Culture Density Research Articles

Can the Culture Density of the Marine Copepod Pseudodiaptomus nihonkaiensis be Improved by Installing an Artificial Substrate?

The understanding of the increasing population density of copepods in culture tanks contributes to establish their stable cultures at high productions for aquaculture industry, which are preferred live feeds for fish larvae. In the present study, the semi-benthic copepod Pseudodiaptomus nihonkaiensis were used as target species. This genus is often found from brackish water and its tolerance to a wide range of salinity and water quality has attracted attention as a potential commercial live feed for aquaculture. Population growth and egg production in the semi-benthic copepod P. nihonkaiensis are investigated. Copepods were cultured in tanks without mesh, with mesh lining tank walls, and with 2, 4 or 8 mesh partitions in each tank to identify the effect of substratum surface area on copepod population density. Linear regression revealed peaks in nauplius, copepodite, adult and total densities, and the cumulative number of eggs for all sampling events to correlate positively with substratum surface area (p<0.01). Greater substratum area within a culture tank may improve egg production by increasing habitat for ovigerous females. The simple installation of inexpensive artificial substrata in existing copepod culture tanks might improve the feasibility of using copepods as a live food in commercial-scale aquaculture.

An upgraded Myxococcus xanthus chassis with enhanced growth characteristics for efficient genetic manipulation

Myxobacteria are well known for multicellular social behaviors and valued for biosynthesis of natural products. Myxobacteria social behaviors such as clumping growth severely hamper strain cultivation and genetic manipulation. Using Myxococcus xanthus DK1622, we engineered Hu04, which is deficient in multicellular behavior and pigmentation. Hu04, while maintaining nutritional growth and a similar metabolic background, exhibits improved dispersed growth, streamlining operational procedures. It achieves high cell densities in culture and is promising for synthetic biology applications.

Centroid of the bacterial growth curves: a metric to assess phage efficiency

Phage replication can be studied using various approaches, including measuring the optical density (OD) of a bacterial culture in a liquid medium in the presence of phages. A few quantitative methods are available to measure and compare the efficiency of phages by using a single index based on the analysis of OD curves. However, these methods are not always applicable to non-canonical OD curves. Using the concept of center of area (centroid), we developed a metric called Centroid Index (CI), sensitive to the trend of the growth curves (OD distribution) including bacterial regrowth, which is not considered by the methods already available. We also provide a user-friendly software to facilitate the calculation of CI. This method offers an alternative and more precise way to determine phage efficiency by considering the OD variations over time, which may help in the selection of phages for biocontrol applications.

Genome-Scale Investigation of the Regulation of azoR Expression in Escherichia coli Using Computational Analysis and Transposon Mutagenesis

Bacterial azoreductases are enzymes that catalyze the reduction of ingested or industrial azo dyes. Although azoreductase genes have been well identified and characterized, the regulation of their expression has not been systematically investigated. To determine how different factors affect the expression of azoR, we extracted and analyzed transcriptional data from the Gene Expression Omnibus (GEO) resource, then confirmed computational predictions by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results showed that azoR expression was lower with higher glucose concentration, agitation speed, and incubation temperature, but higher at higher culture densities. Co-expression and clustering analysis indicated ten genes with similar expression patterns to azoR: melA, tpx, yhbW, yciK, fdnG, fpr, nfsA, nfsB, rutF, and chrR (yieF). In parallel, constructing a random transposon library in E. coli K-12 and screening 4320 of its colonies for altered methyl red (MR)-decolorizing activity identified another set of seven genes potentially involved in azoR regulation. Among these genes, arsC, relA, plsY, and trmM were confirmed as potential azoR regulators based on the phenotypic decolorization activity of their transposon mutants, and the expression of arsC and relA was confirmed, by qRT-PCR, to significantly increase in E. coli K-12 in response to different MR concentrations. Finally, the significant decrease in azoR transcription upon transposon insertion in arsC and relA (as compared to its expression in wild-type E. coli) suggests their probable involvement in azoR regulation. In conclusion, combining in silico analysis and random transposon mutagenesis suggested a set of potential regulators of azoR in E. coli.

Low Initial Cell Density Promotes the Differentiation and Maturation of Human Pluripotent Stem Cells into Erythrocytes.

