Long-Term Culture of Human Pluripotent Stem Cells in Xeno-Free Condition Using Functional Polymer Films.

Human pluripotent stem cells (hPSCs), encompassing human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold immense potential in regenerative medicine, offering new opportunities for personalized cell therapies. However, their clinical translation is hindered by the inevitable reliance on xenogeneic components in culture environments. This study addresses this challenge by engineering a fully synthetic, xeno-free culture substrate, whose surface composition is tailored systematically for xeno-free culture of hPSCs. A functional polymer surface, pGC2 (poly(glycidyl methacrylate-grafting-guanidine-co-carboxylic acrylate)), offers excellent cell-adhesive properties as well as non-cytotoxicity, enabling robust hESCs and hiPSCs growth while presenting cost-competitiveness and scalability over Matrigel. This investigation includes comprehensive evaluations of pGC2 across diverse experimental conditions, demonstrating its wide adaptability with various pluripotent stem cell lines, culture media, and substrates. Crucially, pGC2 supports long-term hESCs and hiPSCs expansion, up to ten passages without compromising their stemness and pluripotency. Notably, this study is the first to confirm an identical proteomic profile after ten passages of xeno-free cultivation of hiPSCs on a polymeric substrate compared to Matrigel. The innovative substrate bridges the gap between laboratory research and clinical translation, offering a new promising avenue for advancing stem cell-based therapies.

EnduroBone: A 3D printed bioreactor for extended bone tissue culture.

Studies of the effects of external stimuli on bone tissue, disease transmission mechanisms, and potential medication discoveries benefit from long-term tissue viability ex vivo. By simulating the in-vivo environment, bioreactors are essential for studying bone cellular activity throughout biological processes. We present the development of an automated 3D-printed bioreactor EnduroBone designed to sustain the ex-vivo viability of 10 mm diameter cancellous bone cores for an extended period. The device is supplied with two critical parameters for maintaining bone tissue viability: closed-loop continuous flow perfusion of 1 mL/min for nutrient diffusion and waste removal and direct mechanical stimulation with cyclic compression at 13.2 RPM (revolutions per minute) to promote cell viability which can lead to improved tissue stability during ex vivo culturing. The bioreactor addresses several limitations of existing systems and provides a versatile open-source platform for bone cancer research, orthopedic device testing, and other related applications. To validate the bioreactor, fresh swine samples were cultured ex-vivo, and their cell viability was determined to be maintained for up to 28 days. Periodic cell viability assessment through live/dead cell staining and confocal imaging at the start (0 days) and at several time points throughout the culture period (7, 14, 21, and 28 days) was used to demonstrate EnduroBone effectiveness in sustaining bone cell health for the extended period tested.

BDNF and TRiC-inspired reagent rescue cortical synaptic deficits in a mouse model of Huntington's disease.

Synaptic changes are early manifestations of neuronal dysfunction in Huntington's disease (HD). However, the mechanisms by which mutant HTT protein impacts synaptogenesis and function are not well understood. Herein we explored HD pathogenesis in the BACHD mouse model by examining synaptogenesis and function in long term primary cortical cultures. At DIV14 (days in vitro), BACHD cortical neurons showed no difference from WT neurons in synaptogenesis as revealed by colocalization of a pre-synaptic (Synapsin I) and a post-synaptic (PSD95) marker. From DIV21 to DIV35, BACHD neurons showed progressively reduced colocalization of Synapsin I and PSD95 relative to WT neurons. The deficits were effectively rescued by treatment of BACHD neurons with BDNF. The recombinant apical domain of CCT1 (ApiCCT1) yielded a partial rescuing effect. BACHD neurons also showed culture age-related significant functional deficits as revealed by multielectrode arrays (MEAs). These deficits were prevented by BDNF, whereas ApiCCT1 showed a less potent effect. These findings are evidence that deficits in BACHD synapse and function can be replicated in vitro and that BDNF or a TRiC-inspired reagent can potentially be protective against these changes in BACHD neurons. Our findings support the use of cellular models to further explicate HD pathogenesis and potential treatments.

