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CN-121975720-A - Model construction method based on animal endometrium epithelium organoid and application thereof

CN121975720ACN 121975720 ACN121975720 ACN 121975720ACN-121975720-A

Abstract

The invention discloses a model construction method based on animal endometrium epithelium organoids and application thereof, relating to the technical fields of cell engineering and organoids, the method takes endometrium tissues of healthy cows as raw materials, the method comprises the steps of pretreatment, enzymolysis, cell cleaning and screening, seed gum culture, passage, cryopreservation and recovery and the like to construct a model, and verifying the morphological integrity through H & E staining, and detecting E-cadherin expression through IHC staining to confirm the cell specificity. The animal organ model reduces three-dimensional microenvironment and species specificity in vivo, overcomes the limitations of the traditional two-dimensional culture and mouse model, can accurately simulate the pathological process of endometritis, is applied to research on pathogenesis of endometritis of cows, can efficiently screen anti-inflammatory drugs and evaluate drug effect, provides a reliable in vitro tool for disease control, and helps healthy development of cow breeding industry.

Inventors

  • SONG LIANGLI
  • WANG GUANQING
  • Tu Jiusu
  • WANG LINNAN
  • ZHANG LINGJIE

Assignees

  • 宁夏大学

Dates

Publication Date
20260505
Application Date
20251226

Claims (10)

