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Zusammenfassung Hintergrund Ein Hornhauttransplantat aus Organkultur darf i. d. R. nur verwendet werden, wenn präoperativ eine Endothelzelldichte von mindestens 2000 Zellen pro mm2 nachgewiesen ist. Die Messung der Endothelzelldichte ist dadurch erschwert, dass in der Phasenkontrastmikroskopie die Zellgrenzen der Endothelzellen nicht immer gut sichtbar sind und die zählbare Fläche durch quellungsbedingte Descemet-Falten limitiert ist. Bislang ist daher keine automatische Methode zur Endothelzelldichtebestimmung in Hornhauttransplantaten verfügbar. Das neuronale Netzwerk U-Net hat sich in der vollautomatischen Analyse von spiegelmikroskopischen Aufnahmen des Hornhautendothels bewährt. Ziel dieser Studie war die Anwendung des U-Net in der Qualitätssicherung von Hornhauttransplantaten. Material und Methoden Das U-Net wurde zunächst anhand von 100 manuell markierten Befundungsbildern von Hornhäuten aus der Lions-Hornhautbank Baden-Württemberg trainiert. Für die Prüfung der Messgenauigkeit des U-Net wurden 100 weitere Befundungsbilder von der o. g. Hornhautbank zur Verfügung gestellt. Diese wurden jeweils von a) einer erfahrenen Befunderin und b) einem weniger erfahrenen Augenarzt manuell gezählt. Die identischen Bilder wurden vollautomatisch über das trainierte U-Net ausgezählt und dieses Ergebnis mit beiden manuellen Analysen mittels Pearson-Korrelation verglichen. Ergebnisse Der Korrelationskoeffizient zwischen dem U-Net und der erfahren Untersucherin als „Goldstandard“ betrug 0,90. Der Korrelationskoeffizient des weniger erfahrenen Augenarztes mit dem Goldstandard betrug nur 0,81. Beide Korrelationen erwiesen sich als statistisch hochsignifikant (p Schlussfolgerung Die enge Korrelation des U-Net mit dem „Goldstandard“ deutet darauf hin, dass mit dem U-Net, die medizintechnische Zulassung vorausgesetzt, eine effektive Unterstützung in der Qualitätsbeurteilung von Hornhauttransplantaten in der Hornhautbank möglich ist. Dies hätte Vorteile für die Objektivität und die Arbeitseffizienz.

For 2 centuries, researchers have studied ex vivo perfusion intending to preserve the physiologic function of isolated organs. If it were indeed possible to maintain ex vivo organ viability for days, transplantation could become an elective operation with clinicians methodically surveilling and reconditioning allografts before surgery. To this day, experimental reports of successfully prolonged (≥24 hours) organ perfusion are rare and have not translated into clinical practice. To identify the crucial factors necessary for successful perfusion, this review summarizes the history of prolonged normothermic ex vivo organ perfusion. By examining successful techniques and protocols used, this review outlines the essential elements of successful perfusion, limitations of current perfusion systems, and areas where further research in preservation science is required.

Light exerts profound effects on cognitive functions across species, including humans. However, the neuronal mechanisms underlying the effects of light on cognitive functions are poorly understood. In this study, we show that long-term exposure to bright-light treatment promotes spatial memory through a di-synaptic visual circuit related to the nucleus reuniens (Re). Specifically, a subset of SMI-32-expressing ON-type retinal ganglion cells (RGCs) innervate CaMKIIα neurons in the thalamic ventral lateral geniculate nucleus and intergeniculate leaflet (vLGN/IGL), which in turn activate CaMKIIα neurons in the Re. Specific activation of vLGN/IGL-projecting RGCs, activation of Re-projecting vLGN/IGL neurons, or activation of postsynaptic Re neurons is sufficient to promote spatial memory. Furthermore, we demonstrate that the spatial-memory-promoting effects of light treatment are dependent on the activation of vLGN/IGL-projecting RGCs, Re-projecting vLGN/IGL neurons, and Re neurons. Our results reveal a dedicated subcortical visual circuit that mediates the spatial-memory-promoting effects of light treatment.

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the increasing use of organoids as a culturing method for primary patient material in the last years, efficient transfer methods of components for genetic engineering in this 3D culture system are in need. Especially for organoids, the efficiency of genetic manipulations depends on a successful transfection. Thus, this protocol was developed to facilitate the electroporation of organoids and to prove its universal functionality in different entities. Human colorectal, pancreatic, hepatic and gastric cancer organoids were successfully electroporated with small and large plasmids in comparison. Based on GFP encoding vectors, the transfection efficiency was determined by FACS. No extensive preparation of the cells or special, cost-intensive electroporation buffers are necessary, and the protocol can be performed within one day.

The technique of in vitro propagation of Arbuscular mycorrhizal fungi has been developed over the past few decades and opens up areas of studying plant-fungi interactions. It is a scientific break through, especially for the study of the Arbuscular mycorrhizal fungi, since these obligate symbionts depend on host plant. The objective of this paper is to find out the in vitro culture of Arbuscular Mycorrhizal Fungi using Root Organ Culture technique. Ascertain of root colonization of these fungi could be affected in vitro without undertaking complex and complicated culture conditions. This could form an economically viable technique for root organ culture of Arbuscular mycorrhizal fungi.

