Culture Vessel Research Articles

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System.

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

Effect of Air Sparging on Ethanol Production from Xylose and Glucose in Continuous Chemostat Fermentation Process Utilizing High Cell Density of <i>Candida intermedia</i> 4-6-4T2

Reducing fermentation periods and increasing ethanol productivity are cost effective for ethanol production from lignocellulosic biomass. Increasing the density of cells for fermentation typically increases ethanol productivity, but also increases the concentration of dissolved carbon dioxide (dCO2) in the fermented broth. Such accumulated dCO2 sometimes reduces ethanol production. The Continuous Chemostat Fermentation (CCF) process utilizing high density of Candida intermedia 4-6-4T2 with and without air sparging was evaluated for the effect on ethanol production and rapid fermentation using 24-h cycles. Synthetic fermentation solution without nitrogen sources containing 20 g/L xylose and 30 g/L glucose plus 5 g/L acetic acid as fermentation inhibitor was supplemented into a culture vessel at 15 mL/h, and fermented broth was recovered from the same flask at 15 mL/h. Various conditions were tested to reduce the accumulated dCO2 in the fermented broth, but air sparging at 0.056 vvm was the most effective for ethanol production in the CCF process. For the 24-h startup-batch and 6-cycle CCF process (144 h), the ethanol yield was 0.4 g/g and the cell density of the used C. intermedia 4-6-4T2 for one cycle was one-third compared to that of sequential batch fermentation.

A Practical Procedure for the In Vitro Generation of Human Osteoclasts and Their Characterization.

Osteoclasts are multinucleated cells derived from the hematopoietic monocyte/macrophage lineage that possess the unique capacity to resorb bone. Due to the crucial role of osteoclasts in maintaining bone homeostasis and pathologies, this cell type is pivotal in multiple research areas dedicated to bone physiology in health and disease. Although numerous methods for generation of human osteoclasts are already available, those rely either on cell labeling-based purification or an intermediate adhesion step after which cells are directly differentiated toward osteoclasts. While the former requires additional reagents and equipment, the latter harbors the risk of variable osteoclast formation due to varying numbers of osteoclast precursors available for different donors. In this study, we report a facile and reliable three-step method for the generation of human osteoclasts from blood-derived precursor cells. Monocytes were obtained after adhering peripheral blood-derived mononuclear cells to plastic substrates followed by macrophage induction and proliferation resulting in a homogeneous population of osteoclast precursors. Finally, macrophages were seeded into suitable culture vessels and differentiated toward osteoclasts. Osteoclastogenesis was monitored longitudinally using nondestructive techniques, while the functionality of mature osteoclasts was confirmed after 14 days of culture by analysis of functional (e.g., elevated tartrate-resistant acid phosphatase [TRAP]-activity, resorption) and morphological (e.g., presence of TRAP, actin ring, and integrin β3) characteristics. Furthermore, we propose to use combinatory staining of three morphological osteoclast markers, rather than previously reported staining of a single or maximal two markers, to clearly distinguish osteoclasts from undifferentiated mononuclear cells. Impact statement Research related to bone biology requires a standardized and reliable method for in vitro generation of human osteoclasts. We here describe such a procedure which avoids shortcomings of previously published protocols. Further, we report on nondestructive methods to qualitatively and quantitatively monitor osteoclastogenesis longitudinally, and on analysis of osteoclast generation and functionality after 14 days. Specifically, we recommend assessment of morphological human osteoclast characteristics using combinatory staining of three markers to confirm successful osteoclast generation.

Engineered Microvessel for Cell Culture in Simulated Microgravity.

As the number of manned space flights increase, studies on the effects of microgravity on the human body are becoming more important. Due to the high expense and complexity of sending samples into space, simulated microgravity platforms have become a popular way to study these effects on earth. In addition, simulated microgravity has recently drawn the attention of regenerative medicine by increasing cell differentiation capability. These platforms come with many advantages as well as limitations. A main limitation for usage of these platforms is the lack of high-throughput capability due to the use of large cell culture vessels. Therefore, there is a requirement for microvessels for microgravity platforms that limit waste and increase throughput. In this work, a microvessel for commercial cell culture plates was designed. Four 3D printable (polycarbonate (PC), polylactic acid (PLA) and resin) and castable (polydimethylsiloxane (PDMS)) materials were assessed for biocompatibility with adherent and suspension cell types. PDMS was found to be the most suitable material for microvessel fabrication, long-term cell viability and proliferation. It also allows for efficient gas exchange, has no effect on cell culture media pH and does not induce hypoxic conditions. Overall, the designed microvessel can be used on simulated microgravity platforms as a method for long-term high-throughput biomedical studies.

