| Sow production is the foundation of pig breeding industry,and improving the reproductive performance of sows is one of the keys to improve the efficiency of pig breeding.Embryo attachment is the process of blastocyst implantation into the maternal uterus,which is a key stage affecting the reproductive performance of sows.At present,studies on embryo implantation in pigs mainly use live animals as models,which have problems such as long cycle time,high cost,low efficiency and difficulty in dynamic observation of maternal-fetal intercourse.Therefore,there is an urgent need for new technical methods to realize in vitro simulation of porcine uterus structure and function,and to provide physiologically relevant,accurate and controllable in vitro models for porcine embryo attachment studies.Organ-on-a-chip is a bionic system that simulates the structure and function of animal organs on a microfluidic chip,mainly through precise microstructure design,cell manipulation and microenvironment control to reproduce the three-dimensional structure,cellular composition and physiological functions of organs in vitro.In view of this,this study constructs a microfluidic chip containing a three-dimensional gel scaffold to support and guide the growth and arrangement of porcine endometrial epithelial cells and vascular endothelial cells,and establishes a porcine in vitro uterine chip model with a uterine gland-like structure,containing three-dimensional microvasculature,and a perfusable uterine cavity,using the physiological structure and cellular composition of the porcine endometrium as a reference.The porcine uterine microarray model constructed in this study can provide an innovative in vitro platform and research ideas based on organ level for early embryo attachment,endometrial tolerance regulation in sows,and porcine embryo development.The main results of this study are as follows:Experiment 1:Design and preparation of porcine uterine microarraysIn this study,using the physiological structure and cell composition of porcine endometrium as a reference,we constructed a microfluidic chip containing a 3D hydrogel scaffold using soft lithography and 3D printing technology,and optimized the surface modification and collagen solidification time of the chip.The results showed that the 3D hydrogel scaffold has the best stability and can support the subsequent long-term cell culture when using PEI-GA to co-modify the gel pool of the chip body and the collagen solidification time of 30 min.Experiment 2:Culture and characterization of endometrial epithelial cells in porcine uterine microarraysIn the uterine microarrays constructed in this study,PEEC were inoculated on the surface of the 3D hydrogel scaffold and grown adductively.The effects of different modification conditions on the hydrogel surface on the proliferation and apposition status of PEEC were investigated by cell counting and CK18 immunofluorescence staining,and the continuous culture time of PEEC in the chip was characterized.And the m RNA expression levels of cell adhesion-related genes E-cadherin,luminal epithelial marker genes STC1 and IGFBP2 were compared by RT-q PCR in PEEC in well plates,in microarrays alone and in co-cultured microarrays.The results showed that:（1）The best PEEC culture results were obtained by using m Tg（4%）cross-linking technique to modify type IV collagen（10 mg/m L）on the gel surface.（2）The PEEC in the microarray maintained good growth for 9 days in culture,which could meet the requirements of subsequent porcine embryo attachment studies.（3）PEEC co-cultured with PIEC in the microarray were more tightly connected and had the best growth condition,and the traits of PEEC in co-culture with PIEC in the microarray were more similar to those of luminal epithelium,which was advantageous in the study of embryo attachment process.Experiment 3:Construction and characterization of 3D microvessels in uterine microarraysIn the present study,PIEC were inoculated in hollow lumen channels and grown on the gel surface inside the lumen to achieve in vitro simulation of the three-dimensional microvascular structure of porcine endometrium.Laser confocal fluorescence imaging results showed that PIEC could grow uniformly and densely inside the lumen to form a 3D vascular-like structure.Microvascular permeability was examined using 1 mg/m L of 40k Da FITC-dextran dye passed into the lumen to characterize its barrier function.The results showed that microvessels formed by two-sided implantation of PIEC had permeability coefficients close to those of real blood vessels in vitro(～6×10-6 cm/s)at day 9,while those formed by four-sided implantation reached permeability coefficients close to those of real blood vessels in vitro(～5×10-6 cm/s)by day 3 of incubation.The four-sided implantation method is more conducive to the formation of microvascular barrier function and the maintenance of cellular activity.Experiment 4:Embryonic trophoblast cell adhesion study based on porcine uterine microarrayIn this study,we investigated the effect of maternal cell environment on the attachment process of porcine embryos by comparing the adhesion rate of PTC in porcine uterine microarrays containing different cell types.Hoechst-stained PTC were inoculated into the central chamber of the uterine microarrays and washed after 3 h.The PTC in the microarrays after inoculation and after washing were counted,and the adhesion rate of PTC in the uterine microarrays was calculated.The results showed that PTC had a high adhesion rate in the chip cultured with PEEC,which was consistent with the binding of embryonic trophoblast cells to the endometrial epithelium during the real attachment process,and co-culture of PIEC could further promote the adhesion of PTC on PEEC monolayer. |
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