  1. 1. A model construction method based on animal endometrium epithelium organoids is characterized by comprising the following steps: S1, selecting healthy cows to pick up uterus, cleaning the tissue surface of the uterus by precooled tissue cleaning liquid, removing fat, connective tissue and residual blood, and shearing to obtain tissue fragments with the diameter of 0.5-1 mm; S2, placing the tissue fragment suspension into a centrifuge tube for centrifugal treatment, adding 10mL of enzymolysis liquid I for resuspension after supernatant is discarded after centrifugation, transferring the suspension into a 6cm culture dish for enzymolysis at 37 ℃, and observing under a lens after blowing with a Pasteur pipette every 3-10 min until no obvious tissue mass exists and a large number of dissociated glandular structures appear, thus obtaining tissue suspension; S3, transferring the tissue suspension to a 15mL centrifuge tube, supplementing the tissue washing liquid to 10mL, stopping enzymolysis, centrifuging, removing supernatant liquid, repeatedly washing for 2-3 times by the tissue washing liquid, centrifuging, removing the supernatant liquid after washing, adding 500-1000 mu L of erythrocyte lysate into cell sediment after washing and centrifuging, blowing and mixing uniformly, standing on ice, adding 5 times of precooled organoid washing liquid to stop lysis, centrifuging, and collecting the cell sediment; S4, re-suspending the cell sediment by using the organoid cleaning solution, collecting the cell mass subjected to complete enzymolysis by using a natural sedimentation method, filtering by using a 100 mu m cell sieve to remove extracellular matrix impurities to obtain filtrate, transferring the filtrate to a centrifuge tube, centrifuging, removing supernatant, and adding 1mL of organoid culture medium to re-suspend cells to obtain a cell suspension; S5, preparing a50 mu L gel drop from each hole of a 24-hole low-adhesion culture plate serving as a carrier, adding the cell suspension and matrigel into the holes of the culture plate in an equal volume, pre-solidifying for 5min at 37 ℃, overturning and incubating for 20-30 min to enable the matrigel to be completely solidified, adding >500 mu L of organoid culture medium containing 1X additive I along the hole wall of the culture plate after complete solidification, culturing for 48h, and replacing the organoid culture medium with the organoid culture medium without the additive I, and then replacing the organoid culture medium every 2-3 days; S6, primary culturing the organoid for 7-15 days, wherein the organoid is 500 and has the size of 100-200 mu m, the boundary is clear, the organoid can be passaged in a good growth state, the culture solution in the culture holes of the culture plate is sucked, the organoid cleaning solution is added to clean the gel drop, after the cleaning solution is sucked, 500 mu L of organoid recovery solution is added to each hole, the matrigel is blown away, standing and incubating for 5min at room temperature, a mixture is obtained, the mixture is collected into a 15mL centrifuge tube, residual cells in the holes are cleaned by the organoid cleaning solution and transferred into the centrifuge tube, and the heterocells are removed through passage of 3-4 generations, so that the organoid is obtained.
  2. 2. The method for constructing a model of an animal endometrium epithelial organoid according to claim 1, wherein said organoid dissociation comprises the steps of: adding 1mL of organoid culture medium containing 1X additive I to the organoid cells for resuspension, and blowing for 5-50 times for observation under a rear mirror; If the cells are single cells and small cell clusters, the dissociation is completed, if the gland organoids growing in a solid form exist in the cells, 500 mu L of enzymolysis solution II is added for resuspension, the cells are transferred to a 24-hole low-adhesion culture plate, the culture plate is subjected to standing digestion for 2 to 15min in a culture box with the temperature of 37 ℃ and blown for 5 to 10 times every 1 to 3min until the cells are small cell clusters, 9.5mL of precooled organoid cleaning solution is added for stopping the digestion, supernatant is removed by centrifugation, and 1mL of organoid culture medium containing 1X additive I is used for resuspension, so that the dissociation is completed.
  3. 3. The method for constructing a model of an animal endometrium epithelial organoid according to claim 1, wherein the organoid model comprises a post-processing step, specifically comprising the steps of: After 3-5 days of culture after passage, collecting and dissociating the organoids to obtain small cell clusters, centrifuging, removing supernatant, adding the obtained organoids into a freezing tube, adding 500-1000 mu L of organoids freezing solution into the freezing tube, placing the freezing tube into a program cooling box, cooling for 24 hours at the temperature of-80 ℃, and transferring to a liquid nitrogen tank for preservation; When the organoid is recovered, the freezing tube is taken out from the liquid nitrogen tank, thawed and blown in a water bath kettle with the temperature of 37 ℃ for 3-5 times, evenly mixed, the organoid suspension is transferred to a centrifuge tube containing 5mL of organoid cleaning liquid, centrifuged for 5min under the conditions of the temperature of 4 ℃ and the centrifugal force of 200-300 g, the supernatant is discarded, and the recovery of the organoid is completed by repeating the steps S4-S6.
  4. 4. The method for constructing a model of an animal endometrium epithelial organoid according to claim 1, wherein said model identification of organoid construction comprises the steps of: h & E staining, namely fixing, embedding and slicing the organoid sample, placing the organoid sample in a 65 ℃ oven for baking slices for 1H, sequentially placing three cylinders of dimethylbenzene for dewaxing, treating each cylinder for 15min, hydrating the sample by using 100%, 95% and 75% gradient ethanol for 3-5 min, staining the sample by using hematoxylin dye liquor for 5min after hydrating, and flushing the sample by using tap water for 5min; Differentiating the mixture for 3 to 5 seconds by using 1 percent hydrochloric acid alcohol after dyeing is finished, returning blue for 1 to 3 seconds in ammonia water, dyeing for 1min by using eosin dye liquor, washing by using tap water, dehydrating by using 75 percent, 95 percent and 100 percent gradients of ethanol, and dehydrating by using each concentration of ethanol for 3min; finally, after the transparent xylene for 3min, dripping a neutral resin sealing piece, observing the morphology of the organoid under a mirror, and confirming the success or failure of the organoid construction; IHC staining, namely dewaxing the organoid slice, carrying out antigen retrieval by using ER2 antigen retrieval liquid at 100 ℃ for 20min, treating the organoid slice by using 3% hydrogen peroxide solution for 10min after retrieval to block endogenous peroxidase, and adding a sealing liquid to seal for 10min; Then adding E-cadherin antibody diluted by 1:5000, incubating for 60min at room temperature, adding goat anti-rabbit HRP secondary antibody, and incubating for 30min at room temperature; and finally, developing by using DAB working solution for 5min, counterstaining for 5min by using hematoxylin, flushing by using tap water, repeating the steps of dehydration, transparency and sealing of H & E staining after flushing, and observing E-cadherein expression under a lens to confirm success or failure of organoid construction.
  5. 5. The method for constructing the model based on the animal endometrium epithelial organoids according to claim 1, wherein the uterus is picked up within 30min after slaughtering of cows, and is put into a tissue preservation solution for preservation after washing by using normal saline, and the tissue preservation solution is kept at 4 ℃ for cold preservation and transportation; the tissue shearing operation of the uterus body is carried out under a sterile environment, and under the condition of shearing 100-200 by using sterile scissors, the diameter of the tissue fragments is controlled to be 0.5-1 mm, and no oversized tissue block remains.
  6. 6. The method for constructing a model of an animal endometrium epithelial organoid according to claim 1, wherein in step S3, the centrifugation parameters are controlled to be at 4℃and the centrifugal force is controlled to be 200-300 g and the centrifugation time is controlled to be 5min each time; And after the erythrocyte lysate is added, blowing for 5-10 times to ensure that the cell sediment is completely and uniformly mixed, and standing on ice for 3min to avoid excessive damage to target cells due to lysis.
  7. 7. The method for constructing the model based on the animal endometrium epithelial organoids according to claim 1, wherein the sedimentation time of the natural sedimentation method for cell screening in the step S4 is 10-15 min, and the natural sedimentation method is used for collecting the enzymolysis complete cell mass with high density; And (3) adopting aseptic operation during the filtration of the cell sieve, flushing the screen mesh of the cell sieve with the organoid cleaning liquid for 2 times after the filtration, and ensuring that target cells on the cell sieve have no residues.
  8. 8. The method for constructing a model of an animal endometrium epithelial organoid according to claim 1, wherein said culture plate of cell seed gel in step S5 is preheated at 37℃for 30min before culturing; blowing for 3-5 times when the cell suspension is mixed with the matrigel, wherein the time for blowing and mixing is not more than 2min, so that the matrigel is prevented from being solidified in advance; The glue drops are added into the culture plate, movement is avoided when the culture plate is placed for incubation for 5min and pre-solidification is carried out, and the glue drops with the cell density of 10000-50000/50 mu L are controlled in each hole of the culture plate.
  9. 9. The method for constructing a model of an animal endometrium epithelial organoid according to claim 1, wherein in step S6, the mixed cells comprise fibroblasts and uterine muscle cells, and the mixed cells are removed by a method of standing for 2-4 hours after inoculation, wherein the method comprises the following steps: Standing the culture plate for 2-4 h after subculture, and after the fibroblasts and the myometrial cells are attached to the wall of the culture plate, gently sucking supernatant in the holes and the non-attached organoid cell mass, supplementing organoid culture medium without additive I for continuous culture, and removing the miscellaneous cells after 3-4 passages of subculture, thereby obtaining the organoid of the high-purity endometrial gland.
  10. 10. An animal endometrium epithelial organoid model constructed by the animal endometrium epithelial organoid-based model construction method according to any one of claims 1-9, the use of said animal endometrium epithelial organoid model in research of pathogenesis of endometritis in cows, screening of anti-inflammatory drugs against endometritis in cows and evaluation of drug efficacy.

