Maintenance Medium Research Articles

Fabrication of Dentin-Pulp-Like Organoids Using Dental-Pulp Stem Cells.

We developed a novel dentin-pulp-like organoid. It has both stem-cell and odontoblast characteristics using a mesenchymal cell lineage of human dental-pulp stem cells (hDPSCs). The mixture of hDPSCs and Matrigel was transferred into the maintenance medium (MM) and divided into four different groups according to how long they were maintained in the odontogenic differentiation medium (ODM). All organoids were harvested at 21 days and analyzed to find the optimal differentiation condition. To assess the re-fabrication of dentin-pulp-like organoid, after dissociation of the organoids, it was successfully regenerated. Additionally, its biological activity was confirmed by analyzing changes of relevant gene expression and performing a histology analysis after adding Biodentine® into the ODM. The organoid was cultured for 11 days in the ODM (ODM 11) had the most features of both stem cells and differentiated cells (odontoblasts) as confirmed by relevant gene expression and histology analyses. Micro-computed tomography and an electron microscope also showed mineralization and odontoblastic differentiation. Finally, ODM 11 demonstrated a biologically active response to Biodentine® treatment. In conclusion, for the first time, we report the fabrication of a dentin-pulp-like organoid using mesenchymal stem cells. This organoid has potential as a future therapeutic strategy for tooth regeneration.

Nursery method of Jatimbulan Tilapia (Oreochromis niloticus) in Pasuruan, East Java

Jatimbulan Tilapia (Oreochromis niloticus) is the third generation of the crossover six parental strain of tilapia. The nursery method for Jatimbulan Tilapia (Oreochromis niloticus) fry is carried out in soil and concrete-walled pond, as well as seeds having an average length of 2.3 cm and an average weight of 0.181 grams. The treatment used is feeding pellets by feeding frequency two times a day with a 30% feeding rate of the biomass. Treatment significantly generating good growth and survival; SGR (1.12%) and SR (97%). Water quality in the maintenance media contained in a reasonable range for raising Jatimbulan Tilapia.

Effects of octreotide on autophagy markers and cell viability markers related to metabolic activity in rat pituitary tumor cells.

The aim of this work was to investigate possible direct effects of the somatostatin analog octreotide on autophagy markers and markers of cellular metabolic activity using in vitro cultured rat pituitary tumor cells (GH3 cell line). We measured two markers of the autophagic flux in cell lysates by Western blot and MTT reductive activity, total cellular ATP levels, pyruvate dehydrogenase (PDH) complex activity in cells lysates as markers of cell viability related to metabolic activity. Octreotide (100nM) treatment induced autophagy activation (increased LC3-I protein lipidation) and enhanced the autophagic flux (SQSTM1/p62 protein downregulation) in GH3 cells in different incubation media, in detail in Hank's balanced salt solution (HBSS) as well as in maintenance medium with serum. We did not observe any decrease of redox activity and energy production related to the induction of autophagy by octreotide. On the other hand, short-term treatments with octreotide in HBSS tended to enhance MTT reduction activity and to increase PDH complex enzymatic activity and ATP levels measured in GH3 cell lysates. We provided evidence that octreotide can affect autophagy in pituitary tumor cells. The observed effects of octreotide were not related to a decrease of cellular metabolic activity. Finally, the induction of autophagy was either short-lived or overshadowed by other factors in the long term and this limit does not help clarifying their real impact on the pharmacological activity of somatostatin analogs.

Accelerated neuronal and synaptic maturation by BrainPhys medium increases A\u03b2 secretion and alters A\u03b2 peptide ratios from iPSC-derived cortical neurons

