{"product_id":"porcine-intestine-organoid-culture-abs9890","title":"Porcine Intestine Organoid  Culture","description":"\u003ch4\u003eProduct Specification\u003c\/h4\u003e\u003cdiv class=\"responsive-table product-detail-table details-table\"\u003e\u003ctable\u003e\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u003cstrong\u003eUsage\u003c\/strong\u003e\u003c\/td\u003e\n\u003ctd\u003e\n\u003cstrong\u003e With 24 Aperture plate as an example \u003c\/strong\u003e\u003cbr\u003e\u003cstrong\u003e I. Pre-sample processing: \u003c\/strong\u003e\u003cbr\u003e1 Experimental materials: Primary culture buffer ( 4 ℃ Pre-cooling), tissue preservation solution, sampling tube, tissue transport box, ice pack. \u003cbr\u003e2 Sample sampling and transportation: The tissue should be in vitro. 30 min Insert tissue preservation solution and wash with primary culture buffer. 3-5 Next, the blood on the tissue surface is washed clean, placed in tissue preservation solution, and the sampling tube is then placed in... 4 ℃ Cryogenic storage and transportation ( 72 h inside). \u003cbr\u003e\u003cbr\u003e\u003cstrong\u003e 2. Primary culture of organoids: \u003c\/strong\u003e\u003cbr\u003e1 Experimental materials: \u003cbr\u003e （ 1 Materials required: Matrix glue (in advance) 24 h Put in 4 ℃ (Refrigerator melting), tweezers ( 10 cm ), pointed ophthalmic surgical scissors \/ Surgical blade, disposable 60 mm Petri dishes, 1.5 mL\/15 mL\/50 mL EP tube, 100 um a cell screen, 3 mL pasteurized straw \/1000 μL pipetting gun, 24 Well cell culture plate, metal ice box, primary culture buffer ( 4 ℃ ), primary tissue digestive juice ( 37 ℃ ）。 \u003cbr\u003e （ 2 Reagent: Porcine normal intestinal organoid culture medium (room temperature or 37 ℃ ）。 \u003cbr\u003e2 Organoid Construction: \u003cbr\u003e （ 1 Organizational recommendations after data collection are as follows:  2-8 ℃ Store and transport under these conditions, and quickly transfer to a clean laboratory for the experimental process of constructing normal pig intestinal organoids. Organize photography and register detailed information. \u003cbr\u003e （ 2 After sterilizing the sampling tube, the tissue was removed from the ultra-clean bench, placed in a petri dish, and primary culture buffer was added. 3 mL pasteurized straw or 1000 μL Pipette gun blow cleaning, repeat cleaning operation 3 Number of times or more. \u003cbr\u003e （ 3 Remove tissue impurities using ophthalmic scissors or a surgical blade, and transfer them to... 1.5 mL EP In the tube, after washing with primary buffer three times, the villi were scraped off, and the tissue was further mechanically dissociated with ophthalmic scissors into a volume of approximately  1-3 mm\u003csup\u003e3\u003c\/sup\u003e The tissue block was transferred to 15 mL EP In the tube, add 5 mL Digestive fluid of normal intestinal primary tissue in pigs  4 ℃ oscillation digestion 15-25 min 。 Each digestive process 10 min The tissue digestion was observed under a microscope. A small amount of digestive juice was taken and observed under the microscope, and a larger amount was observed. 70 μm The following cell clusters or individual cells are followed by the next step. \u003cstrong\u003e Note: \u003c\/strong\u003e If the amount of tissue is too small or the biopsy tissue is used 1 mL Digestive fluid from normal intestinal primary tissue of pigs 1.5 mL EP Tubular digestion. \u003cbr\u003e （ 4 Digestion is completed and the tissue passes through 100 μm The cell sieve with a pore size is filtered, the filtrate is collected, and added... 3 Double volume of primary culture buffer   Rinsing terminates digestion. 300 g enrichment centrifugation 5 min Abandoning the supernatural; If the cell pellet contains red blood cells, add 1-2 mL erythrocyte lysate 1-2 min Then, dilute to 10 mL ， 300 g enrichment centrifugation 5 min They abandoned their superiors. \u003cstrong\u003e Note: \u003c\/strong\u003e Tumor organoid culture is performed directly when there is too little sedimentation or no red blood cells. \u003cbr\u003e （ 5 Observe the volume of cell pellets collected by centrifugation and add... 25 Double the volume of matrix glue is resuspended, forming 3D Cultivate the spatial structure to avoid bubbles during the resuspension process. The cell pellet volume is shown in the figure below. Add the cell pellet volume according to the figure. 