Resumen Introduccion La aplasia cutis congenita (ACC) es un padecimiento dermico de presentacion poco frecuente que se caracteriza por la ausencia de piel al nacimiento y se asocia a malformaciones craneales, faciales, dermicas y oseas. El diagnostico es fundamentalmente clinico. Caso clinico Masculino de 5 dias de vida extrauterina, producto unico de madre primigesta y sin antecedentes familiares de relevancia. La exploracion fisica revelo defectos simetricos y bilaterales en la piel de ambas extremidades inferiores caracterizados por fragilidad cutanea, costras y ulceras recubiertas de pseudomembranas, asociados a una disminucion del espacio interdigital de ortejos de pie izquierdo, retraccion del pie y genu varum . Se manejo con aloinjertos de epidermis cultivado in vitro , cuidados generales de las heridas y seguimiento clinico posterior. Discusion La ACC asociada a epidermolisis ampollosa es una de las formas de presentacion menos frecuente. Es necesario descartar otro tipo de patologias dermicas. Se recomienda manejo clinico con cubierta cutanea biologica o sintetica, prevencion de infecciones, tratamiento oportuno de las complicaciones y seguimiento clinico.

Zusammenfassung Hintergrund Die Anwendung von Serumaugentropfen (AS) stellt eine alternative Behandlungsmethode für therapieresistente korneale Epitheldefekte dar. Bei persistierenden Epitheldefekten könnten Zytokine, die von Keratozyten im Stroma produziert werden, eine entscheidende Rolle bei der epithelialen Wundheilung spielen. Ziel dieser Studie ist es, Transforming Growth Factor β1 (TGF-β1), Keratinocyte Growth Factor (KGF), Hepatocyte Growth Factor (HGF) und Fibroblast Growth Factor basic (FGFb) im Kulturüberstand von Keratozyten mit humanem Serum (HS) in vitro zu untersuchen. Material und Methoden Serumaugentropfen von 10 Patienten wurden nach der Standardmethode der LIONS Hornhautbank Saar-Lor-Lux präpariert und bei − 80 °C tiefgefroren. Primäre humane Keratozyten wurden durch enzymatische Behandlung mit Kollagenase A (1 mg/ml) aus humanen Korneoskleralscheiben isoliert (n = 1) und in DMEM/Hamʼs Kulturmedium, versetzt mit 5% fetalem Kälberserum (FCS), kultiviert. Für den Testansatz wurden die Keratozyten mit 15 oder 30% HS (in DMEM/F14 ohne FCS) inkubiert und nach 24 h die Konzentration von TGF-β1, KGF, HGF und FGFb mittels Enzyme-linked Immunoabsorbent Assay (ELISA) aus dem Kulturüberstand bestimmt. Als Kontrolle wurden 15 oder 30% HS ohne Keratozyten nach 24 h Inkubationszeit (unter den gleichen Bedingungen wie die Keratozyten) verwendet. Ergebnisse Die HGF-Konzentration mit beiden HS-Konzentrationen war im Kulturüberstand von Keratozyten signifikant höher, im Vergleich zur HS-Kontrolle (ohne Keratozyten) nach 24 h Inkubationszeit (p Schlussfolgerungen Durch die Anwesenheit von Keratozyten steigt die Konzentration von HGF und FGFb im Kulturmedium mit humanem Serum innerhalb von 24 Stunden an. Diese Konzentrationsänderungen könnten die Wundheilung bei Epitheldefekten beeinflussen.

The organotypic co-culture skin model has been providing an advanced approach to the in vitro investigation of the skin. Mast cells, containing various mediators such as tryptase and chymase, are thought to contribute to many physiological and pathological events of the skin interactively with other cells. Here, we introduce an organotypic co-culture skin model which successfully integrates human dermal mast cells for further study of mast cell interactions with fibroblasts and keratinocytes.

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Background: Bisphenol S (BPS) has been widely substituted for bisphenol A (BPA) on thermal papers, but little is known about its skin absorption. Objectives: We compared the percutaneous absorption and biotransformation of BPS and BPA in vitro and in a controlled human trial. Methods: Absorption and biotransformation of BPS and BPA were monitored across reconstructed human epidermis at two environmentally relevant doses over 25 h. In the human trial, five male participants handled thermal receipts containing BPS and washed their hands after 2 h. Urine (0–48 h) and serum (0–7.5h) were analyzed for target bisphenols, and one participant repeated the experiment with extended monitoring. BPS data were compared with published data for isotope-labeled BPA (BPA-d16) in the same participants. Results: At doses of 1.5 and 7.7 μg/cm2 applied to reconstructed human epidermis, the permeability coefficient of BPS (0.009 and 0.003cm/h, respectively) was significantly lower than for BPA (0.036 and 0.033cm/h, respectively), and metabolism of both bisphenols was negligible. In participants handling thermal receipts, the quantities of BPS and BPA-d16 on hands was significantly correlated with maximum urinary event flux (μg), but the slope was lower for BPS than BPA (β=0.12 and 1.1, respectively). As a proportion of total urinary bisphenol, free BPS [mean±standard deviation (SD): 6.9±2.8%] was higher than for free BPA (2.7±1.9%). Postexposure maximum urinary BPS concentrations (0.93 to 3.0 ng/mL; n=5) were in the 93–98th percentile range of BPS in background Canadians (0.91–3.2 ng/mL; n=467). Conclusion: Both the in vitro and human studies suggested lower percutaneous absorption of BPS compared with BPA, but a lower biotransformation efficiency of BPS should also be considered in its evaluation as a BPA substitute. https://doi.org/10.1289/EHP5044

Conventional three-dimensional (3D) printing techniques have been growing in importance in the field of reconstructive surgery. Three-dimensional bioprinting is the adaptation of 3D printing techniques to tissue engineering, through the use of a bio-ink containing living cells and biomaterials. We hereby describe the principles of bioprinting, its main current limitations, and the prospects of this technique. A PubMed/MEDLINE search was performed. A total of 40 publications were included. To date, most of the tissues have been printed with promising results in vitro (e.g., skin, cartilage, and muscle). The first animal studies are promising for small-scale defects. Vascularization issues are the main limitation to printing large constructs. Once the barrier of vascularization is overcome, printing organs and composite tissues of any size could be possible, opening the doors for personalized medicine based on medical imaging. Printing custom-made autologous grafts or flaps could minimize donor site morbidity and maximize the morphological results. Considering the potential future applications of bioprinting in the field of reconstructive surgery, one has to be aware of this tool, which could drastically change our practice.

