An in vitro construction method of a Chinese panda small intestine organoid model

Abstract

This invention relates to the field of in vitro intestinal organoid culture technology, and discloses an in vitro method for constructing a small intestinal organoid model of the Chinese red panda. The method involves collecting ileum tissue from Chinese red pandas that have died due to disease or accident, and then using PBS... + After rinsing, the tissue was placed in tissue transport buffer for low-temperature transport; the following steps were performed sequentially: intestinal lumen rinsing, removal of external tissue, longitudinal incision, scraping of intestinal villi onto a glass slide, and pre-cooling with PBS. + The tissue was repeatedly inverted and washed until the supernatant was clear. It was then minced, digested with EDTA digestion solution, and the crypts were separated by mechanical pipetting. After filtration through a sieve, the crypt precipitate was collected by centrifugation. The crypt precipitate was resuspended, its concentration adjusted, and mixed with Matrigel. It was then inoculated into culture plates, and after the Matrigel solidified, complete culture medium was added for further culture to obtain small intestinal organoids from the Chinese red panda. A small intestinal organoid model was successfully constructed and can be stably passaged in vitro, filling a gap in the technology for constructing small intestinal organoids from the Chinese red panda.

Description

Technical Field

[0001] This invention relates to the field of in vitro culture technology for intestinal organoids, and more particularly to a method for in vitro construction of a small intestinal organoid model of the Chinese red panda. Background Technology

[0002] The Chinese red panda (Ailurus styani), belonging to the family Ailurusidae in the order Carnivora, is a Class II protected wild animal in China. Due to habitat destruction and population decline, this species was listed as endangered by the International Union for Conservation of Nature (IUCN) in 2015. Similar to the giant panda, the Chinese red panda is a highly specialized herbivorous carnivore, retaining the structural characteristics of carnivores in its digestive tract and lacking the digestive tract structure characteristic of herbivores. To protect this species, ex-situ and in-situ conservation measures have been implemented. With the expansion of captive populations and the deepening of research on related diseases, the harm of bacterial diseases to the health of Chinese red pandas has gradually become apparent, especially intestinal bacterial diseases, whose incidence rate in captive populations is significantly higher than that of other systemic diseases. However, due to the unique intestinal structure of the Chinese red panda and the lack of suitable research models, the pathogenesis of its intestinal diseases remains unclear.

[0003] Currently, cell lines and animal models are commonly used in the study of intestinal physiological and pathological processes. While traditional cell line models are simple to operate, they typically contain only a single cell type, lack intercellular interactions, and have unstable genomes. Animal models often use mice, but these differ from the red panda in intestinal tissue structure and physiological function, making it difficult to accurately reflect the in vivo physiological environment. Intestinal organoids, on the other hand, are in vitro tissue models formed by the proliferation and differentiation of stem cells or crypts containing stem cells. They resemble intestinal epithelial tissue in structure and function and can be generated from embryonic stem cells, induced pluripotent stem cells, or adult stem cells in a matrix gel containing specific growth factors. They include all intestinal epithelial cell types, such as intestinal epithelial cells, enteroendocrine cells, goblet cells, and Paneth cells, and are highly similar in structure to in vivo intestinal tissue, possessing the absorptive and secretory functions of intestinal epithelial cells. Compared to traditional cell lines and animal models, intestinal organoid models not only better simulate the in vivo microenvironment, intercellular interactions, and in vivo biological processes, but also can be continuously passaged in vitro and maintain stable phenotypic and genetic characteristics.

[0004] With the maturation of intestinal organoid culture technology, intestinal organoids from humans, mice, and livestock have been successfully constructed. However, there are currently no reported technical solutions for constructing small intestinal organoids from the Chinese red panda, either domestically or internationally. Therefore, there is an urgent need to develop an in vitro method for constructing small intestinal organoids from the Chinese red panda, providing an in vitro research model that falls between cells and tissues for studying the mechanisms of intestinal diseases in the Chinese red panda, constructing pathogen infection models, and screening drugs, thereby addressing the technical problem of the lack of suitable research models in existing technologies. Summary of the Invention

[0005] The purpose of this invention is to provide an in vitro construction method for a small intestinal organoid model of the Chinese red panda, which solves the problem of the lack of in vitro research models with similar intestinal structure and function to the Chinese red panda in the prior art.