One of the neuropathological hallmarks of Alzheimer’s disease (AD) is cerebral deposition of amyloid plaques composed of amyloid β (Aβ) peptides and the cerebrospinal fluid concentrations of those peptides are used as a biomarker for AD. Mature induced pluripotent stem cell (iPSC)-derived cortical neurons secrete Aβ peptides in ratios comparable to those secreted to cerebrospinal fluid in human, however the protocol to achieve mature neurons is time consuming. In this study, we investigated if differentiation of neuroprogenitor cells (NPCs) in BrainPhys medium, previously reported to enhance synaptic function of neurons in culture, would accelerate neuronal maturation and, thus increase Aβ secretion as compared to the conventional neural maintenance medium. We found that NPCs cultured in BrainPhys displayed increased expression of markers for cortical deep-layer neurons, increased synaptic maturation and number of astroglial cells. This accelerated neuronal maturation was accompanied by increased APP processing, resulting in increased secretion of Aβ peptides and an increased Aβ38 to Aβ40 and Aβ42 ratio. However, during long-term culturing in BrainPhys, non-neuronal cells appeared and eventually took over the cultures. Taken together, BrainPhys culturing accelerated neuronal maturation and increased Aβ secretion from iPSC-derived cortical neurons, but changed the cellular composition of the cultures.

Fetal Calf Serum Exerts an Inhibitory Effect on Replication of Duck Hepatitis A Virus Genotype 1 in Duck Embryo Fibroblast Cells.

Among the causative agents of duck viral hepatitis, duck hepatitis A virus genotype 1 (DHAV-1) is the most common virus reported in most outbreaks worldwide. How to propagate DHAV-1 in cell cultures efficiently remains a problem to be explored. Here, we aimed to test the effect of serum type on DHAV-1 replication in duck embryo fibroblast (DEF) cells. Comparative studies involved virus culture and passage, observation of cytopathic effect (CPE), virus quantification, and plaque formation assay. From the results of these investigations, we conclude that use of chicken serum (CS) in maintenance medium allows DHAV-1 to establish productive, cytocidal infection in DEF cells, whereas FCS exerts inhibitory effects on DHAV-1 replication, CPE development, and plaque formation. By using a neutralization test, we found that the direct action of FCS on virions is likely to play a key role in inhibiting DHAV-1 replication in DEF cells. Mechanism analyses revealed that FCS inhibits DHAV-1 replication at virus adsorption and reduces extracellular virus yields. The present work may shed light on a new perspective for antiviral agent development, and have provided a virus–host cell system for further studies on molecular mechanism involved DHAV-1 replication and pathogenesis.

Evaluating a DOE screen to reduce hyperhydricity in the threatened plant, Cycladenia humilis var. jonesii

In vitro methods for propagation and preservation are critical when seed banking is not an option for the ex situ conservation of an endangered species. Such methods rely on the ability to maintain normal growth for the ultimate goal of producing plants. Previous studies revealed that the threatened Cycladenia humilis var. jonesii can exhibit extreme hyperhydricity when cultured in vitro, but that this phenotype can be largely normalized by venting of the cultures. A design of experiments (DOE) approach was used to investigate factors contributing to hyperhydricity in this species. Using a 6-factor fractional factorial design, the effects of nitrogen, CaCl2, BAP, gelling agent, and the presence of venting on the phenotype were tested. Venting was the strongest driver decreasing hyperhydricity, but gelling agent and other factors had significant effects, depending on the response measured. A revised medium was then designed and compared with the original maintenance medium through 2–3 subcultures. Venting was again the strongest driver decreasing hyperhydricity, but the revised medium also significantly affected some responses, even in the absence of venting. The revised medium was not optimal, as shoots became progressively smaller with each transfer, but despite their size, hyperhydricity decreased. These results demonstrate the usefulness of DOE for simultaneous testing of multiple factors, but also the need for follow-up optimization experiments. DOE can be particularly useful in working with rare species. By making efficient use of limited resources, it can facilitate the development of in vitro propagation protocols for such species and help ensure their conservation.