Changes in pregnenolone and corticosterone production by the adrenal glands of normoglycemic rats receiving the course of intraperitoneal mifepristone or NaCl administration were co-directed, but differed in magnitude. In rats with hyperglycemia, corticosteroid production increased after NaCl administration over 5 days and returned to the initial values after 15-day administration. On the contrary, pregnenolone and corticosterone production was suppressed after 5-day course of mifepristone, but significantly increased after mifepristone administration for 15 days. Intraperitoneal mifepristone administration almost completely abolished the response of rat adrenal glands with normo- and hyperglycemia to ACTH in vitro; this effect did not depend on the duration of administration.

Various experimental model designs have been used to analyze the inflammatory pathways in human gastrointestinal illnesses. Traditionally, analytical techniques and animal models are popular experimental tools to study the inflammation process of intestinal diseases. However, the comparison of results between animal and human models is difficult for the inconsistency of outcomes. Although there are different animal models for studying the intestinal diseases, none of them fully represents the physiological and environmental conditions typical of the human species. Also, there is currently a concerted effort to decrease the experimental use of animals. On the converse, experimental protocols using the culture of gut mucosa had become popular with the advent of endoscopy which allows explanting multiple fragments from the intestine. The peculiar characteristic of this model is the ability to preserve in vitro the features that we found in vivo, thus also the response to various stimuli that differs from person to person. The aim of the present paper is to provide a review of some of the possible uses of the organ intestinal mucosa culture.

We studied the protective effect of bioregulators isolated from the liver and blood serum of mammals under conditions of manifest fibrosis. Fibrosis was induced by CCl4 administration for 30 days and then, the liver was cultured in a roller organotypic culture for 30 days in the presence of bioregulators. Hepatoprotective effect of bioregulators was evaluated on histological sections of the liver at different terms of culturing. Experiments with roller organotypic culture of the liver isolated from animals with in vivo CCl4-induced fibrosis demonstrated the protective effect of bioregulator of the liver origin, while bioregulator isolated from the blood was ineffective.

Gaultheria trichophylla Royle (Ericaceae) and related species have been used in the traditional system of medicine for the treatment of diarrhoea, pain and inflammation.The present investigation explores G. trichophylla for its potential activity in hyperactive gut disorders.Antidiarrheal activity was evaluated on castor oil induced diarrhoea in mice with reference to standard drug verapamil. Gut modulatory activity was performed on isolated jejunum tissue preparations on spontaneous and high potassium induced contractions. Butyrylcholinesterase (BChE) inhibitory activity was performed with an in vitro study. Extract was tested for toxicity in mice.In the in vivo studies, the methanol extract of G. trichophylla and verapamil significantly (p 0.05, 0.01, 0.001) inhibited the frequency of defecation as well as wetting of faeces when compared with the negative control. The methanol (Gt. MeOH) extract of G. trichophylla caused a dose-dependent inhibitory effect on spontaneous contractions in isolated rabbit jejunum preparations and exhibited a partial inhibitory effect against high KThis study provides evidence that G. trichophylla possesses combinations of inhibitory and stimulatory effects mediated through possible cholinergic and less potent calcium blocking constituents, respectively. The latter may be responsible for the antidiarrheal effect.

Organ culture systems are used to elucidate the mechanisms of pattern formation in developmental biology. Various organ culture techniques have been used, but the lack of microcirculation in such cultures impedes the long-term maintenance of larger tissues. Recent advances in microfluidic devices now enable us to utilize self-organized perfusable capillary networks in organ cultures. In this review, we will overview past approaches to organ culture and current technical advances in microfluidic devices, and discuss possible applications of microfluidics towards the study of developmental biology.

While studies of cultured cells have led to new insights into biological control, greater understanding of human pathophysiology requires the development of experimental systems that permit analysis of intercellular communications and tissue-tissue interactions in a more relevant organ context. Human organs-on-chips offer a potentially powerful new approach to confront this long-standing problem.

The human body can be viewed as an organism consisting of a variety of cellular and non-cellular materials interacting in a highly ordered manner. Its complex and hierarchical nature inspires the multi-level recapitulation of the human body in order to gain insights into the inner workings of life. While traditional cell culture models have led to new insights into the cellular microenvironment and biological control in vivo, deeper understanding of biological systems and human pathophysiology requires the development of novel model systems that allow for analysis of complex internal and external interactions within the cellular microenvironment in a more relevant organ context. Engineering organ-on-chip systems offers an unprecedented opportunity to unravel the complex and hierarchical nature of human organs. In this chapter, we first highlight the advances in microfluidic platforms that enable engineering of the cellular microenvironment and the transition from cells-on-chips to organs-on-chips. Then, we introduce the key features of the emerging organs-on-chips and their proof-of-concept applications in biomedical research. We also discuss the challenges and future outlooks of this state-of-the-art technology.