Rapid fabrication of hydrogel micropatterns by projection stereolithography for studying self-organized developmental patterning.

Self-organized patterning of mammalian embryonic stem cells on micropatterned surfaces has previously been established as an in vitro platform for early mammalian developmental studies, complimentary to in vivo studies. Traditional micropatterning methods, such as micro-contact printing (μCP), involve relatively complicated fabrication procedures, which restricts widespread adoption by biologists. Here, we demonstrate a rapid method of micropatterning by printing hydrogel micro-features onto a glass-bottomed culture vessel. The micro-features are printed using a projection stereolithography bioprinter yielding hydrogel structures that geometrically restrict the attachment of cells or proteins. Compared to traditional and physical photomasks, a digitally tunable virtual photomask is used in the projector to generate blue light patterns that enable rapid iteration with minimal cost and effort. We show that a protocol that makes use of this method together with LN521 coating, an extracellular matrix coating, creates a surface suitable for human embryonic stem cell (hESC) attachment and growth with minimal non-specific adhesion. We further demonstrate that self-patterning of hESCs following previously published gastrulation and ectodermal induction protocols achieves results comparable with those obtained with commercially available plates.

Quality Verification with a Cluster-Controlled Manufacturing System to Generate Monocyte-Derived Dendritic Cells.

Dendritic cell (DC) vaccines for cancer immunotherapy have been actively developed to improve clinical efficacy. In our previous report, monocyte−derived DCs induced by interleukin (IL)−4 with a low−adherence dish (low−adherent IL-4−DCs: la−IL-4−DCs) improved the yield and viability, as well as relatively prolonged survival in vitro, compared to IL-4−DCs developed using an adherent culture protocol. However, la−IL-4−DCs exhibit remarkable cluster formation and display heterogeneous immature phenotypes. Therefore, cluster formation in la−IL-4−DCs needs to be optimized for the clinical development of DC vaccines. In this study, we examined the effects of cluster control in the generation of mature IL-4−DCs, using cell culture vessels and measuring spheroid formation, survival, cytokine secretion, and gene expression of IL-4−DCs. Mature IL-4−DCs in cell culture vessels (cluster−controlled IL-4−DCs: cc−IL-4−DCs) displayed increased levels of CD80, CD86, and CD40 compared with that of la−IL-4−DCs. cc−IL-4−DCs induced antigen−specific cytotoxic T lymphocytes (CTLs) with a human leukocyte antigen (HLA)−restricted melanoma antigen recognized by T cells 1 (MART−1) peptide. Additionally, cc−IL-4−DCs produced higher levels of IFN−γ, possessing the CTL induction. Furthermore, DNA microarrays revealed the upregulation of BCL2A1, a pro−survival gene. According to these findings, the cc−IL-4−DCs are useful for generating homogeneous and functional IL-4−DCs that would be expected to promote long−lasting effects in DC vaccines.

Advanced Sequencing of Kidney Tubule Hypoxia Regulated Epithelial‐to‐Mesenchymal Transition

Hypoxia is a known regulator of epithelial-to-mesenchymal transition in kidney tubule cells. However, culture systems have struggled to observe these transitions at high levels to capture transcriptional signatures due to the detachment of cells from culture vessels. Using the Renal Proximal Tubule Epithelial Cell stabile cell line (RPTEC-TERT1) we developed a shear stress culture system that when exposed to hypoxia (1% oxygen) result in cell dysmorphology instead of detachment, allowing for a large number of cells to be generated for transcriptomic studies. With these cells exposed to hypoxia, we performed both traditional polyA RNAseq using both Illumina and Nanopore technologies and newer direct RNAseq using Nanopore. The polyA RNAseq suggests regulation changes for HIF-1/hypoxia, Nonsense mediated decay, ruffle assembly, and energetic regulated genes. Direct RNAseq, a strategy of running mRNA molecules across Nanopore to detect RNA base modifications transcriptome wide, revealed genes that are acutely modified related to glycolysis, innate immunity, oxidation-reduction, and mitochondrial biology. Cells exposed to hypoxia for 48 hours followed by 48 hours of normoxia, return the modifications back to control levels suggesting there is a major source of hypoxia cell signaling through RNA posttranscriptional modifications. Using the differentially regulated genes of the hypoxia culture model we have also shown that it is possible to filter genetic models systems such as human chronic/acute kidney disease or rat heterogenous stock proteinuria to narrow down novel mechanisms for genetic influence to hypoxia response. In doing this, we nominated and validated the role of SEPTIN8 in hypoxia response cell integrity, where dysregulation of SEPTIN8 can further propagate complications of hypoxia to disease progression.

