsp;S. Braam ,&nbsp;M. Argenziano","doi":"10.1016/j.jcyt.2025.03.040","DOIUrl":"10.1016/j.jcyt.2025.03.040","url":null,"abstract":"<div><h3>Background and Aims</h3><div>Hematopoietic stem cells (HSCs) and microglia are vital for blood, immune, and central nervous system (CNS) development, offering significant potential for cell therapy and neurodegenerative disease treatment. However, producing these cells from human induced pluripotent stem cells (hiPSCs) with consistent quality and scalability remains challenging due to complex differentiation processes. Ncardia has developed robust protocols enabling large-batch, scalable differentiation of HSCs and microglia using stirred tank bioreactors.</div></div><div><h3>Methodology</h3><div>We successfully transitioned small-scale 2D adherent differentiation into 250mL stirred tank bioreactors, aiming to scale up to 1L and 3L systems. A Design of Experiments (DoE) approach was used to optimize critical process parameters (CPPs), including dissolved oxygen, agitation speed, and inoculation density. Parallel protocol testing and in-process monitoring, supported by data-driven analysis, enabled rapid optimization and high reproducibility.</div></div><div><h3>Results</h3><div>Our protocols generated high-quality HSCs and microglia with key functional properties. hiPSC-derived HSCs displayed typical morphology, expressed essential markers (&gt;90% CD34, CD45, CD43), and differentiated into multiple immune cell lineages such as NK cells, macrophages, and microglia, reinforcing their cell therapy potential. Similarly, Ncyte® Microglia expressed core markers (&gt;80% IBA1, TMEM119, CX3CR1) and exhibited functional capabilities like phagocytosis and cytokine secretion in response to inflammatory stimuli. Their ability to co-culture with neurons further enhances their applications in drug discovery and toxicity testing.</div></div><div><h3>Conclusion</h3><div>By integrating expertise in stem cell biology with advanced bioreactor technology, Ncardia enables scalable, high-quality production of HSCs and microglia. This innovation accelerates cell therapy development while supporting regenerative medicine, immunotherapy, and neurodegenerative disease research.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S27"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143887590","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detection of very small embryonic-like stem cells (VSELs) in the peripheral blood of rabbits subjected to induced retinal vein occlusion as a proposed early detection tool for the disease","authors":"E. Gounari ,&nbsp;A. Komnenou ,&nbsp;E. Kofidou ,&nbsp;V. Karampatakis ,&nbsp;G. Koliakos","doi":"10.1016/j.jcyt.2025.03.041","DOIUrl":"10.1016/j.jcyt.2025.03.041","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Very Small Embryonic-like Stem Cells (VSELs) have been successfully detected in human peripheral blood following mobilization under stressful conditions (Bhartiya et al.2016). The aim of the present study is the detection of VSELs in a pharmaceutically induced animal model and the correlation of their number with pro-inflammatory cytokine levels andanimals’ clinical evaluation.</div></div><div><h3>Methodology</h3><div>14 New Zealand rabbits were divided into group I-RVO and group II-control. For the RVO induction group I received intravitreal injections of PD0325901 at a dose of 0.1mL/eye. Successful RVO induction was confirmed through clinical evaluation, histological examinations, quantification of secreted cytokines in the vitreous fluid, and molecular analysis of retinal tissues according to our group's recently published results (Gounari et al.2022).</div><div>Peripheral blood was collected at days 0,7,14 and 36 while VSELs isolated and counted as we have previously described (Gounari et al.2018).</div></div><div><h3>Results</h3><div>Counting of VSELs showed a statistically significant increase in the number of VSELs in the first days compared to the control group (*P=0.03), which was markedly reduced after 2 weeks which proves the direct mobilization of VSELs into the peripheral blood. H&amp;E staining of cell smears in RVO-induced animal group revealed very small sized cells with high nucleo-cytoplasmic ratio able to form embryonic bodies (EBs) in hanging-drop cultures. The positive alkaline phosphatase staining, in combination with the overexpression of the Oct3/4, Nanog and Sox-2 transcription factors confirmed the existence of undifferentiated cells with embryonic like features as a response to damage. Secreted levels of TNF-α, IL-6 and IL-8 measured in plasma samples are related to the numbers of detected-VSELs.</div></div><div><h3>Conclusion</h3><div>We here present a method for VSELs isolation and quantification from RVO-induced animals’ blood. Further research is needed in order to develop an optimized protocol based on VSELs detection able to be applied for the early diagnosis of RVO or other retinal vascular diseases.