Human cardiac drug discovery and disease modeling face challenges in recapitulating cellular complexity and animal-to-human translation due to the limitations of conventional 2D cell culture and animal models. The development of human cardiac organoid technologies could help in stimulating and maintaining differentiated cell functions for extended periods of time. By closely mimicking in vivo organ functions in vitro they could thereby help in overcoming the obstacles mentioned above. Through the construction of human cardiac organoids from pluripotent stem cell-derived cells, derived from patients with specific known genotypes and phenotypes, more complex and robust in vitro tools have recently become available for disease modeling. In this review, we will describe the relevance and importance of evolving organoid platforms in disease biology. We further provide examples of cardiac organoid platforms, which may lead the way toward future personalized medicine and drug discovery.

Donor-to-host transmission of infectious agents is a rare but well-recognised complication of corneal transplantation and may carry a grave visual prognosis. In this case series, we describe the clinical features and risk factors of using culture-positive donor corneas for transplantation. Retrospective chart review of a series of patients who underwent either penetrating keratoplasty (PK) or Descemet’s stripping automated endothelial keratoplasty (DSAEK) with positive microbiology cultivation during routine assessment of donor corneal tissue obtained at the time of surgery. Donor and recipient characteristics, tissue preparation and surgical parameters, clinical signs and outcomes were registered. Eleven patients who received culture-positive corneal grafts were identified: six with Candida, three with Gram-positive bacteria and two with Gram-negative bacteria. Three patients developed clinical keratitis after routine DSAEK using corneas contaminated with Candida species. The median death-to-preservation time (DPT) of these three donor corneas was 18.08 (range 18.08 to 20.90) h, while in the remaining eight donors, it was 12.27 (range 9.32 to 20.47) h. Despite the initiation of antifungal treatment, all three cases required explantation of the graft and a subsequent re-DSAEK. The use of donor corneas that are culture-positive for Candida carries a risk for developing postoperative keratitis and the risk may be higher in DSAEK. Unlike the cold storage technique employed for donor corneas described in this case series, organ culture technique requires microbiological screening and supplementation of an antifungal agent which may reduce the risk of donor-to-host transmission of fungal infection.

BACKGROUND: The use of nonsteroidal anti-inflammatory drugs (NSAIDs) among neonates is associated with a broad spectrum of life-threatening adverse effects on the gastrointestinal tract. Indeed, we have previously demonstrated that NSAIDs, such as indomethacin (INDO), induce damaging effects on the immature intestine (Perron et al. Genomics, 2013). Hydrogen sulfide (H(2)S) has been reported to exert a number of cytoprotective and anti-inflammatory effects in many organ systems. Thus, in order to minimize the deleterious effects caused by NSAIDs, several H(2)S-releasing derivatives have been developed, including ATB-352, an H(2)S-releasing derivative of ketoprofen. AIMS: In the present study, we determined the effects of ketoprofen (KETO) and its H(2)S-releasing derivative on the human mid-gestational intestine using serum-free organ culture, and compared them with those previously described for INDO. METHODS: The representative genes involved with the deleterious effect of INDO were used as reference to compare the effect of KETO and/or its derivative ATB-352 on the mid-gestation human small intestine after a treatment of 48 hours. Gene expression levels were measured by qRT-PCR. RESULTS: By determining the gene expression of COX2 in cultured human small intestinal explants, we observed two distinct patterns of response that allowed to divide the specimens into two groups: 1) those with an increased expression of COX2 mRNA (KETO-responder group), and 2) those where COX2 remained unchanged (non-responder group). For the non-responder group, no significant change was observed compared to control. Although the inflammatory response observed between INDO and the KETO-responder group was similar (CXCL14 and TFF1), the negative effects on oxidoreductase activity (DUOX2, NOS2, SOD2) and intestinal permeability (OCLN, CLDN1) induced by INDO were not found with KETO, suggesting a beneficial effect of KETO on the small intestinal mucosa. In addition, the use of the H(2)S-releasing derivative of KETO did not induce significant changes compared to KETO on these metabolic pathways. CONCLUSIONS: Our results show that KETO (responder group) has beneficial effects on oxidoreductase activity and epithelial permeability in the immature human small intestine compared to INDO. However, the lack of beneficial effects of H(2)S on the small intestinal mucosa in organ culture suggests a systemic type mechanism rather than a local one. FUNDING AGENCIES: CIHR

Background: Application of cold atmospheric pressure plasmas (CAPs) in or on the human body was termed ‘plasma medicine'. So far, plasmas were utilized for sterilization of implants, other heat-sensitive products, or employed for chemical surface modifications. By now, CAPs are further used effectively for wound treatment. The present study analyses the effect of a plasma jet with air or nitrogen as process gas, previously evaluated for antimicrobial efficacy, on human cells using a 3D skin model. Methods: CAP treatment of 3D skin models consisting of a keratinocyte-containing epidermal layer and a fibroblast/collagen dermal matrix was performed using the Tigres plasma MEF technology. To evaluate the effects on the 3D skin models, the following plasma parameters were varied: process gas, input power, and treatment time. Results: Low CAP doses exhibited good cell compatibility. Increasing input power or elongating treatment intervals led to detrimental effects on 3D skin model morphology as well as to release of inflammatory cytokines. It was further observed that air as process gas was more damaging compared to nitrogen. Conclusions: Treatment of 3D skin models with the plasma MEF nozzle using air or nitrogen is reported. A clearly dose- and time-dependent effect of CAPs could be observed in which the CAP based on nitrogen exhibited higher cell compatibility than the CAP generated from air. These settings might be recommended for medical in vivo applications such as wound decontamination.

