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.

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.

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.

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.

Derivation of hormone-responsive human endometrial organoids and stromal cells from cryopreserved biopsies.

The human endometrium is a dynamic tissue that undergoes cyclic changes in response to sex steroid hormones to provide a receptive status for embryo implantation. Disruptions in this behavior may lead to implantation failure and infertility; therefore, it is essential to develop an appropriate in vitro model to study endometrial changes in response to sex hormones. In this regard, the first choice would be human endometrial cells isolated from biopsies that could be used as monolayer cell sheets or to generate endometrial organoids. However, the need for fresh samples and short-time viability of harvested endometrial biopsy limits these approaches. In order to overcome these limitations, we sought to develop an efficient, simple, robust and reproducible method to cryopreserve human endometrial biopsies that could be stored and/or shipped frozen and later thawed to generate endometrial organoids and endometrial stromal cells (EnSCs). These cryopreserved biopsies could be thawed and used to generate simple endometrial organoids or organoids for co-culture with matched stromal cells that are functionally responsive to sex hormones as similar as the organoids generated from fresh biopsy. An optimal endometrial tissue cryopreservation method would allow the possibility for endometrial tissue biobanking to enable future organoid generation from both healthy tissues and pathological conditions, and open new venues for generate endometrial assembloids, consisting of epithelial organoids and primary stromal cells.

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.

A pediatric case of Gordonia otitidis bacteremia detected by long-term blood culture.

For immunocompromised patients receiving chemotherapy or bone mallow transplantation, slow-growing bacteria should also be considered one of the pathogenic microorganisms. However, there is no evidence pertaining to the microbiological tests associated with a patient with febrile neutropenia before peripheral blood stem cell harvest (PBSCH). We report a case of a 4-year-old cancer-bearing female presenting with a catheter-related bloodstream infection due to Gordonia otitidis. We detected G. otitidis from long-term blood cultures for approximately 6 days and prevented iatrogenic bacteremia by identifying the same organism from the culture of the PBSC sample and postponing the scheduled PBSCH. If febrile neutropenia occurs before PBSCH, we should collect multiple sets of blood cultures and culture them for a longer period.

Spontaneous Transformation of a p53 and Rb-Defective Human Fallopian Tube Epithelial Cell Line after Long Passage with Features of High-Grade Serous Carcinoma.

Ovarian cancer is one of the most lethal gynecological cancers, and 80% are high-grade serous carcinomas (HGSOC). Despite advances in chemotherapy and the development of targeted therapies, the survival rate of HGSOC has only moderately improved. Therefore, a cell model that reflects the pathogenesis and clinical characteristics of this disease is urgently needed. We previously developed a human fallopian tube epithelial cell line (FE25) with p53 and Rb deficiencies. After long-term culture in vitro, cells at high-passage numbers showed spontaneous transformation (FE25L). This study aimed to compare FE25 cells cultured in vitro for low (passage 16-31) and high passages (passage 116-139) to determine whether these cells can serve as an ideal cell model of HGSOC. Compared to the cells at low passage, FE25L cells showed increased cell proliferation, clonogenicity, polyploidy, aneuploidy, cell migration, and invasion. They also showed more resistance to chemotherapy and the ability to grow tumors in xenografts. RNA-seq data also showed upregulation of hypoxia, epithelial-mesenchymal transition (EMT), and the NF-κB pathway in FE25L compared to FE25 cells. qRT-PCR confirmed the upregulation of EMT, cytokines, NF-κB, c-Myc, and the Wnt/ß-catenin pathway. Cross-platform comparability found that FE25L cells could be grouped with the other most likely HGSOC lines, such as TYKNU and COV362. In conclusion, FE25L cells showed more aggressive malignant behavior than FE25 cells and hence might serve as a more suitable model for HGSOC research.

Vitamin C alleviates the senescence of periodontal ligament stem cells through inhibition of Notch3 during long-term culture.

Periodontal ligament stem cells (PDLSCs), as potential "seed cells" for periodontal tissue repair and regeneration, require to be expanded in vitro for a large scale. Senescence of PDLSCs occurred during long-term culture may compromise the therapeutic effects of PDLSCs. Medium supplements may be useful in antisenescence. However, the effects and mechanisms of vitamin C (Vc) treatment on PDLSCs during long-term culture are still unclear. In this study, we identified that Vc-treated PDLSCs cells maintained a slender morphology, higher growth rate and migration capacity, stemness, and osteogenic differentiation capability during a long-term culture. Moreover, we also identified that Notch3 was significantly upregulated during the cell senescence, and Vc treatment alleviated the senescence of PDLSCs through inhibition of Notch3 during long-term culture. In summary, Vc treatment suppressed PDLSCs senescence by reducing the expression of Notch3 and might be a simple and useful strategy to inhibit cellular senescence during the cell long-term culture.