Transmission of Desulfovibrio salexigens DSM2638 Bacteria in Pachyseris involuta-Infection Rate and changes in Coral Morphology at different temperatures

Desulfovibrio salexigens is one of the bacteria that infect corals with black band disease (BBD). These bacteria develop fast and cause damage to corals. Therefore, there is a monitor disease transmissinon, especially BBD, in healthy corals to determine the extent to which pathogens can infect coral tissue and the impact of these bacteria on infected coral populations. This study was conducted at the Marine Station Laboratory on Barrang Lompo Island, South Sulawesi, Indonesia. Bacteria were cultured in the Laboratory of Microbiology, Faculty of Medicine, Hasanuddin University. Corals were acclimatised in controlled containers, followed by transmission tests using aquaria (24 units, 45cmx30 cm x30 cm) filled with 15 litres of sterile seawater. A multifactorial approach was used with treatments comprising different incubation temperature (29⁰C and 31⁰C) and different bacterial concentration in the maintenance media (102, 104, and 106 UNIT), as well as the interaction between the effects of temperature and bacterial concentration. Trial parameters were observed every day at intervals of 5 hours. Changes in the appearance diameter of corals infected with pathogenic bacteria were analysed descriptively using underwater photography (Nikon Collfix P7100 camera) and the corals were measured with callipers (precision 0.1 mm). The study concluded that the infection rate of the bacterium D. salexigens strain DSM 2638 in Pachyseris involuta was higher at 31⁰C-29⁰C. There was no interaction between the effects of temperature and bacterial concentration on the infection rate of infected coral fragments. Bacterial attack was more severe on Pachyseris involuta fragments maintained at 31⁰C than at 29⁰C.

Population Dietary Variation of Drosophila Melanogaster Associated with Different Yeasts

Objective: To analyze the importance of the quality of the growth medium as to the development of Drosophila melanogaster grown in the laboratory, in order to assess changes in physical fitness and in the survival and fertility rates of the studied populations, both of the White and wild strains, according to the yeast. provided as a food resource. Method: To do this, different yeasts were used as food resources (Saccharomyces cerevisiae, Dekkera bruxelensis and Meyerozyma caribbica) in the standard medium and in order to evaluate the variables, the RING test and analysis of survival and fertility rates were performed. Results: Through the data, the growth medium with Saccharomyces cerevisiae was that with all analyzed rates were within the expected value and that the individuals were able to complete their life cycle, while the other resources did not obtain the expected values. Conclusion: The efficiency of Saccharomyces cerevisiae for maintenance medium is confirmed, in addition to this fact, with the intriguing result of the other two yeasts, it is necessary to add different evaluation methods so that the results become increasingly robust.

Development of Selective Enrichment Medium for Clinical Isolates of Vibrio vulnificus Based upon Virulence Correlating Genes

The objective of this study was to find out if different enrichment media would influence the selection of clinical and environmental strains of Vibrio vulnificus. Therefore, traditional enrichment and maintenance medium for V. vulnificus were tested for selective preference for clinical or environmental strains. In the laboratory, Alkaline Peptone Water (APW) and Brain Heart Infusion Broth (BHIB), with adjusted salinity to that of APW, were inoculated with equal concentrations of clinical and environmental strains and incubated for 20 hr at 35 °C. BHIB selected for clinical strains (85%) as indicated by percentage of vcgC to vvhA genes, enumerated with quantitative PCR. In APW, the ratio of clinical (47%) to environmental strains (53%) was roughly equal. Enrichments of seawater with BHIB, APW, and Luria Bertani broth (LB) resulted in higher percentage of clinical strains in BHIB but not in APW or LB, in most samples. However seasonal differences in predominance of clinical or environmental strains in the seawater samples influenced the enrichment process. Our study suggests that BHIB10 might be advantageous for studying virulence factors and APW10 could be suitable for isolation and MPN enumeration in V. vulnificus. The selective preference of the BHIB10 medium for clinical strains indicates that iron plays an important role in the infection of the human host.

Effect of allithiamine on the level of hyperglycaemia-induced advanced glycation end products

Diabetes mellitus is a rapidly growing public health burden in both developed and developing countries. Diabetes mellitus is accompanied by hyperglycaemia, which can cause tissue injury by several mechanisms. One of these is the formation of advanced glycation end-products (AGEs). In this study, the effect of allithaimine, a fat-soluble thiamine derivative, was investigated on hyperglycaemia-induced AGEs levels using human umbilical cord vein endothelial cells (HUVECs) as a hyperglycaemic model. HUVECs were isolated by enzymatic digestion, characterized by flow cytometer and treated 30 mM glucose plus allithaimine or thiamine or cell maintenance medium as control. Allithiamine was synthesized and purified. The structure of the synthesized and isolated compound was verified by reverse phase HPLC and MALDI-TOF. AGEs were evaluated by ELISA. Collectively, our results indicate that allithiamine can reduce level of the hyperglycaemia-induced AGEs similar to thiamine.