Identification of Periostin Positive Cell Population During the Long-Term Culture of Mouse Tendon-Derived Cells in Late Passage.

Tendon-derived cells exhibit phenotypic changes and gradually lose their proliferative capacity during serial passages in vitro. This study aimed to characterize the changes in the growth and stem cell characteristics of tendon-derived cells over a long-term culture. Mouse flexor digitorum profundus tendon-derived cells were obtained by enzymatic digestion and seeded at an initial density of 5,000/cm2. Cells were characterized by morphology, growth, senescence staining, trilineage differentiation assays, real-time polymerase chain reaction, immunocytochemistry, flow cytometry, and RNA sequencing analysis. Tendon-derived cells underwent a proliferative stage in the first three passages, followed by a gradual senescence. However, a novel cell population expressing periostin (Postn+) emerged during the long-term culture from passages 5-8, which possessed a high rate of proliferation without significant senescence over successive passages. Compared to early passage cells, Postn+ cells exhibited enhanced osteogenic differentiation potential and attenuated chondrogenic differentiation potential, decreased expression of SSEA-1, Oct3/4, tenomodulin, scleraxis, CD90.2, CD73, CD105, Sca-1, and CD44, and increased expression of collagen III and CD34. RNA-sequencing analysis of Postn+ and early passage cells identified 908 differentially expressed genes, with extracellular matrix-receptor interaction and focal adhesion as the top pathways, and integrins as hub genes. This study highlights the dynamics of tendon-derived cells during serial passages. We identify a Postn+ cell population during long-term culture in late passages, with high proliferative ability and prominent osteogenic differentiation potential. Further investigations are needed to elucidate the origin and potential applications of Postn+ tendon-derived cells.

In vitro aging alters the gene expression and secretome composition of canine adipose-derived mesenchymal stem cells.

Introduction: Canine adipose-derived mesenchymal stem cells (cAD-MSCs) hold therapeutic promise due to their regenerative potential, particularly within their secretome. However, concerns arise regarding the impact of in vitro cultivation necessitated for storing therapeutic doses, prompting this study to comprehensively explore the impact of in vitro aging on gene expression and secretome composition. Methods: The study involved collecting abdominal adipose tissue samples from nine healthy female dogs, from which cAD-MSCs were extracted and cultured. Stem cells were validated through trilineage differentiation assays and flow cytometry immunophenotyping. Gene expression profiling using RT-qPCR array, and cAD-MSCs secretome LC-MS/MS analysis, were conducted at passages 3 and 6 to reveal gene expression and protein composition alterations during in vitro culture. Results and Discussion: The results demonstrate that the gene expression and secretome composition of cAD-MSCs were impacted by in vitro aging. Among many alterations in gene expression between two passages, two significant downregulations were noted in the MSC-associated PTPRC and IL10 genes. While the majority of proteins and their functional characteristics were shared between passages, the influence of cell aging on secretome composition is highlighted by 10% of proteins being distinctively expressed in each passage, along with 21 significant up- and downregulations. The functional attributes of proteins detected in passage 3 demonstrated a greater inclination towards supporting the regenerative capacity of cAD-MSCs. Moreover, proteins in passage 6 exhibited a noteworthy correlation with the blood coagulation pathway, suggesting an elevated likelihood of coagulation events. To the best of our knowledge, this study presents the first original perspective on the changes in secretome composition that occur when cAD-MSCs age in vitro. Furthermore, it contributes to broadening the currently restricted knowledge base concerning the secretome of cAD-MSCs. In conclusion, our findings show that the regenerative potential of cAD-MSCs, as well as their secretome, may be compromised by in vitro aging. Therefore, our study suggests a preference for earlier passages when considering these cells for therapeutic applications.

Injectable Micro-Hydrogel for DNA Delivery: A Promising Therapeutic Platform.