A Membrane Filter-Assisted Mammalian Cell-Based Biosensor Enabling 3D Culture and Pathogen Detection.

We have developed a membrane filter-assisted cell-based biosensing platform by using a polyester membrane as a three-dimensional (3D) cell culture scaffold in which cells can be grown by physical attachment. The membrane was simply treated with ethanol to increase surficial hydrophobicity, inducing the stable settlement of cells via gravity. The 3D membrane scaffold was able to provide a relatively longer cell incubation time (up to 16 days) as compared to a common two-dimensional (2D) cell culture environment. For a practical application, we fabricated a cylindrical cartridge to support the scaffold membranes stacked inside the cartridge, enabling not only the maintenance of a certain volume of culture media but also the simple exchange of media in a flow-through manner. The cartridge-type cell-based analytical system was exemplified for pathogen detection by measuring the quantities of toll-like receptor 1 (TLR1) induced by applying a lysate of P. aeruginosa and live E. coli, respectively, providing a fast, convenient colorimetric TLR1 immunoassay. The color images of membranes were digitized to obtain the response signals. We expect the method to further be applied as an alternative tool to animal testing in various research areas such as cosmetic toxicity and drug efficiency.

[Design and experiment of online monitoring system for long-term culture of embryo].

In the study of embryo development process, the morphological features at different stages are essential to evaluate developmental competence of the embryo, which can be used to optimize and improve the system for in-vitro embryo culture. In this paper, an online monitoring system was designed for long-term culture of embryos, based on a monitoring strategy of low-magnification search and high-magnification observation. Three optical modules of 4× phase contrast, 10× and 20× Hoffman modulation phase contrast were configured in this system to meet the requirements of different fields of view, especially when the size of the embryo increases during the culture. Using an optomechanical system matching design, an error control and alignment test, the resolution of optical imaging was guaranteed, and a relief stereoscopic imaging with high contrast of embryos was obtained. Through low-magnification field of view to identify and locate embryos and high-magnification field of view to capture the details, the system realized online tracking and monitoring of embryos. In addition, we developed and verified an embryo identifying and locating algorithm based on image contour area and definition evaluation. The online monitoring system of in-vitro embryo culture proposed in this paper can track and record the morphological features of embryos without affecting the embryo development, providing a basis for the assessment of embryo development and the optimization of in-vitro culture system.

Maintenance and differentiation of human ES cells on polyvinylidene fluoride scaffolds immobilized with a vitronectin-derived peptide.

Polyvinylidene fluoride (PVDF) is biocompatible, easy to fabricate, and has piezoelectric properties; it has been used for many biomedical applications including stem cell engineering. However, long-term cultivation of human embryonic stem cells (hESCs) and their differentiation toward cardiac lineages on PVDF have not been investigated. Herein, PVDF nanoscaled membrane scaffolds were fabricated by electrospinning; a vitronectin-derived peptide-mussel adhesive protein fusion (VNm) was immobilized on the scaffolds. hESCs cultured on the VNm-coated PVDF scaffold (VNm-PVDF scaffold) were stably expanded for more than 10 passages while maintaining the expression of pluripotency markers and genomic integrity. Under cardiac differentiation conditions, hESCs on the VNm-PVDF scaffold generated more spontaneously beating colonies and showed the upregulation of cardiac-related genes, compared with those cultured on Matrigel and VNm alone. Thus, VNm-PVDF scaffolds may be suitable for the long-term culture of hESCs and their differentiation into cardiac cells, thus expanding their application in regenerative medicine.

Effects of cryopreservation and long-term culture on biological characteristics and proteomic profiles of human umbilical cord-derived mesenchymal stem cells.