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages S27-S28"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143887591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Stem Cell Therapy as a Potentially Effective Treatment for End-Stage Kidney Failure in Cats","authors":"Y. Lee ,&nbsp;E. Kang ,&nbsp;J. Ju","doi":"10.1016/j.jcyt.2025.03.098","DOIUrl":"10.1016/j.jcyt.2025.03.098","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Stem cell transplantation is an emerging therapy for feline chronic kidney disease (CKD), showing promising results in many cases. It has been associated with significant improvements in clinical signs and blood test results. Recently, intravenous stem cell injection has been explored as a potential treatment option for managing feline CKD. This therapy has shown benefits in improving appetite, mobility, renal function, and coat condition, as well as slowing the progression of CKD.</div><div>Blood test data from this study demonstrated the effectiveness of stem cell injections across various stages of CKD, classified using the 2023 modified International Renal Interest Society (IRIS) CKD staging guidelines. These stages range from Stage 1 (early) to Stage 4 (end-stage disease).</div></div><div><h3>Methodology</h3><div>This study aimed to evaluate the outcomes of three series of stem cell injections by analyzing key renal indicators—Blood Urea Nitrogen (BUN), Creatinine, Phosphorus, and Symmetric Dimethylarginine (SDMA)—from blood samples, based on the 2023 modified IRIS CKD staging guidelines. The felines were categorized into four groups according to their CKD stages: <strong>Group 1:</strong> Felines at CKD Stage 1, <strong>Group 2:</strong> Felines at CKD Stage 2, <strong>Group 3:</strong> Felines at CKD Stage 3, <strong>Group 4:</strong> Felines at CKD Stage 4</div></div><div><h3>Results</h3><div>A total of 29 felines, all showing elevated concentrations of BUN, Creatinine, Phosphorus, and SDMA prior to treatment, were included in the study. Each feline received three series of intravenous stem cell injections, with intervals of 1 to 3 weeks between injections.</div><div>The results demonstrated a significant reduction in key renal biomarkers (BUN, Creatinine, Phosphorus, and SDMA) following stem cell treatment, with the most pronounced improvements observed in felines at Stage 4 of CKD.</div></div><div><h3>Conclusion</h3><div>This suggests that stem cell injections offer a stable and effective approach for managing CKD, leading to marked clinical improvements and enhanced quality of life. These findings support the potential of stem cell therapy as a complementary treatment to traditional therapies for feline CKD.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S57"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143887598","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Breakthroughs in 3D printing: functional human islets in an alginate-dECM bioink","authors":"Q. Perrier ,&nbsp;W. Jeong ,&nbsp;A. Rengaraj ,&nbsp;L. Byers ,&nbsp;G. Gonzalez ,&nbsp;E. Peveri ,&nbsp;J. Miller ,&nbsp;E. Opara ,&nbsp;R. Bottino ,&nbsp;A. Mikhailov ,&nbsp;C. Fraker ,&nbsp;A. Tomei ,&nbsp;S. Lee ,&nbsp;G. ORLANDO ,&nbsp;A. Asthana","doi":"10.1016/j.jcyt.2025.03.052","DOIUrl":"10.1016/j.jcyt.2025.03.052","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Bioprinting human islets (HI) for beta-cell replacement therapy for treating type 1 diabetes (T1D) holds immense potential but faces challenges due to shear-induced HI damage, reduced functionality, and HI aggregation. Previous studies have mainly focused on animal-derived islets, with limited research addressing HI-specific bioink optimization and bioprinting parameters. This study aims to develop a scalable HI bioprinting methodology that preserves islet viability, functionality, and construct structural integrity for clinical applications.</div></div><div><h3>Methodology</h3><div>HI were bioprinted in clinically applicable alginate-based bioinks, supplemented with decellularized human pancreatic extracellular matrix (dECM). Bioinks were optimized for rheological behavior, shear-thinning properties, and mechanical stability. Constructs were printed with an extrusion-based bioprinter using optimized parameters to minimize HI shear stress. Viability and functionality were assessed via live/dead assays, glucose-stimulated insulin secretion (GSIS), and immunostaining over 21 days in culture. Construct pore size and mechanical stability were evaluated for structural integrity, while high-density bioprinting (10,000 iEQ/mL) addressed scalability challenges.</div></div><div><h3>Results</h3><div>The optimized bioprinting parameters resulted in over 90% viability of HI (2,500 iEQ/mL, N=3 donors), with stable stimulus index (SI) comparable to free islets over 7 days. High-density printing demonstrated the potential for volumetric tissue manufacturing. The printing conditions resulted in over 90% viability and a significantly higher SI on day 21 compared to free islets (3.7 ± 0.6 vs. 2.6 ± 0.4). dECM-enriched constructs demonstrated long-term HI viability, with a significant absence of glucagon-insulin co-expressing cells.</div></div><div><h3>Conclusion</h3><div>This study has established a HI bioprinting platform for T1D therapy, by optimizing bioink formulations and printing parameters that resulted in enhanced HI viability, functionality, and construct structural stability. Functional high-density HI constructs were also successfully printed, paving the way for therapeutic applications. This strategy could address the delivery system requirements of various groups developing clinically relevant beta-cell replacement therapies.