Culture Vessel Research Articles

Hatchery practices for Patella aspera and P. ordinaria: evaluating culture vessels requirements

Innovation in limpet production requires knowledge of the optimal qualities and limitations of the culture vessels. Two limpet species from Madeira fishery, Patella aspera and P. ordinaria, were used to evaluate the influence of culture vessel shape (microtubes, test tubes, centrifuge tubes, specimen cups, disposable cups, vials, flasks and beakers), composition (polypropylene and glass), volume (1.5 ml to 1.5 L) and stocking density on oocyte incubation and larval culture through nine assays. Moreover, three assays evaluated the use of microtubes and the isolation of trochophores, instead of pediveligers, for eventual settlement induction. The results showed effective hatching in most culture vessels regardless of composition or volume, except when the shape of the bottom caused oocyte clustering. Larval culture was not influenced by any variable under scrutiny (shape, composition, volume or rearing density). Microtubes were useful for settlement, while the placement of trochophores in the settling vessels, instead of pediveligers, proved inefficient and even led to failed cultures. The best settlement was obtained when the pediveligers were isolated from the water column of the beakers and transferred to clean vessels with renewed water and coralline algae as substrate. The current findings could be extrapolated to other marine invertebrates that share similar life stage requirements.

In vitro proliferation of Mytilus edulis male germ cell progenitors.

Our understanding of basic cellular processes has mostly been provided by mammalian cell culture, and by some non-mammalian vertebrate and few invertebrate cell culture models. Developing reliable culture conditions for non-model organisms is essential to allow investigation of more unusual cellular processes. Here, we investigate how cells isolated from different tissues of the marine mussel Mytilus edulis thrive and survive in vitro in the hope of establishing a suitable laboratory model for the investigation of cellular mechanisms specific to these bivalve mollusks. We found that cells dissociated from mantle tissue attached to the culture vessels and proliferated well in vitro, whereas cells isolated from gills, although remaining viable, did not maintain divisions over three to four weeks in culture. We used antibodies against the germ-line marker DEAD-box helicase 4 (DDX4), also known as VASA, and the epithelial cell marker cytokeratin to distinguish different cell types in culture. DDX4-positive cells were predominant in 25-day-old cultures from male mantles. Cells from other tissues remained in low numbers and did not seem to change in composition over time. Overall, the culture conditions described here allow an efficient selection of male germ cells that could be used to study specific cellular mechanisms in vitro.

Stable and efficient generation of functional iPSC-derived neural progenitor cell rosettes through regulation of collective cell-cell behavior.

Although the potential of stem cells to differentiate into several cell types has shown promise in regenerative medicine, low differentiation efficiency and poor reproducibility significantly limit their practical application. We developed an effective and robust differentiation strategy for the efficient and robust generation of neural progenitor cell rosettes from induced pluripotent stem cells (iPSCs) incorporating botulinum hemagglutinin (HA). Treatment with HA suppressed the spontaneous differentiation of iPSCs cultured under undirected differentiation conditions, resulting in the preservation of their pluripotency. Moreover, treatment with HA during neural progenitor differentiation combined with dual SMAD inhibition generated a highly homogeneous population of PAX6-and SOX1-expressing neural progenitor cells with 8.4-fold higher yields of neural progenitor cells than untreated control cultures. These neural progenitor cells formed radially organized rosettes surrounding the central lumen. This differentiation method enhanced the generation of functional iPSC-derived neural progenitor cell rosettes throughout the culture vessel, suggesting that the regulation of collective cell-cell behavior using HA plays a morphogenetically important role in rosette formation and maturation. These findings show the significance of HA in the suppression of spontaneous differentiation through spatial homogeneity. The study proposes a novel methodology for the efficient derivation of functional iPSC-derived neural progenitor cell rosettes.