BACKGROUND: Human umbilical cord-derived MSCs (hUC-MSCs) have been identified as promising seeding cells in tissue engineering and clinical applications of regenerative medicine due to their advantages of simple acquisition procedure and the capability to come from a young tissue donor over the other MSCs sources. In clinical applications, large scale production is required and optimal cryopreservation and culture conditions are essential to autologous and allogeneic transplantation in the future. However, the influence of cryopreserved post-thaw and long-term culture on hUC-MSCs remains unknown, especially in terms of specific protein expression. Therefore, biological characteristics and proteomic profiles of hUC-MSCs after cryopreserving and long-term culturing were investigated. METHODS: Firstly, hUC-MSCs were isolated from human umbilical cord tissues and identified through morphology, surface markers and tri-lineage differentiation potential at passage 3, and then the biological characteristics and proteomic profiles were detected and compared after cryopreserving and long-term culturing at passage 4 and continuously cultured to passage 10 with detection occurring here as well. The proteomic profiles were tested by using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique and differential protein were confirmed by mass spectrometry. RESULTS: The results showed no significant differences in phenotypes including morphology, surface marker and tri-lineage differentiation potential but have obvious changes in translation level, which is involved in metabolism, cell cycle and other pathways. CONCLUSION: This suggests that protein expression may be used as an indicator of hUC-MSCs security testing before applying in clinical settings, and it is also expected to provide the foundation or standardization guide of hUC-MSCs applications in regenerative medicine.

Optimization of 3D hydrogel microenvironment for enhanced hepatic functionality of primary human hepatocytes.

Although primary human hepatocytes (PHHs) are the gold standard in drug efficacy and metabolism studies, long-term survival of PHHs and maintenance of their hepatic function are still challenging. In this study, we focused on the effect of the initial microenvironment on upregulation and long-term preservation of hepatic function of PHHs encapsulated within biodegradable hydrogel systems. PHHs were encapsulated in RGD-functionalized hybrid hydrogels with various degrees of degradability, and their hepatic functionality was analyzed. Regardless of the hydrogel elastic modulus, the combination with nondegradable hydrogels had a predominantly negative effect on the prompt engraftment of PHHs, whereas a degradable hydrogel with intermediate initial degradability was most effective in maintaining hepatic function. Efficient network formation by PHHs and cocultured cells, along with the control of hydrogel degradation, governed the hepatic functionality at an early stage and upon long-term cultivation. Under optimized conditions, expression of genes involved in biological processes such as focal adhesions, cell survival, cytoskeleton formation, and extracellular matrix interactions was significantly higher than that in a control with relatively delayed initial degradation. Thus, we suggest that the orchestrated control of initial cellular remodeling may play an important role in the maintenance of hepatic function in a three-dimensional PHH culture.

Establishment of ornithine transcarbamylase deficiency-derived primary human hepatocyte with hepatic functions.

A long-term hepatocyte culture maintaining liver-specific functions is very essential for both basic research and the development of bioartificial liver devices in clinical application. However, primary hepatocytes rapidly lose their proliferation and hepatic functions over a few days in culture. This work is to establish an ornithine transcarbamylase deficiency (OTCD) patient-derived primary human hepatocyte (OTCD-PHH) culture with hepatic functions for providing an in vitro cell model. Liver tissue from an infant with OTCD was dispersed into single cells. The cells were cultured using conditional reprogramming. To characterize the cells, we assessed activities and mRNA expression of CYP3A4, 1A1, 2C9, as well as albumin and urea secretion. We found that the OTCD-PHH can be subpassaged for more than 15 passages. The cells do not express mRNA of fibroblast-specific maker, whereas they highly express markers of epithelial cells and hepatocytes. In addition, the OTCD-PHH retain native CYP3A4, 1A1, 2C9 activities and albumin secretion function at early passages. The OTCD-PHH at passages 2, 6, 9 and 13 have identical DNA fingerprint as the original tissue. Furthermore, under 3D culture environment, low urea production and hepatocyte marker staining of the OTCD-PHH were detected. The established OTCD-PHH maintain liver-specific functions at early passages and can be long-term cultured in vitro. We believe the established long-term OTCD-PHH culture is highly relevant to study liver diseases, particularly in infants with OTCD.

Protocol for the long-term culture of human primary keratinocytes from the normal colorectal mucosa.

