Culture Vessel Research Articles

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.

Advancement in Plant Tissue Culture-Based Research for Sustainable Exploitation of Well-Known Medicinal Herb <i>Bacopa Monnieri</i>

The current review focuses on the plant Bacopa monnieri, one of prominent medical herbs in Indian ayurvedic system. The plant is well known for its cognitive and memory enhancing capabilities. The plant contains many useful alkaloids and secondary metabolites. Studies have shown that it has various promising pharmacological properties which have the potential to treat many illnesses and disorders such as asthma, bronchitis, rheumatism also in for renal disease, water retention, blood cleaning etc. This leads to the over exploitation of the plant which puts a stress on the naturally available stock of the plant, therefore, it becomes a necessity to find optimum methods for mass production of the plant and its important secondary metabolites. This review attempts to compile and to discuss the advancements in methods and techniques including type of culture vessels, plant growth regulators (PGRs), effect of stress, plant growth promoting rhizobacteria (PGPRs) interactions; for in vitro propagation of Bacopa monnieri and the enhanced production of its important bioactive (bacoside) for its sustainable exploitation.

Recent advances in upstream process development for production of recombinant adeno-associated virus.

Recombinant adeno-associated virus (rAAV) is rapidly emerging as the preferred delivery vehicle for gene therapies, with promising advantages in safety and efficacy. Key challenges in systemic in-vivo rAAV gene therapy applications are the gap in production capabilities versus potential market demand and complex production process. This review summarizes currentavailable information on rAAV upstream manufacturing processes and proposed optimizations for production. The advancements in rAAV production media were reviewed with proposals to speed up the cell culture process development. Furthermore, major methods for genetic element delivery to host cells were summarized with their advantages, limitations, and future directions for optimization. In addition, culture vessel selection criteria were listed based on production cell system, scale, and development stage. Process control at the production step was also outlined with an in-depth understanding of production kinetics and quality control.

Investigation and evaluation of a 3D-printed optical modified cultivation vessel for improved scattered light measurement of biotechnologically relevant organisms.

In the field of bioprocess development miniaturization, parallelization and flexibility play a key role reducing costs and time. To precisely meet these requirements, additive manufacturing (3D-printing) is an ideal technology. 3D-printing enables rapid prototyping and cost-effective fabrication of individually designed devices with complex geometries on demand. For successful bioprocess development, monitoring of process-relevant parameters, such as pH, dissolved oxygen (DO), and biomass, is crucial. Online monitoring is preferred as offline sampling is time-consuming and leads to loss of information. In this study, 3D-printed cultivation vessels with optical prisms are evaluated for the use in upstream processes of different industrially relevant microorganisms and cell lines. It was shown, that the 3D-printed optically modified well (OMW) is of benefit for a wide range of biotechnologically relevant microorganisms and even for mammalian suspension cells. Evaluation tests with Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Chinese hamster ovary (CHO) cells were performed, providing highly reproducible results. Growth behavior of OMW cultures was comparable to behavior of shake flask (SF) cultivations and the signal to noise ratio in online biomass measurement was shown to be reduced up to 95.8% by using the OMW. Especially the cultivation phases with low turbidity respective optical densities below 1.0rel.AU could be monitored accurately for the first time. Furthermore, it was demonstrated that the 3D-printed optics are transferable to different well geometries and sizes, enabling efficient biomass monitoring for individual requirements with tailor-made 3D-printed cultivation vessels in small scale.

COMPARATIVE STUDY OF THE INFLUENCE OF BREWERY EFFLUENT ON THE GROWTH OF TWO MARINE MICROALGAE

A growing global trend is the use of microalgae for the treatment of industrial effluents due to their capacity to consume contaminants, sequester carbon, and provide biomass for high-value products. Thalassiosira sp. and Chaetoceros gracilis, two marine microalgae, are the subjects of this study, which seeks to ascertain the effect of brewery effluent on their growth. The test microalgae were grown in seven culture vessels which were in triplicates and different concentrations; control (without effluent), 5, 10, 15, 20, 25, and 30% of brewery effluent. On a visible spectrophotometer, optical density at 750 nm was used to quantify growth responses, and several physicochemical variables were studied at the start and end using standard methods. Using Microsoft Excel 2010 and the social sciences statistical tool SPSS20, descriptive statistics, inferential (one way analysis of variance) ANOVA repeated measures, paired t-tests, and Tukey tests were performed. The findings revealed a significant difference in Thalassiosira sp. and Chaetoceros gracilis with growth response (p < 0.05). The growth of Thalassiosira sp. was stimulated by different concentrations of brewery effluent, Tukey test showed that 20, 25 and 30 % concentrations had higher mean differences, followed by 15 and 10% concentrations while, there was no mean difference between the control and 5% concentration. While the results for Thalassiosira sp. showed that, 25 and 30 % concentrations had higher mean differences, followed by 10, 15 and 20% concentrations. There was no mean difference between the control and 5% concentration. The results of physicochemical variables indicated that only TOC and COD of brewery effluent fell at lower concentrations and stayed constant at higher concentraions, a decrease in temperature was seen across all concentrations. Thalassiosira sp. had more stimulatory effect than Chaetoceros gracilis making it a better option in the bioremediation of brewery wastewater when applied in higher concentrations.