Human mesenchymal stromal cell adhesion and expansion on fluoropolymer surfaces modified with oxygen and nitrogen-rich plasma polymers

Fluorinated ethylene propylene (FEP) vessels are of significant interest for therapeutic cell biomanufacturing applications due to their chemical inertness, hydrophobic surface, and high oxygen permeability. However, these properties also limit the adhesion and survival of anchorage-dependent cells. Here, we develop novel plasma polymer coatings to modify FEP surfaces, enhancing the adhesion and expansion of human mesenchymal stromal cells (hMSCs). Similar to commercially available tissue culture polystyrene vessels, oxygen-rich or nitrogen-rich surface chemistries can be achieved using this approach. While steam sterilization increased the roughness of the coatings and altered the surface chemistry, the overall wettability and oxygen or nitrogen-rich nature of the coatings were maintained. In the absence of proteins during initial cell attachment, cells adhered to surfaces even in the presence of chelators, whereas adhesion was abrogated with chelator in a protein-containing medium, suggesting that integrin-mediated adhesion predominates over physicochemical tethering in normal protein-containing cell seeding conditions. Albumin adsorption was more elevated on nitrogen-rich coatings compared to the oxygen-rich coatings, which was correlated with a higher extent of hMSC expansion after 3 days. Both the oxygen and nitrogen-rich coatings significantly improved hMSC adhesion and expansion compared to untreated FEP. FEP surfaces with nitrogen-rich coatings were practically equivalent to commercially available standard tissue culture-treated polystyrene surfaces in terms of hMSC yields. Plasma polymer coatings show significant promise in expanding the potential usage of FEP-based culture vessels for cell therapy applications.

Sistema de vedação em frascos de cultura afeta o desenvolvimento in vitro de mãe-de-mil (Kalanchoe delagoensis Ecklon e Zeyher)

Abstract Sealing systems that provide greater permeability to gasses can favor plant development in vitro. This study aimed to evaluate the effectiveness of a low-cost sealing system that allows different CO2-exchange rates [CO2-ER] to promote the in vitro growth of mother-of-millions (Kalanchoe delagoensis). Nodal segments of K. delagoensis were surface sterilized with sodium hypochlorite (2.5% active chlorine) and inoculated in culture vessels with Murashige and Skoog (MS) medium, sealed with rigid polypropylene lids with one (code: 1M; CO2-ER 21 μL L−1s−1) or two (code: 2M; CO2-ER 25 μL L−1s−1) gas-permeable membranes. In the control, the culture vessels were sealed with rigid polypropylene lids without membrane (code: 0M; CO2-ER 14 μL L−1s−1). After 45 days of culture, growth parameters were measured. Plants kept in culture vessels with 0M and 1M lids had a significant increase in the total plant length, main root, aerial part, and number of nodes when compared to the 2M sealing system. The number of leaves and plantlets along the leaf margin was 35.52% and 43.69% higher in plants grown in culture vessels with a 1M sealing system. In conclusion, sealing systems that allow gas exchange rates of up to 21 μL L−1s−1 (0M and 1M) provided the greatest in vitro development of mother-of-millions.

ABOUT MINIATURE VESSELS OF KOBAN CULTURE

ABOUT MINIATURE VESSELS OF KOBAN CULTURE

PEI-Mediated Transient Gene Expression in CHO Cells.

We describe a method for polyethyleneimine (PEI)-mediated transient transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells for protein expression applicable at scales from 2mL to 2L. The method involves transfection at a high cell density (5×106 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31°C with agitation by orbital shaking. This method requires 0.3mg/L of coding pDNA, 2.7mg/L of nonspecific (filler) DNA, and 15mg/L of PEI. The production phase is performed at 31°C in the presence of 0.25% N,N-dimethylacetamide (DMA). If desired, the method can be modified to avoid use of DMA by increasing the amount of coding DNA. We also provide information on culture vessel options, recommended working volumes, and recommended shaking speeds for transfections at scales from 2mL to 2L.

Measuring Pericellular Oxygen Tension for In Vitro Cell Culture.

Cell culture, the process of growing cells in conditions that mimic those in the body, is a key technique in biomedical research. Oxygen is not controlled in conventional cell culture, although chambers that control oxygen in the surrounding gas phase are commercially available. In both cases, it is valuable to understand the pericellular oxygen tension (i.e., the oxygen concentration that cells experience) in cultures. Herein we describe a procedure for using commercial optical sensor spots to measure pericellular oxygen for adherent and suspension cultures. Spots are placed on surfaces on which cells are grown, and optical cables are attached to the outside of the cell culture vessels and connected to a computer. Associated software allows for the real-time monitoring of pericellular oxygen during cell culture experiments. This procedure enhances the reproducibility and control of cell culture.