Human pluripotent stem cell (hPSC)-derived red blood cells (RBCs) possess great potential for compensating shortages in transfusion medicine. For better RBC generation from hPSCs, we compared the cell seeding density in the embryoid body formation-based hPSC induction protocol. In the selection of low- and high-density inoculation conditions, we found that low-density culture performed better in the final RBC product with more cell output and increased average cellular hemoglobin content. An elaborate study using flow cytometry demonstrated that low inoculation density promoted endothelial-to-hematopoietic transition, followed by improved hematopoietic progenitor formation and erythrocyte generation. The improved transformation from glycolysis to mitochondrial oxidation and reduced apoptosis might be responsible for this effect. Hints from RNA sequencing suggested that molecules involved in microenvironment interaction and metabolic regulation might respond for the different developmental potential. The possible mediators between outer message and intracellular response could be the nutrition sensors FOXO, PRKAA1 (AMPK), and MTOR genes. It is possible that low inoculation density triggered metabolic regulation signals, promoted mitochondrial oxidation, and resulted in enhanced cell amplification and hematopoietic differentiation. The low cell culture density will improve RBC generation from hPSCs.

Performances of two rapid LAMP-based techniques for the intrapartum detection of Group B Streptococcus vaginal colonization

PurposeGroup B Streptococcus (GBS) is the leading cause of invasive infections in newborns. The prevention of GBS neonatal disease relies on the administration of an intrapartum antibiotic prophylaxis to GBS-colonized women. In recent years, rapid intrapartum detection of GBS vaginal colonization using real-time nucleic acid amplification tests (NAATs) emerged as an alternative to antenatal culture screening methods.MethodsWe compared the performances of two loop-mediated isothermal amplification (LAMP) tests, the Ampliflash® GBS and the PlusLife® GBS tests, to standard culture for GBS detection in vaginal specimens from pregnant women. The study was conducted from April to July 2023 in a French hospital of the Paris area.ResultsA total of 303 samples were analyzed, including 85 culture-positive samples (28.1%). The Ampliflash® GBS test and the PlusLife® GBS tests gave a result for 100% and 96.3% tests, respectively. The performances of the tests were as follows: sensitivity 87.1% (95% confidence interval (CI) 78.3–92.6) and 98.7% (95% CI 93.0-99.8), specificity 99.1% (95% CI 96.7–99.8), and 91.9% (95% CI 87.3–95.0), respectively. False negative results of the Ampliflash® GBS test correlated with low-density GBS cultures. Time-to-results correlated with GBS culture density only for the PlusLife® GBS test (p < 0.001).ConclusionBoth techniques provide excellent analytical performances with high sensitivity and specificity together with a short turnaround time and results available in 10 to 35 min. Their potential to further reduce the burden of GBS neonatal disease compared with antenatal culture screening needs to be assessed in future clinical studies.

ISOLATION OF BONE MARROW MESENCHYMAL STEM CELLS EMBEDDED IN NATIVE TISSUE STROMA YIELDS ENRICHED HARVEST, IMPROVED ADHERENCE AND PROLIFERATION, AND UNIQUE SECRETOME

Background: The field of orthobiologics traditionally utilizes cellular products, including bone-marrow aspirate concentrate (BMAC), micronized adipose tissue, and platelet preparations to address pain from degenerative processes, orthopedic injuries and medical conditions characterized by chronic inflammation and tissue degradation. For BMAC, maximizing the concentration of mesenchymal stem cells (MSCs) in a reduced volume is thought to allow for the therapeutic delivery of the cellular concentrate, secretome, and extracellular vesicles to a site of orthopedic injury or surgical repair. The extracellular matrix (ECM) within the bone marrow stroma contains collagens and proteoglycans known to regulate cell proliferation, migration, differentiation, and cell-cell communication among resident bone marrow cells. This study aimed to evaluate the cellular effects on MSC health and function when harvested to retain their native tissue stroma. Methods: We evaluated a novel and unique processing method and device (BMAX™) to mechanically generate a purified MSC product derived from bone marrow in a nonenzymatic manner. BMAX™ products, including cells and stroma, were plated in MSC culture media and incubated for 3–14 days (P0-P1) before evaluation with flow cytometry for cell phenotyping and immunoassays for secretome profiling. Results: The orthobiologic product containing three-dimensional stromal components can be produced in minutes using an automated bedside device requiring minimal benchtop space. We found increased MSC adherence, improved proliferative density in culture, and significantly elevated enrichment of stromal-derived MSCs versus traditional BMAC centrifugation-based preparations. Further, we demonstrate a unique secretome profile in BMAX™ versus traditional BMAC centrifugation-based preparations. Conclusions: These qualities provide a novel and unique platform for autologous and allogeneic bone-marrow-derived therapy to better address inflammatory and destructive processes that may improve bone-marrow-derived cell therapies’ efficacy.