200 μL 、 150 μL 、 100 μL 、 50 μL Matrix glue. \u003cdiv\u003e\n\u003cimg style=\"display: block; margin-left: auto; margin-right: auto;\" src=\"https:\/\/absin.oss-cn-shanghai.aliyuncs.com\/absin-china\/20240411\/318431bcb65e4dc9a8be5eed4bcf2962.png\" alt=\"\" width=\"250\" height=\"127\"\u003e\u003cbr\u003e\u003cp style=\"text-align: justify;\"\u003e Example: 24 well cell culture plate according to 25 μL-30 μL\/ hole   Dispensing glue, \u003cstrong\u003e matrix rubber \u003c\/strong\u003e\u003cstrong\u003e whole course \u003c\/strong\u003e\u003cstrong\u003e Maintain 0-4\u003c\/strong\u003e\u003cstrong\u003e \u003c\/strong\u003e\u003cstrong\u003e℃ operation under conditions \u003c\/strong\u003e 。 cell culture plate placement 37 ℃ Incubator 10-15 min After the matrix gum has set, add to each well. 500-750 μL  Porcine normal intestinal organoid medium (in advance) 37 ℃ (Preheating) for cultivation. \u003cbr\u003e\u003cbr\u003e\u003cstrong\u003e III. Organoid subculture: \u003cbr\u003e\u003c\/strong\u003e1 Experimental materials: \u003cstrong\u003e\u003cbr\u003e\u003c\/strong\u003e （ 1 Materials required: Matrix glue (in advance) 24 h Put in 4 ℃ (Refrigerator melting) 1.5 mL\/15 mL EP tube, 3 mL pasteurized straw \/1000 μL pipetting gun, 24 Well cell culture plate, metal ice box, organoid subculture buffer ( 4 ℃ ), organoid passage digestive fluid D （ 37 ℃ ）。 \u003cbr\u003e （ 2 Reagent: Porcine normal intestinal organoid culture medium (room temperature or 37 ℃ ）。 \u003cbr\u003e2 Organoid passage: \u003cbr\u003e （ 1 Select appropriate organoids for passage, generally after about a week of growth, under a microscope. 10X You can see more below 20 Organoids, or size 100-200 μm Organoids. Aspirate off the culture medium, add an equal volume of organoid subculture buffer to each well, gently blow away the matrix gel with a pipette, and collect... 15 mL EP In the tube, each 6-8 The hole is transferred to one EP tube, 4 ℃ standing 10-15 min 。 \u003cbr\u003e （ 2 Whether digestion and passage are necessary is determined based on the growth of the organoid. \u003cstrong\u003e Note: \u003c\/strong\u003e If there is little precipitation at the bottom of the tube after centrifugation, no cells are seen, and the matrix glue is not delaminated, it can be resuspended again to increase the centrifugal force and centrifuge again. \u003cstrong\u003e\u003cbr\u003e\u003c\/strong\u003ea:  When the number of organoids is insufficient or the volume is small, 300 g centrifuge 5 min Abandon the Shangqing Dynasty \u003cbr\u003eb:  When the number of organoids is high or the volume is large, 300 g centrifuge 5 min Discard the supernatant and select digestive juice for digestion. \u003cbr\u003e     Digestive juice digestion: Add 1-2 mL organoid passage digestive fluid D After the cell pellet is blown out, it is digested at room temperature. 2-4 min Blow every minute and observe under a microscope until digested (Figure). A-B It can be stopped when in the ) state. Add 3 Organoid subculture buffer with twice the volume of organoid digestive fluid   Termination of digestion, 300 g centrifuge 5 min Abandon the Shangqing. \u003c\/p\u003e\n\u003cimg style=\"display: block; margin-left: auto; margin-right: auto;\" src=\"https:\/\/absin.oss-cn-shanghai.aliyuncs.com\/absin-china\/20240517\/62b26839db5e44ec8033ebc7b28d2001.png\" alt=\"\" width=\"428\" height=\"134\"\u003e\u003cbr\u003e （ 3 Observe the volume of organoid pellets collected by centrifugation. If there is very little pellet, it can be reserved. 1 Dispense the supernatant with twice the precipitation volume; a large amount of precipitated supernatant can be absorbed. Add... 25 The organoids were resuspended in an amount of matrix gel twice the organoid pellet volume. Example: 24 well cell culture plate according to 25 μL-30 μL\/ hole   Dispensing glue, \u003cstrong\u003e The matrix adhesive is maintained throughout the 0-4 ℃ operation under conditions \u003c\/strong\u003e 。 cell culture plate placement 37 ℃ Incubator 10-15 min After the matrix gum has set, add to each well. 500-750 μL  Porcine normal intestinal organoid medium (in advance) 37 ℃ (Preheating) for cultivation. \u003cbr\u003e\u003cbr\u003e\u003cstrong\u003e IV. Cryopreservation of organoids: \u003c\/strong\u003e\u003cbr\u003e1 Experimental materials: \u003cstrong\u003e\u003cbr\u003e\u003c\/strong\u003e （ 1 Materials required: 15 mL EP Tubes, cell cryopreservation tubes, programmed cooling cassettes, pipetting guns, organoid subculture buffer ( 4 ℃ ), organoid cryopreservation solution. \u003cbr\u003e2 Organoid cryopreservation: \u003cbr\u003e Organoids that are temporarily unused should be frozen and stored in a low-temperature environment. \u003cbr\u003e （ 1 Aspirate off the culture medium, add an equal volume of organoid subculture buffer to each well, gently blow the matrix gel with a pipette, and collect... 