Reducing Cancer Cell Adhesion using Microtextured Surfaces.

For the past century, trypsin has been the primary method of cell dissociation, largely without any major changes to the process. Enzymatic cell detachment strategies for large-scale cell culturing processes are popular but can be labor-intensive, potentially lead to the accumulation of genetic mutations, and produce large quantities of liquid waste. Therefore, engineering surfaces to lower cell adhesion strength could enable the next generation of cell culture surfaces for delicate primary cells and automated, high-throughput workflows. In this study, a process for creating microtextured polystyrene (PS) surfaces to measure the impact of microposts on the adhesion strength of cells is developed. Cell viability and proliferation assays show comparable results in twocancer cell lines between micropost surfaces and standard cell culture vessels. However, cell image analysis on microposts reveals that cell area decreases by half, and leads to an average twofold increase in cell length per area. Using a microfluidic-based method up to a seven times greater percentage of cells are removed from micropost surfaces than the flat control surfaces. These results show that micropost surfaces enable decreased cell adhesion strength while maintaining similar cell viabilities and proliferation as compared to flat PS surfaces.

Development of a bioreactor for in-vitro compression cycling of tissue engineered meniscal implants

Injuries to the meniscus are common and can impair physical activities. Bioprinted meniscal tissue offers an attractive alternative to donor tissue for meniscal repair but achieving the strength of native tissue is a challenge. Here we report the development of a tissue engineering bioreactor designed to apply repetitive force which may lead to an increase in the compressive modulus and durability of bioprinted meniscal tissues. The modular bioreactor system is composed of a sterilizable tissue culture vessel together with a dock that applies and measures mechanical force. The culture vessel allows for simultaneous compression cycling of two anatomically sized menisci. Using a hybrid linear actuator with a stepper motor, the dock can apply up to 300 N of force at speeds up to 20 mm/s, corresponding to the upper limits of anatomical force and motion in the knee. An interchangeable 22 N load cell was mated between the culture vessel and the dock to log changes in force. Both the culture vessel and dock are maintained in a standard cell culture incubator to provide heat and CO2, while the dock is powered and controlled externally using a step motor drive and customized software.

Clinorotation inhibits myotube formation by fluid motion, not by simulated microgravity

To study processes related to weightlessness in ground-based cell biological research, a theoretically assumed microgravity environment is typically simulated using a clinostat – a small laboratory device that rotates cell culture vessels with the aim of averaging out the vector of gravitational forces. Here, we report that the rotational movement during fast clinorotation induces complex fluid motions in the cell culture vessel, which can trigger unintended cellular responses. Specifically, we demonstrate that suppression of myotube formation by 2D-clinorotation at 60 rpm is not an effect of the assumed microgravity but instead is a consequence of fluid motion. Therefore, cell biological results from fast clinorotation cannot be attributed to microgravity unless alternative explanations have been rigorously tested and ruled out. We consider two control experiments mandatory, i) a static, non-rotating control, and ii) a control for fluid motion. These control experiments are also highly recommended for other rotation speed settings and experimental conditions. Finally, we discuss strategies to minimize fluid motion in clinorotation experiments.