Utilizing the immune system as a strategy for disease prevention and treatment is promising, especially with dendritic cells (DCs) playing a central role in adaptive immune responses. The unique properties of DCs drive interest in developing materials for cell-based therapy and immune modulation. Injectable systems require syringe-compatible scaffolds, while hydrogels, like alginate, known for their programmability and biocompatibility, offer a versatile platform for immune medicine enhancement through easy preparation and room-temperature cross-linking. In this study, we synthesized alginate balls loaded with DCs or cytosine-phosphorothioate-guanine deoxyribonucleotide (CpG DNA) microparticles, aiming for long-term immune cell culture with potential immune stimulation effects. Encapsulated DCs exhibited proliferation within the alginate balls for up to 7 days, and CpG MPs were uniformly dispersed, which can facilitate uptake by DCs. This was supported by the result that DCs effectively phagocytosed CpG microparticles in a 2D environment. After the uptake of CpG MPs, the alginate balls with CpG-MP-uptaken DCs were synthesized successfully. The injectable properties of the alginate balls were easily modulated by adjusting the syringe needle gauges. This innovative strategy holds substantial promise for advancing medical treatments, offering effective and comfortable solutions for controlled immune modulation.

Evaluation of stability and safety of equine mesenchymal stem cells derived from amniotic fluid for clinical application.

Amniotic fluid mesenchymal stem cells (AF-MSCs), which can be obtained from fetal tissue, reportedly have self-renewal capacity and multi-lineage differentiation potential. The aim of this study was to identify the biological characteristics of AF-MSCs and evaluate their stability and safety in long-term culture. To confirm the biological characteristics of AF-MSCs, morphology, proliferation capacity, karyotype, differentiation capacity, gene expression level, and immunophenotype were analyzed after isolating AF-MSCs from equine amniotic fluid. AF-MSCs were differentiated into adipocytes, chondrocytes, and osteocytes. Immunophenotype analyses revealed expression levels of ≥95% and ≤ 2% of cells for a positive and negative marker, respectively. Analysis of the MSCs relative gene expression levels of AF-MSCs was approximately at least twice that of the control. The endotoxin level was measured to verify the safety of AF-MSCs and was found to be less than the standard value of 0.5 EU/ml. AF-MSCs were cultured for a long time without any evidence of abnormalities in morphology, proliferation ability, and karyotype. These results suggest that amniotic fluid is a competent source for acquiring equine MSCs and that it is valuable as a cell therapy due to its high stability.

Molecular cytogenetic analysis of multi-miscarriage products of conception in clinical cases from Al-Anbar Governorate, west of Iraq.

Most clinical miscarriages often occur throughout the first trimester of pregnancy, with fetal chromosomal abnormalities being identified as the primary reason for such occurrences. The objective is to analyze the fetal chromosomal aberrations in the product of conception among Iraqi patients suffering from recurrent miscarriages. The cross-sectional study was performed on 60 cases of products of conception in women suffering from multiple miscarriages, obtained from Department of Obstetrics and Gynecology is located in Ramadi Teaching Hospital for Child and Maternity, as well as other Private Clinics in the Ramadi City. Long-term culture of conventional cytogenetic analysis using the G-banding technique was employed to determine the chromosomal disorder of fetal tissue part or villus samples. Fetal chromosomal abnormalities were detected in 86.7 %. Numerical chromosomal abnormalities were revealed in 98.1 %, while structural abnormalities were detected in 1.9 %. Additionally, the commonest gestation loss occurs in parents under 35 years in the first trimester (92.3 %). Trisomy 21 was the most frequent (46.2 %) in gestational loss. Fetal chromosomal abnormalities have been linked with gestational loss in Iraqi couples. Therefore, it is recommended that cytogenetic analysis should be performed to identify the genetic cause of recurrent miscarriage. This is important for providing appropriate genetic counseling and educating couples about the risk of future pregnancies.

