Developing a reproducible protocol for culturing functional confluent monolayers of differentiated equine oviduct epithelial cells

We describe the development of two methods for obtaining confluent monolayers of polarized, differentiated equine oviduct epithelial cells (EOEC) in Transwell inserts and microfluidic chips. EOECs from the ampulla were isolated post-mortem and seeded either (1) directly onto a microporous membrane as differentiated EOECs (direct seeding protocol) or (2) first cultured to a confluent de-differentiated monolayer in conventional wells, then trypsinized and seeded onto a microporous membrane (re-differentiation protocol). Maintenance or induction of EOEC differentiation in these systems was achieved by air-liquid interface introduction. Monolayers cultured via both protocols were characterized by columnar, cytokeratin 19-positive EOECs in Transwell inserts. However, only the re-differentiation protocol could be transferred successfully to the microfluidic chips. Integrity of the monolayers was confirmed by transepithelial resistance measurements, tracer flux and the demonstration of an intimate network of tight junctions. Using the direct protocol, 28% of EOECs showed secondary cilia at the apical surface in a diffuse pattern. In contrast, re-differentiated polarized EOECs rarely showed secondary cilia in either culture system (>90% of the monolayers showed <1% ciliated EOECs). Occasionally (5–10%), re-differentiated monolayers with 11–27% EOECs with secondary cilia in a diffuse pattern were obtained. Additionally, nuclear progesterone receptor expression was found to be inhibited by simulated luteal phase hormone concentrations, and sperm binding to cilia was higher for re-differentiated EOEC monolayers exposed to estrogen-progesterone concentrations mimicking the follicular rather than luteal phase. Overall, a functional equine oviduct model was established with close morphological resemblance to in vivo oviduct epithelium.

Research and Application of Evaluation Methods for Functional Characteristics of Oil-Based Drilling Fluid in Shale Gas Wells

Intelligent unconventional reservoir optimal production control technology is a comprehensive technology, involving geology, reservoir simulation, and efficient drilling and completion. Efficient drilling and completion provides a flow channel for unconventional oil and gas exploitation and a wellbore with good integrity for reservoir transformation, which is an important link in the efficient development of unconventional oil and gas. The application of industry standard method to evaluate the performance of oil-based drilling fluid has the problem of poor correlation. It cannot reflect the difference of performance among oil-based drilling fluid systems, which lacks the significance for field construction. Based on shale expansion, rolling dispersion experiment, and microporous membrane filtration loss test, the physicochemical mechanism of borehole wall instability in shale formation was investigated. The evaluation methods of shale lubrication, antiaccretion test, slake durability, buck hardness test, etc. are put forward, and the formula of oil-based drilling fluid is optimized. The lubrication and antiaccretion experiment method can effectively and intuitively characterize the cleaning and lubrication effect of drilling fluid on drilling tools. The slake durability evaluation method simulates the collision between drill cuttings and the drill string and well wall. The bucking hardness experiment is through testing the cuttings and the hardness change after drilling fluid action reflects its inhibitory effect. The new methods were used to evaluate the oil-based drilling fluid used in 4 wells in the Changning block. It was found that the drilling fluid of CN209H2 well adhered to the steel column with at least 0.41 g of cuttings; the recovery rate of the drilling fluid resistance of CN209H1 was up to 87.70%, and YX1200 oil-based drilling fluid plugging agent was selected through the microporous membrane experiment. In the process of drilling the well CN209H5, the new oil-based drilling fluid formulation improved the lubrication performance by 44%, accompanied by 95.48% recovery rate and less than 10 mL HTHP fluid loss at the same time. The research results show that the oil-based drilling fluid system optimized according to the new method can significantly inhibit shale hydration and dispersion and can effectively solve the problem of unstable performance of traditional oil-based drilling fluids in the Changning block.

Effects of different fertilization rates on growth, yield, quality and partial factor productivity of tomato under non-pressure gravity irrigation

To select the optimum fertilizer application under specific irrigation levels and to provide a reliable fertigation system for tomato plants, an experiment was conducted by using a microporous membrane for water-fertilizer integration under non-pressure gravity. A compound fertilizer (N:P2O5:K2O, 18:7:20) was adopted for topdressing at four levels, 1290 kg/ha, 1140 kg/ha, 990 kg/ha, and 840 kg/ha, and the locally recommended level of 1875 kg/ha was used as the control to explore the effects of different fertilizer application rates on growth, nutrient distribution, quality, yield, and partial factor of productivity (PFP) in tomato. The new regime of microporous membrane water-fertilizer integration under non-pressure gravity irrigation reduced the fertilizer application rate while promoting plant growth in the early and intermediate stages. Except for the 990 kg/ha fertilizer treatment, yields per plant and per plot for each fertilizer application rate were higher than or equal to those of the control. The new regime could effectively improve PFP and reduce soil nutrient enrichment. Fertilizer at 840 kg/ha showed the optimum results by increasing PFP by 75.72% as compared to control. In conclusion, the fertilizer rate at 840 kg/ha has not only maintained the productivity of soil but also tomato growth and quality of fruit which makes the non-pressure gravity irrigation a potential and cost-effective way for fertilizer application.

Large-scale investigation of single cell activities and response dynamics in a microarray chip with microfluidics-fabricated microporous membrane

Microengineering technology involving microfabrication, micropatterning and microfluidics enables promising advances in single cell manipulation and analysis. Herein, we decribe a parallel, large-scale, and temporal investigation of diverse single cell activities...