{"product_id":"chicken-intestine-organid-culture-abs90067","title":"Chicken intestine Organid 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 I. Pre-organization processing: \u003cbr\u003e\u003c\/strong\u003e1 Experimental materials: \u003cbr\u003e Primary buffer needs to be prepared in advance B （ 4 ℃ Pre-cooling), tissue preservation solution E Sampling tube, tissue transport box, ice pack. \u003cbr\u003e2 Organization acquisition and transportation: \u003cbr\u003e Tissue sampling and transportation are the first and most easily overlooked steps in the successful construction of organoids. Improper preservation of tissues in the early stages can lead to problems such as poor cell activity, contamination, and few normal cells, reducing the success rate of organoid construction. \u003cbr\u003e The tissue should be in vitro 30 min Place tissue preservation solution inside E , primary buffer B cleaning 3-5 Next, the blood on the tissue surface is washed clean and placed in tissue preservation solution. E In the sampling tube 4 ℃ Cryogenic storage and transportation ( 72 h inside). \u003cbr\u003e\u003cstrong\u003e II. Primary organoid culture (with 24 (Taking orifice plates as an example) \u003c\/strong\u003e\u003cbr\u003e1 Experimental materials: \u003cbr\u003e Primary buffer needs to be prepared in advance B （ 4 ℃ ), special primary tissue digestive fluid C （ 4℃ ), matrix glue (in advance 24 h Put in 4 ℃ (Refrigerator melting)   chicken small intestine organoid culture medium A (Room temperature or 37 ℃ ), tweezers ( 10㎝ ), pointed ophthalmic surgical scissors \/ Surgical blade, disposable 60 mm Petri dishes, 1.5 mL\/15 mL\/50 mL centrifuge tube, 100㎛ a cell screen, 3 mL pasteurized straw \/1000 uL pipetting gun, 24 Well cell culture plate, metal ice box. \u003cbr\u003e2 Construction of chicken small intestine organoids: \u003cbr\u003e2.1 Treatment of tissues \u003cbr\u003e Chicken small intestines after being harvested ( 9-11 Day-old embryos or chicks around two weeks old) tissue recommendations  2 – 8 ℃ Store and transport under these conditions, and quickly transfer to a clean laboratory for further processing.   Chicken small intestine organoid construction experimental procedure, organizing photography and registering detailed information. \u003cbr\u003e （ 1 Tissue cleansing \u003cbr\u003e After the sampling tube is sterilized, the tissue is removed from the ultra-clean table, placed in a petri dish, and primary buffer is added.  B , use 3 mL pasteurized straw or 1000 uL The pipette gun is blown and cleaned, and the cleaning operation is repeated three or more times. \u003cbr\u003e （ 2 Dissociation and digestion of tissues \u003cbr\u003e Tissue impurities are removed with ophthalmic scissors or a surgical blade, and the forceps are transferred to 1.5 mL EP In the tube, after washing with primary buffer three times, the intestine is cut open, the upper villi are scraped off, and the tissue is further mechanically dissociated into approximately [volume missing] with ophthalmic scissors. 3-5 mm3 The tissue block was transferred to 15 mL EP In the tube, add 5 mL primary tissue digestive fluid 4℃ oscillation digestion 15-25 min 。 Each digestive process 10 min The tissue digestion was observed under a microscope, and a small amount of digestive fluid was taken for observation under a microscope, and more crypts were observed. The degree of tissue digestion is shown in the figure 1 。 \u003cbr\u003e (Vortex after digestion) 10-20s ） \u003cbr\u003e\u003cstrong\u003e If the amount of tissue is too small or the biopsy tissue is used 1mL primary tissue digestive fluid C In 1.5 mL EP Tubular digestion. \u003c\/strong\u003e\u003cbr\u003e\u003cdiv style=\"text-align: center;\"\u003e\n\u003cimg style=\"display: block; margin-left: auto; margin-right: auto;\" src=\"https:\/\/absin.oss-cn-shanghai.aliyuncs.com\/absin-china\/20250122\/6643ae7bf61c43bfb263db4eeca92923.png\" alt=\"\" width=\"387\" height=\"150\"\u003e Figure 1   Tissue digestion into multiple crypts \u003c\/div\u003e （ 3 ) Tissue filtration \u003cbr\u003e Digestion completes tissue passage 100 μm The cell sieve with a pore size is filtered, the filtrate is collected, and added... 3 Double the volume of primary buffer B 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. \u003cbr\u003e （ 4 Organoid culture \u003cbr\u003e\u003cdiv\u003e 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. 2 Add the cell pellets according to the amount shown in the figure. 