The first arctic strain of Rhizochromulina: Morphology, ultrastructure, and position in the evolutionary tree of the order Rhizochromulinales (Heterokontophyta, Dictyochophyceae)

Rhizochromulina marina is a unicellular amoeboid alga capable of forming flagellate cells; it is a single validly named species in the genus. Besides, there are numerous environmental sequences and undescribed strains designated as Rhizochromulina sp. or R. marina. The biogeography of the genus is understudied: rhizochromulines from the Indian, Southern, and Arctic Oceans are unknown. Here, we present the description of Rhizochromulina sp. B44, which was for the first time isolated from an arctic habitat. Biofilms of this microalga grow at the bottom of a culture vessel, where neighbouring amoeboid cells form associations through a common network of pseudopodia, i.e. meroplasmodia. Pseudopodia branch, anastomose mainly during meroplasmodia formation, and are supported by microtubules that arise from the perinuclear zone. Actin filaments are localized in the cytoplasm and can be revealed only near the bases of pseudopodia. We succeeded in inducing the transformation of amoeboid cells into flagellates using a prolonged agitation of cultures. Morphological and molecular analyses revealed that the studied strain is most closely related to the type strain of R. marina. At the same time, 18S rDNA sequences of early branching-off rhizochromulinids differ significantly from Rhizochromulina sp. B44, suggesting a high divergence at the genus level.

Traditions of creating the form and ornamentation of the pottery vessels of the Alakul Culture in the Alakul burial ground (a case study for the burial mounds 1 and 14)

This paper reports on the results of the analysis of the form and ornamentation of the pottery vessels of the Late Bronze Age Alakul Culture from the burial mounds 1 and 14 of the Alakul burial ground (the Trans-Urals). The study was carried out within the framework of the historical-cultural approach developed by A.A. Bobrinsky. The work is aimed at the identification of pottery-making traditions of creating the form and ornamentation of pottery vessels amongst the population who built the burial mounds 1 and 14. In the result of the analysis, there has been identified the cultural core of the traditions of the form-making of the pottery vessels in the population who built the burial mounds 1 and 14. The vessels are primarily of medium proportions and quinquepartite structure — ‘lip + neck + brachium + body + base’. Also, the dominant parameters of the functional parts ap-peared to be similar amongst the vessels from the different burial mounds. These data indicate relatively high uniformity of the composition of the consumers of the ware. The study of the ornamentation revealed that smooth stamp, which was used in the techniques of stamping and drawing, served as a traditional tool for the application of ornament. Toothed stamp was less fre-quently used in the techniques of stamping and drawing. The main ornamental elements were a ‘short straight line’ and a ‘long straight line’. Twenty two ornamental patterns have been identified; the widespread ones were the ‘horizontal zigzag, ‘horizontal straight line’, ‘cross-hatched isosceles triangle with point upwards’, and ‘cross-hatched isosceles triangle with point downwards’. Most of the identified leading and infrequent ornamental patterns functioned as a main motif. In the complementary motif, the widespread one was the pattern ‘horizontal straight line’. In the composition of the ornament, traditional was the placement of the ornamental motifs in the upper part of the neck and on the upper and lower parts of the body, with the allocation of a “free band” in the lower part of the neck and brachium. In the result of the correlation of the obtained data on the pottery vessels from the burial mounds 1 and 14, certain dissimilarities have been noted. In the burial mound 14, a large quantity of the vessels have a relatively higher neck; the vessels with the lowest index of the proportionality and the slope angle of the body are also present; in the ornamentation, toothed stamp was employed more frequently in the technique of drawing, which was used to apply only ‘horizontal zigzag’ and ‘horizontal straight lines’ patterns. The correlation of the data on the form and ornamentation of the ves-sels for the burial mounds 1 and 14 revealed that toothed stamp in the technique of drawing was used to apply ornaments mainly to the vessels with lower indices of proportionality and angel of the body. A hypothesis has been put forward on the in-creasing complexity of the composition of the studied group of the Alakul Culture population during the period of construction of the burial mound 14, which may explain the noted nonuniformity of the pottery-making skills.