A scalable human islet 3D-culture platform maintains cell mass and function long-term for transplantation.

Present-day islet culture methods provide short-term maintenance of cell viability and function, limiting access to islet transplantation. Attempts to lengthen culture intervals remain unsuccessful. A new method was developed to permit the long-term culture of islets. Human islets were embedded in polysaccharide 3D-hydrogel in cell culture inserts or gas-permeable chambers with serum-free CMRL 1066 supplemented media for up to 8 weeks. The long-term cultured islets maintained better morphology, cell mass, and viability at 4 weeks than islets in conventional suspension culture. In fact, islets cultured in the 3D-hydrogel retained ß cell mass and function on par with freshly isolated islets in vitro and, when transplanted into diabetic mice, restored glucose balance similar to fresh islets. Using gas-permeable chambers, the 3D-hydrogel culture method was scaled up over 10-fold and maintained islet viability and function, although the cell mass recovery rate was 50%. Additional optimization of scale-up methods continues. If successful, this technology could afford flexibility and expand access to islet transplantation, especially single-donor islet-after-kidney transplantation.

Encapsulated human islets in alginate fiber maintain long-term functionality.

Maintenance of islet function after in vitro culture is crucial for both transplantation and research. Here we evaluated the effects of encapsulation in alginate fiber on the function of human islets which were distributed by the Alberta Islet Distribution Program. Encapsulated human islets from 15 deceased donors were cultured under 5.5 or 25 mM glucose conditions in vitro. The amounts of C-peptide and glucagon secreted from encapsulated islets into the culture media were measured periodically, and immunohistochemical studies were performed. Encapsulated islets maintained C-peptide and glucagon secretion for more than 75 days in 5 cases; in two cases, their secretion was also successfully detected even on day 180. α- and ß-cell composition and ß-cell survival in islets were unaltered in the fiber after 75 or 180 days of culture. The encapsulated islets cultured with 5.5 mM glucose, but not those with 25 mM glucose, exhibited glucose responsiveness of C-peptide secretion until day 180. We demonstrate that alginate encapsulation enabled human islets to maintain their viability and glucose responsiveness of C-peptide secretion after long-term in vitro culture, potentially for more than for 180 days.

Optimal temperature for the long-term culture of adult porcine islets for xenotransplantation.

Porcine islet xenotransplantation represents a promising therapy for severe diabetes mellitus. Long-term culture of porcine islets is a crucial challenge to permit the on-demand provision of islets. We aimed to identify the optimal temperature for the long-term culture of adult porcine islets for xenotransplantation. We evaluated the factors potentially influencing successful 28-day culture of islets at 24°C and 37°C, and found that culture at 37°C contributed to the stability of the morphology of the islets, the proliferation of islet cells, and the recovery of endocrine function, indicated by the expression of genes involved in pancreatic development, hormone production, and glucose-stimulated insulin secretion. These advantages may be provided by islet-derived CD146-positive stellate cells. The efficacy of xenotransplantation using islets cultured for a long time at 37°C was similar to that of overnight-cultured islets. In conclusion, 37°C might be a suitable temperature for the long-term culture of porcine islets, but further modifications will be required for successful xenotransplantation in a clinical setting.

Organoid Establishment of Long-Term Culture Using Primary Mouse Hepatocytes and Evaluation of Liver Function.