A Hydrogel Based 3D Culture System for Hematopoietic Differentiation of Induced Pluripotent Stem Cells

The generation of hematopoietic stem and progenitor cells (HSPCs) from induced pluripotent cells (iPSCs) holds a great potential in development of cell therapies, modeling hematological malignancies, and testing new drugs. Hematopoiesis is regulated by bone marrow (BM) microenvironmental factors such as BM architecture, and cell-cell and cell-matrix interactions. The current methods for iPSC differentiation into HSPCs rely on cell-cell interaction (embryoid body formation and feeder cell co-cultures) or cell-matrix interaction (ECM coated dishes). Generation of a 3-dimensional (3D) culture environment attempts to capture both cell-cell and cell matrix interactions in studying the differentiation of the iPSCs. This study reports development of a 3D culture system for hematopoietic differentiation of iPSCs. iPSC colonies were encapsulated in 3D Matrigel or polyethylene glycol (PEG) based hydrogels containing synthetic integrin binding peptide (GRGDSPC) and enzymatically degradable peptide (GGPQGIWGQGKG) and cultured in feeder-free cell culture and maintenance medium (mTeSR1™, Stem Cell Technology). There were notable morphological differences between the 3D encapsulated and 2D cultured iPSC colonies (Fig 1A). The 3D encapsulated colonies were more compact with a spheroid morphology in PEG compared to Matrigel, whereas colonies in 2D were more diffused. Viability of the encapsulated colonies was evaluated in situ using cytotoxicity kit. The 3D encapsulation did not have an adverse effect on the viability and growth of iPSCs (Fig 1B). The encapsulated iPSCs maintained the pluripotent phenotype in 3D as assessed by TRA-1-60 staining (Fig 1C). To test the efficiency of these 3D model systems to generate HSPCs, the encapsulated iPSCs were subjected to hematopoietic differentiation using STEMdiff™ Hematopoietic Kit (Fig 1D). Following differentiation, the single cells were collected and immunophenotype analysis was performed by flow cytometry. Above 50% population stained positive for HSPC surface marker CD34 both in PEG hydrogel and Matrigel (Fig 1E). To test the robustness of the 3D system in inducing hematopoietic differentiation, we encapsulated the isogenic trisomic 21 (Tri21) iPSCs and subjected to hematopoietic differentiation and flow cytometry analysis as described above. As reported previously in 2D system (Banno et al., 2016, Cell Reports), Tri21 iPSCs showed higher percentage of hematopoietic commitment into erythroid and megakaryocytic lineages compared to disomic 21 (Di21), as characterized by the presence of respective surface markers (Fig 1F). In conclusion, 3D culture system was suitable for inducing hematopoietic differentiation of iPSCs in isogenic disomic and trisomic lines. The 3D model can be used to generate patient-specific HSPCs. Figure 1 Disclosures No relevant conflicts of interest to declare.

Expansion of Transdifferentiated Human Hepatocytes in a Serum-Free Microcarrier Culture System.