[0006] To achieve the above objectives, this invention provides an in vitro method for constructing a small intestinal organoid model of the red panda, comprising the following steps: Ileal tissue was collected from red pandas that died from disease or accident, and processed using PBS. + After rinsing the intestinal contents, the contents were placed in tissue transport buffer and transported to the laboratory at low temperature. Ileal tissue was soaked in pre-cooled PBS + Rinse the intestinal cavity 1-3 times and remove the membranes, blood vessels and adipose tissue attached to the outside of the intestine. Make a longitudinal incision in the intestine so that the intestinal lumen is facing upwards, and scrape the villi on the surface of the intestinal lumen in one direction with a sterile glass slide. Ileal tissue after villi removal was placed in pre-cooled PBS + Repeat the inverting and washing process 15-25 times until the supernatant is clear; Cut the cleaned ileum tissue into 1-2 mm pieces. 3 Small pieces were added to pre-cooled PBS and allowed to settle by gravity. The supernatant was then aspirated. Add EDTA digestion solution to the tissue precipitate and digest in a 37°C water bath. During digestion, use a pipette to blow up and down the tissue fragments every 5 minutes to separate the crypts from the basal layer. Digestion was terminated when microscopic examination revealed a large number of crypt structures. The filtrate was filtered through a 100 µm cell sieve, and the crypt precipitate was collected after centrifugation. Resuspend the crypt precipitate in pre-cooled PBS, take the suspension for crypt counting, and adjust the crypt suspension concentration. Mix the adjusted crypt suspension with Matrigel at a volume ratio of 1:1 and mix thoroughly by pipetting on ice. Take the crypt-matrix mixture and inoculate it into the center of the bottom of the well of the preheated multi-well culture plate. During the inoculation process, continuously mix the suspension. After inoculation, place the culture plate in a 37°C, 5% CO2 incubator and let it stand until the matrix gel solidifies. Add preheated complete culture medium (37°C) along the well wall to each well, place in an incubator, and change the culture medium as needed to obtain small intestinal organs from the Chinese red panda.

[0007] Among these, ileum tissue was collected from Chinese red pandas that died due to disease or accident, and processed using PBS. + After flushing the intestinal contents, the contents are placed in tissue transport buffer and transported at low temperature to the laboratory, specifically including: The tissue transport buffer contains Advanced DMEM / F-12, 1% Antibiotic-Antimycotic, 50 µg / mL Primocin, 10 µmol / L Y-27632 and 20% FBS.

[0008] The process involves adding EDTA digestion solution to the tissue precipitate and digesting it in a 37°C water bath. During digestion, the tissue fragments are agitated with a pipette every 5 minutes to separate the crypts from the basal layer. Specifically, this includes: During digestion, tissue fragments were agitated by pipetting with a pre-washing solution, which was a PBS solution containing 1% BSA.

[0009] Digestion was terminated when microscopic examination revealed numerous crypt structures. The sample was then filtered through a 100 µm cell sieve, and the filtrate was centrifuged and the crypt precipitate was collected. Specifically, the precipitate included: The centrifugation conditions were 1500 rpm for 5 minutes.

[0010] The process of resuspending the crypt precipitate in pre-cooled PBS, performing crypt counting on the suspension, and adjusting the crypt suspension concentration specifically includes: The concentration of the crypt suspension was adjusted to 200 crypts / µL.

[0011] In this process, preheated culture medium at 37°C was added along the well wall to each well, and the wells were incubated in an incubator. The culture medium was changed according to the growth of the intestinal organs to obtain small intestinal organs from the red panda, specifically including: The complete culture medium includes Advanced DMEM / F12 basal medium, and the following added to it: R-spondin-1 500 ng / mL, Noggin 100 ng / mL, Wnt3a 150 ng / mL, N2 1×, B27 supplement 1×, N-acetylcysteine ​​1 mM, Nicotinamide 10 mM, A83-01 500 nM, SB202190 10 µM, hEGF 50 ng / mL, hGastrin I 5 nM, Primocin 100 µg / mL, CHIR-99021 3 µM, HEPES 10 mM, and GlutaMAX 10 mM.