Potchevashsky ceramic complex from the Dolgovskoye 1 settlement (based on the materials of the 1997 excavations)

The object of the study was a representative collection of ceramic vessels (324 specimens) of the Potchevash culture of the VI–IX centuries, obtained during archaeological research in 1997 at the Dolgovskoye 1 settlement in the Tobolsk Irtysh region. The purpose of the study: to determine the main morphological and decorative features of the ceramic complex of the Potchevash culture based on the materials of the collection of the Dolgovskoye 1 settlement, to determine the place of the ceramic complex of the settlement in the system of archaeological cultures of Western Siberia. Based on the purpose of the study, the following tasks were put forward: characterization of the main morphological, decorative and stylistic characteristics of the ceramic collection; comparison with the materials of synchronous monuments of adjacent territories, determination of the degree of their similarities and differences. The relevance of the work is determined by the small number of publications in which statistical calculations on Potchevash ceramics are given. To solve the tasks set in the work, traditional research methods are used for historical science: comparative-historical, typological, comparative-typological, formal-stylistic, descriptive method of analyzing material sources, the method of analogies. As a result of the study, it was found that the dominant features of the studied complex include the numerical predominance of pot-shaped vessels with a high neck and a sharp transition to a slightly expanded body; the ornamentation is characterized by a combination of dimpled pearl decor, combed stamp and horizontal drawn grooves, as well as a small proportion of curly stamps. A comparative analysis showed the proximity of the ceramics of the Dolgovskoye 1 settlement with the collections of a number of Potchevash monuments of the South Taiga Irtysh region and the Barabinsk forest-steppe, as well as its significant difference from both the Potchevash ceramic complexes from the Priishimye region and ceramics from the monuments of the Zelenogorsky stage from the Surgut Ob region. A certain similarity with the ceramics of the Kuchiminsky stage, which is expressed in the rare use of figured stamps, a large proportion of grooves, fixed on ceramics from the 1997 excavation from the Dolgovskoye 1 settlement, allows us to suggest its synchronicity with the Kuchiminskaya and date it within the VIII–IX centuries AD.

Lipid Nanoparticle-Mediated Gene Editing of Human Primary T Cells and Off-Target Analysis of the CRISPR-Cas9 Indels

Background and Aims: The CRISPR/Cas9 system has emerged as a powerful tool for gene editing of primary cells. In our previous work, we demonstrated a novel lipid nanoparticle (LNP) reagent for the multi-step engineering of gene-edited CAR T cells. We showed high cell viabilities and potent CAR-T mediated killing, even after multiple genetic manipulations. Here, we extend this work by assessing potential off-target editing effects in both LNP-treated T cells and T-cells where the CRISPR reagents were delivered by electroporation. Further, we evaluated multiple Cas9 variants and guide RNA targets. Methods: LNPs encapsulating wild type or high fidelity S.p. Cas9 mRNA and various TRAC and CD52 targeted guide RNAs (sgRNAs) were produced using our scalable NanoAssemblrTM microfluidics platform. Concurrently, electroporation was performed to deliver equivalent cargoes. Purified primary T cells were cultured, activated, and expanded in serum-free media in plates, flasks, or small bioreactors. The LNPs were added to cells by direct addition for gene editing. Gene expression and cell viability were measured using flow cytometry or colorimetric assays. Multi-target performance of CRISPR-Cas9 editing was evaluated through rhAmpSeq-based targeted next-generation sequencing (NGS) and indels analyzed for with CRISPRAltRations. Results: TCR or CD52 targeted Cas9 mRNA-LNP addition or electroporation yielded high single and double knockout efficiencies. High-throughput NGS analysis showed strong agreeance to flow cytometry for on-target analysis. We tested a range of sgRNA targets, wild-type and high-fidelity Cas9 mRNAs, and determined off-target editing for all targets and variants investigated. Similar results were obtained when comparing different LNP batch sizes (micro to milligram RNA) and cell culture vessels (0.1 to 45 million cells), demonstrating scalability of both LNP production and cell treatment. Conclusions: The results from this study further support the utility RNA-LNPs for the genetic engineering of primary T cells. The simple and gentle nature of LNP cell treatment allows for multiple genetic engineering steps for simultaneous expression and deletion of proteins for the next cell therapies. These LNPs can be easily manufactured from small-scale screening of RNA libraries to rapid scale-up for clinical translation.