Comparison of THP-1 Macrophages Viability in Different Types of Culture Vessel

The ox-LDL generated apoptotic bodies using THP-1 macrophage is a useful tool to study foam cell formation in atherosclerosis. However, the common problem is the cells in the negative control (i.e., absence of ox-LDL) undergo apoptosis. Therefore, the type of cell culture vessel was proposed to be the key factor contributing to cell apoptosis. The THP-1 cells were differentiated into M1 macrophages using 10 ng/μL PMA, 5 ng/μL LPS, and 20 ng/μL IFN-? while 5 ng/μL PMA, 20 ng/μL IL-4 and 20 ng/μL IL-13 were used to differentiate THP-1 into M2 macrophages. Two types of cell culture vessels (6-well plate and T25 flask) were used to culture the macrophages. The cells were subsequently stained using Annexin V-FITC and Propidium Iodide prior to flow cytometry analysis. Interestingly, both M1 and M2 macrophages cultured in the T25 flask resulted in a significantly higher percentage of cell viability compared to macrophages cultured in 6-well plate [M1: 84.15% ± 4.39 vs 8.02% ± 1.55, p < 0.0001; M2: 95.95% ± 1.74 vs 10.50% ± 0.05, p < 0.0001]. In summary, the type of culture vessel is a vital factor in determining cell viability attributed to the surface area and cell seeding density in different types of vessels.

Investigation of the production and dietary features of oyster mushrooms for a planned lunar farm

In our previous work, we organized a project mainly to design a lunar mushroom farm. In this work, we proceeded to study the features of the production and consumption of oyster mushrooms in that project. Oyster mushrooms were grown in cultivation vessels containing a sterilized substrate. The fruit yield and mass of the spent substrate in the cultivation vessels were measured. A three-factor experiment was carried out with the subsequent application of the steep ascent method and correlation analysis in the R program. These factors included the density of the substrate in the cultivation vessel, its volume, and the number of harvesting flushes. The data obtained was used to calculate the process parameters: productivity, speed and degree of substrate decomposition, and biological efficiency. The consumption and dietary features of oyster mushrooms were modeled in Excel using the Solver Add-in. In the three-factor experiment, the highest productivity amounting to 272 g of fresh fruiting bodies/(m3*day) was obtained with a substrate density of 500 g/L, a cultivation vessel volume of 3 L, and two harvest flushes. The application of the method of steep ascent showed that it is possible to increase the productivity by increasing the substrate density and reducing the volume of the cultivation vessel. In production, there is a need to tally the substrate decomposition speed with the substrate decomposition degree and the biological efficiency of growing oyster mushrooms, since these process parameters have a negative correlation. Most of the nitrogen and phosphorus passed from the substrate into the fruiting bodies. These biogenic elements could limit the yield of oyster mushrooms. It is safe to set the daily intake of oyster mushrooms at 100–200 g while maintaining the antioxidant capacity of the food set.

Structural alterations of Cymbopogon citratus (DC.) Stapf leaves and roots caused by silicon nanoparticles during in vitro propagation

Cymbopogon citratus (DC.) Stapf (lemongrass or citronella) is a herb of therapeutic potential highly sought by various industries due to the presence of essential oil. In vitro propagation of commercial crops is highly desirable; it is an alternative approach for the production of quality planting materials. But the plantlets encounter certain morpho-structural and physiological disorders under in vitro conditions resulting in suppressed growth and reduced survival. The present study aimed to analyze the effect of silicon nanoparticles (SiNPs) on the in vitro morphometric and structural developments, rooting, and acclimatization efficiency in C. citratus. Murashige and Skoog (MS) medium supplemented with 4.0 mg/L of 6-benzylaminopurine (BAP) was effective for bud breaking. The shoot proliferation was further accelerated with the addition of 0.25 mg/L NAA (α-naphthalene acetic acid) in subsequent subcultures (after 3rd subculture, 21.0 shoots with 4.9 cm length per explants per culture vessel). Such shoots were characterized by thin leaf blades, reduced leaf area, and shoot tip necrosis, which negatively affected the life of cultures, ex vitro rooting, and acclimatization efficiency. Microscopic evaluation of those leaves showed underdeveloped stomata, epidermis, and reduced photosynthetic and vascular tissues. The incorporation of optimal concentration of SiNPs (40 mg/L) with the optimized growth regulators (4.0 mg/L BAP + 0.25 mg/L NAA) significantly increased shoot proliferation (38.0), shoot length (6.4 cm), robust leaf, and biomass (5.2 g fresh weight; 1.9 g dry weight). Shoot tip necrosis was reversed by the addition of SiNPs, and the developed shoots were morphologically thick with well-developed tissue systems. These C. citratus shoots performed better during ex vitro rooting (100% rooting, 8.0 roots/shoot) when treated with indole-3-butyric acid (IBA) at the concentration of 200 mg/L and acclimatization.