STUDY HYPOTHESIS: Are the placental aquaporins (AQPs) involved in the apoptosis of human trophoblast? STUDY FINDING: The general blocking of placental AQPs with HgCl2 and, in particular, the blocking of AQP3 activity with CuSO4 abrogated the apoptotic events of human trophoblast cells. WHAT IS KNOWN ALREADY: Although apoptosis of trophoblast cells is a natural event involved in the normal development of the placenta, it is exacerbated in pathological processes, such as pre-eclampsia, where an abnormal expression and functionality of placental AQPs occur without alterations in the feto-maternal water flux. Furthermore, fluctuations in O2 tension are proposed to be a potent inducer of placental apoptotic changes and, in explants exposed to hypoxia/reoxygenation (H/R), transcellular water transport mediated by AQPs was undetectable. This suggests that AQPs might be involved in processes other than water transport, such as apoptosis. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Explants from normal term placentas were maintained in culture under conditions of normoxia, hypoxia and H/R. Cell viability was determined by assessing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide incorporation. For the general or specific inhibition of AQPs, 0.3 mM HgCl2, 5 mM CuSO4, 0.3 mM tetraethylammonium chloride (TEA) or 0.5 mM phloretin were added to the culture medium before explants were exposed to each treatment. Oxidative stress parameters and apoptotic indexes were evaluated in the presence or absence of AQPs blockers. AQP3 expression was confirmed by western blot and immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: First, we observed that in H/R treatments cell viability decreased by 20.16 ± 5.73% compared with those explants cultured in normoxia (P = 0.009; n = 7). Hypoxia did not modify cell viability significantly. Both hypoxia and H/R conditions induced oxidative stress. Spontaneous chemiluminescence and thiobarbituric acid reactive substance levels were significantly increased in explants exposed to hypoxia (n = 6 per group, P = 0.0316 and P = 0.0009, respectively) and H/R conditions (n = 6 per group, P = 0.0281 and P = 0.0001, respectively) compared with those cultured in normoxia. Regarding apoptosis, H/R was a more potent inducer of trophoblast apoptosis than hypoxia alone. Bax expression and the number of apoptotic nuclei were significantly higher in explants cultured in H/R compared with normoxia and hypoxia conditions (n = 12, P = 0.0135 and P = 0.001, respectively). DNA fragmentation was only observed in H/R and, compared with normoxia and hypoxia, the activity of caspase-3 was highest in explants cultured in H/R (n = 12, P = 0.0001). In explants exposed to H/R, steric blocking of AQP activity with HgCl2 showed that DNA degradation was undetectable (n = 12, P = 0.001). Bax expression and caspase-3 activity were drastically reduced (n = 12, P = 0.0146 and P = 0.0001, respectively) compared with explants cultured in H/R but not treated with HgCl2. Similar results were observed in explants exposed to H/R when we blocked AQP3 activity with CuSO4. DNA degradation was undetectable and the number of apoptotic nuclei and caspase-3 activity were significantly decreased compared with explants cultured in H/R but not treated with CuSO4 (n = 12, P = 0.001 and P = 0.0001, respectively). However, TEA and phloretin treatments, to block AQP1/4 or AQP9, respectively, failed in abrogate apoptosis. In addition, we confirmed the expression and localization of AQP3 in explants exposed to H/R. LIMITATIONS, REASONS FOR CAUTION: Our studies are limited by the number of experimental conditions tested, which do not fully capture the variability in oxygen levels, duration of exposure and alternating patterns of oxygen seen in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that any alteration in placental AQP expression might disturb the equilibrium of the normal apoptotic events and may be an underlying cause in the pathophysiology of placental gestational disorders such as pre-eclampsia. Furthermore, the dysregulation of placental AQPs may be one of the crucial factors in triggering the clinical manifestations of pre-eclampsia. Fil: Szpilbarg, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Castro-Parodi, Mauricio Omar. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Reppetti, Julieta. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Repetto, M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentina Fil: Maskin, B.. Hospital Nacional "Prof. Dr. Alejandro Posadas"; Argentina Fil: Martinez, Nora Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Damiano, Alicia Ermelinda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentina

White Guinea yam (Dioscorea rotundata Poir.) is an important staple to millions of people in West Africa. Obtaining good quality planting material for yam cultivation is a major challenge. Multiplication ratios are low, and seed tubers are prone to contamination with pests and pathogens in the traditional systems of production. Some approaches to producing quality seed of yam are as follows: farmers select small whole tubers from a ware crop harvest; stimulate the production of seed tubers by ‘milking’ ware tubers while the leaves of the plant are still green (double harvest system); cut ware tubers into setts about the same sizes as regular seed tubers; or use the ‘Anambra’ system where smaller setts are cut and used to produce seed tubers. New methods that have been developed to address some of the challenges of quantity and quality of seed tubers are not yet widely applied, so farmers continue to use traditional methods and save seed from a previous harvest to plant the ware crop. This document presents an overview of traditional and modern methods of seed yam production and gives a perspective for the future. Among the modern methods of seed yam production, only the minisett technique, which uses 25–100 g tuber pieces, is currently used at farmer level, although on a limited scale. While tissue and organ culture techniques are the most rapid methods of multiplying disease free propagules, their limitations include high costs, need for skilled personnel and specialized equipment. The aeroponics and temporary immersion bioreactor methods of producing seed yam are relatively new, and still need more research. To build and sustain a viable seed yam production system, a multiplication scheme is required that combines two or more methods including tissue culture for cleaning the seed stock.

