Duck small intestinal crypt stem cell isolation method and duck small intestinal organoid culture method

Abstract

This invention discloses a method for isolating duck small intestinal crypt stem cells and a method for culturing duck small intestinal organoids. It belongs to the field of animal tissue isolation and culture technology, and particularly relates to a method for isolating duck small intestinal crypt stem cells, comprising: isolating small intestinal segments from ducklings aged 1-28 days; pre-treating, followed by shaking digestion 1-3 times; retaining the precipitate based on the number of single cells or the presence of crypt cell clusters in the supernatant after shaking digestion; adding pre-cooled DPBS solution containing antibiotics; manually filtering to obtain the filtrate; separating the filtrate into turbid and clear filtrates based on the presence of white flocculent matter and crypt density; collecting the white flocculent matter as duck small intestinal crypt stem cells. This invention establishes a stable method for isolating small intestinal crypt stem cells from 1-28 day-old ducklings and a 3D duck small intestinal organoid culture method, breaking the limitation of traditional meat duck intestinal organoids that can only be extracted at the hatching stage.

Description

Technical Field

[0001] This invention belongs to the field of animal tissue isolation and culture technology, and particularly relates to a method for isolating duck small intestinal crypt stem cells and a method for culturing duck small intestinal organoids. Background Technology

[0002] Intestinal organoids are three-dimensional cell complexes that resemble the structure and function of intestinal epithelial tissue, formed by the continuous proliferation and differentiation of stem cells or crypts containing stem cells in vitro. They represent an emerging in vitro research model. Currently, intestinal organoid culture technology is well-established as an in vitro model for studying intestinal inflammation mechanisms and drug targeting. Compared to traditional 2D cell models, organoids better reflect the unique tissue structure and physiological functions of the intestine. Intestinal organoids can simulate the complex structure and function of the intestine, providing a powerful tool for intestinal development and disease research. Through the culture of intestinal organoids, the differentiation process of intestinal stem cells, as well as the regeneration and repair mechanisms of intestinal tissue, can be observed. The introduction of intestinal organoids will significantly reduce the use of laboratory animals, has positive ethical implications, and will promote research progress in intestinal nutrition and pathology.

[0003] The establishment of in vitro models of intestinal organoids in animals such as pigs and chickens has provided new tools for intestinal health research in the field of livestock and poultry nutrition. It breaks through the limitations of previous studies that could only observe villus height and crypt depth phenotypes, extending to the level of functional cell units such as stem cells, absorptive cells, goblet cells, endocrine cells, and Paneth cells. This lays the foundation for better revealing the interaction mechanisms between feed environmental factors (nutrients and toxins, etc.) and the host gut. However, the construction methods for duck intestinal organoid models are still immature. Currently available methods for extracting and culturing duck small intestinal organoids are limited to extraction from newly hatched duck embryos. However, in actual production and scientific research, more attention is paid to the intestinal health of ducklings aged 1-28 days. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a method for isolating duck small intestinal crypt stem cells and a method for culturing duck small intestinal organoids. The aim is to establish a stable method for isolating small intestinal crypt stem cells from 1-28 day-old ducklings and a 3D method for culturing duck small intestinal organoids, breaking the limitation that traditional meat duck intestinal organoids can only be extracted at the hatching stage. This provides a new platform for promoting the evaluation of the activity of meat duck nutrients and functional feed additives, drug screening, and the analysis of the mechanisms of meat duck intestinal diseases.

[0005] To achieve the above objectives, the present invention provides a method for isolating duck small intestinal crypt stem cells, comprising the following steps: 1) Separate small intestine segments from ducklings aged 1-28 days; 2) Pre-treat the small intestinal segment obtained in step 1), and then perform shaking digestion 1 to 3 times. Observe the number of single cells or whether crypt cell clusters appear in the upper suspension after shaking digestion. When the number of single cells present is greater than the preset single cell number threshold or crypt cell clusters appear, remove the upper suspension and retain the precipitate. 3) Add the precipitate obtained in step 2) to a 10% DPBS solution pre-cooled to 4°C, shake by hand 1-3 times, filter, and obtain the filtrate. Based on the presence of white flocculent matter and crypt density in the filtrate, the filtrate is divided into turbid filtrate and clear filtrate. Turbid filtrate is filtrate with white flocculent matter or filtrate without white flocculent matter but with crypt density ≥ preset crypt density threshold. Clear filtrate is filtrate without white flocculent matter. 4) Spread the turbid filtrate from step 3) evenly in a culture dish and collect the white flocculent material, which is duck small intestinal crypt stem cells; separate the clear filtrate from step 3) where the crypt density is less than the preset crypt density threshold, centrifuge to remove the supernatant, and obtain a centrifuged precipitate. Resuspend the precipitate in F12 medium with a penicillin-streptomycin double antibiotic solution at 4°C and centrifuge to obtain a resuspension. Take the resuspension where the number of intact crypt structures is greater than the preset threshold for the number of intact crypt structures and the number of contaminating cells is less than the preset threshold for the number of contaminating cells, centrifuge, and the resulting precipitate is duck small intestinal crypt stem cells.