[0012] In this process, preheated culture medium at 37°C was added along the well wall to each well, and the wells were incubated in an incubator. The culture medium was changed according to the growth of the intestinal organs to obtain small intestinal organs from the red panda, specifically including: During primary culture, the concentration of Primocin in the complete medium was adjusted to 200 µg / mL, and 10 µM of Y-27632 was added to the complete medium during primary culture and the first culture after subculture.

[0013] This invention discloses an in vitro method for constructing a small intestinal organoid model of the Chinese red panda, which involves collecting ileum tissue from Chinese red pandas that have died due to disease or accident, and then using PBS... + After rinsing with PBS solution containing 5% Antibiotic-Antimycotic and 50 µg / mL Primocin, the tissue was transported at low temperature in tissue transport buffer. The following steps were performed sequentially: intestinal lumen rinsing, removal of external tissue, longitudinal incision, scraping of intestinal villi onto a slide, and pre-cooling PBS. + The tissue was repeatedly inverted and washed 15-25 times until the supernatant was clear. The tissue was then minced, digested with EDTA digestion solution, and the crypts were separated by mechanical pipetting. After filtration through a 100 µm sieve, the crypt precipitate was collected by centrifugation. The crypt precipitate was resuspended and the concentration was adjusted to 200 crypts / µL. It was then mixed with Matrigel at a 1:1 volume ratio and inoculated into culture plates. After the Matrigel solidified, complete culture medium containing R-spondin-1, Noggin, and Wnt3a was added for further culture to obtain small intestinal organoids from the Chinese red panda. A small intestinal organoid model containing intestinal epithelial cells, enteroendocrine cells, goblet cells, and Paneth cells with a crypt-villi structure was successfully constructed. This model can be stably passaged in vitro, filling the gap in small intestinal organoid construction technology for the Chinese red panda. It solves the technical problem of lacking an in vitro research model with a similar intestinal structure and function to that of the Chinese red panda, providing a reliable in vitro research platform for studying the mechanisms of intestinal diseases in the Chinese red panda, constructing pathogen infection models, and screening drugs. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0015] Figure 1 This is a growth diagram of intestinal organs according to the present invention.

[0016] Figure 2 This is an image of the intestinal organ of the present invention.

[0017] Figure 3 This is a flowchart illustrating the steps of the in vitro construction method of the small intestinal organoid model of the Chinese red panda according to the present invention. Detailed Implementation

[0018] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0019] Please refer to Figures 1 to 3 This invention provides an in vitro method for constructing a small intestinal organoid model of the Chinese red panda, comprising the following steps: S101: Collect ileum tissue from Chinese red pandas that died due to disease or accident, and use PBS. + After rinsing the intestinal contents, the contents were placed in tissue transport buffer and transported to the laboratory at low temperature. S102: Ileal tissue was rinsed with pre-cooled PBS + Rinse the intestinal cavity 1-3 times and remove the membranes, blood vessels and adipose tissue attached to the outside of the intestine. Make a longitudinal incision in the intestine so that the intestinal lumen is facing upwards, and scrape the villi on the surface of the intestinal lumen in one direction with a sterile glass slide. S103: Ileal tissue after villi removal was placed in pre-cooled PBS. + Repeat the inverting and washing process 15-25 times until the supernatant is clear; S104: Cut the cleaned ileum tissue into 1-2 mm pieces. 3 Small pieces were added to pre-cooled PBS and allowed to settle by gravity. The supernatant was then aspirated. S105: Add EDTA digestion solution to the tissue precipitate and digest in a 37°C water bath. During digestion, use a pipette to blow up and down the tissue fragments every 5 minutes to separate the crypts from the basal layer. S106: Digestion was terminated when microscopic examination revealed a large number of crypt structures. The crypt precipitate was then filtered through a 100 µm cell sieve, centrifuged, and collected after centrifugation. S107: Add pre-cooled PBS to the crypt precipitate and resuspend it. Take the suspension to count crypts and adjust the concentration of the crypt suspension. S108: Mix the adjusted crypt suspension with Matrigel at a volume ratio of 1:1 and mix thoroughly by blowing on ice. S109: Take the crypt-matrix mixture and inoculate it into the center of the bottom of the well of the preheated multi-well culture plate. During the inoculation process, continuously mix the suspension. After inoculation, place the culture plate in a 37℃, 5% CO2 incubator and let it stand until the matrix gel solidifies. S110: Add preheated complete culture medium (37°C) along the well wall to each well, place in an incubator, and change the culture medium according to the growth of the intestinal organs to obtain the small intestinal organs of the Chinese red panda.