Description

Model construction method based on animal endometrium epithelium organoid and application thereof Technical Field The invention relates to the technical fields of cell engineering and organoids, in particular to a model construction method based on animal endometrial epithelium organoids and application thereof. Background The organoid (Organoid) is a micro-organ model formed by self-organization of cells through an in vitro culture technology, and the core principle is that the organoid gradually develops into a three-dimensional structure with organ characteristics in a culture environment containing specific growth factors and matrix materials by utilizing the self-renewal and directional differentiation capability of stem cells or multipotent progenitor cells, can simulate the structure and the function of a real organ, is formed by differentiating stem cells or specific tissue cells under specific conditions, has cell types, spatial arrangement and partial physiological functions similar to those of natural organs, and is mainly used in the fields of disease research, drug testing, regenerative medicine and the like. At present, research and development of pathogenic mechanism and anti-inflammatory drugs for cow endometritis still mainly depend on traditional two-dimensional cell culture and a mouse model, the traditional two-dimensional cell culture and mouse model can not simulate three-dimensional structures and intercellular interaction microenvironment of endometrium in vivo, the traditional two-dimensional cell culture and mouse model and the cow model have obvious species difference, so that pathological reaction simulation distortion, pertinence of test results and insufficient reliability are caused, disease pathogenesis is difficult to be accurately revealed, anti-inflammatory drugs suitable for cows are efficiently screened, and further, the breakthrough of cow endometritis prevention and treatment technology is restricted, and huge economic losses such as reproductive performance reduction, milk yield reduction and the like are continuously brought to cow breeding industry. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a model construction method based on animal endometrial epithelium organoids and application thereof, and solves the problems mentioned in the background art. In order to achieve the aim, the invention is realized by the following technical scheme that the model construction method based on the animal endometrium epithelium organoid comprises the following steps: S1, selecting healthy cows to pick up uterus, cleaning the tissue surface of the uterus by precooled tissue cleaning liquid, removing fat, connective tissue and residual blood, and shearing to obtain tissue fragments with the diameter of 0.5-1 mm; S2, placing the tissue fragment suspension into a centrifuge tube for centrifugal treatment, adding 10mL of enzymolysis liquid I for resuspension after supernatant is discarded after centrifugation, transferring the suspension into a 6cm culture dish for enzymolysis at 37 ℃, and observing under a lens after blowing with a Pasteur pipette every 3-10 min until no obvious tissue mass exists and a large number of dissociated glandular structures appear, thus obtaining tissue suspension; S3, transferring the tissue suspension to a 15mL centrifuge tube, supplementing the tissue washing liquid to 10mL, stopping enzymolysis, centrifuging, removing supernatant liquid, repeatedly washing for 2-3 times by the tissue washing liquid, centrifuging, removing the supernatant liquid after washing, adding 500-1000 mu L of erythrocyte lysate into cell sediment after washing and centrifuging, blowing and mixing uniformly, standing on ice, adding 5 times of precooled organoid washing liquid to stop lysis, centrifuging, and collecting the cell sediment; S4, re-suspending the cell sediment by using the organoid cleaning solution, collecting the cell mass subjected to complete enzymolysis by using a natural sedimentation method, filtering by using a 100 mu m cell sieve to remove extracellular matrix impurities to obtain filtrate, transferring the filtrate to a centrifuge tube, centrifuging, removing supernatant, and adding 1mL of organoid culture medium to re-suspend cells to obtain a cell suspension; S5, preparing a50 mu L gel drop from each hole of a 24-hole low-adhesion culture plate serving as a carrier, adding the cell suspension and matrigel into the holes of the culture plate in an equal volume, pre-solidifying for 5min at 37 ℃, overturning and incubating for 20-30 min to enable the matrigel to be completely solidified, adding >500 mu L of organoid culture medium containing 1X additive I along the hole wall of the culture plate after complete solidification, culturing for 48h, and replacing the organoid culture medium with the organoid culture medium without the additive I, and then replacing the organoid culture medium every 2-3 days; S6, primary cu