Primary hepatocytes and various animal models have traditionally been used in liver function tests to assess the effects of nutrients. However, these approaches present several limitations such as time consumption, high cost, the need for facilities, and ethical issues in primary mouse hepatocytes and animal models. In this study, we constructed liver organoids from primary mouse hepatocytes (OrgPH) to replace primary hepatocytes and animal models. We isolated primary mouse hepatocytes from 6- to 10-week-old male C57BL/6J mice using the two-step collagenase method, and generated liver organoids by clustering the cells in Matrigel. To assess the hepatic function of OrgPH, we examined specific liver markers and gene expressions related to hepatic glucose, ethanol, and cholesterol metabolism. Over a 28-day culture period, liver-specific markers, including Alb, Arg1, G6pc, and Cyp1a1, increased or remained stable in the OrgPH. However, they eventually decreased in primary hepatocytes. Glucose and ethanol metabolism-related gene expression levels exhibited a similar tendency in AML12 cells and OrgPH. However, the expression levels of cholesterol metabolism-related genes displayed an opposite trend in OrgPH compared with those in AML12 cells. These results agree with those of previous studies involving in vivo models. In conclusion, our study indicates that OrgPH can retain liver function and mimic the hepatocytic physiology of mouse in vivo models. Therefore, organoids originating from primary mouse hepatocytes are potentially useful as an animal-free method for evaluating the safety and toxicity of health functional foods and a replacement for animal models.

Ex vivo Culture and Contractile Force Measurements of Non-human Primate Heart Slices.

Cardiovascular diseases are the leading cause of death and morbidity worldwide. Patient mortality has been successfully reduced by nearly half in the last four decades, mainly due to advances in minimally invasive surgery techniques and interventional cardiology methods. However, a major hurdle is still the translational gap between preclinical findings and the conversion into effective therapies, which is partly due to the use of model systems that fail to recapitulate key aspects of human physiology and disease. Large animal models such as pigs and non-human primates are highly valuable because they closely resemble humans but are costly and time intensive. Here, we provide a method for long-term ex vivo culture of non-human primate (NHP) myocardial tissue that offers a powerful alternative for a wide range of applications including electrophysiology studies, drug screening, and gene function analyses.

Maintenance of Human Primordial Germ Cell-Like Cells in a Long-Term Culture System.

Primordial germ cells (PGCs) are the earliest form of mammalian germ lineage. In humans, PGCs are present during a very early and limited window in development, limiting the ability to study fundamental developmental steps in human reproductive biology. However, recent advancements in generating in-vitro models of gametogenesis have allowed the field to generate human primordial germ cell-like cells (hPGCLCs). In this chapter, we will review the generation of hPGCLCs using the incipient mesoderm-like cell (iMeLC) protocol and the subsequent expansion of hPGCLCs in a long-term culture system.

Establishment of Continuous In Vitro Culture of Babesia gibsoni by Using VP-SFM Medium with Low-Concentration Serum.

The establishment of in vitro culture methods has greatly facilitated the research of Babesia. However, the current Babesia gibsoni in vitro culture medium requires high concentrations of canine serum, which intensively limits the culture and is unable to satisfy the demands of long-term studies. In this study, AlbuMAX I (2 mg/mL) and 2.5% dog serum (vol/vol) were added to VP-SFM medium to develop a low-concentration serum culture medium named VP-SFMAD (2.5%), and the effectiveness of this medium was assessed by the growth of B. gibsoni. The results showed that VP-SFMAD (2.5%) could support the continuous growth of the parasite, and the parasitemia has no difference with the cultivation in RPMI 1640 with 20% dog serum. In contrast, either a low concentration of dog serum or absence of AlbuMAX I will significantly lower the parasite growth or fail to maintain B. gibsoni growth in the long term. The strategy of reducing the hematocrit was also evaluated, and VP-SFMAD (2.5%) improved the parasitemia to over 50% within 5 days. The high parasitemia is helpful for larger numbers of parasite collection, which is valuable for studying the biology, pathogenesis, and virulence of Babesia and other intraerythrocytic parasites. In addition, VP-SFMAD (2.5%) medium was successfully used for monoclonal parasite screening, which obtained monoclonal strains with parasitized erythrocytes about 3%, which is similar to RPMI-1640D (20%) medium that obtains monoclonal strains on the 18th day. Those results showed that VP-SFMAD can be applied to B. gibsoni continuous long-term, expansion culture, and subclone culture. IMPORTANCE The VP-SFM as a base medium supplemented with AlbuMAX I and a low concentration of canine serum (2.5%) allowed the continuous in vitro culture of Babesia gibsoni at both small and large volumes, which was to meet different experimental needs, such as long-term culture and obtaining high parasitemia and subclone culture. The establishment of in vitro culture systems allows researchers to better understand the metabolism and growth patterns of Babesia. Importantly, several technical problems impeding such studies have been overcome.