Three-dimensional glass scaffolds improve the In Vitro maturation of porcine cumulus-oocyte complexes and subsequent embryonic development after parthenogenetic activation

In vitro maturation (IVM) methods for porcine oocytes are still deficient in achieving full developmental capacity, as the currently available oocyte in vitro culture systems still have limitations. In vitro embryo production must also improve the porcine oocyte IVM system to acquire oocytes with good developmental potential. Herein, we tested a three-dimensional (3D) glass scaffold culture system for porcine oocyte maturation. After 42 h, we matured porcine cumulus-oocyte complexes (COCs) on either two-dimensional glass dishes (2D-B), two-dimensional microdrops (2D-W), or 3D glass scaffolds. The 3D glass scaffolds were tested for porcine oocyte maturation and embryonic development. Among these culture methods, the extended morphology of the 3D group maintained a 3D structure better than the 2D-B and 2D-W groups, which had flat COCs that grew close to the bottom of the culture vessel. The COCs of the 3D group had a higher cumulus expansion index and higher first polar body extrusion rate, cleavage rate, and blastocyst rate of parthenogenetic embryos than the 2D-B group. In the 3D group, the cumulus-expansion-related gene HAS2 and anti-apoptotic gene Bcl-2 were significantly upregulated (p < 0.05), while the pro-apoptotic gene Caspase3 was significantly downregulated (p < 0.05). The blastocysts of the 3D group had a higher relative expression of Bcl-2, Oct4, and Nanog than the other two groups (p < 0.05). The 3D group also had a more uniform distribution of mitochondrial membrane potential and mitochondria (p < 0.05), and its cytoplasmic active oxygen species content was much lower than that in the 2D-B group (p < 0.05). These results show that 3D glass scaffolds dramatically increased porcine oocyte maturation and embryonic development after parthenogenetic activation, providing a suitable culture model for porcine oocytes.

426 Forages Containing Plant Secondary Compounds Can Improve Digestibility, Rumen Ammonia and Rumen Metabolites of Pasture: an in Vitro Continuous Culture Case Study

Abstract Forages containing plant secondary metabolites (PSM) have been investigated for use in reducing nitrogen waste in feedlot and dairy cattle diets but use as supplements to grazing beef cattle to capture the positive effects of PSM is unclear. Here we investigate potential improvement in rumen digestibility and metabolome, rumen ammonia production and flow from feed protein due to PSM bioactivity in forages used to supplement pasture grass using in vitro methods. Can PSM-containing forages offer a practical way to improve range nutrition and provide delivery of phytochemical benefits? Phytochemical- containing forages birdsfoot trefoil (BFT), sainfoin (SAN), small burnet (SMB) and cicer milkvetch (CMV) supplemented grass pasture diets (200 g/kg of DM). In vitro true dry matter digestibility was determined using 2-stage in vitro methods. Randomly assigned diets of PAS supplemented with one of the PSM-containing forage diets (PAS+X) or not supplemented (PAS) were randomly applied to in vitro dual flow continuous culture (CC) vessels inoculated with grazing beef cow rumen fluid as a metabolic host. For 2-stage digestibility, SMB digestibility exceeded that of CMV, followed by BFT and SAN. The grass diet (PAS) was the least digestible (Table 1). Saponin was detected in CMV samples, which is a new finding. For CC, compared with PAS, minimum culture pH for PAS+CMV was highest (P &amp;lt; 0.02) but no differences were observed in pH between PAS, and PAS+BFT and PAS+SAN. Compared with PAS, the molar proportion of the fatty acid propionate increased with PAS+CMV(P = 0.03) in contrast to the acetate- to- propionate ratio found highest for PAS and decreased for all diets with supplementation of PSM forages. Concentration of rumen NH3–N flow to rumen fluid per gram of protein intake was lower (P &amp;lt; 0.01) with PAS+CMV and PAS+SMB, compared with grass only. Feeding CMV and SMB to grazing cattle may improve nutrient utilization, metabolome, and environmental sustainability of beef cattle production by improving diet digestibility and reducing ammonia waste.

Clonal micropropagation of some species of the genus Thymus L.