Abstract 1785: Multi-step engineering of gene-edited CAR T cells using RNA lipid nanoparticles

Abstract Autologous chimeric antigen receptor (CAR) T therapies utilize patient cells and can be limited by cell quality, and the high manufacturing burden of viral vectors. As such, there is a need for allogeneic, “off-the-shelf” CAR T cells to make these transformative treatments widely available. However, allogeneic therapies require multiple genetic engineering steps to express CAR and to delete proteins responsible for graft-versus-host disease. Messenger RNA (mRNA) is a promising approach for expression of therapeutic proteins and gene editing nucleases. In this work, we demonstrate a new method for multi-step engineering of gene-edited CAR T cells using RNA lipid nanoparticles (LNPs). LNPs encapsulating Spy-Cas9 mRNA, TCR and CD52 guide RNA (sgRNA), and CAR mRNA were produced using microfluidics. The CAR construct contained an anti-CD19 scFv binding domain and CD3ζ/4-1BB co-stimulatory domains. Microgram quantities of RNA LNPs were produced to optimize LNP packaging, cargo ratios, and sgRNA combinations. Lead candidates were scaled to milligrams. Purified human primary T cells were cultured, activated, and expanded in serum-free media in plates, flasks and bioreactors. CAR+, TCR− or CD52− cells were generated by addition of the corresponding LNP to activated cells. Cytotoxic killing was determined by co-culture assays with leukemia cells. Gene knockout, CAR expression, viability and cell killing were measured using flow-cytometry. CD19 CAR was selected as a relevant protein for expression, with TCR and CD52 proteins as gene knockout targets. Single-step addition of CAR LNPs to T cells resulted in transfection efficiencies of 95.0 ± 2.1% and high protein expression. Upon TCR or CD52 LNP addition to T cells, the onset of gene editing was within 48 hours, reaching single target knockout efficiencies of 92.3 ± 3.0% (TCR−), and double knockouts (TCR−/CD52−) of 74.5 ± 6.1%. Similar results were obtained when comparing different LNP batch sizes (microgram to milligram RNA) and cell culture vessels (125,000 to 45 million cells), demonstrating scalability of both the LNP production and cell treatment. Cell viabilities above 90% were maintained at all steps and for all RNA LNPs. Finally, as proof-of-concept for multi-step engineering, sequential addition of TCR LNPs and CAR LNPs resulted in simultaneous CAR expression and TCR gene knockout. These “off-the-shelf” gene-edited CAR T cells were functionally equivalent to non-edited cells in a B cell killing assay, efficiently clearing over 80% of leukemia target cells at a 1:1 ratio. Our findings demonstrate the advantages of LNPs for RNA delivery to T cells. The simple and gentle nature of LNP cell treatment allows for multiple genetic engineering steps for simultaneous expression and deletion of proteins. Furthermore, LNPs can be easily manufactured using microfluidics, enabling small-scale screening of RNA libraries and rapid scale-up of lead candidates for clinical translation. Citation Format: Samuel Clarke, R Geczy, A Balgi, S Park, R Zhao, M Swaminathan, R Tieu, N Hoang, C Webb, E Watt, M Wong, M Fujisawa, N Jain, Angela Zhang, Anitha Thomas. Multi-step engineering of gene-edited CAR T cells using RNA lipid nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1785.

Transcriptomic and proteomic studies suggest the establishment of advanced zonation-like profiles in human-induced pluripotent stem cell-derived liver sinusoidal endothelial cells and carboxypeptidase M-positive liver progenitor cells cocultured in a fluidic microenvironment.

Hepatic zonation is a physiological feature of the liver, known to be key in the regulation of the metabolism of nutrients and xenobiotics and the biotransformation of numerous substances. However, the reproduction of this phenomenon remains challenging in vitro as only part of the processes involved in the orchestration and maintenance of zonation are fully understood. The recent advances in organ-on-chip technologies, which allow for the integration of multicellular 3D tissues in a dynamic microenvironment, could offer solutions for the reproduction of zonation within a single culture vessel. An in-depth analysis of zonation-related mechanisms observed during the coculture of human-induced pluripotent stem cell (hiPSC)-derived carboxypeptidase M-positive liver progenitor cells and hiPSC-derived liver sinusoidal endothelial cells within a microfluidic biochip was carried out. Hepatic phenotypes were confirmed in terms of albumin secretion, glycogen storage, CYP450 activity, and expression of specific endothelial markers such as PECAM1, RAB5A, and CD109. Further characterization of the patterns observed in the comparison of the transcription factor motif activities, the transcriptomic signature, and the proteomic profile expressed at the inlet and the outlet of the microfluidic biochip confirmed the presence of zonation-like phenomena within the biochips. In particular, differences related to Wnt/β-catenin, transforming growth factor-β, mammalian target of rapamycin, hypoxia-inducible factor-1, and AMP-activated protein kinase signaling, to the metabolism of lipids, and cellular remolding were observed. The present study shows the interest in combining cocultures of hiPSC-derived cellular models and microfluidic technologies for reproducing in vitro complex mechanisms such as liver zonation and further incites the use of those solutions for accurate reproduction of in vivo situations.