Purpose To evaluate the impact of lubricant eyedrops on the corneal healing process and corneal toxicity. Methods Optive® and Cationorm® were tested regarding corneal irritability against Vismed Multi® and 0.01% benzalkonium chloride as negative and positive control, respectively. Formulas were applied on rabbit corneas (n = 5) cultured on artificial anterior chambers (EVEIT system) hourly over 3 days. Initially, 4 corneal abrasions (2–5.4 mm2) were induced. All defects were monitored during drug application by fluorescein stains and photographs. To ensure corneal vitality, glucose and lactate concentrations were determined photometrically in artificial anterior chamber fluids. Corneal fluorescein sodium permeability was tested as an indicator of the corneal barrier function. Results Optive® and Vismed Multi® showed a complete corneal healing on day 2. In one cornea (Optive®), erosion reoccurred on day 3. Erosion sizes of Cationorm®-treated corneas increased significantly from 12.20 mm2 to a subtotal erosion of 51.89 mm2 on day 3. Histology revealed epithelial loss and severe alterations of the superficial stroma for Cationorm®. Glucose and lactate concentrations did not change after application of Optive® and Vismed Multi®. In contrast, Cationorm®- and BAC-treated corneas showed a significant increase in lactate concentrations. Conclusions Vismed Multi® application resulted in rapid corneal healing. Whether the toxicity seen for Optive® in one cornea is a valid result should be examined further. Cationorm® showed considerable corneal toxicity that could be caused by its additive, cetalkonium chloride. Otherwise, the electrostatic properties of Cationorm® led to a drug film on the area of epithelial loss that could hinder epithelial cell migration and adhesion in order to heal the lesion.

Background Corticosteroids increase the expression of β2-adrenoceptors (β2-ARs) and protect them against down-regulation. Conversely, β2-AR agonists improve the anti-inflammatory action of corticosteroids. Nevertheless, it is still uncertain whether adding a long-acting β2-AR agonist (LABA) to an inhaled corticosteroid (ICS) results in an additive effect, or there is true synergy. Therefore, the aim of this study was to pharmacologically characterize the interaction between the ICS beclomethasone diproprionate (BDP) and the LABA formoterol fumarate (FF) in a validated human ex vivo model of bronchial asthma. Methods Human medium and small airways were stimulated by histamine and treated with different concentrations of BDP and FF, administered alone and in combination at concentration-ratio reproducing ex vivo that of the currently available fixed-dose combination (FDC; BDP/FF 100:6 combination-ratio). Experiments were performed in non-sensitized (NS) and passively sensitized (PS) airways. The pharmacological interaction was assessed by using Bliss Independence and Unified Theory equations. Results BDP/FF synergistically increased the overall bronchorelaxation in NS and PS airways (+ 15.15% ± 4.02%; P

For more than a decade pharmaceutical R&D has been hampered by considerable attrition rates during clinical trials. The main reasons for drug failure is related to the lack of efficacy, limitations with respect to ADME (absorption, distribution, metabolism and excretion) properties, and—in approximately 30% of the cases—unforeseen toxicity (Kola and Landis 2004). The majority of adverse drug reactions observed in the clinical phase refer to organ injuries, e.g. of the cardiovascular system, the liver, the central nervous system and skeletal muscle (Cook et al. 2014). This clearly demonstrates the limited predictive accuracy of current preclinical models such as the rodent bioassay in evaluating repeated dose toxicity for predicting human toxic risks. It has been argued that overall, only 43% of toxic effects in humans may be correctly predicted by applying rodent-based safety evaluation protocols due to the fact that these assays tend to generate relatively large numbers of false negative as well as false positive read outs (Hartung 2009). ISSN:0340-5761 ISSN:1432-0738

Mammalian embryogenesis requires the coordination of embryonic and extra-embryonic tissues to enable implantation into the uterus and post-implantation development to establish the body plan. Mouse embryonic stem cells (ESCs) are a useful tool for studying pluripotent embryonic tissue in vitro. However, they cannot undertake correct embryogenesis alone. Many attempts to model the early embryo in vitro involve the aggregation of ESCs into spheroids of variable size and cell number that undertake germ-layer specification but fail to recapitulate the characteristic architecture and arrangement of tissues of the early embryo. Here, we describe a protocol to generate the first embryo-like structures by directing the assembly of mouse ESCs and extra-embryonic trophoblast stem cells (TSCs) in a 3D extracellular matrix (ECM) into structures we call 'polarized embryo-like structures'. By establishing the medium and culture conditions needed to support the growth of both stem cell types simultaneously, embryonic architecture is generated within 4 d of co-culture. This protocol can be performed by those proficient in standard ESC culture techniques and can be used in developmental studies to investigate the interactions between embryonic and extra-embryonic tissues during mammalian development.

Electrical impedance myography (EIM) is an electrophysiological technique for neuromuscular evaluation that is impacted by subcutaneous fat (SF). Exploiting the differing frequency dependences of muscle and fat, we assessed a 2-frequency EIM phase ratio in Duchenne muscular dystrophy (DMD) boys.Twenty-eight DMD boys aged 2-13years underwent EIM and the 6-minute walk test (6MWT). For each subject, 50kHz phase data was input into the numerator while 20-500kHz phase values were input into the denominator. We then performed correlation analyses seeking to identify the denominator frequency that simultaneously optimized SF and 6MWT correlations. This optimized ratio was then tested in 24 healthy boys.50kHz phase correlated to 6MWT in DMD boys with R=0.52, p=0.0066, and to SF thickness with R=-0.67, p0.001. An optimized ratio of 50/200kHz phase reduced the correlation of SF thickness to R=-0.075, p=0.45 while improving the relationship to the 6MWT (R=0.60, p=0.001). In normal subjects, the optimization decreased SF correlation from R=0.61 from R=0.16 with 6MWT correlation remaining unchanged.The 50/200kHz EIM phase ratio removes the impact of SF while maintaining EIM's association with function.The use of a phase ratio may enhance EIM's application for evaluation of neuromuscular disease.