The aim of the work was to study the influence of cultivation factors on the explants development to optimize the protocol of in vitro clonal micropropagation of four species of the genus Thymus L., valuable essential oil and medicinal plants. Conditions that ensure the production of up to 94.2 % of sterile explants were selected. It has been established that, when introduced into in vitro culture, it is advisable to use shoot tips and stem segments with a node (isolated from donor plants) and cultivate them in foil-covered test tubes. The optimal culture media for the first stage of micropropagation were determined: Murashige and Skoog (MS) medium with 1.0 mg/l benzylaminopurine (BAP) for Thymus serpyllum L. or MS with 1.0 mg/l kinetin and 1.0 mg/l gibberellic acids (GA3) for T. vulgaris L. ‘Gornyy Balzam’, T. × citriodorus (Pers.) Schreb. ‘Doone Valley’, T. caucasicus Willd. ex Ronniger. At the second stage of clonal micropropagation, it is advisable to cultivate T. caucasicus and T. serpyllum explants during 40 days, but T. vulgaris and T. × citriodorus – during 60 days. The maximum multiplication index at the recommended cultivation duration was obtained for T. × citriodorus (9.9) on MS medium without hormones, T. caucasicus (16.1) and T. vulgaris (10.0) – on MS medium with 1.0 mg/l kinetin., and T. serpyllum (6.8) on MS medium with 1.0 mg/l BAP. When comparing different culture vessels (tubes, jars and flasks), the effectiveness of jars was revealed. This type of culture vessels made it possible to increase the multiplication index up to 2.1–5.7 times. It was revealed that when microshoots of four thyme species were cultivated on MS medium with 1.0 mg/l IBA, the rooting rate reached 84.6–98.9 %. The frequency of adaptation ex vitro when using a mixture of peat and perlite (1:1) ranged from 62.8 to 89.5 %.

The effect of light intensity, spectrum, and photoperiod on the physiological performance of Asparagopsis taxiformis tetrasporophytes

Over the last several years there has been increased interest in large scale cultivation of the red seaweed Asparagopsis taxiformis due to its ability to reduce methane emissions in ruminant animals. However, research focused on developing strategies to optimize culture conditions for this alga is still relatively scarce. Both light quality and quantity are widely known to differentially influence any alga's photosynthetic rates, metabolic budgets and ultimately biomass and bio-active compound production. Optimization of cultivation conditions for algae requires the understanding and fine-tuning of light parameters including Photon Flux Density (PFD), light spectrum, and photoperiod, as well as culture density which largely governs light quantity (PFD) and quality (spectrum) within the culture. Here we explore the effects of light intensity, spectral light distribution, and day length on the physiological performance of the tetrasporophyte stage of A. taxiformis in culture using a series of experiments. The results were used to characterize the optimal light and biomass density range for cultivation, and a model was developed to optimize the light path in a cultivation vessel. Spectral light optimization revealed a significant reduction in cyanobacterial contamination for cultures illuminated with blue LED light. Artificially extending light duration longer than 12 h per day did not improve algal growth. The insights gained from these experimental results may be used to inform the design of tank-based A. taxiformis cultivation systems to support high yield and stable production of this emerging commercial crop.

Experimental Work on the Production of Ceramics of the Chirik-Rabat Culture

The article introduces the results, descriptions and analysis of the experiment on the reconstruction of the molding mass and the manufacture of ceramic vessels of the Chirik-Rabat culture (3rd–2nd centuries BC). The site adjacent to the pottery workshops of the Babish-mola 7 archaeological complex (Eastern Aral Sea region) was used as a landfill. There is the Akkyr hill nearby, abounding in reserves of clay raw materials. The experiment was conducted during the field research of the Chirik-Rabat archaeological expedition of the Margulan Institute of Archaeology in 2022. The main objective of this experiment was to determine all aspects of the production cycle for the manufacture of ceramic vessels – from collecting raw materials for the manufacture of ceramics, designing vessels, firing and ending with obtaining the result. During the experimental work, a search was carried out for clay deposits near the Babish-mola oasis along the riverbeds. The process of collecting organic matter (cattle manure) was carried out in the nearest settlement, located 60 km from the Babish-mola monument, for the admixture of chamotte, featureless fragments of ceramics from the monument were used. The vessels were formed on a single-disc turntable.

