Biotechnic & Histochemistry最新文献

{"title":"Therapeutic potential of tanshinone IIA in folic acid-induced acute kidney injury: a network pharmacology approach.","authors":"Peijian Chen, Ping He, Jieting Liu, Ran Guo, Yanhui Chu, Yang Xiao, Minglu Ding","doi":"10.1080/10520295.2025.2589264","DOIUrl":"10.1080/10520295.2025.2589264","url":null,"abstract":"<p><p>Acute kidney injury (AKI) is a clinical syndrome that can be caused by a variety of factors, leading to rapid decline of kidney function and increased morbidity and mortality, whilst also exerting significant economic burden on the affected patient. <i>Salvia miltiorrhiza</i> is a highly valued plant in traditional Chinese medicine (TCM). Tanshinone IIA (Tan IIA) is an important active compound that can be extracted from salvia miltiorrhiza, which has reported anti-inflammatory effects. The objective of the present investigation was to explore the potential effects of Tan IIA on folic acid-induced AKI and elucidate its underlying mechanism. A comprehensive analysis was conducted utilizing the TCM Systematic Pharmacology Database and Analytical Platform database to screen for chemical components and their corresponding targets. Subsequently, by using network pharmacology techniques and Cytoscapes 3.7.2 software, a protein-protein interaction (PPI) network was constructed and analyzed. Through Venn diagram analysis of the DeGeNET, OMIM, PharmGKB, and GeneCards databases using the key word \"acute kidney injury,\" a total of 76 overlapping targets were obtained. Building upon this, a compound-target gene network was constructed and analyzed by Cytoscapes 3.7.2 software, revealing TP53, STAT3, CASP3, VEGFA, and JUN to be pivotal therapeutic targets. Subsequently, an AKI mouse model was established to investigate the renal effects of Tan IIA. By immunohistochemistry, Western blot results showed the Tan IIA ameliorated kidney function by alleviating inflammation, mitigating necrosis of renal tubular cells, promoting their proliferation and attenuating kidney injury. These beneficial effects were found to be achieved by inhibiting the PI3K/AKT signaling pathway and inhibiting the expression of TP53 by Western blot. In conclusion, TP53may be a potential target for folic acid-induced AKI, whilst Tan IIA exerts its renoprotective effects and improves renal function by PI3K/AKT signaling pathways.</p>","PeriodicalId":8970,"journal":{"name":"Biotechnic & Histochemistry","volume":" ","pages":"69-83"},"PeriodicalIF":1.4,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145699726","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The effect of stromal vascular fraction cells derived from epididymal and inguinal adipose tissues on <i>in vitro</i> spermatogenesis.","authors":"Nilgün Öksel, Merve Bulut, Selin Önen, Merve Gizer, Zeliha Cansel Özmen, Petek Korkusuz","doi":"10.1080/10520295.2025.2604573","DOIUrl":"10.1080/10520295.2025.2604573","url":null,"abstract":"<p><p>Infertility affects 10-20% of sexually active couples and male-related factors contribute to 30-50% of all cases of infertility. The development of three-dimensional in vitro approaches to promote the differentiation of spermatogonial stem cells (SSCs) into functional spermatozoa is an essential step for the treatment of male infertility. Stromal vascular fraction (SVF) cells derived from adipose tissue are known to have a regenerative effect on spermatogenesis and testicular regeneration after testicular damage. The study assessed the effect of SVF cells from epididymal and inguinal adipose tissue on in vitro spermatogenesis. Testicular cells from prepubertal mice were cultured alone as control and co-cultured with SVF cells from epididymal (ESVF) and inguinal (ISVF) adipose tissue in experimental groups by an air-liquid interface system. Spermatogenic progression was evaluated histomorphometrically and immunohistochemically at weeks 1, 3, 4, and 6. ESVF increased formation of tubule-like structures at week 1 and ISVF had a similar effect at week 4. The ISVF group showed higher numbers of ID4(+) (Inhibitor of DNA Binding 4) SSCs than control at all time points. At weeks 3 and 4, the ISVF exhibited increased number of SCP3(+) (Synaptonemal Complex Protein 3) spermatocytes compared to the control group and the ESVF showed a similar increase at week 6. The presence of ACR(+) (Acrosin) spermatids was observed in all groups at week 3. At week 4, the ISVF group had more ACR(+) spermatids than the control and ESVF groups. Our findings demonstrated that SVF cells effectively supported in vitro spermatogenesis. Notably, inguinal-derived SVF cells led to a higher production of ACR(+) spermatids than edidiymal-derived SVF cells. In conclusion, inguinal derived SVF cells can be used as a new co-culture method to preserve the SSC pool and promote in vitro spermatogenesis in infertile patients.