Importance Descemet membrane endothelial keratoplasty (DMEK) is a challenging procedure for the surgeon, particularly because of deficient visibility of the delicate tissue due to the natural en face view through the operating microscope. A cross-sectional view would greatly enhance intraoperative overview and enable the surgeon to better control the procedure. Objective To retrospectively analyze the use of intraoperative optical coherence tomography (iOCT) for improving the safety of DMEK. Design Intraoperative OCT during DMEK was performed in 26 eyes of 26 patients. We retrospectively analyzed imaging and video data. Setting Department of Ophthalmology, University of Cologne. Participants Seven men and 19 women aged 39 to 93 years with corneal endothelial dysfunction undergoing DMEK. Exposure Descemet membrane endothelial keratoplasty. Main Outcomes and Measures Visibility of surgical steps, overall duration of DMEK, overall time for complete intraoperative air filling of the anterior chamber, and correlation between donor age and Descemet rolling behavior. Results Intraoperative OCT enables visualization of all steps of the DMEK procedure. Overall mean (SD) duration of the DMEK procedure was 25.7 (6.9) minutes when using iOCT. Overall mean (SD) complete intraoperative anterior chamber air-filling time was 236 (108) seconds in contrast to 60 to 90 minutes for standard air-filling time. Descemet membrane rolling behavior showed significant inverse correlation between donor age (range, 39-93 years) and the extent of rolling ( R 2 = 0.5 [ P = .006]). Conclusions and Relevance Intraoperative OCT enhances the visibility of graft orientation and unfolding, thereby improving safety of the DMEK procedure. Overall, iOCT is a helpful device that may support surgeons in all steps of DMEK procedures.

Three-dimensional aggregation and organ culture methods are critical for recreating in vivo cellular phenomena outside the body. Previously, we used the conventional gas liquid interphase organ culture method to induce complete mouse spermatogenesis. After incorporating microfluidic systems, we achieved a significant increase in efficiency and duration of spermatogenesis. One of the major drawbacks preventing the popularization of microfluidics, however, is the use of a power-pump to generate medium flow. In this study, we produced a pumpless microfluidic device using hydrostatic pressure and a resistance circuit to facilitate slow, longer lasting medium flow. During three months of culture, results in induction and maintenance of spermatogenesis showed no difference between pumpless and pump-driven devices. Correspondingly, the spermatogonial population was favorably maintained in the pumpless device compared to the conventional method. These results show the advantage of using microfluidic systems for organ culture experiments. Our pumpless device could be applied to a variety of other tissues and organs, and may revolutionize organ culture methods as a whole.

Background The evidence on the role of gut microbiota in post-infectious irritable bowel syndrome (PI-IBS) is convincing. Lactobacillus spp. positively affect IBS symptoms, although the mechanisms through which probiotics exert their beneficial effects are largely unknown. The aim of the study is to evaluate the role of Lactobacillus casei DG (LC-DG) and its postbiotic (PB) in modulating the inflammatory/immune-response in PI-IBS in an ex-vivo organ culture model. Methods Ex vivo cultures of ileal and colonic mucosa from 10 PI-IBS, diarrhea predominant subtype (D) patients, and 10 healthy controls (HC) were treated with LPS, LC-DG and PB. Interleukin (IL)-1α, IL-6, IL-8 and IL-10 mRNA levels were assessed by real-time PCR and Toll like receptor 4 (TLR-4) protein expression by Western blotting. Results At baseline, IL-1α, IL-6 and IL-8 mRNA levels as well as TLR-4 protein expression were significantly higher while IL-10 mRNA levels were lower in PI-IBS D than in HC in both ileum and colon. LC-DG and PB significantly reduced the mRNA levels of pro-inflammatory cytokines and TLR-4 while increased that of IL-10 after LPS stimulation. The protective effect was more pronounced for PB than LC-DG treatment. Conclusion LC-DG and its PB attenuate the inflammatory mucosal response in an ex-vivo organ culture model of PI-IBS D.

This protocol describes the preparation of hippocampal slice cultures from rat or mouse pups using sterile conditions that do not require the use of antibiotics or antimycotics. Combining very good optical and electrophysiological accessibility with a lifetime approaching that of the intact animal, many fundamental questions about synaptic plasticity and long-term dynamics of network connectivity can be addressed with this preparation.

The ability of bone repair scaffolds to form bone is traditionally evaluated using cell culture and animal experiments. Mouse calvarial organ culture maintains the natural cell-to-cell and cell-to-matrix relationships as well as the anatomical order, and this model has been used to study the biological behavior of intramembranous bones. The aim of this study was to evaluate the potential of mouse calvarial organ culture to be used as an in vitro model to study the bone regenerative ability of bone repair polymer scaffolds. Critical size defects (CSD) were created in the parietal bones. Electrospun poly(e-caprolactone) scaffolds were placed into one group of defects. The remaining defects served as a control. The bones were cultured for 38 days and analyzed with μCT, phase-contrast microscopy, dissecting microscopy, scanning electron microscopy, and energy-dispersive X-ray analyses. This organ culture technique is easily available and could permit researchers to quickly establish a valuable database of candidate bone repair scaffolds. Microsc. Res. Tech. 77:1037–1043, 2014. © 2014 Wiley Periodicals, Inc.

PurposeTo explore the potential of non-invasive reverse iontophoresis transdermal extraction of iohexol as a marker of glomerular filtration rate.MethodsA series of in vitro experiments were undertaken to establish the feasibility of iohexol reverse iontophoresis and to determine the optimal conditions for the approach. Subsequently, a pilot study in paediatric patients was performed to provide proof-of-concept.ResultsThe iontophoretic extraction fluxes of iohexol in vitro were proportional to the marker subdermal concentration and the reverse iontophoretic technique was able to track changes dynamically in simulated pharmacokinetic profiles. Reverse iontophoresis sampling was well tolerated by the four paediatric participants. The deduced values of the iohexol terminal elimination rate constant from transdermal reverse iontophoresis sampling agreed with those estimated by conventional blood sampling.ConclusionsReverse iontophoretic transdermal extraction fluxes mirrored the subdermal concentration profiles of iohexol, a relatively large neutral marker of glomerular filtration both in vitro and in vivo. The efficiency of extraction in vivo was well predicted by the in vitro model used.