Methanooceanicella nereidis gen. nov., sp. nov., the first oceanic Methanocellaceae methanogen, isolated from potential methane hydrate bearing area offshore southwestern Taiwan.

A novel mesophilic, hydrogenotrophic methanogen, strain CWC-04T, was obtained from a sediment sample extracted from a gravity core retrieved at station 22 within the KP-9 area off the southwestern coast of Taiwan during the ORIII-1368 cruise in 2009. Cells of strain CWC-04T were rod-shaped, 1.4-2.9 µm long by 0.5-0.6 µm wide, and occurred singly. Strain CWC-04Tutilized formate, H2/CO2, 2-propanol/CO2 or 2-butanol/CO2 as catabolic substrates. The optimal growth conditions were 42 °C, 0.17 M NaCl and pH 5.35. The genomic DNA G+C content calculated from the genome sequence of strain CWC-04T was 46.19 mol%. Phylogenetic analysis of 16S rRNA gene revealed that strain CWC-04T is affiliated with the genus Methanocella. The 16S rRNA gene sequences similarities within strains Methanocella arvoryzae MRE50T, Methanocella paludicola SANAET and Methanocella conradii HZ254T were 93.7, 93.0 and 91.3 %, respectively. In addition, the optical density of CWC-04T culture dropped abruptly upon entering the late-log growth phase, with virus-like particles (150 nm in diameter) being observed on and around the cells. This observation suggests that strain CWC-04T harbours a lytic virus. Based on these phenotypic, phylogenetic and genomic results, we propose that strain CWC-04T represents a novel species of a novel genus in the family Methanocellaceae, for which the name Methanooceanicella nereidis gen. nov., sp. nov. is proposed. The type strain is CWC-04T (=BCRC AR10050T=NBRC 113165T).

Co-cultured of red hybrid tilapia and Gracilaria changii (Gracilariaceae: Rhodophyta): Effects on water quality, growth performance and algal density

ABSTRACT We evaluated the potential for co-cultured of red hybrid tilapia with Gracilaria changii in two different densities in zero water exchange conditions. Five treatments were performed, consisting of three monoculture systems as controls: algae at two different densities (M1 and M2 with 1 g L−1 density and 3 g L−1 density, respectively) and fish without algae (Mfish). For comparison purposes, the other two treatments were co-cultured fish with algae at densities of 1 g L−1 (P1) and 3 g L−1 (P2). The effects of different culture systems and densities on the water quality, growth performance, nutrient content and C/N ratio of the algae were determined. During the experimental period, the dissolved oxygen (DO) and pH values in the algal tanks (monoculture and co-culture) were significantly higher than those in the Mfish tank. The ammonia and nitrate concentrations in P1 and P2 were significantly lower compared to initial measurements. The water parameters indicated that the algae improved the water quality of the culture system. Compared to the fish monoculture (Mfish), the fish grew better with algae (P1 and P2) maybe due to the more favourable water conditions in the tanks. On day 20, the algae growth rates in the co-culture tanks (P1 and P2) were significantly higher than those in the monoculture tanks. Additionally, P1 and P2 improved the nutrient composition and C/N ratio of the algae. This study found that although the co-culture system resulted in better water quality and nutrient content in the alga, the algal density had no significant effect on fish or algal growth.

Demography and movement patterns of a freshwater ciliate: The influence of oxygen availability.