[0006] Preferably, the small intestinal segment mentioned in step 1) is the duodenal and jejunal segments.

[0007] Preferably, the pretreatment in step 2) specifically involves: placing the small intestinal segment into a 10% DPBS solution containing penicillin-streptomycin antibiotics at 4°C pre-cooled; peeling off the attached mesentery and adipose tissue; cleaning the contents; then cutting open the small intestinal segment, washing, cutting it into pieces, and collecting it in a fresh 10% DPBS solution containing penicillin-streptomycin antibiotics at 4°C pre-cooled; washing until there is no obvious turbidity; and removing the supernatant.

[0008] Preferably, the shaking digestion in step 2) specifically involves: shaking digestion of the pretreated small intestinal segment in cell lysis buffer, wherein the ratio of the pretreated small intestinal segment to the cell lysis buffer is 1g:8mL, the shaking digestion temperature is 37℃, the shaking digestion speed is 40~60r / min, and the shaking digestion time is 15~25min.

[0009] Preferably, the preset single-cell number threshold in step 2) is a single-cell number that accounts for 5% to 15% of the total cell structure in the field of view.

[0010] Preferably, the hand-cranking time in step 3) is 2 to 5 minutes.

[0011] Preferably, the preset crypt density threshold in step 3) is 200~500 crypts / pore.

[0012] Preferably, the preset threshold for the number of complete crypt structures in step 4) is 50-150 per 10x magnification field; the preset threshold for the number of miscellaneous cells in step 4) is 10-40 per 10x magnification field.

[0013] The present invention also provides a method for culturing duck small intestinal organoids using duck small intestinal crypt stem cells obtained by the aforementioned method for isolating duck small intestinal crypt stem cells, comprising the following steps: The duck small intestinal crypt stem cells were resuspended in duck intestinal organoid culture medium, then pre-chilled on ice was added and mixed well. The mixture was allowed to stand until the pre-chilled on ice had fully solidified, and then duck intestinal organoid culture medium was added. The duck intestinal organoid culture medium was replaced every 2-3 days and cultured continuously at 37°C with 5% CO2.