[0020] Specifically, the reagents and formulations used in this invention are as follows: (1) Relevant reagents: Advanced DMEM / F-12 (catalog number: 12634-010), Antibiotic-Antimycotic (catalog number: 15240-062), FBS (catalog number: 10099141C), and PBS (C20012500BT) were purchased from Gibco. Primocin (catalog number: ant-pm-1) was purchased from InvivoGen. BSA (catalog number: CLB-SL1061) was purchased from Cooler Master. Y-27632 (catalog number: 72304) was purchased from STEMCELL. Matrigel (catalog number: 356231) was purchased from Corning.

[0021] (2) Relevant formula: Tissue transport buffer: Advanced DMEM / F-12 + 1% Antibiotic-Antimycotic + 50 µg / ml Primocin + 10 µmol / L Y-27632 + 20% FBS.

[0022] Washing solution: PBS solution containing 1% BSA.

[0023] PBS + : 5% Antibiotic-Antimycotic + 50 µg / ml Primocin + PBS.

[0024] The complete small intestine culture medium for red pandas in this invention includes the following steps: (1) The complete culture medium includes the following components: (2) The concentration of Primocin in the primary culture medium was adjusted to 200 ug / ml for the first completion, and the medium was replaced with new complete medium after 24 hours. 10 uM of Y-27632 was added to the primary and subculture culture mediums for the first completion.

[0025] The method for isolating the small intestinal crypts of the red panda in this invention includes the following steps: (1) Collect the ileum interruption from individuals who died from disease or accident, rinse the intestinal contents with PBS+, and transfer them to a 50 mL centrifuge tube containing tissue transport buffer after rinsing. Transport the tube to the laboratory at low temperature for the next step of the experiment.

[0026] (2) Transfer the 50 mL centrifuge tube containing ileal tissue to the cell culture room, sterilize its surface, and then transfer it to the clean bench.

[0027] (3) Remove the ileum tissue and place it in a container of pre-cooled PBS. +In a 10 cm culture dish, use a 10 mL sterile syringe to draw pre-cooled PBS. + Insert it into the opening at one end of the intestine and gently flush the intestinal lumen 1-3 times until all the intestinal contents are flushed out.

[0028] (4) Transfer the ileum tissue to a new culture dish, and use sterile pointed forceps and ophthalmic scissors to remove the membranes, blood vessels, and fat outside the intestine. Use pre-cooled PBS. + Rinse and transfer to a new petri dish.

[0029] (5) Use sterile ophthalmic scissors to make a longitudinal incision, open the intestinal lumen upwards, rinse the intestinal tissue three times with pre-cooled PBS, and transfer it to a new culture dish.

[0030] (6) Spread the ileum tissue flat in a culture dish with the intestinal lumen facing upwards. Hold one end down with forceps and gently scrape away the villi on the surface of the intestinal lumen in one direction using a sterile glass slide. After the villi are cleaned, transfer the ileum tissue to a container of pre-cooled PBS. + Wash in the culture dish, repeating the washing process 3 times.

[0031] (7) Transfer the ileum tissue to a 50 mL centrifuge tube containing 30 mL of pre-cooled PBS+, repeatedly invert and wash, remove the ileum tissue, rinse and transfer it to the next 50 mL centrifuge tube containing 30 mL of pre-cooled PBS+, repeat the operation 15-25 times until the supernatant is clear.

[0032] (8) Take out the ileum tissue and place it in a culture dish. Cut the tissue into small pieces of about 1-2 mm3 with scissors. Add an appropriate amount of pre-cooled PBS and transfer it to a 15 mL centrifuge tube that has been pre-washed with rinsing solution. Let the fragment settle by gravity and then gently aspirate the supernatant. Repeat the addition of PBS once.

[0033] (9) Add 10 mL of pre-cooled PBS to a 15 mL centrifuge tube containing the precipitated tissue, centrifuge at 1100 rpm for 3 min and remove the supernatant.