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages S32-S33"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888000","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of Frozen-thawed Allogenic Fibroblast Sheets for Esophageal Anastomotic Leakage","authors":"K. Hamano ,&nbsp;R. Sakamoto ,&nbsp;K. Ueno ,&nbsp;Y. Takemoto ,&nbsp;H. Kurazumi ,&nbsp;Y. Tanaka ,&nbsp;K. Yoshida ,&nbsp;W. Kawai ,&nbsp;T. Takami","doi":"10.1016/j.jcyt.2025.03.093","DOIUrl":"10.1016/j.jcyt.2025.03.093","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Gastrointestinal anastomotic leakage is a severe postoperative complication, and its effective preventive measures remain unestablished. We have developed allogeneic multilayered fibroblast sheets, which promote wound healing in various organs. When considering clinical applications, the cryopreserved cell sheets can be transplanted immediately after thawing. However, frozen-thawed allogenic fibroblast sheets have not been investigated for gastrointestinal anastomotic leakage. This study examined the prevention of anastomotic leakage by transplanting frozen-thawed allogeneic multilayered fibroblast sheets into an esophageal anastomosis model.</div></div><div><h3>Methodology</h3><div>Multilayered fibroblast sheets were prepared by culturing the oral mucosa of Sprague–Dawley rats for 9 days. The esophageal anastomosis model was established by transecting the esophagus of Wistar/ST rats and performing reanastomosis with three-point interrupted sutures. The multilayered fibroblast sheets were cryopreserved in an ultralow temperature freezer after freezing in a three-dimensional freezer. The rats were categorized into the control (suture only) and fibroblast sheet groups (in which two fibroblast sheets were transplanted) (Fig. 1). The incidence of anastomotic leakage and burst pressure was evaluated 3 and 5 days after transplantation. Furthermore, growth factors secreted by the fibroblast sheets and the collagen expression levels in the tissue were assessed.</div></div><div><h3>Results</h3><div>The incidence of anastomotic leakage was significantly lower in the fibroblast sheet group (allogeneic multilayered fibroblast sheet transplantation) than in the control group (suture only) (Table 1).Furthermore, the burst pressure was significantly higher in the fibroblast sheet group than in the control group (day 5: 78 ± 26 mmHg vs 124 ± 30 mmHg; <em>p</em> &lt; 0.01).The mRNA expression level of type I collagen 5 days postoperatively was significantly higher in the fibroblast sheet group than in the control group (control group: 8.35 ± 3.77 vs cell sheet group: 19.58 ± 10.28; <em>p</em> = 0.05). Additionally, the quantitative analysis of type I collagen 5 days postoperatively revealed significantly higher levels in the fibroblast sheet group than in the control group (control group: 0.09 ± 0.04 μg vs fibroblast sheet group: 0.14 ± 0.03 μg; <em>p</em> = 0.04).</div></div><div><h3>Conclusion</h3><div>The transplantation of frozen-thawed allogeneic multilayered fibroblast sheets reduced the incidence of anastomotic leakage and burst pressure, indicating their potential to help prevent esophageal anastomotic leakage.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S55"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888035","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"US2-mediated Immune Evasion: A Novel Strategy For Generating Hypoimmunogenic iPSC Lines","authors":"J.J. Novoa ,&nbsp;L.H. Gaykema ,&nbsp;J. Peerlings ,&nbsp;R.Y. van Nieuwland ,&nbsp;A.R. van der Slik ,&nbsp;M.F. Goncalves ,&nbsp;C. van den Berg ,&nbsp;T.J. Rabelink ,&nbsp;A. Zaldumbide","doi":"10.1016/j.jcyt.2025.03.076","DOIUrl":"10.1016/j.jcyt.2025.03.076","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Mismatches in HLA haplotypes between donors and recipients significantly increase the risk of graft failure due to immune rejection. While knocking out beta-2 microglobulin (B2M) is the current standard for preventing HLA class I surface expression and protecting stem cell-derived products from allogeneic rejection, the complete ablation of HLA-I molecules can impair natural killer (NK) cell “self” recognition and disrupt critical immune-regulatory interactions.</div></div><div><h3>Methodology</h3><div>To address these challenges, we developed a novel hypoimmunogenic induced pluripotent stem cell (iPSC) line by inserting the cytomegalovirus-derived US2 encoding sequence into the AAVS1 safe harbor locus. Transcriptomic analysis of the modified cells, confirmed that US2 expression preserves stemness and differentiation capacity into both endothelial cells (ECs) and kidney organoids.</div></div><div><h3>Results</h3><div>Flow cytometry (FACS) analysis revealed that US2-expression abrogates HLA-A2 cell surface expression while retaining low levels of non-classical HLA molecules. In co-culture assays, US2 expression abolishes HLA-A2 alloreactive T cell activation and significantly lowers NK cell activation and degranulation compared to B2M KO ECs, demonstrating superior evasion of \"missing self\"-mediated NK cell responses.</div></div><div><h3>Conclusion</h3><div>These findings establish US2-mediated \"stealth\" technology as a refined alternative to B2M knockout, offering selective modulation of HLA expression to mitigate immune rejection while preserving critical immune-regulatory interactions.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S47"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888046","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}