ADVANCE STUDIES ON MICROPROPAGATION OF DATE PALM.

Date palm is one of the most important fruit crops in the present and past, as the cultivated areas increase due to its high economics and grown in most lands and its tolerance to difficult conditions and therefore the demand for its offshoots was abundant. This study aimed to micropropagate date palm cultivar Amri through direct organogenesis, improving the growth of plantlets and establish a protocol for photoautotrophic and photomixotrophic as a new micropropagation system in order to obtain highest success percentage and lowest the loss percentage in the acclimatized plantlets during the acclimatization stage, this was done by performing two main experimental groups In-vitro and Ex-vitro Where the effect of different concentrations of auxins, cytokinins and AC. during the direct organogenesis stage was studied, as well as the study of some substances affecting the growth and improvement of plants during the elongation and rooting stages, such as the use of different concentrations of CNTs, spirulina platensis algae extract and Kafom-Cu, PEG and SiO2 and Kafom-Mg. The effect of ventilation rates in cultivation vessels with different concentrations of sucrose in the rooting medium and under CO2 enriched conditions (photoautotrophic and photomixtrophic) or under normal conditions for micro-propagation in the in vitro during the rooting and pre-acclimatization stages in addition to the acclimatization stage were studied in which different systems were studied. For greenhouse cultivation, it also affects different alcohol sugar concentrations of sorbitol and salicylic acid concentrations. Anatomical and chemical studies were also conducted on the best treatments during the rooting and pre-acclimatization stages. The following results were obtained: Adding 0.2 mg l-1 2,4-D + 0.12 mg l-1 NAA + 0.12 mg l-1 IAA + 0.1 mg l-1 2iP + 0.15 mg l-1 BA + 0.5 g l-1 AC to the cultivation medium encouraged the formation of direct shoots. While planting the plants on a medium containing MS added to 20 mg l-1 of spirulina extract increased the length of the shoots, the addition of K-Mg at 1.2 mg l-1 to the rooting medium achieved the highest values ​​of vegetative growth parameters and root system, with the plantlets and media free from contamination. Addition of SiO2 at 20 mg l-1 improved the trunk thickness. It also showed the use of the rate of ventilation 4 holes in the planting pot + 15 g l-1 of sucrose added to the rooting medium under the conditions of enriched with CO2 at 1500 ppm achieved the highest growth vigor and the highest percentage of survival and increased the content of the leaves of amino acids and reducing sugars and their content of Chlorophyll a + b. cultivation plantlets in mixture of Peat moss with Perlite (1: 1 v / v) plus 3 g/pot Barbary-Plante with foliar spray and watered by sorbitol at 30 g l-1 plus salicylic acid at 40 mg l-1 gave the highest values ​​for survival percentage, leaf width and growth vigor (91%, 2.73 cm, 4 respectively).

Normalized chlorophyll fluorescence imaging: A method to determine irradiance and photosynthetically active radiation in phytoplankton cultures

In order to expand microalgae's use as a mainstream biotechnological resource, the efficiency of light capturing in culture vessels must be optimised. As a response to the complex nature of microalgal culture vessel modelling, it is important that a robust method to assess irradiance in photobioreactors (PBR's) is developed. Several hardware based or mathematical models to predict irradiance exist; however, these methods can be impractical as they are time-consuming, depend on growth vessel geometry or species to be cultivated. In this work, a chlorophyll a fluorescence imaging method was developed to assess both light penetration and utilisation in PBR's. This new method, termed normalized chlorophyll fluorescence imaging (NCFI), allows users to obtain images (light maps) that reflect irradiance as a function of depth into the PBR. This information is then combined with biophysical measurements to provide both 2D maps and 3D models of wavelength dependent light utilisation in PBR's. The methods presented in this work aim to demonstrate how these kinds of measurements are undertaken, the potential artefacts one may encounter and its utility for designing efficient PBR's that are optimised for maximal photosynthetic productivity.