[0034] (10) Add appropriate EDTA digestion solution to the precipitated tissue and place in a 37°C water bath for digestion. Shake the centrifuge tube several times every 5 min. During digestion, use a 5 mL-1 mL pipette (pre-washed with rinsing solution) to pipette up and down several times to allow intestinal tissue fragments to repeatedly pass through the tip of the pipette, generating mechanical shear force to separate the crypts and basal layer. Starting from the 15th min, take 10 μL of cell suspension every 5 min for microscopic examination. When a large number of tissue fragments can pass through the tip of a 1000 µL pipette and there are a large number of crypt structures in the cell suspension, stop pipetting and add PBS to terminate digestion.

[0035] (11) Rinse a 100 µm cell sieve and a 50 mL centrifuge tube with rinsing solution. Filter the digested tissue suspension through a 100 µm cell sieve. Wash the intestinal tissue fragments in the cell sieve several times with pre-cooled PBS. Transfer the filtrate to a 15 mL centrifuge tube after rinsing.

[0036] (12) Centrifuge the filtrate at 1500 rpm for 5 min, remove the supernatant, and obtain the crypt precipitate.

[0037] The method for culturing small intestinal organoids of the red panda in this invention includes the following steps: (1) Add 10 mL of Advanced DMEM / F-12 medium to the crypt pellet to resuspend the cell pellet, centrifuge at 1500 rpm for 5 min, remove the supernatant, and add 500 µL of pre-cooled PBS to the pellet. Take 10 µL of the suspension and place it on a glass slide for microscopic examination and crypt counting.

[0038] (2) Adjust the suspension to a concentration of 200 crypts / µL. Mix the adjusted cell suspension with Matrigel at a ratio of 1:1, transfer to ice, and pipette dozens of times to resuspend the precipitate, avoiding the generation of air bubbles.

[0039] (3) Use a pre-cooled pipette to draw 50 µL of the crypt suspension from the previous step and inoculate it into a 24-well plate that has been preheated in a CO2 incubator for 2 h. During inoculation, mix the crypt suspension frequently, bring the tip of the pipette close to the center of the bottom of the plate but do not touch the bottom, and gently press the pipette stopper to avoid generating air bubbles.

[0040] (4) After the inoculation is completed, place the culture plate in a CO2 incubator (37℃, 5% CO2) and let it stand for about 5 minutes. After gently shaking the gel droplets and there is no obvious flow, carefully invert it and continue to place it in the CO2 incubator until the Matrigel is fully solidified.

[0041] (5) Remove the solidified culture plate and gently add 500 µL of preheated 37°C complete intestinal organoid culture medium to each well using a pipette along the well wall, avoiding damage to the droplets. Add 500 µL of sterile PBS to the wells without droplets to maintain humidity during incubation.

[0042] (6) Cover the culture plate with the lid, mark it, and put it in a CO2 incubator for incubation. Change the culture medium according to the growth of intestinal organs.

[0043] A small intestinal organoid model containing intestinal epithelial cells, enteroendocrine cells, goblet cells, and Paneth cells with a crypt-villi structure was successfully constructed. This model can be stably passaged and cultured in vitro, filling the gap in the technology for constructing small intestinal organoids from the Chinese red panda. It solves the technical problem of lacking an in vitro research model with a similar intestinal structure and function to that of the Chinese red panda, and provides a reliable in vitro research platform for studying the mechanism of intestinal diseases in the Chinese red panda, constructing pathogen infection models, and screening drugs.

[0044] The above-disclosed embodiments are merely one or more preferred embodiments of this application and should not be construed as limiting the scope of this application. Those skilled in the art can understand that all or part of the processes for implementing the above embodiments and equivalent changes made in accordance with the claims of this application still fall within the scope of this application.