300 uL 、 250 uL 、 150 uL 、 100uL Matrix glue. \u003cimg style=\"display: block; margin-left: auto; margin-right: auto;\" src=\"https:\/\/absin.oss-cn-shanghai.aliyuncs.com\/absin-china\/20250122\/0e9c03d683074cdf9abb16e1cca8b5a1.png\" alt=\"\" width=\"331\" height=\"150\"\u003e \u003cbr\u003e                                                                                                       Figure 2   cell pellet volume \u003cbr\u003e\u003cbr\u003e24 well cell culture plate according to 25 uL-30 uL\/ When dispensing the adhesive through the holes, the matrix adhesive is 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 uL  chicken small intestine organoid culture medium A (Preheat) for incubation. \u003cbr\u003e\u003cstrong\u003e III. Organoid subculture (with 24 (Taking orifice plates as an example) \u003cbr\u003e\u003c\/strong\u003e1. Experimental Materials: \u003cbr\u003e passage buffer G （ 4 ℃ ), organoid passage digestive fluid D (Room temperature or 37 ℃ ), matrix glue (in advance 24 h Put in 4 ℃ (Refrigerator melting)   chicken small intestine organoid culture medium A (Room temperature or 37 ℃ ）、 1.5 mL\/15 mL centrifuge tube, 24 Well cell culture plate, ice box. \u003cstrong\u003e\u003cbr\u003e\u003c\/strong\u003e2. Organoid Passage: \u003cbr\u003e Select suitable organoids for passage, generally after about a week of growth, microscope 10X You can see more below 20 Organoids, or size 100-200㎛ Organoids. \u003cbr\u003e Aspirate off the medium and add an equal volume of passage buffer to each well G The pipette gently blows away the matrix glue, collecting it from... 15 mL In the centrifuge tube, each 6-8 The hole is transferred to a centrifuge tube, 4 ℃ standing 10-15 min 。 \u003cbr\u003e2.1 organoid digestion \u003cbr\u003e The need for digestive passaging is determined based on the growth of the organoid. 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. \u003cbr\u003e When the number of organoids is insufficient or the volume is small, 300 g centrifuge 5 min Abandon the superiors and the Qing \u003cbr\u003e When the number of organoids is high or the volume is large, 300 g centrifuge 5 min Discard the supernatant, and you can choose digestive juice digestion or mechanical 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. 1min Blow every minute and observe under a microscope until digested (Figure). A-B It can be stopped when in the ) state. Add 3 Passage buffer with twice the volume of organoid digestive fluid G Termination of digestion, 300 g centrifuge 5 min Abandon. \u003cbr\u003e\u003cbr\u003e\u003cstrong\u003e                                                                         A                                                                                     B\u003c\/strong\u003e\u003cbr\u003e                                                    \u003cimg src=\"https:\/\/absin.oss-cn-shanghai.aliyuncs.com\/absin-china\/20250122\/a01c8fc3663449e797260b8e4b073a70.png\" alt=\"\" width=\"249\" height=\"150\"\u003e                   \u003cimg src=\"https:\/\/absin.oss-cn-shanghai.aliyuncs.com\/absin-china\/20250122\/28687b40045a40f8a97f9752268060bf.png\" alt=\"\" width=\"249\" height=\"150\"\u003e\u003cbr\u003e\u003cbr\u003e                                                                                            Figure 3   Organoid passage digestion degree diagram \u003cbr\u003e\u003cbr\u003e2.2 passage organoid culture \u003cbr\u003e 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. The volume of matrix rubber can be referenced. “ Organoid primary culture operation Figure 2 ” 。 \u003cbr\u003e24 well cell culture plate according to 25 uL-30 uL\/ When dispensing the adhesive through the holes, 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 uL  chicken small intestine organoid culture medium A (Preheat) for incubation. \u003cbr\u003e\u003cstrong\u003e\u003cstrong\u003e four \u003c\/strong\u003e Organoid cryopreservation (with 24 (Taking orifice plates as an example) \u003c\/strong\u003e\u003cbr\u003e1 Experimental materials: \u003cbr\u003e passage buffer G （ 4 ℃ ), organoid cryopreservation solution F （ 4 ℃ ）、 15 mL Centrifuge tube, cell cryopreservation tube, program cooling box, pipette gun. \u003cbr\u003e2 Organoid cryopreservation: \u003cbr\u003e Organoids that are temporarily unused should be frozen and stored in a low-temperature environment. \u003cbr\u003e Aspirate off the medium and add an equal volume of passage buffer to each well G The pipette gently blows away the matrix glue, collecting it from... 15 mL In the centrifuge tube, each 6-8 The hole is transferred to a centrifuge tube, 4 ℃ standing 10-15 min 。 