Optimization of culture conditions for human bone marrow-derived mesenchymal stromal cell expansion in macrocarrier-based Tide Motion system.

With high cell doses required for mesenchymal stromal cell (MSC) clinical trials, there is a need to upgrade technologies that facilitate efficient scale up of MSCs for cell therapy. Conventional expansion with 2D culture vessels becomes the bottleneck when large cell dosages are required. Tide Motion bioreactors offer a robust, scalable platform using BioNOC II macrocarriers developed for the production of adherent cells. We evaluated the growth and expansion of bone marrow-derived MSCs (BM-MSCs) on the macrocarrier-based culture system by optimizing key parameters such as cell seeding densities, culturing conditions, and harvesting procedures to achieve optimal cell growth. BM-MSCs expanded in conventional 2D adherent cultures were seeded into BioNOC II macrocarriers and grown in serum-containing or serum-free medium. BM-MSCs attained a maximum cell density of 0.49 ± 0.07×106 cells/carrier after 12 days of culture in BioNOC II macrocarriers with cell viability > 86% while retaining MSC specific characteristics such as surface marker expression, tri-lineage differentiation potential, immunosuppressive properties, and potency. These results reveal the feasibility of BM-MSC expansion in the scalable macrocarrier-based Tide Motion system both under serum and serum-free conditions and represent an important step for the large-scale production system of BM-MSC based cellular therapies.

Better light spectral quality and thermal amplitude inside the greenhouse stimulate growth and improve acclimatization of in vitro–grown Cattleya warneri T. Moore

Growth and photosynthetic capacity of Cattleya warneri T. Moore seedlings cultivated in vitro were evaluated in two environments: (1) growth room (GR) with constant light, humidity, and temperature; and (2) greenhouse (GH) with variable humidity, temperature, and light intensity and quality. In both environments, two different tissue culture vessel lids were used: transparent plastic lids (TCVplastic) and non-transparent metal lids (TCVmetal). After 11 months of in vitro cultivation, five seedlings from each tissue culture vessel were evaluated for growth and photosynthetic capacity, while the other five seedlings of each tissue culture vessel were transferred to a greenhouse for acclimatization. Increased biomass production in vitro was observed in GH and GR (GH>GR). However, the photosynthetic capacity was not altered by the GH environment, since the net photosynthetic rate at 300 μmol m−2 s−1 (NPR300) was low in the final of period in all treatments. Therefore, the increase in biomass production in C. warneri was mostly dependent on the exogenous carbon source through the addition of sucrose to the culture medium. The use of TCVplastic in vitro improved seedlings’ growth in both GR and GH, showing an advantage in relation to TCVmetal. GH environment with quality light spectrum and thermal amplitude, and use of TCVplastic, increased biomass production in vitro and improved the acclimatization process of C. warneri seedlings, which also reduced electricity costs since the use of artificial light and air conditioning is not required.

Human Adipose Stem Cells (hASCs) Grown on Biodegradable Microcarriers in Serum- and Xeno-Free Medium Preserve Their Undifferentiated Status.

Human adipose stem cells (hASCs) are promising candidates for cell-based therapies, but they need to be efficiently expanded in vitro as they cannot be harvested in sufficient quantities. Recently, dynamic bioreactor systems operated with microcarriers achieved considerable high cell densities. Thus, they are a viable alternative to static planar cultivation systems to obtain high numbers of clinical-grade hASCs. Nevertheless, the production of considerable biomass in a short time must not be achieved to the detriment of the cells’ quality. To facilitate the scalable expansion of hASC, we have developed a new serum- and xeno-free medium (UrSuppe) and a biodegradable microcarrier (BR44). In this study, we investigated whether the culture of hASCs in defined serum-free conditions on microcarriers (3D) or on planar (2D) cell culture vessels may influence the expression of some marker genes linked with the immature degree or the differentiated status of the cells. Furthermore, we investigated whether the biomaterials, which form our biodegradable MCs, may affect cell behavior and differentiation. The results confirmed that the quality and the undifferentiated status of the hASCs are very well preserved when they grow on BR44 MCs in defined serum-free conditions. Indeed, the ASCs showed a gene expression profile more compatible with an undifferentiated status than the same cells grown under standard planar conditions.