Bioartificial livers (BALs) have attracted much attention as potential supportive therapies for liver diseases. A serum-free microcarrier culture strategy for the in vitro high-density expansion of human-induced hepatocyte-like cells (hiHeps) suitable for BALs was studied in this article. hiHeps were transdifferentiated from human fibroblasts by the lentiviral overexpression of FOXA3, HNF1A, and HNF4A. Cells were cultured on microcarriers, their proliferation was evaluated by cell count and CCK-8 assays, and their function was evaluated by detecting liver function parameters in the supernatant, including urea secretion, albumin synthesis, and lactate dehydrogenase levels. The expressions of hepatocyte function-associated genes of hiHeps were measured by qRT-PCR in 2D and 3D conditions. The expression of related proteins during fibronectin promotes cell adhesion, and proliferation on microcarrier was detected by western blotting. During microcarrier culture, the optimal culture conditions during the adherence period were the use of half-volume high-density inoculation, Cytodex 3 at a concentration of 3mg/mL, a cell seeding density of 2.0 × 105 cells/mL, and a stirring speed of 45rpm. The final cell density in self-developed, chemically defined serum-free medium (SFM) reached 2.53 × 106 cells/mL, and the maximum increase in expansion was 12.61-fold. In addition, we found that fibronectin (FN) can promote hiHep attachment and proliferation on Cytodex 3 microcarriers and that this pro-proliferative effect was mediated by the integrin-β1/FAK/ERK/CyclinD1 signaling pathway. Finally, the growth and function of hiHeps on Cytodex 3 in SFM were close to those of hiHeps on Cytodex 3 in hepatocyte maintenance medium (HMM), and cells maintained their morphology and function after harvest on microcarriers. Serum-free microcarrier culture has important implications for the expansion of a sufficient number of hiHeps prior to the clinical application of BALs.

A Reproducible and Simple Method to Generate Red Blood Cells from Human Pluripotent Stem Cells

Erythroid cells generated from human pluripotent stem cells (hPSCs) can potentially offer an unlimited and safe supply of red blood cells (RBCs) for transfusion. Human PSC-derived erythroid cells at various stages of differentiation can also be used to model blood diseases, test new drug candidates, and develop cellular and genetic therapies. Although several protocols for deriving RBCs from hPSCs have been described, these are typically complex, involving multiple culture steps that may include co-culture with feeder cells, and exhibit large variability in erythroid cell yields between hPSC lines and replicate experiments. We have developed a straightforward, serum-free and feeder-free culture method to generate erythroid cells from hPSCs with high yields and high purity. The method has been validated on multiple human embryonic stem (ES) cell lines (H1, H7, H9) and induced pluripotent stem (iPS) cell lines (WLS-1C, STiPS-F016, STiPS-B004). The protocol involves two steps: hematopoietic specification of hPSCs, followed by differentiation of hPSC-derived hematopoietic stem and progenitor cells (HSPCs) into erythroid cells. Lineage specification and differentiation is driven by only three supplements that combine cytokines and other factors to support optimal differentiation efficiency and cell yield across cell lines. In the first step, small hPSC aggregates routinely maintained in feeder-free maintenance medium, are plated onto matrigel-coated microwells, and specification to mesoderm and subsequent hematoendothelial differentiation is induced by addition of successive expansion supplements. This phase promotes extensive hematopoietic progenitor cell generation, with a single hPSC producing on average 142 HSPCs (range: 50 - 360, n = 3 experiments) by day 10 across all six ES and iPS cell lines tested. The average frequency of cells expressing CD43, an embryonic pan-hematopoietic marker, is 92% (range: 85 - 95%), and the frequency of CD34+ cells ranges between 24-55%. In the second, erythroid differentiation step, hPSC-derived HSPCs expand on average 300-fold (range: 80 - 1000) within 10 - 14 days, and the average frequency of GlyA+ cells is 75% (range: 70 - 85%). Cumulatively, this results in the generation of on average 30,000 GlyA+ cells (range: 10,000 - 80,000) per initial hPSC after 20 - 24 days. Further maturation in 7-day cultures containing EPO and human serum resulted in a > 90% pure population of GlyA+ erythroid cells. Notably, no cell loss was observed during the maturation phase, resulting in an average yield of 50,000 GlyA+ cells (range: 10,000 - 220,000) per single initial hPSC on day 31. Differentiated cells were characterized by orthochromatic normoblast morphology and decreased CD71 expression, consistent with erythroid maturation. Erythroid cells generated in this differentiation culture system expressed a mix of 'primitive' and 'definitive' hemoglobin types, but with adult and fetal hemoglobin being expressed at higher levels than embryonic hemoglobin. The observed enucleation rates of hPSC-derived erythroid cells are consistent with current reports and are subject to further optimization. In summary, we have developed a two-step, serum- and feeder-free erythroid differentiation method to generate large numbers of erythroid cells from multiple hPSC lines. This culture system provides a simple, standardized and reproducible platform to generate RBCs from hPSCs with high yields and efficiency for basic and translational research. Disclosures Walasek: STEMCELL Technologies, Inc: Employment. Chau:STEMCELL Technologies, Inc: Employment. Barborini:STEMCELL Technologies, Inc: Employment. Richardson:STEMCELL Technologies, Inc: Employment. Szilvassy:STEMCELL Technologies, Inc: Employment. Louis:STEMCELL Technologies, Inc: Employment. Thomas:STEMCELL Technologies, Inc: Employment. Eaves:STEMCELL Technologies, Inc: Employment. Wognum:STEMCELL Technologies, Inc: Employment.