</p>","PeriodicalId":8970,"journal":{"name":"Biotechnic & Histochemistry","volume":" ","pages":"133-143"},"PeriodicalIF":1.4,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145899302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expression of programmed death-ligand 1 protein in head and neck squamous cell carcinoma and its clinicopathological correlates.","authors":"Abdullah Ahmad Bader, Manal A Abbas, Maher A Sughayer","doi":"10.1080/10520295.2025.2583966","DOIUrl":"10.1080/10520295.2025.2583966","url":null,"abstract":"<p><p>The ability to escape immune surveillance is a hallmark of malignancy. Programmed death ligand 1 (PD-L1) facilitates tumor progression by binding to the immune inhibitory receptor known as programmed cell death protein 1 (PD1) on immune cells, resulting in suppression of the cytotoxic T lymphocyte function. The degree of PD-L1 expression may have a prognostic value in some cancer types, and it may vary according to the genetic makeup and the ethnicity of patients. The expression level of PD-L1 in 63 cases of primary head and neck squamous cell carcinoma (HNSCC) tumor tissues was evaluated using immunohistochemistry (IHC). Also, PD-L1 association with various clinicopathologic characteristics and overall survival was studied. The positive expression rate of PD-L1 in HNSCC was 85.7%, 60.3%, and 52.3% of the total number of cases using combined positive score (CPS)<i> ≥ </i>1, CPS<i> ≥ </i>5, and CPS <i>≥ </i>20 cutoff values, respectively. Statistical analysis revealed no significant relationship between the expression of PD-L1 protein and clinicopathological features except for tobacco use using a cutoff CPS ≥ 20. The log-rank chi-square results showed that PD-L1 was not a significant factor affecting the 4-year overall survival of HNSCC patients. Also, the overall survival rate was not significantly affected by the patient's age, tumor differentiation, tumor size, and lymphovascular invasion. However, survival curves demonstrated lower overall survival in HNSCC female patients, disease recurrence, and positive perineural invasion. Our findings showed relatively high PDL-1 expression in most HNSCC patients. No significant association was found between PD-L1 protein expression and overall survival.</p>","PeriodicalId":8970,"journal":{"name":"Biotechnic & Histochemistry","volume":" ","pages":"21-29"},"PeriodicalIF":1.4,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145647260","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Stereological analysis of spleen alterations in streptozotocin-nicotinamide-induced diabetic rats.","authors":"Teresa Lucretia, Cherry Azaria, Imelda Imelda, Dewi Kartikawati Paramita, Rina Susilowati","doi":"10.1080/10520295.2025.2587290","DOIUrl":"10.1080/10520295.2025.2587290","url":null,"abstract":"<p><p>Studies have indicated that spleen and white pulp atrophy develops within 5 weeks following hyperglycemia onset in streptozotocin-induced diabetic rats. This study aimed to delineate the histopathological alterations in the spleen across two stages of diabetes progression using design-based stereology. Twenty-six rats were categorized into four groups based on condition (normal control [NC] or diabetic model [DM]) and observation period post-induction (5 or 10 weeks): NC5, DM5, NC10, and DM10. Diabetes was induced using streptozotocin-nicotinamide combination. Histological evaluations were performed using standard staining techniques, whereas spleen compartment volumes were quantitatively assessed through point-counting methods on histological sections. Additionally, immunohistochemistry (IHC) and flow cytometry analyses were utilized to determine the distribution and percentages of T and B lymphocytes. Compared to its NC5 control, the DM5 group exhibited inflammatory responses, including polymorphonuclear leukocyte infiltration, but no significant atrophy. DM5 showed a significantly elevated IHC score for T lymphocytes (<i>p</i> < 0.01) and a higher percentage of CXCR5 + B lymphocytes (<i>p</i> < 0.05) compared to NC5, suggesting an active adaptive immune response. In contrast to the NC10 group, the DM10 group displayed significant spleen atrophy (<i>p</i> = 0.005), with marked reductions in total white pulp volume (<i>p</i> = 0.015) and marginal zone volume (<i>p</i> = 0.008). Furthermore, compared to NC10, DM10 exhibited an increased connective tissue volume fraction (<i>p</i> < 0.001). Across all groups, spleen atrophy was directly correlated with reductions in body weight. These findings underscore an initial inflammatory phase characterized by immune cell recruitment in the spleen during early diabetes, subsequently evolving into significant atrophy, reduced white pulp and marginal zone volumes, and an increased connective tissue volume fraction in advanced stages of the disease, all proportional to body weight loss.