Chlorophyll a and non-pigmented biomass are sufficient predictors for estimating light attenuation during cultivation of Dunaliella viridis

Characterizing light in microalgal cultivation vessels is needed for modeling and optimizing microalgal growth for large-scale cultivation. Dynamic changes in light intensity over space due to geometry, refraction/reflection, and the interactive impacts of algal growth and their biocomponents with light make this characterization challenging. Understanding which biocomponents within microalgal cultures are key variables in accurately estimating light attenuation is fundamentally important, yet, inconsistent and wide-ranging applications of the Beer-Lambert law are often used to estimate light attenuation.This research rigorously evaluated which biocomponents (total biomass, cell count, and chl a, chl b, and total photosynthesizing pigments), or biocomponent combinations, serve as best predictors for light attenuation when modeling with the Beer-Lambert law. Calibration and validation experiments were performed using salt-water species Dunaliella viridis microalgal cultures grown in 3-L flat-plate PBRs with continuous light monitoring. Results at the various light and nitrogen levels tested showed Beer-Lambert's law predicted photosynthetic light attenuation well when both biomass and chlorophyll a were considered as distinct attenuating components, providing light estimates with less than 6% error on average over validation experiments. If the model included only one component as a predictor for attenuation, pigments were best, with a 20% error in estimating light, as compared to ~70%, 60%, 40% for models that used solely biomass, cells, or chlorophyll a as an attenuating component., respectively. These results suggest that when using the Beer-Lambert's law to estimate photosynthetic light attenuation in microalgal cultures, both a chlorophyll a and biomass component should be consistently included.

Spore germination and ectomycorrhizae formation of Tricholoma matsutake on pine root systems with previously established ectomycorrhizae from a dikaryotic mycelial isolate of T. matsutake.

In vitro ectomycorrhizal synthesis of Tricholoma matsutake with host plants has been widely conducted to elucidate fungal symbiotic properties for future cultivation practices. Here, we report on the importance of basidiospore inocula for this fungus to provide ectomycorrhizal seedlings in vitro. Ectomycorrhizal pine seedlings synthesized in vitro with cultured mycelium of T. matsutake (isolate #45 or #84) in a 250-mL culture vessel (soil volume) were transplanted to a large 1-L culture vessel. Fresh basidiospores of this fungus were aseptically inoculated on the ectomycorrhizal root system. The ectomycorrhizal seedlings in the 1-L vessel were grown for 9 months, and some plants were further grown for 6 more months under non-aseptic conditions in 4.1-L jars. The ectomycorrhizal seedlings previously inoculated with isolate #84 in the 1-L vessel showed significant ectomycorrhizal biomass (mycorrhizal root length) after spore inoculation. The ectomycorrhizal seedlings in the 4.1-L vessel showed large shiro structures (> 10 cm in diameter). PCR amplification of intergenic spacer 1 of the rRNA gene and long terminal repeat retroelement of T. matsutake in ectomycorrhizal root tips in both the 1-L vessels and 4.1-L jars revealed the presence of amplicons of the previously inoculated culture isolate of T. matsutake and the new genet(s) that established via germination of the inoculated basidiospores. This is the first report that inoculated basidiospores of T. matsutake germinated and colonized the host root to generate ectomycorrhizae in vitro.

Biomimetic microsystems for cardiovascular studies.

Traditional tissue culture platforms have been around for several decades and have enabled key findings in the cardiovascular field. However, these platforms failed to recreate the mechanical and dynamic features found within the body. Organs-on-chips (OOCs) are cellularized microfluidic-based devices that can mimic the basic structure, function, and responses of organs. These systems have been successfully utilized in disease, development, and drug studies. OOCs are designed to recapitulate the mechanical, electrical, chemical, and structural features of the in vivo microenvironment. Here, we review cardiovascular-themed OOC studies, design considerations, and techniques used to generate these cellularized devices. Furthermore, we will highlight the advantages of OOC models over traditional cell culture vessels, discuss implementation challenges, and provide perspectives on the state of the field.