Three-Dimensional Bioprinting of Organoid-Based Scaffolds (OBST) for Long-Term Nanoparticle Toxicology Investigation.

The toxicity of nanoparticles absorbed through contact or inhalation is one of the major concerns for public health. It is mandatory to continually evaluate the toxicity of nanomaterials. In vitro nanotoxicological studies are conventionally limited by the two dimensions. Although 3D bioprinting has been recently adopted for three-dimensional culture in the context of drug release and tissue regeneration, little is known regarding its use for nanotoxicology investigation. Therefore, aiming to simulate the exposure of lung cells to nanoparticles, we developed organoid-based scaffolds for long-term studies in immortalized cell lines. We printed the viscous cell-laden material via a customized 3D bioprinter and subsequently exposed the scaffold to either 40 nm latex-fluorescent or 11-14 nm silver nanoparticles. The number of cells significantly increased on the 14th day in the 3D environment, from 5 × 105 to 1.27 × 106, showing a 91% lipid peroxidation reduction over time and minimal cell death observed throughout 21 days. Administered fluorescent nanoparticles can diffuse throughout the 3D-printed scaffolds while this was not the case for the unprinted ones. A significant increment in cell viability from 3D vs. 2D cultures exposed to silver nanoparticles has been demonstrated. This shows toxicology responses that recapitulate in vivo experiments, such as inhaled silver nanoparticles. The results open a new perspective in 3D protocols for nanotoxicology investigation supporting 3Rs.

Long term culture promotes changes to growth, gene expression, and metabolism in CHO cells that are independent of production stability.

Phenotypic stability of Chinese hamster ovary (CHO) cells over long term culture (LTC) presents one of the most pressing challenges in the development of therapeutic protein manufacturing processess. However, our current understanding of the consequences of LTC on recombinant (r-) CHO cell lines is still limited, particularly as clonally-derived cell lines present distinct production stability phenotypes. This study evaluated changes of culture performance, global gene expression, and cell metabolism of two clonally-derived CHO cell lines with a stable or unstable phenotype during the LTC (early [EP] vs. late [LP] culture passages). Our findings indicated that LTC altered the behavior of CHO cells in culture, in terms of growth, overall gene expression, and cell metabolism. Regardless whether cells were categorized as stable or unstable in terms of r-protein production, CHO cells at LP presented an earlier decline in cell viability and loss of any observable stationary phase. These changes were parallelled by the upregulation of genes involved in cell proliferation and survival pathways (i.e., MAPK/ERK, PI3K-Akt). Stable and unstable CHO cell lines both showed increased consumption of glucose and amino acids at LP, with a parallel accumulation of greater amounts of lactate and TCA cycle intermediates. In terms of production stability, we found that decreased r-protein production in the unstable cell line directly correlated to the loss in r-gene copy number and r-mRNA expression. Our data revealed that LTC produced ubiquitious effects on CHO cell phenotypes, changes that were rooted in alterations in cell transcriptome and metabolome. Overall, we found that CHO cells adapted their cellular function to proliferation and survival during the LTC, some of these changes may well have limited effects on overall yield or specific productivity of the desired r-product, but they may be critical toward the capacity of cells to handle r-proteins with specific molecular features.

Understanding Intra- and Inter-Species Variability in Neural Stem Cells' Biology Is Key to Their Successful Cryopreservation, Culture, and Propagation.