[0014] Preferably, the duck intestine organoid culture medium comprises the following components in appropriate amounts: Wnt3a 100ng / mL, R-spondin1 200ng / mL, Noggin 100ng / mL, B27 additive stock solution 1mL, N2 additive stock solution 0.5mL, N-acetylcysteine ​​1.25mM, epidermal growth factor EGF 50ng / mL, SB202190 10μM, Y27632 10μM, nicotinamide 10mM, A83-01 500nM, 5% penicillin-streptomycin (100×), DMEM / F12 basal solution 46mL.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides a method for isolating duck small intestinal crypt stem cells and culturing duck small intestinal organoids. Small intestinal segments are isolated from ducklings under sterile conditions. The isolated segments are repeatedly washed with DPBS solution containing antibiotics and anti-inflammatory drugs (antibiotics) pre-cooled to 4°C. Through repeated treatments using a specific culture solution combined with shaking digestion and observation, further observation and processing are performed using an optical microscope. The isolation process for duck small intestinal crypt stem cells is optimized by adjusting the digestion time, rotation speed, cell lysis buffer, and filtration times on a horizontal shaker. This method overcomes the traditional limitation of extracting duck intestinal organoids only at the hatching stage, resulting in more complete morphology, higher density, and a cleaner background for the obtained duck small intestinal crypt stem cells, thus achieving higher product density and purity. This invention, by controlling digestion time and using multiple resuspension and filtration methods, overcomes the time limitation of extracting poultry intestinal organoids at the hatching stage, greatly improving the applicability of duck organoid extraction and culture. Simultaneously, the optical microscope observation method provided in this invention facilitates microscopic inspection of the extracted organoid quality, effectively ensuring organoid growth. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of the original liquid and filtrate after shaking and digestion in Example 1 under an optical microscope. In this diagram, A is the original liquid, B is the first filtrate, C is the second filtrate, D is the third filtrate, E is the fourth filtrate, and F is the fifth filtrate. Figure 2 This is a schematic diagram of the duck small intestinal crypt stem cells obtained in Example 1; Figure 3 This is a schematic diagram of a 3D organoid of the intestine obtained under a 4X objective lens on the second day of culture of duck small intestinal crypt stem cells obtained in Example 2; Figure 4 This is a schematic diagram of a 3D organoid of the intestine obtained under a 10X objective lens on the second day of culture of duck small intestinal crypt stem cells obtained in Example 2. The scale bar is 200μm. Figure 5 This is a schematic diagram of organoids extracted from duck small intestinal crypt stem cell clusters without microscopic examination. Figure 6 This is a schematic diagram showing the observation of primary intestinal organoids of ducks obtained from duck small intestinal crypt stem cell culture for 1-5 days in Example 3. In the diagram, A is day 1, B is day 2, C is day 3, D is day 4, and E is day 5. Figure 7 This is a schematic diagram showing the culture of duck small intestinal crypt stem cells obtained in Comparative Example 2, Example 1, and Example 2. In this diagram, A represents the first day of culture of duck small intestinal crypt stem cells obtained in Comparative Example 2; B represents the third day of culture of duck small intestinal crypt stem cells obtained in Comparative Example 2; C represents the fifth day of culture of duck small intestinal crypt stem cells obtained in Comparative Example 2; D represents the first day of culture of duck small intestinal crypt stem cells obtained in Example 1; E represents the third day of culture of duck small intestinal crypt stem cells obtained in Example 1; F represents the fifth day of culture of duck small intestinal crypt stem cells obtained in Example 1; G represents the first day of culture of duck small intestinal crypt stem cells obtained in Example 2; H represents the third day of culture of duck small intestinal crypt stem cells obtained in Example 2; and I represents the fifth day of culture of duck small intestinal crypt stem cells obtained in Example 2. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The materials used in this invention were sourced from: penicillin-streptomycin (100×) purchased from Yiaobang, DPBS solution purchased from Yiaobang, Gentle Cell Dissociation Reagent cell lysis buffer purchased from STEMCELL, F12 culture medium purchased from Gibico, Matrigel gel purchased from Corning, B27 additive stock solution (50×) purchased from Beyotime, and N2 additive stock solution (100×) purchased from Beyotime.

[0024] Example 1 1) The duodenum and jejunum segments of freshly euthanized 15-day-old ducklings were isolated under sterile conditions.

[0025] 2) Place segments of the duodenum and jejunum into a 4°C pre-chilled 10% DPBS solution containing penicillin-streptomycin. Peel off the attached mesenteric and adipose tissue. Use a 1mL pipette tip to aspirate the 4°C pre-chilled 10% DPBS solution to clear the intestinal contents until the excreted fluid is clear. Then, dissect the duodenum and jejunum segments, wash them, and cut them into 1cm × 3cm pieces. Collect the pieces in a 15mL centrifuge tube containing fresh 4°C pre-chilled 10% DPBS solution. Agitate and wash until no significant turbidity remains. Remove the supernatant, leaving just enough 4°C pre-chilled 10% DPBS solution to cover the pre-treated duodenum and jejunum segments. The solutions used before the agitation digestion process should not contain calcium or magnesium ions.

[0026] Add 1g of pretreated duodenal and jejunal segments to 8mL of Gentle Cell DissociationReagent cell lysate was shaken and digested at 37°C, 50 rpm, and for 20 minutes. The number of single cells or the presence of crypt cell clusters in the supernatant after shaking and digestion were observed using an optical microscope. If the number of single cells exceeded a preset single-cell count threshold (single cells comprising 10% of the total cell structure in the field of view) or crypt cell clusters were present, the supernatant was removed, and the precipitate was retained. If the number of single cells was less than or equal to the preset single-cell count threshold or no crypt cell clusters were present, shaking and digestion was repeated at 37°C, 50 rpm, and for 20 minutes, until the number of single cells exceeded the preset single-cell count threshold or crypt cell clusters were present. The supernatant was then removed, and the precipitate was retained. Shaking digestion can separate crypt stem cells from tissues without damaging the crypt structure.