</p>","PeriodicalId":8970,"journal":{"name":"Biotechnic & Histochemistry","volume":" ","pages":"49-66"},"PeriodicalIF":1.4,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145699745","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimization of culture conditions for the differentiation of endothelial progenitor cells from mouse blood <i>in vitro</i>.","authors":"Jianguo Wu, Qian Chen, Tao Liu, Biao Yang, Tianhang Luo","doi":"10.1080/10520295.2025.2577997","DOIUrl":"10.1080/10520295.2025.2577997","url":null,"abstract":"<p><p>Endothelial progenitor cells (EPCs) play a crucial role in neovascularization and tissue repair, with significant therapeutic potential in ischemic diseases, tumor therapy, and as gene carriers. However, the current methods for isolating and culturing EPCs are not standardized, leading to inconsistencies in cell numbers and functionality. This study aimed to optimize the in vitro culture conditions for EPCs using an orthogonal design, focusing on four main factors: cell density, culture medium, fetal bovine serum (FBS) concentration, and vascular endothelial growth factor (VEGF) concentration. The most effective conditions among those tested were a cell density of 1 × 10⁶/cm², EGM-2 medium, 10% FBS, and 20 ng/mL VEGF. Under these conditions, EPCs exhibited significantly enhanced proliferation, migration, and pro-angiogenic (paracrine) capacity. Immunohistochemistry and fluorescent staining confirmed high expression of EPC-specific markers, such as CD133 and KDR, and the ability to uptake DiI-ac-LDL and bind FITC-UEA-1. Angiogenesis assays showed that most effective conditions among those tested significantly increased the number of vessel-like structures. Additionally, the migration rate and proliferative activity of EPCs were significantly higher under the most effective conditions among those tested compared to conventional conditions. These findings provide a robust foundation for further refining in vitro EPC culture and pave the way for more effective clinical applications. Future studies should validate these optimized conditions in in vivo models to fully realize the therapeutic potential of EPCs.</p>","PeriodicalId":8970,"journal":{"name":"Biotechnic & Histochemistry","volume":" ","pages":"1-10"},"PeriodicalIF":1.4,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145408307","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Role of sitagliptin in diabetes-induced testicular damage via the Fas/FasL signalling pathway.","authors":"Onur Ersoy, Gulnur Kizilay","doi":"10.1080/10520295.2025.2583963","DOIUrl":"10.1080/10520295.2025.2583963","url":null,"abstract":"<p><p>Clinical and experimental studies have shown that sitagliptin regulates blood glucose levels. This study was designed because it is thought that sitagliptin may reduce diabetes-induced apoptosis in testes by affecting blood glucose levels and may have a beneficial effect on spermatogenesis by regulating hormonal activity. Thirty-four male Sprague-Dawley rats were divided into three groups: Control group (n = 10), given citrate buffer only; Diabetes group (n = 12), after 2 weeks of the high-fat diet, given a single dose of 35 mg/kg streptozotocin (STZ, dissolved in citrate buffer, intraperitoneally); Diabetes + Sitagliptin group (n = 12), after 2 weeks of the high-fat diet, diabetes was induced with STZ and 10 mg/kg sitagliptin (intragastric) was administered daily for 6 weeks. At the end of the experiment, blood glucose levels measured in the sitagliptin-treated group were found to be significantly lower than in the diabetes group. Serum testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, seminiferous tubule diameter, Johnsen score, and proliferation indices were significantly lower in the diabetic groups compared to the control group, while no significant difference was found between the diabetes and sitagliptin groups. Basement membrane thickness, apoptotic cell and apoptotic tubul indexes, Fas, FasL, and caspase8 immunoreactivities were higher in diabetic groups compared to the control group, while no difference was found between the diabetic and sitagliptin groups. In conclusion, although 10 mg/kg sitagliptin reduced blood glucose levels in diabetes-induced hyperglycemia, it did not alter serum testosterone, FSH and LH levels, and did not appear to have a beneficial effect on diabetes-induced apoptosis and proliferation in the testes.