Procedures for in vitro culturing of human primary keratinocytes from normal colon mucosa specimens have not been fully feasible, thus far. The protocol described herein allows primary keratinocytes from small tissue fragments of colorectal mucosa biopsies to grow in vitro. The procedure develops in three steps: (a) the enzymatic digestion of the tissue biopsy; (b) the use of cloning rings to purify primary keratinocyte colonies, (c) a defined keratinocyte medium to grow these cells in long-term culture. Our cultural method enables normal primary keratinocytes to be obtained by simple and rapid techniques. In our culture condition, primary keratinocytes express specific epithelial markers. Colorectal mucosa keratinocyte colonies require approximately 2 weeks to grow. Compared with previous approaches, our protocol provides a valuable model of study for human primary keratinocytes from normal colorectal (NCR) mucosa both at the cellular and molecular levels. It is well known, that different mutations occurring during the multistep process of carcinogenesis in the NCR mucosa, are strictly associated to the onset/progression of the colorectal carcinoma. On this ground, normal keratinocytes grown with our protocol, may represent an innovative tool to investigate the mechanisms that lead to colorectal carcinoma and other diseases. Our innovative procedure may allow to perform comparative investigations between normal and pathological colorectal cells.

Long-term culture of human lung adenocarcinoma A549 cells enhances the replication of human influenza A viruses.

Long-term culture of the human lung adenocarcinoma cell line A549 promotes the differentiation of these cells toward an alveolar type II cell phenotype. Here, we evaluated the susceptibility of long-term cultured A549 cells to human influenza viruses. A549 cells were cultured continuously for 25 days (D25-A549) or 1 day (D1-A549) in Ham's F12K medium. Six human influenza A viruses grew much faster in D25-A549 cells than in D1-A549 cells; however, two influenza B viruses replicated poorly in both cell types. Two avian influenza viruses replicated efficiently in both cell types, with similar titres. Expression levels of human virus receptors were higher in D25-A549 cells than in D1-A549 cells. D25-A549 cells thus more efficiently support the replication of human influenza A viruses compared with D1-A549 cells. Our data suggest that long-term cultured A549 cells will be useful for influenza A virus research.

Activation of transposable elements and genetic instability during long-term culture of the human fungal pathogen Candida albicans.

It has been repeatedly reported that transposable elements (TE) become active and/or mobile in the genomes of replicatively and stress-induced senescent mammalian cells. However, the biological role of senescence-associated transposon activation and its occurrence and relevance in other eukaryotic cells remain to be elucidated. In the present study, Candida albicans, a prevalent opportunistic fungal pathogen in humans, was used to analyze changes in gene copy number of selected TE, namely Cirt2, Moa and Cmut1 during long-term culture (up to 90 days). The effects of stress stimuli (fluconazole, hydrogen peroxide, hypochlorite) and ploidy state (haploid, diploid, tetraploid cells) were also considered. An increase in copy number of Cirt2 and Moa was the most accented in tetraploid cells after 90 days of culture that was accompanied by changes in karyotype patterns and slightly more limited growth rate compared to haploid and diploid cells. Stress stimuli did not potentiate TE activity. Elevation in chromosomal DNA breaks was also observed during long-term culture of cells of different ploidy, however this was not correlated with increased TE activity. Our results suggest that increased TE activity may promote genomic diversity and plasticity, and cellular heterogeneity during long-term culture of C. albicans cells.

Optical Investigation of Action Potential and Calcium Handling Maturation of hiPSC-Cardiomyocytes on Biomimetic Substrates.

Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are the most promising human source with preserved genetic background of healthy individuals or patients. This study aimed to establish a systematic procedure for exploring development of hiPSC-CM functional output to predict genetic cardiomyopathy outcomes and identify molecular targets for therapy. Biomimetic substrates with microtopography and physiological stiffness can overcome the immaturity of hiPSC-CM function. We have developed a custom-made apparatus for simultaneous optical measurements of hiPSC-CM action potential and calcium transients to correlate these parameters at specific time points (day 60, 75 and 90 post differentiation) and under inotropic interventions. In later-stages, single hiPSC-CMs revealed prolonged action potential duration, increased calcium transient amplitude and shorter duration that closely resembled those of human adult cardiomyocytes from fresh ventricular tissue of patients. Thus, the major contribution of sarcoplasmic reticulum and positive inotropic response to ß-adrenergic stimulation are time-dependent events underlying excitation contraction coupling (ECC) maturation of hiPSC-CM; biomimetic substrates can promote calcium-handling regulation towards adult-like kinetics. Simultaneous optical recordings of long-term cultured hiPSC-CMs on biomimetic substrates favor high-throughput electrophysiological analysis aimed at testing (mechanistic hypothesis on) disease progression and pharmacological interventions in patient-derived hiPSC-CMs.