[0027] 3) Add the obtained precipitate to 7 mL of 10% DPBS solution pre-cooled at 4℃, shake once for 3 min. The solution will become cloudy instantly. Filter the supernatant through a 70 μm filter to obtain the first filtrate. Add another 7 mL of 10% DPBS solution pre-cooled at 4℃, shake once for 3 min until the surface becomes cloudy. Filter the supernatant through a filter to obtain the second filtrate. Add another 7 mL of 10% DPBS solution pre-cooled at 4℃, shake once for 3 min until the surface becomes cloudy. Filter the supernatant through a filter to obtain the third filtrate. Add another 7 mL of PBS solution pre-cooled at 4℃, shake once for 3 min until the surface becomes cloudy. Filter the supernatant through a filter to obtain the third filtrate. Add another 7 mL of PBS solution pre-cooled at 4℃, shake once for 3 min until the surface becomes cloudy. Filter the supernatant through a filter to obtain the third filtrate. Pre-cooled penicillin-streptomycin antibiotic solution (10% DPBS, volume concentration) at 4℃ was added, and the mixture was shaken once for 3 minutes until the surface became cloudy. The supernatant was collected and filtered through a sieve to obtain four filtrates. Then, 7 mL of pre-cooled penicillin-streptomycin antibiotic solution (10% DPBS, volume concentration) at 4℃ was added, and the mixture was shaken once for 3 minutes until the surface became cloudy. The supernatant was collected and filtered through a sieve to obtain five filtrates. Each filtrate was collected in a different test tube and observed until intestinal tissue began to suspend on the surface. 1 mL of the filtrate from each test tube was then transferred to a 6-well plate. The results are as follows. Figure 1 China A~ Figure 1 As shown in Figure F. Under an optical microscope, the primary, secondary, and tertiary filtrates were divided into turbid and clear filtrates based on the presence of white flocculent matter and the crypt density. Turbid filtrate was defined as filtrate containing white flocculent matter or filtrate without white flocculent matter but with a crypt density ≥ a preset crypt density threshold (300 crypts / well). Clear filtrate was defined as filtrate without white flocculent matter.

[0028] 4) Spread the turbid filtrate evenly in a sterile bacterial culture dish and collect the white flocculent material, which is the duck small intestine crypt stem cells. In the turbid filtrate, the crypt cell clusters can generate a large surface tension in the sterile bacterial culture dish, causing them to aggregate due to this surface tension. Therefore, the collected white flocculent material is a relatively pure crypt cell cluster. The light microscopic appearance of the turbid filtrate obtained in this example in a sterile culture dish is as follows... Figure 2 As shown, the crypt stem cell clusters are tightly aggregated due to water surface tension.

[0029] Separate the filtrate where the crypt density is less than the preset crypt density threshold (300 crypts / well), discard the filtrate, and do not collect the crypt cell clusters.

[0030] Separate the filtrate with a crypt density ≥ the preset crypt density threshold (300 crypts / well). Add the filtrate to a clean 50mL centrifuge tube, add 10% DPBS solution (pre-chilled at 4℃) to make a volume of 7mL, and centrifuge at 200×g for 3min at 4℃ in an ultra-low temperature centrifuge. Discard the supernatant to obtain the centrifuged precipitate. Resuspend the precipitate in 7mL of 5% F12 medium (pre-chilled at 4℃) and centrifuge to obtain the resuspension. Place 1mL of the resuspension in a 6-well plate and observe under an optical microscope. Select the resuspension with a number of intact crypt structures > the preset threshold (100 crypt structures / 10x field) and a number of contaminating cells < the preset threshold (20 contaminating cells / 10x field) and centrifuge at 290×g for 3min at 4℃ in an ultra-low temperature centrifuge. The resulting precipitate is the duck small intestinal crypt stem cells. Under an inverted biological microscope, the isolated duck small intestinal crypt stem cells were found to be morphologically intact and numerous, indicating that the method of this invention has a good isolation effect. The DPBS solution was replaced with F12 medium to prevent the crypt cell clusters from being deprived of nutrients for an extended period and becoming inactive.

[0031] Example 2 1) The duodenum and jejunum segments of a 25-day-old duckling were isolated under sterile conditions.

[0032] 2) Place the duodenal and jejunal segments into a 4°C pre-cooled 10% DPBS solution containing penicillin-streptomycin. Peel off the attached mesenteric and adipose tissue. Use a 1mL pipette tip to aspirate the 4°C pre-cooled 10% DPBS solution to clean the intestinal contents until the excreted fluid is clear. Then, cut open the duodenal and jejunal segments, wash them, and cut them into 1cm×3cm pieces. Collect the pieces in a 15mL centrifuge tube containing a fresh 4°C pre-cooled 10% DPBS solution containing penicillin-streptomycin. Aspirate and wash until there is no obvious turbidity. Remove the supernatant, leaving just enough 4°C pre-cooled 10% DPBS solution to cover the pretreated duodenal and jejunal segments.