ВЛИЯНИЕ РАЗЛИЧНЫХ КОНЦЕНТРАЦИЙ ГЛЮКОЗЫ В ПИТАТЕЛЬНОЙ СРЕДЕ НА МОРФОГЕНЕЗ РАСТЕНИЙ ВИНОГРАДА IN VITRO

To study the organic nutrition of grape plants in in vitro culture, we conducted research on the effect of glucose introduced into the nutrient medium on the morphogenesis of test-tube grape plants. The experiments were carried out on two model varieties (Sibirkovyi and Krasnostop Karpi) selected by us from the laboratory collection of in vitro healthy grape plants. These varieties, in addition to differences in genotypes, most noticeably differ in the features of development in in vitro culture, which we noted earlier when they were cultivated and deposited in the collection. The Sibirkovyi variety is one of the fastest growing, and Krasnostop Karpi is one of the slowest developing varieties in in vitro culture. In the experiment, glucose concentrations from 0.0 (sucrose control 10.0 g/l) to 40.0 g/l were used. The varietal response of the model varieties in the experiment with different concentrations of glucose in the nutrient medium was different. The variety Sibirkovyi was already one and a half times ahead of the variety Krasnostop Karpi in development in the first terms of accounting. At the same time, for the Sibirkovyi variety, the optimal range of concentrations for the development and preservation of microplants according to most indicators was the range from 5 to 20 g/l, and for the Krasnostop Karpi variety, the optimal range was narrower – 0-20 g/l. At high glucose concentrations of 30,0-40,0 g/l, the plants of both varieties experienced stress, which was manifested in the intensive release of phenols into the nutrient medium (this was most clearly manifested in the Sibirkovyi variety), and often the plants did not reach the top of the cultivation vessel during ontogenesis. besides, they showed earlier aging processes. At the same time, at high concentrations of glucose, a slowdown in growth processes and its ripening of shoots in individual microplants was noted.

Effect of morphological change on the maturation of human induced pluripotent stem cell-derived cardiac tissue in rotating flow culture

IntroductionUnderstanding the critical factors for the maturation of human induced pluripotent stem cell (hiPSC)-derived cardiac tissue is important for further development of culture techniques. Rotating flow culture, where the tissues float in the culture medium by balancing its gravitational settling and the medium flow generated in rotating disk-shaped culture vessels, is one of culture systems used for tissue engineering. It has previously been demonstrated that rotating flow culture leads to the formation of matured cardiac tissue with higher levels of function and structure than the other culture systems. However, the detailed mechanisms underlying the maturation of cardiac tissue remain unclear. This study investigated the maturation process of hiPSC-derived cardiac tissue in rotating flow culture with a focus on morphological changes in the tissue, which is a trigger for maturation. MethodsThe cardiac tissue, which consisted of cardiomyocytes derived from hiPSCs, was cultured on the 3D scaffold of poly (lactic-co-glycolic) acid (PLGA)-aligned nanofibers, in rotating flow culture for 5 days. During the culture, the time profile of projected area of tissue and formation of maturation marker proteins (β-myosin heavy chain and Connexin-43), tissue structure, and formation of nuclear lamina proteins (Lamin A/C) were compared with that in static suspension culture. ResultsThe ratio of the projected area of tissue significantly decreased from Day 0 to Day 3 due to tissue shrinkage. In contrast, Western blot analysis revealed that maturation protein markers of cardiomyocytes significantly increased after Day 3. In addition, in rotating flow culture, flat-shaped nuclei and fiber-like cytoskeletal structures were distributed in the surface region of tissue where medium flow was continuously applied. Moreover, Lamin A/C, which are generally formed in differentiated cells owing to mechanical force across the cytoskeleton and critically affect the maturation of cardiomyocytes, were significantly formed in the tissue of rotating flow culture. ConclusionsIn this study, we found that spatial heterogeneity of tissue structure and tissue shrinkage occurred in rotating flow culture, which was not observed in static suspension culture. Moreover, from the quantitative analysis, it was also suggested that tissue shrinkage in rotating flow culture contributed its following tissue maturation. These findings showed one of the important characteristics of rotating flow culture which was not revealed in previous studies.

A feast: Indigenous production and interregional exchange reflected by the bronze vessels unearthed from Shuangyuan Village Cemetery in southwestern China

AbstractSurprising combinations of bronze vessels with multiple cultural elements have been unearthed in Shu tombs in southwestern China, indicating a significant connection with the middle and lower reaches of the Yangtze River. However, there is currently no comprehensive evidence for the provenance of these foreign style vessels, and little emphasis has been given to the diachronic changes in the interaction between Shu culture and its surroundings. This article investigates the bronze vessels unearthed from the Shuangyuan Village Cemetery in Chengdu during the Eastern Zhou Dynasty and aims to deeply reveal the production and exchange of bronze vessels unearthed from Shuangyuan Village Cemetery from the perspective of lead sources through typological analysis and scientific analysis. The study clarified the ages of the local style Shu culture vessels and foreign style vessels, and the latter exhibited a strong influence of the Chu and Yue cultures. Lead isotope analysis shows that the lead source characteristics of foreign style vessels are consistent with those of vessels from Chu‐Yue culture at the same time, and they may be imported from Chu directly. The specific interaction modes of Shu and Chu underwent a clear transformation during the middle Warring States period.