Cannabis sativa L. photoautotrophic micropropagation: a powerful tool for industrial scale in vitro propagation

Global demands for an in vitro culture of cannabis have never been more sought after as countries shift their paradigm towards legalization. Cannabis conventional (photomixotrophic) micropropagation has not been suitable enough for large-scale propagation due to a high degree of plant hyperhydricity, low growth rate, poor rooting, and acclimation efficiency. In the present study, cannabis photoautotrophic micropropagation method is introduced with the purpose of overcoming the difficulties that conventional micropropagation entails when conducted at a large scale. The roles of rockwool medium pH and moisture content, cutting length, basal wounding methods, light intensity, and culture vessel gas exchange capacity were assessed with the intention of increasing productivity of micropropagation method. The results showed 300 mL per vessel of fertilizer solution containing 5-mM MES buffer stabilized medium pH and increased rooting success. Both 5- and 7-cm cutting lengths significantly increased the percent of rooted plants compared to 3-cm cutting length. However, the basal wounding methods did not significantly improve or impede the rooting success. The highest rooting success was also obtained with 150 μmol m−2 s−1 of photosynthetic photon flux density compared to 50 and 100 μmol m−2 s−1. Increasing gas exchange rates either using more permeable vessels or aeration practices significantly improved the rooting success. Overall, more than 90% of cannabis plantlets grown in photoautotrophic micropropagation are rooted in 2 wk of culture followed by 4 d ex vitro acclimation period, which was remarkably shorter than any other available method in cannabis micropropagation. This study not only optimizes a method for cannabis photoautotrophic micropropagation using passive ventilation for the first time but also scales up in vitro clonal propagation for in vitro commercial production.

In vitro Regeneration of Clematis Plants in the Nikita Botanical Garden via Somatic Embryogenesis and Organogenesis.

The effects of growth regulators, namely, 6-benzylaminopurine (BAP) and thidiazuron (TDZ), on the morphogenic capacity of 13 cultivars of clematis plants, in terms of their morphological structure formation, shoot regeneration, and somatic embryo development, are presented. The clematis cultivars ‘Alpinist,’ ‘Ay-Nor,’ ‘Bal Tsvetov,’ ‘Crimson Star,’ ‘Crystal Fountain,’ ‘Kosmicheskaya Melodiya,’ ‘Lesnaya Opera,’ ‘Madame Julia Correvon,’ ‘Nevesta,’ ‘Nikitsky Rosovyi,’ ‘Nikolay Rubtsov,’ ‘Serenada Kryma,’ and ‘Vechniy Zov’ were taken in collection plots of the Nikita Botanical Gardens for use in study. After explant sterilization with 70% ethanol (1 min), 0.3–0.4% Cl2 (15 min), and 1% thimerosal (10 min), 1-cm long segments with a single node were introduced to an in vitro culture. The explants were established on the basal MS medium supplemented with BAP (2.20–8.90 μM) and 0.049 μM NAA, or TDZ (3.0; 6.0, and 9.0 μM) with 30 g/L sucrose and 9 g/L agar. The medium with 0.89 μM BAP served as the control. Culture vessels and test tubes with the explants were maintained in plant growth chamber-controlled conditions: with a 16-h photoperiod, under cool-white light fluorescent lamps with a light intensity of 37.5 μmol m–2 s–1, at a temperature of 24 ± 1°C. Histological analysis demonstrated that adventitious bud and somatic embryo formation in studied clematis cultivars occurred at numerous areas of active meristematic cell zones. The main role of plant growth regulators and its concentrations were demonstrated. It was determined that maximum adventitious microshoot regeneration without any morphological abnormalities formed on the media supplemented with BAP or TDZ. 4.40 μM BAP, or 6.0 μM TDZ were optimal cytokinin concentrations for micropropagation. The explants of ‘Alpinist,’ ‘Ay-Nor,’ ‘Crimson Star,’ ‘Crystal Fountain,’ ‘Nevesta,’ and ‘Serenada Kryma’ cultivars displayed high morphogenetic capacity under in vitro culturing. During indirect somatic embryogenesis, light intensity 37.5 μmol m–2 s–1 stimulated a higher-number somatic embryo formation and a temperature of 26°C affected somatic embryo development. Active formation of primary and secondary somatic embryos was also demonstrated. 2.20 μM BAP with 0.09 μM IBA affected the high-number somatic embryo formation for eight cultivars. Secondary somatic embryogenesis by the same concentration of BAP was induced. The frequency of secondary somatic embryogenesis was higher in ‘Crystal Fountain’ (100%), ‘Crimson Star’ (100%), ‘Nevesta’ (97%), and ‘Ay-Nor’ (92%) cultivars. Based on these results, the methodology for direct somatic embryogenesis and organogenesis of studied clematis cultivars has been developed.