15 mL EP In the tube, each 6-8 The hole is transferred to one EP tube, 4 ℃ standing 10-15 min 。 300 g centrifuge 5 min Discard the supernatant and add it every three holes. 2 mL Organoid cryopreservation solution, gently blown and mixed, transferred to cell cryopreservation tubes, each tube... 1 mL 。 \u003cbr\u003e （ 2 Mark the information, place it in the program cooling box, and move it to... -80 ℃ In the refrigerator, 48 h Afterwards, it was placed in a liquid nitrogen tank for storage. Or put in 4 ℃ refrigerator 40 min Then, put in -20 ℃ in the refrigerator 2 h , move to -80 ℃ In the refrigerator, 48 h Afterwards, it was placed in a liquid nitrogen tank for storage. \u003cbr\u003e\u003cbr\u003e\u003cstrong\u003e V. Organoid resuscitation culture: \u003c\/strong\u003e\u003cbr\u003e1 Experimental materials: \u003cstrong\u003e\u003cbr\u003e\u003c\/strong\u003e （ 1 Materials required: Matrix glue (in advance) 24 h Put in 4 ℃ (Refrigerator melting) 15 mL EP Pipe, water bath, pipette gun, 24 Well cell culture plate, ice box. \u003cbr\u003e （ 2 Kit reagents: Organoid subculture buffer ( 4 ℃ ), porcine normal intestinal organoid culture medium. \u003cbr\u003e2 Organoid resuscitation culture: \u003cbr\u003e （ 1 The frozen organoids were removed from the cryogenic environment and quickly placed in... 37 ℃ The cryopreservation tube needs to be gently shaken during the water bath thawing process to ensure that the cryopreservation liquid is completely thawed in a short time. Rapid transfer of thawed organoids to 15 mL EP Gently blow on the tube using a pipette. 6-8 next time, 300 g  centrifuge 5 min Abandon the Shangqing; Add an appropriate amount of passage buffer G Resuspend, move in 1.5 mL EP tube 300 g centrifuge 5 min They abandoned the Shangqing \u003cbr\u003e （ 2 ) Add per cryopreservation tube 100 μL The matrix glue is resuspended, 24 well cell culture plate according to 25 μL-30 μL\/ hole   Dispensing, the matrix adhesive is maintained throughout the entire process. 0-4 ℃ Operate under these conditions. cell culture plate placement 37 ℃ Incubator 10-15 min After the matrix gum has set, add to each well. 500-750 μL  Porcine normal intestinal organoid medium (in advance) 37 ℃ (Preheating) for cultivation. \u003c\/div\u003e\n\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u003cstrong\u003eAppearance\u003c\/strong\u003e\u003c\/td\u003e\n\u003ctd\u003esolution\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u003cstrong\u003eGeneral Notes\u003c\/strong\u003e\u003c\/td\u003e\n\u003ctd\u003e1. Pig normal intestinal organoid culture medium can be stored at 4℃ for 3 months. After receiving the goods, store at 4℃. It is recommended to use it up within 1 month. If not used for a long time, it is recommended to store it at -20℃ to avoid repeated freezing and thawing more than twice.\u003cstrong\u003e\u003cstrong\u003e。\u003cbr\u003e\u003c\/strong\u003e\u003c\/strong\u003e2. Tissue preservation solution and digestive solution of normal intestinal primary tissue of pigs contain nutrients that maintain cell activity. In order to maintain the activity of the reagent nutrients, it is recommended to store them at -20℃ without long-term use to avoid repeated freezing and thawing more than twice.\u003cbr\u003e3. Thaw the matrix glue overnight at 2–8℃. When using matrix glue, keep it in an ice box to prevent premature setting. The matrix gel formed a gel within 20 min at 37°C.\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u003cstrong\u003eStorage Temp.\u003c\/strong\u003e\u003c\/td\u003e\n\u003ctd\u003eStore at -20°C with a shelf life of 12 months.\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\u003c\/table\u003e\u003c\/div\u003e","brand":"Absin","offers":[{"title":"100mL","offer_id":43224148377675,"sku":"abs9890-100mL","price":476.0,"currency_code":"USD","in_stock":true},{"title":"500mL","offer_id":43224148410443,"sku":"abs9890-500mL","price":1587.0,"currency_code":"USD","in_stock":true}],"url":"https:\/\/www.antbioinc.com\/products\/porcine-intestine-organoid-culture-abs9890","provider":"AntBio","version":"1.0","type":"link"}