Three-dimensional (3D) bioprinting, a flexible automated on-demand platform for the free-form fabrication of complex living architectures, is a novel approach for the design and engineering of human organs and tissues. Here, we demonstrate the potential of 3D bioprinting for tissue engineering using human skin as a prototypical example. Keratinocytes and fibroblasts were used as constituent cells to represent the epidermis and dermis, and collagen was used to represent the dermal matrix of the skin. Preliminary studies were conducted to optimize printing parameters for maximum cell viability as well as for the optimization of cell densities in the epidermis and dermis to mimic physiologically relevant attributes of human skin. Printed 3D constructs were cultured in submerged media conditions followed by exposure of the epidermal layer to the air-liquid interface to promote maturation and stratification. Histology and immunofluorescence characterization demonstrated that 3D printed skin tissue was morphologically and biologically representative of in vivo human skin tissue. In comparison with traditional methods for skin engineering, 3D bioprinting offers several advantages in terms of shape- and form retention, flexibility, reproducibility, and high culture throughput. It has a broad range of applications in transdermal and topical formulation discovery, dermal toxicity studies, and in designing autologous grafts for wound healing. The proof-of-concept studies presented here can be further extended for enhancing the complexity of the skin model via the incorporation of secondary and adnexal structures or the inclusion of diseased cells to serve as a model for studying the pathophysiology of skin diseases.

Intervertebral disc (IVD) degeneration is a common cause of back pain, and attempts to develop therapies are frustrated by lack of model systems that mimic the human condition. Human IVD organ culture models can address this gap, yet current models are limited since vertebral endplates are removed to maintain cell viability, physiological loading is not applied, and mechanical behaviors are not measured. This study aimed to (i) establish a method for isolating human IVDs from autopsy with intact vertebral endplates, and (ii) develop and validate an organ culture loading system for human or bovine IVDs. Human IVDs with intact endplates were isolated from cadavers within 48 hours of death and cultured for up to 21 days. IVDs remained viable with ~80% cell viability in nucleus and annulus regions. A dynamic loading system was designed and built with the capacity to culture 9 bovine or 6 human IVDs simultaneously while applying simulated physiologic loads (maximum force: 4kN) and measuring IVD mechanical behaviors. The loading system accurately applied dynamic loading regimes (RMS error 85% viable cells throughout the 3 week culture period. This organ culture loading system can closely mimic physiological conditions and be used to investigate response of living human and bovine IVDs to mechanical and chemical challenges and to screen therapeutic repair techniques.

Multi-organ microdevices can mimic tissue–tissue interactions that occur as a result of metabolite travel from one tissue to other tissues in vitro. These systems are capable of simulating human metabolism, including the conversion of a pro-drug to its effective metabolite as well as its subsequent therapeutic actions and toxic side effects. Since tissue–tissue interactions in the human body can play a significant role in determining the success of new pharmaceuticals, the development and use of multi-organ microdevices present an opportunity to improve the drug development process. The devices have the potential to predict potential toxic side effects with higher accuracy before a drug enters the expensive phase of clinical trials as well as to estimate efficacy and dose response. Multi-organ microdevices also have the potential to aid in the development of new therapeutic strategies by providing a platform for testing in the context of human metabolism (as opposed to animal models). Further, when operated with human biopsy samples, the devices could be a gateway for the development of individualized medicine. Here we review studies in which multi-organ microdevices have been developed and used in a ways that demonstrate how the devices' capabilities can present unique opportunities for the study of drug action. We will also discuss challenges that are inherent in the development of multi-organ microdevices. Among these are how to design the devices, and how to create devices that mimic the human metabolism with high authenticity. Since single organ devices are testing platforms for tissues that can later be combined with other tissues within multi-organ devices, we will also mention single organ devices where appropriate in the discussion.

Background Aging is an independent risk factor for cardiovascular and cerebrovascular disease. To date, little is known about the mechanisms of aging of cerebral arteries and whether the aging gene p66 Shc is implicated in it. The present study was designed to assess age-induced vascular dysfunction in cerebral and systemic arteries of wild type (wt) and p66 Shc−/− mice. Methods Basilar arteries and size matched second order femoral arteries of 3-month (3M), 6-month (6M) and 2-year old (2Y) mice were studied in wt and p66 Shc−/− mice. To assess vascular function, arterial rings mounted in a myograph for isometric tension recordings were exposed to increasing concentrations of acetylcholine and sodium nitroprusside. Reactive oxygen species (ROS) generation was assessed in femoral and basilar arteries using the spin trap 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Results In wt mice, endothelial function of the femoral artery was not affected by age unlike in the basilar artery where an age-dependent dysfunction was observed. In p66 Shc−/− a similar response was observed in the femoral artery; however, age-dependent endothelial dysfunction of the basilar artery was blunted as compared to wt. Levels of ROS were comparable in the femoral arteries of 3M and 2Y of wt and p66 Shc−/− mice. Differently, ROS levels in the basilar artery of wt mice were strongly increased by age unlike in p66 Shc−/− mice where they remained comparable irrespective of age. Conclusions Endothelial function in cerebral arteries, but not in size-matched systemic ones, is heavily impaired by aging. This process is paralleled by an increased ROS production and is mediated by the p66 Shc gene.