In freshwater habitats, aerobic animals and microorganisms can react to oxygen deprivation by a series of behavioural and physiological changes, either as a direct consequence of hindered performance or as adaptive responses towards hypoxic conditions. Since oxygen availability can vary throughout the water column, different strategies exist to avoid hypoxia, including that of active 'flight' from low-oxygen sites. Alternatively, some organisms may invest in slower movement, saving energy until conditions return to more favourable levels, which may be described as a 'sit-and-wait' strategy. Here, we aimed to determine which, if any, of these strategies could be used by the freshwater ciliate Tetrahymena thermophila when faced with decreasing levels of oxygen availability in the culture medium. We manipulated oxygen flux into clonal cultures of six strains (i.e. genotypes) and followed their growth kinetics for several weeks using automated image analysis, allowing to precisely quantify changes in density, morphology and movement patterns. Oxygen effects on demography and morphology were comparable across strains: reducing oxygen flux decreased the growth rate and maximal density of experimental cultures, while greatly expanding the duration of their stationary phase. Cells sampled during their exponential growth phase were larger and had a more elongated shape under hypoxic conditions, likely mirroring a shift in resource investment towards individual development rather than frequent divisions. In addition to these general patterns, we found evidence for intraspecific variability in movement responses to oxygen limitation. Some strains showed a reduction in swimming speed, potentially associated with a 'sit-and-wait' strategy; however, the frequent alteration of movement paths towards more linear trajectories also suggests the existence of an inducible 'flight response' in this species. Considering the inherent costs of turns associated with non-linear movement, such a strategy may allow ciliates to escape suboptimal environments at a low energetic cost.

Expression of filaments of the Geobacter extracellular cytochrome OmcS in Shewanella oneidensis.

The physiological role of Geobacter sulfurreducens extracellular cytochrome filaments is a matter of debate and the development of proposed electronic device applications of cytochrome filaments awaits methods for large-scale cytochrome nanowire production. Functional studies in G. sulfurreducens are stymied by the broad diversity of redox-active proteins on the outer cell surface and the redundancy and plasticity of extracellular electron transport routes. G. sulfurreducens is a poor chassis for producing cytochrome nanowires for electronics because of its slow, low-yield, anaerobic growth. Here we report that filaments of the G. sulfurreducens cytochrome OmcS can be heterologously expressed in Shewanella oneidensis. Multiple lines of evidence demonstrated that a strain of S. oneidensis, expressing the G. sulfurreducens OmcS gene on a plasmid, localized OmcS on the outer cell surface. Atomic force microscopy revealed filaments with the unique morphology of OmcS filaments emanating from cells. Electron transfer to OmcS appeared to require a functional outer-membrane porin-cytochrome conduit. The results suggest that S. oneidensis, which grows rapidly to high culture densities under aerobic conditions, may be suitable for the development of a chassis for producing cytochrome nanowires for electronics applications and may also be a good model microbe for elucidating cytochrome filament function in anaerobic extracellular electron transfer.

Phytosulfokine alpha enhances regeneration of transformed and untransformed protoplasts of Brassica oleracea.

Phytosulfokine-α (PSK-α) is a disulfated pentapeptide (YIYTQ) acting as an intercellular signal peptide and growth factor. It was originally isolated from conditioned medium of asparagus mesophyll cell cultures in 1996 and later characterized as a hormone-like signal molecule with important roles in numerous processes of in vivo plant growth and development. It is currently becoming a valuable mitogenic factor in plant breeding and biotechnology due to its stimulatory effect on in vitro cell elongation, proliferation and differentiation. The focus of our work was to review current knowledge about the roles of PSK-α in plant biotechnology and to evaluate its influence on the regeneration of protoplasts of four Brassica oleracea cultivars (two cauliflower and two cabbage) cultured under two distinctive protocols and with different protoplast densities. Protoplast regeneration was studied due to its high value for plant genome editing, which is generally limited by the inefficient regeneration of treated protoplasts of numerous important plant genotypes. Our hypothesis was that the stress related to PEG-mediated protoplast transformation and the following decrease in viable protoplast density in culture could be alleviated by the addition of PSK-α to the culture medium. We therefore tested whether PSK-α could increase cell division at the early stages of culture (5 and 15 days after protoplast isolation) and stimulate the formation of microcallus colonies up to the 30st day of culture and to evaluate its influence on callus organogenesis leading to shoot regeneration. The PSK-α showed a strong stimulatory effect on untransformed protoplast regeneration already during the first days of culture, accelerating cell division up to 5.3-fold and the formation of multicellular microcallus colonies up to 37.0-fold. The beneficial influence was retained at later stages of regeneration, when PSK improved shoot organogenesis even if it was present only during the first 10 days of culture. The highest numbers of shoots, however, were regenerated when PSK was present during the first days of culture and later in solid shoot regeneration medium. Finally, the addition of PSK-α to PEG-transformed protoplasts significantly enhanced their division rate and the formation of microcallus colonies in selection media, up to 44.0-fold.