A Comparison of Two Media Formulations and Two Vented Culture Vessels for Shoot Multiplication and Rooting of Hemp Shoot Tip Cultures

Micropropagation of hemp (Cannabis sativa) is constrained by problems with hyperhydricity and culture decline of microshoots. These problems can be reduced by increasing agar and nutrients in the media during micropropagation stages 1 and 2, respectfully. Performance of microshoots of ‘Abacus’ and ‘Wife’ hemp cultured in Driver and Kuniyuki Walnut medium (DKW) for 15 weeks (6 weeks of stage 1 + 9 weeks of stage 2), with subculturing every 3 weeks during both stages 1 and 2, or in Murashige and Skoog with vitamins medium (MS) for 6 weeks (stage 1) followed by Lubell-Brand Cannabis medium (LBC) for 9 weeks (stage 2), with subculturing every 3 weeks during both stages 1 and 2, was evaluated. In a separate study, microshoot performance of ‘Abacus’ and ‘Wife’ in MS for 3 weeks (stage 1) followed by LBC for 6 weeks (stage 2), with subculturing every 3 weeks, using boxes (Magenta GA-7) with lids featuring a vent with a diameter of 10 mm and a pore size of 0.2 µM or using microboxes (Sac O2 O95/114 + OD95) with lids featuring a filter (Sac O2 #10) were evaluated. Shoot multiplication rate (SMR) and explant height were greater for ‘Abacus’ in LBC than DKW. For ‘Wife’, SMR at 9 weeks was greater in LBC, as LBC provided more nutrients and water than cultures had received in MS initially during stage 1. Culture medium did not influence ex vitro rooting success, which was 75% for ‘Abacus’ and ≥ 90% for ‘Wife’. Microboxes resulted in greater hyperhydricity of shoots and a lower ex vitro rooting percentage than boxes. For cultivars that are highly prone to developing hyperhydricity, like ‘Abacus’, the microboxes were not adequate to control this condition.

Research on the Isobaric Transfer Method and Device for Sediment of Full-Ocean Depth

Abstract In this study, a sediment isobaric transfer device based on the principle of volume displacement was built, and the work process of the device was explained. A physical model was built in hydraulic analysis software AMESim to simulate the actual working conditions. The influences of pre-charge pressure and volume of accumulator, diameter, and length of the hydraulic pipeline and opening pressure of safety valve on the pressure fluctuation and pressure steady-time during transfer process were studied. The transfer system was optimally designed based on the results of parametric analysis. The sea trial verified that the system could transfer sediment with a pressure exceeding 90 MPa into a culture vessel with low-pressure fluctuating pressure.

Case Studies in the Assessment of Microbial Fitness: Seemingly Subtle Changes Can Have Major Effects on Phenotypic Outcomes

Following the completion of an adaptive evolution experiment, fitness evaluations are routinely conducted to assess the magnitude of adaptation. In doing so, proper consideration should be given when determining the appropriate methods as trade-offs may exist between accuracy and throughput. Here, we present three instances in which small changes in the framework or execution of fitness evaluations significantly impacted the outcomes. The first case illustrates that discrepancies in fitness conclusions can arise depending on the approach to evaluating fitness, the culture vessel used, and the sampling method. The second case reveals that variations in environmental conditions can occur associated with culture vessel material. Specifically, these subtle changes can greatly affect microbial physiology leading to changes in the culture pH and distorting fitness measurements. Finally, the last case reports that heterogeneity in CFU formation time can result in inaccurate fitness conclusions. Based on each case, considerations and recommendations are presented for future adaptive evolution experiments.