Claims

1. A method for in vitro construction of a small intestinal organoid model of the red panda, characterized in that, Includes the following steps: Ileal tissue was collected from red pandas that died from disease or accident, and processed using PBS. + After rinsing the intestinal contents, the contents were placed in tissue transport buffer and transported to the laboratory at low temperature. Ileal tissue was soaked in pre-cooled PBS + Rinse the intestinal cavity 1-3 times and remove the membranes, blood vessels and adipose tissue attached to the outside of the intestine. Make a longitudinal incision in the intestine so that the intestinal lumen is facing upwards, and scrape the villi on the surface of the intestinal lumen in one direction with a sterile glass slide. Ileal tissue after villi removal was placed in pre-cooled PBS + Repeat the inverting and washing process 15-25 times until the supernatant is clear; Cut the cleaned ileum tissue into 1-2 mm pieces. 3 Small pieces were added to pre-cooled PBS and allowed to settle by gravity. The supernatant was then aspirated. Add EDTA digestion solution to the tissue precipitate and digest in a 37°C water bath. During digestion, use a pipette to blow up and down the tissue fragments every 5 minutes to separate the crypts from the basal layer. Digestion was terminated when microscopic examination revealed a large number of crypt structures. The filtrate was filtered through a 100 µm cell sieve, and the crypt precipitate was collected after centrifugation. Resuspend the crypt precipitate in pre-cooled PBS, take the suspension for crypt counting, and adjust the crypt suspension concentration. Mix the adjusted crypt suspension with Matrigel at a volume ratio of 1:1 and mix thoroughly by pipetting on ice. Take the crypt-matrix mixture and inoculate it into the center of the bottom of the well of the preheated multi-well culture plate. During the inoculation process, continuously mix the suspension. After inoculation, place the culture plate in a 37°C, 5% CO2 incubator and let it stand until the matrix gel solidifies. Add preheated complete culture medium (37°C) along the well wall to each well, place in an incubator, and change the culture medium as needed to obtain small intestinal organs from the Chinese red panda.

2. The in vitro construction method of the small intestinal organoid model of the red panda as described in claim 1, characterized in that, Ileal tissue was collected from red pandas that died from disease or accident, and processed using PBS. + After flushing the intestinal contents, the contents are placed in tissue transport buffer and transported at low temperature to the laboratory, specifically including: The tissue transport buffer contains Advanced DMEM / F-12, 1% Antibiotic-Antimycotic, 50 µg / mL Primocin, 10 µmol / L Y-27632 and 20% FBS.

3. The in vitro construction method of the small intestinal organoid model of the Chinese red panda as described in claim 1, characterized in that, Add EDTA digestion solution to the tissue precipitate and digest in a 37°C water bath. During digestion, pipette up and down to dissipate tissue fragments every 5 minutes to separate the crypts from the basal layer. Specifically, this includes: During digestion, tissue fragments were agitated by pipetting with a pre-washing solution, which was a PBS solution containing 1% BSA.

4. The in vitro construction method of the small intestinal organoid model of the red panda as described in claim 1, characterized in that, Digestion was terminated when microscopic examination revealed numerous crypt structures. The solution was filtered through a 100 µm cell sieve, and the filtrate was centrifuged and the crypt precipitate was collected, which specifically included: The centrifugation conditions were 1500 rpm for 5 minutes.

5. The in vitro construction method of the small intestinal organoid model of the red panda as described in claim 1, characterized in that, Resuspend the crypt precipitate in pre-cooled PBS, collect the suspension for crypt counting, and adjust the crypt suspension concentration, specifically including: The concentration of the crypt suspension was adjusted to 200 crypts / µL.

6. The in vitro construction method of the small intestinal organoid model of the red panda as described in claim 1, characterized in that, Add preheated (37°C) complete culture medium along the well wall to each well, incubate in an incubator, and change the culture medium as needed to observe the growth of the intestinal organs. This yielded small intestinal organs from the red panda, specifically including: The complete culture medium includes Advanced DMEM / F12 basal medium, and the following added to it: R-spondin-1500 ng / mL, Noggin 100 ng / mL, Wnt3a 150 ng / mL, N2 1×, B27 supplement 1×, N-acetylcysteine ​​1 mM, Nicotinamide 10 mM, A83-01 500 nM, SB202190 10 µM, hEGF 50 ng / mL, hGastrin I 5 nM, Primocin 100 µg / mL, CHIR-99021 3 µM, HEPES 10 mM, and GlutaMAX 10 mM.

7. The in vitro construction method of the small intestinal organoid model of the red panda as described in claim 6, characterized in that, Add preheated (37°C) complete culture medium along the well wall to each well, incubate in an incubator, and change the culture medium as needed to observe the growth of the intestinal organs. This yielded small intestinal organs from the red panda, specifically including: During primary culture, the concentration of Primocin in the complete medium was adjusted to 200 µg / mL, and 10 µM of Y-27632 was added to the complete medium during primary culture and the first culture after subculture.

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