Ascorbic acid predominantly kills cancer stem cell-like cells in the hepatocellular carcinoma cell line Li-7 and is more effective at low cell density and in small spheroids

The development of therapies that target cancer stem cells (CSCs) is an important challenge in cancer research. The antioxidant system is enhanced in CSCs, which may lead to resistance to existing therapies. Ascorbic acid (AA) has the potential to act as both an antioxidant and a pro-oxidant agent, but its effects on CSCs are a subject of current research. Here, we investigated the effect of AA focusing specifically on CSCs with the hepatocellular carcinoma cell line Li-7. The Li-7 cell line is heterogenous consisting of CD166- and CD166+ cells; CD166- cells include CSC-like cells (CD13+CD166- cells) and CD13−CD166- cells that can revert to CD13+CD166- cells. The addition of AA to the culture medium caused cell death in both cell populations in CD166- cells in a concentration dependent manner. In contrast, AA administration had a limited effect on CD166+ non-CSC cells. The level of reactive oxygen species after AA treatment was elevated only in CD166- cells. The effect of AA only occurred at low cell densities in 2D and 3D cultures. In a mouse tumor model injected with Li-7 cells, intraperitoneal administration of AA failed to prevent tumor formation but appeared to delay tumor growth. Our findings shed light on why AA administration has not become a mainstream treatment for cancer treatment; however, they also show the possibility that AA can be used in therapies to suppress CSCs.

Defined co-cultures of glutamatergic and GABAergic neurons with a mutation in DISC1 reveal aberrant phenotypes in GABAergic neurons

BackgroundMutations in the gene DISC1 are associated with increased risk for schizophrenia, bipolar disorder and major depression. The study of mutated DISC1 represents a well-known and comprehensively characterized approach to understand neuropsychiatric disease mechanisms. However, previous studies have mainly used animal models or rather heterogeneous populations of iPSC-derived neurons, generated by undirected differentiation, to study the effects of DISC1 disruption. Since major hypotheses to explain neurodevelopmental, psychiatric disorders rely on altered neuronal connectivity observed in patients, an ideal iPSC-based model requires accurate representation of the structure and complexity of neuronal circuitries. In this study, we made use of an isogenic cell line with a mutation in DISC1 to study neuronal synaptic phenotypes in a culture system comprising a defined ratio of NGN2 and ASCL1/DLX2 (AD2)-transduced neurons, enriched for glutamatergic and GABAergic neurons, respectively, to mimic properties of the cortical microcircuitry.ResultsIn heterozygous DISC1 mutant neurons, we replicated the expected phenotypes including altered neural progenitor proliferation as well as neurite outgrowth, deregulated DISC1-associated signaling pathways, and reduced synaptic densities in cultures composed of glutamatergic neurons. Cultures comprising a defined ratio of NGN2 and AD2 neurons then revealed considerably increased GABAergic synapse densities, which have not been observed in any iPSC-derived model so far. Increased inhibitory synapse densities could be associated with an increased efficiency of GABAergic differentiation, which we observed in AD2-transduced cultures of mutant neurons. Additionally, we found increased neuronal activity in GABAergic neurons through calcium imaging while the activity pattern of glutamatergic neurons remained unchanged.ConclusionsIn conclusion, our results demonstrate phenotypic differences in a co-culture comprising a defined ratio of DISC1 mutant NGN2 and AD2 neurons, as compared to culture models comprising only one neuronal cell type. Altered synapse numbers and neuronal activity imply that DISC1 impacts the excitatory/inhibitory balance in NGN2/AD2 co-cultures, mainly through increased GABAergic input.