Effects of probiotics (rabal) with different doses on the survival, feed conversion, and growth of giant prawns (Macrobrachium rosenbergii)

This study aims to determine the effect of the best RABAL probiotic dose on the media for the maintenance of giant shrimp larvae (Macrobrachium rosenbergii) on the survival, feed conversion, and growth. This research was conducted from March to April 2019 at Brackish Water Aquaculture (Balai Perikanan Budidaya Air Payau, BPBAP) Ujung Batee, Aceh Besar district, Province of Aceh. This study used a Completely Randomized Design (CRD) with 4 levels of treatments and 4 repetitions. The treatments used of RABAL with a dose (A) 0 ppm, (B) 75 ppm, (C) 150 ppm, and (D) 225 ppm. The frequency of administration of RABAL was once a day. The giant prawn larvae used was a larvae stage with an average weight of 0.05 g/larvae. Based on the result of ANOVA test the best value was obtained a treatment D (225 ppm) with absolute weight growth value of 0.16 g ± 0.003, absolute growth length of 1.30 cm ± 0.053, specific growth rate 5.08 ± 0.043, survival rate 96.25 ± 4.787 and feed conversion ratio 0.83 ± 0.013, respectively. The result of this research showed that the administration of RABAL with different doses into maintenance media had a significant effect (P<0.05) on the growth and feed conversion ratio of giant shrimp larvae but did not significant influence (P>0.05) on the survival rate of giant shrimp larvae.

Gene expression profiles of differentiated and undifferentiated adipose derived mesenchymal stem cells dynamically seeded onto a processed nerve allograft

BackgroundDifferentiation of mesenchymal stem cells (MSCs) into Schwann-like cells onto processed nerve allografts may support peripheral nerve repair. The purpose of this study was to understand the biological characteristics of undifferentiated and differentiated MSCs before and after seeding onto a processed nerve allograft by comparing gene expression profiles. MethodsMSCs from Lewis rats were cultured in maintenance media or differentiated into Schwann-like cells. Both treatment groups were dynamically seeded onto decellularized nerve allografts derived from Sprague-Dawley rats. Gene expression was quantified by quantitative polymerase chain reaction (qPCR) analysis of representative biomarkers, including neurotrophic (GDNF, PTN, GAP43, PMP22), angiogenic (CD31, VEGF1), extracellular matrix (ECM) (COL1A1, COL3A1, FBLN1, LAMB2) or cell cycle (CAPS3, CCBN2) genes. Gene expression values were statistically evaluated using a 2-factor ANOVA with repeated measures. ResultsBaseline gene expression of undifferentiated and differentiated MSCs was significantly altered upon interaction with processed nerve allografts. Interaction between processed allografts and undifferentiated MSCs enhanced expression of neurotrophic (NGF, GDNF, PMP22), ECM (FBLN1, LAMB2) and regulatory cell cycle genes (CCNB2) during a 7-day time course. Interactions of differentiated MSCs with nerve allografts enhanced expression of neurotrophic (NGF, GDNF, GAP43), angiogenic (VEGF1), ECM (FBLN1) and regulatory cell cycle genes (CASP3, CCNB2) within one week. ConclusionsDynamic seeding onto processed nerve allografts modulates temporal gene expression profiles of differentiated and undifferentiated MSCs. These changes in gene expressions may support the reparative functions of MSCs in supporting nerve regeneration in different stages of axonal growth.