</p>","PeriodicalId":8970,"journal":{"name":"Biotechnic & Histochemistry","volume":" ","pages":"11-20"},"PeriodicalIF":1.4,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145538692","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effect of beta-glucan on oxidative stress, inflammation, hormonal and histopathological changes in dehydroepiandrosterone-induced polycystic ovary syndrome.","authors":"H Yüce, N Basak Turkmen, M Aydin, A Taşlıdere, D Aşkin Özek, S Şenkal, S Aslan, A Dogan, O Ciftci, S Ünüvar","doi":"10.1080/10520295.2025.2586701","DOIUrl":"10.1080/10520295.2025.2586701","url":null,"abstract":"<p><p>Beta-glucans (βTGs) are a class of dietary fibers and biologically active polysaccharides derived from natural sources, known for their diverse bioactive properties. Their documented effects include anti-tumor, anti-inflammatory, prebiotic, anti-obesity, anti-allergic, anti-microbial, antiviral, anti-osteoporotic, and immunomodulating activities. Despite these well-established benefits, the role of βTG in dehydroepiandrosterone (DHEA)-induced polycystic ovary syndrome (PCOS) remains largely unexplored. This study investigated the protective effects of βTG treatment on PCOS and its potential to reverse PCOS-induced changes. Female Sprague-Dawley (SD) rats were randomly divided into four groups (n = 8 each): control, PCOS, PCOS+βTG, and βTG. We assessed biochemical markers related to oxidative stress, antioxidant status, inflammation, cytokines, and hormone levels. Additional analyses included immunohistochemistry and histopathology. Membrane array analysis was used to profile growth factors, cytokines, and chemokines. However, βTG normalized deviations in the estrous cycle caused by PCOS and positively affected the reproductive system (<i>p</i> < 0.05). It also reduced the inflammatory response in PCOS rats by decreasing inflammatory cytokines (<i>p</i> < 0.05). Furthermore, oxidative stress was significantly reduced, and antioxidant enzyme activities were markedly elevated in the βTG group (<i>p</i> < 0.05). Histopathological alterations were prevented by βTG, which also induced the expression of essential proteins such as beta-nerve growth factor (bNGF), tissue inhibitor of metalloproteinase-1 (TIMP-1), Agrin, cytokine-induced neutrophil chemoattractant-1 (CINC-1), brain-derived neurotrophic factor (BDNF), and basic fibroblast growth factor (FGF-2/bFGF) (<i>p</i> < 0.05). In conclusion, βTG treatment effectively protects against oxidative stress, inflammation, hormone imbalance, and histopathological damage in ovarian tissue caused by PCOS.</p>","PeriodicalId":8970,"journal":{"name":"Biotechnic & Histochemistry","volume":" ","pages":"30-48"},"PeriodicalIF":1.4,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145653505","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ameliorative role of curcumin and ascorbic acid on nephrotoxicity induced by environmentally relevant concentrations of different combinations of lead, fluoride and nitrate in albino rats.","authors":"Chetanjyoti Tuteja, Navdeep Kaur","doi":"10.1080/10520295.2025.2555991","DOIUrl":"10.1080/10520295.2025.2555991","url":null,"abstract":"<p><p>Groundwater pollution with lead, fluoride, and nitrate presents a growing environmental and health challenge. This study aimed to evaluate the nephrotoxic effects of these pollutants in male albino rats and assess the potential ameliorative effects of curcumin and ascorbic acid in counteracting their toxicity for 135 days. A total of ten treatment groups were established viz. control, lead + fluoride + nitrate (BIS), lead + nitrate, lead + nitrate + curcumin + ascorbic acid, lead + fluoride, lead + fluoride + curcumin + ascorbic acid, fluoride + nitrate, fluoride + nitrate + curcumin + ascorbic acid, lead + fluoride + nitrate, lead + fluoride + nitrate + curcumin + ascorbic acid. Exposure to lead, fluoride, and nitrate resulted in a significant decrease in the activity of oxidative stress enzymes viz. superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase, and a notable increase in the lipid peroxidation levels. Further, significantly increased urea and creatinine levels in plasma and renal damage including glomerular shrinkage, widened Bowman's space, and tubular degeneration were also observed. The greatest damage was recorded in the lead + fluoride + nitrate group followed by lead + fluoride, lead + nitrate, and fluoride + nitrate. Co-treatment with curcumin and ascorbic acid demonstrated remarkable protective effects, with improvements in oxidative stress markers, plasma urea, and creatinine levels along with a significant restoration of glomerular structure and normalization of Bowman's space reflecting improved renal function. This research highlights the kidneys' susceptibility to environmental toxicants and the combined efficacy of curcumin and ascorbic acid in mitigating nephrotoxicity.