Assessment of photosynthetic activity in dense microalgae cultures using oxygen production

Microalgae are photosynthetic microorganisms playing a pivotal role in primary production in aquatic ecosystems, sustaining the entry of carbon in the biosphere. Microalgae have also been recognized as sustainable source of biomass to complement crops. For this objective they are cultivated in photobioreactors or ponds at high cell density to maximize biomass productivity and lower the cost of downstream processes.Photosynthesis depends on light availability, that is often not constant over time. In nature, sunlight fluctuates over diurnal cycles and weather conditions. In high-density microalgae cultures of photobioreactors outdoors, on top of natural variations, microalgae are subjected to further complexity in light exposure. Because of the high-density cells experience self-shading effects that heavily limit light availability in most of the mass culture volume. This limitation strongly affects biomass productivity of industrial microalgae cultivation plants with important implications on economic feasibility.Understanding how photosynthesis responds to cell density is informative to assess functionality in the inhomogeneous light environment of industrial photobioreactors. In this work we exploited a high-sensitivity Clark electrode to measure microalgae photosynthesis and compare cultures with different densities, using Nannochloropsis as model organism. We observed that cell density has a substantial impact on photosynthetic activity, and demonstrated the reduction of the cell's light-absorption capacity by genetic modification is a valuable strategy to increase photosynthetic functionality on a chlorophyll-basis of dense microalgae cultures.

Extraction of chlorophyll a from Tetradesmus obliquus—a method upgrade

Nowadays, the use of algae is prevalent for both industrial and agricultural purposes. The determination of chlorophyll (Chl) content is a commonly used method for estimating the phytoplankton abundance in different water bodies or biomass density of algal cultures. The aim of the present work is to optimise the efficiency of the Chl extraction from the green alga Tetradesmus obliquus using methanol as extracting solvent. The extraction efficiency was estimated by measuring the Chl a concentration of the extracts using fluorescence spectroscopy. To increase the extraction yield, glass fibre filters with algal cells on top were treated with 10% (v/v) formalin prior to the extraction. We found that this pretreatment significantly enhanced the extraction yield of Chl without its chemical decomposition. We also found that the optimal cell concentration for Chl determination ranged from 1.44 × 104 to 3.60 × 105 cells/mL and the extraction efficiency was lower when the cell density of the culture was out of this range. These results highlight the importance of the optimization of the pigment extraction for the studied algal species.

Improvement of Plasmid Volumetric Yield by Addition of Glycerol and Phosphate Buffer in Escherichia coli TOP10 Batch Culture

The investigation of mRNA development has gained substantial interest, particularly in the ex vivo and in vivo therapy. mRNA is widely used for the development of gene editing-based therapies and mRNA vaccines. The aim of this study was to optimize the medium and harvest time to increase plasmid DNA production as part of mRNA production. This study modified used a medium modification approach to achieve high density culture of Escherichia coli TOP10 pGEMT-N in batch cultivation method. Various media formulations were assessed, including LB; LB with phosphate buffer (K2HPO4 12.549 g/L and KH2PO4 2.31 g/L); LB with glycerol (50 g/L); LB with glycerol and phosphate buffer; LB with phosphate buffer, glycerol, glucose (15 g/L), and galactose (15 g/L). The effect of additional carbon sources and phosphate buffer on culture density was measured through OD600 and wet cell weight analysis. The highest OD600 and wet cell weight was observed when LB with glycerol and phosphate buffer was used, with OD600 of 4.78±0.14 and wet cell weight of 36.00±0.63 mg/ml. Plasmid DNA was subsequently isolated from these cultures following 5- and 7.5-hour incubation periods. The utilization of LB medium with glycerol and phosphate buffer resulted in a substantial increase in the volumetric concentration of plasmid DNA of 1,516.97±385.00 ng/ml after 5 hours of incubation. In conclusion, a remarkable enhancement in plasmid DNA volumetric yield within 5 hours was achieved by addition of glycerol and phosphate buffer to LB medium, leading to incubation period.