Generation of liver organoids by 3D co-culturing of hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells

Background and Hypothesis: Recent progress with combination of chemicals in cell culture media prolongs hepatocyte (HC) function in vitro. However, without other cells of hepatic lineage that comprise natural liver, HC alone are not suitable for the study of complex liver diseases. Hepatic stellate cells (HSC) and liver sinusoidal endothelial cells (LSEC) play vital roles in physiological and pathological liver function in situ. We hypothesized that coculturing primary HC with HSC and LSEC as 3D liver organoids in the same chemical conditioned media would uphold HC function over time, creating a better physiological 3D liver microenvironment for liver disease research.&#x0D; Experimental Design: Freshly thawed primary human HCs were combined with immortalized human HSC alone, or together with immortalized human LSECs, to generate 3D liver spheroids. Spheroids were formed in HC maintenance media (HMM, Lonza) using low adhesion 96-well plates for 6 days before switching to the media with different combination of chemicals (SB31542, Forskolin, IWP2, DAPT, and LDN193189) for culturing another 14 days. Spheroids characterization, albumin secretion, mRNA transcription (CYP3A4), and histological analysis were performed for HC differentiation, maturation, and function. &#x0D; Results: Both co-cultures of HC:HSC (2.5:1 ratio), and HC:HSC:LSEC (2.5:1:1) formed spheroids in HMM within 4 to 6 days (Fig.1A). The introduction of the different chemical-based media affected the roundness and diameter of spheroids differently (Fig.1B). In the presence of all 5 chemicals (5C), HC function was better maintained up to 21 days in HC:HSC:LSEC spheroids, measured by albumin, CYP3A4, and CK-19 secretion (Fig.1C). Surprisingly, 5C-based media significantly upregulated the expression of CK-19, which is one of the markers for cholangiocytes and liver precursor cells. &#x0D; Conclusion and Potential Impact: 3D liver organoids composed of HC, HSC, and LSEC would create a niche environment mimicking the in vivo condition. Optimization of complex hepatic spheroids, as well as optimization of complex hepatic spheroid culturing media, would allow for the generation of a unique model for studying complex liver diseases.

Water Quality in the Cultivation of Catfish (Clarias gariepinus) and Nile Tilapia (Oreochromis niloticus) in the Aquaponic Biofloc System

Aims: Water quality is one of them being an important role in terms of cultivation. Good water quality can support growth productivity in aquaculture. This is also of great concern when the rest of fish metabolism results that can contaminate fish contained in the maintenance media and if water is changed every day, water carrying toxins can pollute the area around the cultivation. One of the first steps to reduce the toxic content found in the maintenance media and the area around cultivation is to use aquaponic cultivation using biofloc. It is also expected to minimize the toxin content in maintenance media. This research aims to determine the comparison of the polyculture stocking density of sangkuriang catfish and nile tilapia which results in water quality that supports fish productivity.&#x0D; Study Design: The research was conducted experimentally.&#x0D; Place and Duration of Study: This research was carried out for 40 days between March 2019 - April 2019 in the Ciparanje Laboratory of the Faculty of Fisheries and Marine Sciences, Universitas Padjadjaran.&#x0D; Methodology: The method used in this research is experimental using a completely randomized design (CRD), consisting of 5 treatments with 3 replications. The treatments to be given are dense stocking variations with the following treatments: A(75 catfish:75 tilapia), B(100 catfish:50 tilapia), C(125 catfish:25 tilapia), D(150 catfish), E(150 tilapia).&#x0D; Results: Water Quality Measured During The Study Namely, DO, Temperature, Ph, Nitrite, Nitrate And Ammonia Are Still In The Threshold For The Growth Of Test Animals During Maintenance.&#x0D; Conclusion: Based on the results of the research that has been done, it can be concluded that the quality of water in maintenance media using aquaponics biofloc gives good water quality values on maintenance media and is still within the threshold for the growth of catfish and tilapia.

BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single-cell analysis

We describe a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts or genomic regions from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, which are all pre-selected and optimized in silico. By applying the matrices in a novel workflow named Barcode Assembly foR Targeted Sequencing (BART-Seq), we analyze developmental states of thousands of single human pluripotent stem cells, either in different maintenance media or upon Wnt/β-catenin pathway activation, which identifies the mechanisms of differentiation induction. Moreover, we apply BART-Seq to the genetic screening of breast cancer patients and identify BRCA mutations with very high precision. The processing of thousands of samples and dynamic range measurements that outperform global transcriptomics techniques makes BART-Seq first targeted sequencing technique suitable for numerous research applications.

Electrophysiological characterization of drug response in hSC-derived cardiomyocytes using voltage-sensitive optical platforms

IntroductionVoltage-sensitive optical (VSO) sensors offer a minimally invasive method to study the time course of repolarization of the cardiac action potential (AP). This Comprehensive in vitro Proarrhythmia Assay (CiPA) cross-platform study investigates protocol design and measurement variability of VSO sensors for preclinical cardiac electrophysiology assays. MethodsThree commercial and one academic laboratory completed a limited study of the effects of 8 blinded compounds on the electrophysiology of 2 commercial lines of human induced pluripotent stem-cell derived cardiomyocytes (hSC-CMs). Acquisition technologies included CMOS camera and photometry; fluorescent voltage sensors included di-4-ANEPPS, FluoVolt and genetically encoded QuasAr2. The experimental protocol was standardized with respect to cell lines, plating and maintenance media, blinded compounds, and action potential parameters measured. Serum-free media was used to study the action of drugs, but the exact composition and the protocols for cell preparation and drug additions varied among sites. ResultsBaseline AP waveforms differed across platforms and between cell types. Despite these differences, the relative responses to four selective ion channel blockers (E-4031, nifedipine, mexiletine, and JNJ 303 blocking IKr, ICaL, INa, and IKs, respectively) were similar across all platforms and cell lines although the absolute changes differed. Similarly, four mixed ion channel blockers (flecainide, moxifloxacin, quinidine, and ranolazine) had comparable effects in all platforms. Differences in repolarisation time course and response to drugs could be attributed to cell type and experimental method differences such as composition of the assay media, stimulated versus spontaneous activity, and single versus cumulative compound addition. DiscussionIn conclusion, VSOs represent a powerful and appropriate method to assess the electrophysiological effects of drugs on iPSC-CMs for the evaluation of proarrhythmic risk. Protocol considerations and recommendations are provided toward standardizing conditions to reduce variability of baseline AP waveform characteristics and drug responses.

Inhibition of the mTOR pathway: A new mechanism of β cell toxicity induced by tacrolimus.

The mechanisms of tacrolimus-induced β cell toxicity are unknown. Tacrolimus (TAC) and rapamycin (Rapa) both bind to FK506-binding protein 12 (FKBP12). Also, both molecular structures are similar. Because of this similarity, we hypothesized that TAC can also inhibit the mTOR signalling, constituting a possible mechanism of β cell toxicity. Thus, we studied the effect of TAC and Rapa over the mTOR pathway, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), and insulin secretion and content in INS-1 β cells treated with or without glucose and palmitate and in islets from lean or obese rats. TAC and Rapa inhibited the mTOR pathway as reflected by lower levels of phospho-mTOR, phospo-p70S6K, and phospo-S6. The effect of Rapa was larger than TAC. Both drugs reduced the levels of MafA, insulin secretion, and content although these effects were larger with TAC. The changes on MafA and insulin metabolism were observed in cells on glucose and palmitate, in obese animals, and were absent in cells on maintenance medium or in lean animals. In silico docking and immunoprecipitation experiments confirmed that TAC can form a stable noncovalent interaction with FKBP12-mTOR. Thus, the mTOR inhibition may be a mechanism contributing to the diabetogenic effect of TAC.