</p>","PeriodicalId":8970,"journal":{"name":"Biotechnic & Histochemistry","volume":" ","pages":"460-474"},"PeriodicalIF":1.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145184536","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigation of ameliorative effects of indole-3 carbinol on TNBS-induced ulcerative colitis in rats.","authors":"Erkmen Tuğrul Epikmen, Emrah İpek, Mehmet Hesapçıoğlu, Mehmet Karaboğa, Ali Riza Öztürk, Hamdi Avci","doi":"10.1080/10520295.2025.2561682","DOIUrl":"10.1080/10520295.2025.2561682","url":null,"abstract":"<p><p>Ulcerative colitis is a chronic inflammatory condition of the gastrointestinal tract that can predispose patients to colonic neoplasms. Various natural compounds have been explored for their therapeutic potential. Indole-3-carbinol (I3C), a natural compound derived from cruciferous vegetables, is recognized for its tissue-protective and regenerative properties. This study aimed to investigate the effects of I3C on experimental ulcerative colitis in rats. Thirty-two Wistar rats were randomly assigned to four groups: a control group receiving isotonic saline, a TNBS group administered trinitrobenzene sulfonic acid (TNBS) intrarectally, an I3C group receiving I3C via gastric gavage, and a TNBS+I3C group treated with I3C following TNBS induction. After 7 days, all animals were euthanized under anesthesia, and pathological, histochemical, and immunohistochemical evaluations were conducted. The results revealed that I3C mitigated the severity of TNBS-induced colonic lesions and facilitated tissue repair. The I3C-treated group exhibited reduced tissue damage and enhanced mucosal regeneration. Additionally, vessel count, collagen, and myofibroblastic activity were markedly increased following I3C treatment. In conclusion, I3C exhibits both protective and reparative effects in experimental ulcerative colitis, potentially through anti-inflammatory mechanisms and the activation of tissue repair pathways.</p>","PeriodicalId":8970,"journal":{"name":"Biotechnic & Histochemistry","volume":" ","pages":"475-485"},"PeriodicalIF":1.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145211490","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Morphological characterization of 3D cell cultures generated by liquid overlay technique.","authors":"W Metzger, E Oh, L Lemke, M Hannig, F Krull, S Antonyuk, T Pohlemann","doi":"10.1080/10520295.2025.2568063","DOIUrl":"10.1080/10520295.2025.2568063","url":null,"abstract":"<p><p>Cultivating cells in 3D is considered a significant advancement in cell culture models, as it better reflects natural cellular environments compared to 2D cultures. However, analytical methods like standard light microscopy are less effective for 3D cultures. In this study, 3D cell cultures were generated using the liquid overlay technique with 10,000, 50,000, 100,000 and 200,000 Normal Human Dermal Fibroblasts, analyzed on days 1, 2, and 3 post-seeding. We quantified the influence of fixation with paraformaldehyde or glutardialdehyde/dehydration on their morphology compared to living 3D cell cultures. They were analyzed by light microscopy, scanning electron microscopy as well as by digital light microscopy (height profile measurement). Over time, the cultures decreased in size, likely due to cell shrinkage and structural reorganization. The size reduction could be mathematically described by an exponential decay function. The proportion of round spheroids versus indented aggregates depended on cell number, culture age, and fixation method. On day 1, cultures seeded with 10,000 cells formed nearly 100% round spheroids, regardless of fixation. Higher cell numbers led to fewer round spheroids, and fixation further reduced their number. This suggests that large cell quantities sediment in layers due to steric hindrance, forming indentations. Since aldehydes are responsible for cross-linking proteins, we hypothesize that this chemical reaction, combined with low stability of the 3D cell cultures, leads to the increased formation of the indented 3D cell aggregates. This is consistent with an overall increase in the number of round spheroids and a decrease of the negative influence of fixation over time. In summary, it is important to consider the number of seeded cells, the incubation time, as well as the possible fixation effects when generating stable spheroids using the liquid overlay technique for down-stream experiments.</p>","PeriodicalId":8970,"journal":{"name":"Biotechnic & Histochemistry","volume":" ","pages":"494-504"},"PeriodicalIF":1.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145298377","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}