Flowering and Seed Production across the Lemnaceae.

Plants in the family Lemnaceae are aquatic monocots and the smallest, simplest, and fastest growing angiosperms. Their small size, the smallest family member is 0.5 mm and the largest is 2.0 cm, as well as their diverse morphologies make these plants ideal for laboratory studies. Their rapid growth rate is partially due to the family’s neotenous lifestyle, where instead of maturing and producing flowers, the plants remain in a juvenile state and continuously bud asexually. Maturation and flowering in the wild are rare in most family members. To promote further research on these unique plants, we have optimized laboratory flowering protocols for 3 of the 5 genera: Spirodela; Lemna; and Wolffia in the Lemnaceae. Duckweeds were widely used in the past for research on flowering, hormone and amino acid biosynthesis, the photosynthetic apparatus, and phytoremediation due to their aqueous lifestyle and ease of aseptic culture. There is a recent renaissance in interest in growing these plants as non-lignified biomass sources for fuel production, and as a resource-efficient complete protein source. The genome sequences of several Lemnaceae family members have become available, providing a foundation for genetic improvement of these plants as crops. The protocols for maximizing flowering described herein are based on screens testing daylength, a variety of media, supplementation with salicylic acid or ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) (EDDHA), as well as various culture vessels for effects on flowering of verified Lemnaceae strains available from the Rutgers Duckweed Stock Cooperative.

Volume-constrained microcontainers enable myoepithelial functional differentiation in highly parallel mammary organoid culture

SummaryA long-standing constraint on organoid culture is the need to add exogenous substances to provide hydrogel matrix, which limits the study of fully human or fully native organoids. This paper introduces an approach to culture reconstituted mammary organoids without the impediment of exogenous matrix. We enclose organoids in nanoliter-scale, topologically enclosed, fluid compartments surrounded by agar. Organoids cultured in these “microcontainers” appear to secrete enough extracellular matrix to yield a self-sufficient microenvironment without exogenous supplements. In microcontainers, mammary organoids exhibit contractility and a high-level, physiological, myoepithelial (MEP) behavior that has not been previously reported in reconstituted organoids. The presence of contractility suggests that microcontainers elicit MEP functional differentiation, an important milestone. Microcontainers yield thousands of substantially identical and individually trackable organoids within a single culture vessel, enabling longitudinal studies and statistically powerful experiments, such as the evaluation of small effect sizes. Microcontainers open new doors for researchers who rely on organoid models.

Characterization of stem cells from human ovarian follicular fluid; a potential source of autologous stem cell for cell-based therapy.

Human ovarian follicular fluid (HOFF) contains proteins, extracellular matrixes necessary for growth and maturation of oocytes as well as granulosa cells. Epithelial cells and stem cells can be isolated from HOFF. However, information regarding stem cells derived from HOFF is still lacking. The objectives of the present study were to isolate, characterize, and differentiate cells derived from HOFF. HOFF was collected during the routine aspiration of oocytes in an assisted fertilization program and subjected to cell isolation, characterization, and in vitro culture. After 24h of culture, different cell morphologies including epithelial-like-, neural-like- and fibroblast-like cells were observed. Immunocytochemistry reveals the expression of pluripotent stem cell markers (OCT4, NANOG, SSEA4), epithelial marker (CK18), FSH- and LH-receptor. For in vitro culture, the isolated cells were continuously cultured in a growth medium; alpha MEM containing 10% FBS and epidermal growth factor (EGF). After 2weeks of in vitro culture, cells with fibroblast-like morphology dominantly grow in the culture vessels and resemble mesenchymal stem cells (MSCs). HOFF-derived cells exhibited MSC expression of CD44, CD73, CD90, CD105, CD146, and STRO-1, and were capable of differentiation into osteoblasts, chondrocytes, and adipocytes. After induction of neural differentiation, HOFF-derived cells formed spheroidal structures and expressed neural stem cell markers including Nestin, β-tubulin III, and O4. Besides, the oocyte-like structure was observed after prolonged culture of HOFF. In conclusion, cells derived from follicular fluidexhibited stem cell characteristics, which could be useful for regenerative medicine applications and cell-based therapies.