LIFTOSCOPE: development of an automated AI-based module for time-effective and contactless analysis and isolation of cells in microtiter plates

BackgroundThe cultivation, analysis, and isolation of single cells or cell cultures are fundamental to modern biological and medical processes. The novel LIFTOSCOPE technology aims to integrate analysis and isolation into one versatile, fully automated device.MethodsLIFTOSCOPE’s three core technologies are high-speed microscopy for rapid full-surface imaging of cell culture vessels, AI-based semantic segmentation of microscope images for localization and evaluation of cells, and laser-induced forward transfer (LIFT) for contact-free isolation of cells and cell clusters. LIFT transfers cells from a standard microtiter plate (MTP) across an air gap to a receiver plate, from where they can be further cultivated. The LIFT laser is integrated into the optical path of an inverse microscope, allowing to switch quickly between microscopic observation and cell transfer.ResultsTests of the individual process steps prove the feasibility of the concept. A prototype setup shows the compatibility of the microscope stage with the LIFT laser. A specifically designed MTP adapter to hold a receiver plate has been designed and successfully used for material transfers. A suitable AI algorithm has been found for cell selection.ConclusionLIFTOSCOPE speeds up cell cultivation and analysis with a target process time of 10 minutes, which can be achieved if the cell transfer is sped up using a more efficient path-finding algorithm. Some challenges remain, like finding a suitable cell transfer medium.SignificanceThe LIFTOSCOPE system can be used to extend existing cell cultivation systems and microscopes for fully automated biotechnological applications.

Constant and Intermittent Contact with the Volatile Organic Compounds of Serendipita indica Alleviate Salt Stress In Vitro Ocimum basilicum L.

Serendipita indica is a plant growth-promoting fungus. It is a natural soil dweller that can colonize the roots of a wide range of plants, including cultivated crops. S. indica has been reported to improve plant nutrient uptake and increase stress tolerance when inoculated into the soil. The present study was undertaken to study the effect of volatile organic compounds (VOCs) of S. indica on salt-stressed Ocimum basilicum ‘Fin vert’ in vitro, either in a culture vessel with a semi-solid medium or via a modified temporary immersion bioreactor system (SETIS). For all salt concentrations, VOCs of S. indica significantly improved plant growth in both semi-solid medium and SETIS bioreactors. This resulted in heavier and taller plants, more shoots per plant, and longer roots. This was even observed for the control without salt. At 9 g/L NaCl, plants with Serendipita were able to give longer roots than those without (1.2 cm vs. 0.0 and 1.7 cm vs. 1.7 cm) in the semi-solid medium and SETIS, respectively. Nevertheless, the VOCs were not able to make the plant salt tolerant to this high concentration. The increase in total phenolic and flavonoid content and radical scavenging suggest that the antioxidant defense system is triggered by the S. indica VOCs. In the semi-solid system, without VOCs, 1 g/L NaCl led to an increase in total chlorophyll content (TCC) and a significant decrease in TCC was further measured only at 6 g/L NaCl or more. However, when VOCs were added, the bleaching effect of the salt was partially restored, even at 6 and 9 g/L NaCl. A significant decrease in TCC was also measured in the SETIS system at 6 g/L NaCl or more and treatment with VOC did not make any difference. An exception was 9 g/L, where the VOC-treated plants produced more than three times more chlorophyll than the non-treated plants. These findings will encourage the application of Serendipita indica for stress reduction. In addition, the proposed original adaptation of a temporary immersion system will be instrumental to investigate stress reduction associated with volatile compounds and better understand their mechanism of action.

Serial Cultivation of an MSC-Like Cell Line with Enzyme-Free Passaging Using a Microporous Titanium Scaffold.

In vitro studies on adherent cells require a process of passage to dissociate the cells from the culture substrate using enzymes or other chemical agents to maintain cellular activity. However, these proteolytic enzymes have a negative influence on the viability and phenotype of cells. The mesenchymal stem cell (MSC)-like cell line, C3H10T1/2, adhered, migrated, and proliferated to the same extent on newly designed microporous titanium (Ti) membrane and conventional culture dish, and spontaneous transfer to another substrate without enzymatic or chemical dissociation was achieved. The present study pierced a 10 μm-thick pure Ti sheet with 25 μm square holes at 75 μm intervals to create a dense porous structure with biomimetic topography. The pathway of machined holes allowed the cells to access both sides of the membrane frequently. In a culture with Ti membranes stacked above- and below-seeded cells, cell migration between the neighboring membranes was confirmed using the through-holes of the membrane and contact between the membranes as migration routes. Furthermore, the cells on each membrane migrated onto the conventional culture vessel. Therefore, a cell culture system with enzyme-free passaging was developed.