Although clinical trials on human neural stem cells (hNSCs) have already been implemented in the treatment of neurological diseases and they have demonstrated their therapeutic effects, many questions remain in the field of preclinical research regarding the biology of these cells, their therapeutic properties, and their neurorestorative potential. Unfortunately, scientific reports are inconsistent and much of the NSCs research has been conducted on rodents rather than human cells for ethical reasons or due to insufficient cell material. Therefore, a question arises as to whether or which conclusions drawn on the isolation, cell survival, proliferation, or cell fate observed in vitro in rodent NSCs can be introduced into clinical applications. This paper presents the effects of different spatial, nutritional, and dissociation conditions on NSCs' functional properties, which are highly species-dependent. Our study confirmed that the discrepancies in the available literature on NSCs survival, proliferation, and fate did not only depend on intra-species factors and applied environmental conditions, but they were also affected by significant inter-species variability. Human and rodent NSCs share one feature, i.e., the necessity to be cultured immediately after isolation, which significantly maintains their survival. Additionally, in the absence of experiments on human cells, rat NSCs biology (neurosphere formation potential and neural differentiation stage) seems closer to that of humans rather than mice in response to environmental factors.

Establishment and application of four long-term culture cell lines of the olive flounder Paralichthys olivaceus blastocysts.

Four new embryonic cell lines derived from blastocysts of the olive flounder Paralichthys olivaceus, an important commercial marine fish, were established and characterized. They were designated as PoEFCI, PoEFCII, PoEFCIII, and PoEFCIV and were all fibroblastic cells. The cells were cultured in DMEM/F-12 medium supplemented with antibiotics, FBS, and growth factors at temperature of 25 °C and subcultured for >100 passages over 18 months. The origin of the cell lines was confirmed by examining the partial sequences of the cytochrome oxidase c subunit I (COI) gene of the flounder mitochondrial DNA (mtDNA). The four cell lines showed different growth curve patterns. According to the results of gene and protein expression and enzyme activity, the cell lines PoEFCI, PoEFCII, and PoEFC III could be pluripotent. The cells of all four cell lines were also successfully transfected with the green fluorescent protein (GFP) reporter gene, suggesting that they could be used to study gene function in the flounder or other fish. More importantly, PoEFCI-III were sensitive to chromium (Cr) and red sea bream Pagrus major iridovirus (RSIV), so they could be used as a powerful tool for the study of the toxicological investigation of heavy metals and RSIV in fish. Therefore, these cell lines would be useful for biotechnological and toxicological research on marine fish as an in vitro biological system.

Prolonged culture of human pancreatic islets under glucotoxic conditions changes their acute beta cell calcium and insulin secretion glucose response curves from sigmoid to bell-shaped.

AIMS/HYPOTHESIS: The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1-3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration. METHODS: Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3-7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1-3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute stepwise stimulation with glucose [NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)]. RESULTS: Culture of ND-islets for 1-3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were no longer sigmoid but bell-shaped, with maximal stimulation at 5 or 10 mmol/l glucose and rapid sustained inhibition above that concentration. Such paradoxical inhibition was, however, no longer observed when islets were acutely depolarised by 30 mmol/l extracellular K+. The glucotoxic alterations of beta cell function were fully reversible after culture at 5 mmol/l glucose and were mimicked by pharmacological activation of glucokinase during culture at 5 mmol/l glucose. Similar results to those seen in ND-islets were obtained in T2D-islets, except that their rate of insulin secretion during culture at 8 and 20 mmol/l glucose was lower, their cytosolic glutathione oxidation increased after culture at 8 and 20 mmol/l glucose, and the alterations in GSIS and upstream coupling events were greater after culture at 8 mmol/l glucose. CONCLUSIONS/INTERPRETATION: Prolonged culture of human islets under moderate to severe glucotoxic conditions markedly increased their glucose sensitivity and revealed a bell-shaped acute glucose response curve for changes in [Ca2+]c and insulin secretion, with maximal stimulation at 5 or 10 mmol/l glucose and rapid inhibition above that concentration. This novel glucotoxic alteration may contribute to beta cell dysfunction in type 2 diabetes independently from a detectable increase in beta cell apoptosis.