Effects of bovine ova density and culture supplements on cleavage and blastocyst development rates of in vitro embryos

An optimized in vitro culture system is important to maximize bovine blastocyst development. Four experiments were conducted to explore manipulation of culture environments and investigate the benefits of culturing embryos in groups. Follicles were aspirated from abattoir-derived ovaries and selected oocytes were matured, fertilized, vortexed, and randomly placed in culture groups. Experiment 1 evaluated the effects of ova culture density on blastocyst development among embryos cultured individually or in groups of 5, 10, 20, or 50 embryos. Experiment 2 evaluated the effects of conditioned media from previous replicates on embryo development of the current culture. In Experiment 3, cleaved embryos were grouped together after being in culture for 24 hours. Lastly, in Experiment 4, grouped embryos were placed in 10 μl culture drops to evaluate the effect of high density/low media volume on development. Cleavage and blastocyst rates analyzed via Chi-square indicated that although cleavage rates were similar among culture groups (and experiments), blastocyst development was lower (p &lt; 0.05) in 1 embryo culture group compared to other groups. When conditioned culture media from previous replicates were added to original culture media, blastocyst development was similar among original and conditioned culture groups of 20 and 50 embryos. When cleaved embryos were amalgamated and cultured, blastocyst development was higher (p &lt; 0.05) in culture groups of 10 than 1 cleaved embryo groups but similar to controls for both culture groups. When embryos were cultured in 10 μl drops, embryos cultured in groups of 5 had lower development to blastocysts compared to groups of 2, 10, 25, and control. In conclusion, these data indicated an apparent ‘helper effect’ expressed in culture environments of groups, and this effect apparently occurred after cleavage but before blastocyst development. Direct or indirect role(s) that additional cells have on in vitro culture and the mechanism of this helper effect requires further investigation.

In Vitro Culture of Glochidia and Morphological Changes in Juveniles of the Endangered Freshwater Mussel Solenaia oleivora

The artificial propagation of the endangered Solenaia oleivora, with unknown fish hosts, was performed via in vitro culture with bighead carp, grass carp, common carp, bovine, and rabbit sera. The effects of glochidium density on transformation rates were evaluated, and the development of juveniles that metamorphosed successfully was documented. The control group had a transformation rate of 0 and a contamination rate of 0. No significant differences were found in the transformation and contamination rates of the bighead carp, grass carp, and common carp serum groups, and their transformation rates were significantly higher, and contamination rates were significantly lower than those of the bovine and rabbit serum groups. Moreover, no significant differences were observed in the transformation rates of glochidia (culture density, 2000–5000 glochidia/dish) in contamination-free conditions. Specimen shell length/height increased from 1.08 ± 0.01 on the first day to 3.08 ± 0.29 during the 13th week. On the basis of anterior and posterior growth differences, juvenile growth was divided into the following three phases: the rapid anterior growth period, distinct anterior and posterior idiophase, and rapid posterior growth period. This study not only provides technological support for the artificial propagation of S. oleivora but also lays a foundation for resource recovery.

Probing T-cell activation in nanoliter tumor co-cultures using membrane displacement trap arrays.

Immune responses against cancer are inherently stochastic, with small numbers of individual T cells within a larger ensemble of lymphocytes initiating the molecular cascades that lead to tumor cytotoxicity. A potential source of this intra-tumor variability is the differential ability of immune cells to respond to tumor cells. Classical microwell co-cultures of T cells and tumor cells are inadequate for reliably culturing and analyzing low cell numbers needed to probe this variability, and have failed in recapitulating the heterogeneous small domains observed in tumors. Here we leverage a membrane displacement trap array technology that overcomes limitations of conventional microwell plates for immunodynamic studies. The microfluidic platform supports on-demand formation of dense nanowell cultures under continuous perfusion reflecting the tumor microenvironment, with real-time monitoring of T cell proliferation and activation within each nanowell. The system enables selective ejection of cells for profiling by fluorescence activated cell sorting, allowing observed on-chip variability in immune response to be correlated with off-chip quantification of T cell activation. The technology offers new potential for probing the molecular origins of T cell heterogeneity and identifying specific cell phenotypes responsible for initiating and propagating immune cascades within tumors. Insight Box Variability in T cell activation plays a critical role in the immune response against cancer. New tools are needed to unravel the mechanisms that drive successful anti-tumor immune response, and to support the development of novel immunotherapies utilizing rare T cell phenotypes that promote effective immune surveillance. To this end, we present a microfluidic cell culture platform capable of probing differential T cell activation in an array of nanoliter-scale wells coupled with off-chip cell analysis, enabling a high resolution view of variable immune response within tumor / T cell co-cultures containing cell ensembles orders of magnitude smaller than conventional well plate studies.