300 g centrifuge 5 min Discard the supernatant and add it every three holes. 2 mL organoid cryopreservation fluid F Gently blow and mix well, then transfer to cell cryopreservation tubes. Each tube... 1 mL 。 \u003cbr\u003e 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\u003cstrong\u003e V. Organoid resuscitation culture (with 24 (Taking orifice plates as an example) \u003c\/strong\u003e\u003cbr\u003e1 Experimental materials: \u003cbr\u003e passage buffer G 、   chicken small intestine organoid culture medium A Matrix glue (in advance) 24 h Put in 4 ℃ (Refrigerator melting) 24 well cell culture plates, ice boxes, 15mL Centrifuge tube, water bath, 3mL pasteurized straw \/ Pipetting gun. \u003cbr\u003e2 Organoid resuscitation culture: \u003cbr\u003e The frozen organoids were removed from the low-temperature 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 Centrifuge tube, gently blow with 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 centrifuge tube 300 g centrifuge 5 min They abandoned the Shangqing \u003cbr\u003e Add per cryopreservation tube 100 uL The matrix glue is resuspended, 24 well cell culture plate according to 25 uL-30 uL\/ When dispensing the adhesive through the holes, the matrix adhesive is maintained throughout the entire process. 0-4 ℃ Operate under these conditions. cell culture plate placement 39 ℃ Incubator 10-15 min After the matrix gum has set, add to each well. 500-750 uL  chicken small intestine organoid culture medium A (Preheat) for incubation. \u003cbr\u003e\u003cstrong\u003e VI. Use of matrix adhesive \u003cbr\u003e\u003c\/strong\u003e In 2–8 ℃ The matrix glue was thawed overnight under ambient conditions. When using matrix glue, keep it in an ice box to prevent premature setting. Matrix glue in 37 ℃ Below 20 A gel formed within minutes. \u003cstrong\u003e\u003cbr\u003e\u003c\/strong\u003e1. Matrix adhesive characteristics: \u003cbr\u003e （ 1 ） 4 ℃ continuous 14 Good liquidity can still be maintained today. \u003cbr\u003e （ 2 ) Put in 37 ℃ Incubator 10-15 min It can solidify \u003cbr\u003e （ 3 The culture plate is not easily damaged during the cultivation process, and the glue is clean and does not stick to it. \u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u003cstrong\u003eDescription\u003c\/strong\u003e\u003c\/td\u003e\n\u003ctd\u003e\n\u003cp\u003e\u003cbr\u003e\u003cbr\u003eProduct Information:\u003c\/p\u003e\n\u003ctable style=\"border-collapse: collapse; width: 70%; margin-left: auto; margin-right: auto;\" border=\"1\"\u003e\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 5.94921%; text-align: center;\" width=\"111\"\u003e \u003c\/td\u003e\n\u003ctd style=\"width: 23.9414%; text-align: center;\" width=\"111\"\u003eProduct Composition\u003c\/td\u003e\n\u003ctd style=\"width: 14.8998%; text-align: center;\" width=\"224\"\u003eSpecification\u003c\/td\u003e\n\u003ctd style=\"width: 50.2162%; text-align: center;\" width=\"269\"\u003eStorage conditions and periods\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 5.94921%; text-align: center;\"\u003e1\u003c\/td\u003e\n\u003ctd style=\"width: 23.9414%; text-align: center;\"\u003echicken small intestine organoid culture medium\u003c\/td\u003e\n\u003ctd style=\"width: 14.8998%; text-align: center;\"\u003e100 mL\u003c\/td\u003e\n\u003ctd style=\"width: 50.2162%; text-align: center;\"\u003e4℃, 3 months or -20℃, 1 year\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\u003c\/table\u003e\n\u003cbr\u003e\u003cp\u003eReagent arrival processing:\u003cbr\u003eChicken small intestine organoid culture medium can be stored at 4℃ for 3 months. After receiving the goods, it is recommended to use it 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.\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003c\/p\u003e\n\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℃ and shelf life is one year.\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\u003c\/table\u003e\u003c\/div\u003e","brand":"Absin","offers":[{"title":"100mL","offer_id":43224150310987,"sku":"abs90067-100mL","price":476.0,"currency_code":"USD","in_stock":true}],"url":"https:\/\/www.antbioinc.com\/products\/chicken-intestine-organid-culture-abs90067","provider":"AntBio","version":"1.0","type":"link"}