[0033] Add 1g of pretreated duodenal and jejunal segments to 8mL of Gentle Cell DissociationReagent cell lysate was shaken and digested at 37°C, 50 rpm, and for 20 minutes. The number of single cells or the presence of crypt cell clusters in the supernatant after shaking and digestion were observed using an optical microscope. If the number of single cells exceeded a preset single-cell count threshold (single cells comprising 10% of the total cell structure in the field of view) or crypt cell clusters were present, the supernatant was removed, and the precipitate was retained. If the number of single cells was less than or equal to the preset single-cell count threshold or no crypt cell clusters were present, shaking and digestion was repeated at 37°C, 50 rpm, and for 20 minutes, until the number of single cells exceeded the preset single-cell count threshold or crypt cell clusters were present. The supernatant was then removed, and the precipitate was retained.

[0034] 3) Add the obtained precipitate to 7 mL of 10% DPBS solution pre-cooled at 4℃, shake once for 2 min. The solution will become cloudy instantly. Filter the supernatant through a 70 μm filter to obtain the first filtrate. Add another 7 mL of 10% DPBS solution pre-cooled at 4℃, shake once for 2 min until the surface becomes cloudy. Filter the supernatant through a filter to obtain the second filtrate. Add another 7 mL of 10% DPBS solution pre-cooled at 4℃, shake once for 5 min until the surface becomes cloudy. Filter the supernatant through a filter to obtain the third filtrate. Transfer 1 mL of the filtrate from each tube into a 6-well plate. Under an optical microscope, the primary, secondary, and tertiary filtrates were divided into turbid and clear filtrates based on the presence or absence of white flocculent matter and the density of crypts. Turbid filtrate was defined as filtrate containing white flocculent matter or filtrate without white flocculent matter but with a crypt density ≥ the preset crypt density threshold (300 crypts / well). Clear filtrate was defined as filtrate without white flocculent matter.

[0035] 4) Spread the turbid filtrate evenly in a sterile bacterial culture dish and collect the white flocculent material, which is duck small intestinal crypt stem cells. In the turbid filtrate, the crypt cell clusters can form a large water surface tension in the sterile bacterial culture dish, and will aggregate together due to the surface tension of water, so the collected white flocculent material is a relatively pure crypt cell cluster.

[0036] Separate the filtrate where the crypt density is less than the preset crypt density threshold (300 crypts / well), discard the filtrate, and do not collect the crypt cell clusters.

[0037] Separate the filtrate with a crypt density ≥ the preset crypt density threshold (300 crypts / well). Add the filtrate to a clean 50mL centrifuge tube, add 10% DPBS solution (pre-chilled at 4℃) to make a volume of 7mL, and centrifuge at 200×g for 3min at 4℃ in an ultra-low temperature centrifuge. Discard the supernatant to obtain the centrifuged precipitate. Resuspend the precipitate in 7mL of 5% F12 medium (pre-chilled at 4℃) and centrifuge to obtain the resuspension. Place 1mL of the resuspension in a 6-well plate and observe under an optical microscope. Select the resuspension with a number of intact crypt structures > the preset threshold (100 crypt structures / 10x field) and a number of contaminating cells < the preset threshold (20 contaminating cells / 10x field) and centrifuge at 290×g for 3min at 4℃ in an ultra-low temperature centrifuge. The resulting precipitate is the duck small intestinal crypt stem cells.

[0038] The microscopic results obtained on the second day of culture of duck small intestinal crypt stem cells obtained from the turbid filtrate and the clear filtrate in this embodiment are as follows: Figure 3 and Figure 4 As shown, the density and purity of the crypt cells are very high, which can be seen with the naked eye.

[0039] In this embodiment, the organoid extraction process was performed without microscopic examination; the results of direct plate spotting after centrifugation are as follows: Figure 5 As shown, the content of heterogeneous cells is significantly increased, leading to slow or no growth of organoids.

[0040] Example 3 The duck small intestinal crypt stem cells prepared in Example 1 were resuspended in an appropriate amount of duck intestinal organoid culture medium, and then pre-chilled on ice with a matrix gel was added and mixed well. The mixture was then spread on 48-well plates that had been preheated in an incubator for 0.5 hours. The plates were then placed in an incubator containing 5% CO2 for 10 minutes until the pre-chilled matrix gel was fully solidified. The 48-well plates were carefully removed from the incubator and placed in a clean bench. 200 μL of duck intestinal organoid culture medium was then added. The duck intestinal organoid culture medium was replaced every 2-3 days, and the plates were cultured continuously at 37°C with 5% CO2.

[0041] The duck intestine organoid culture medium consists of the following components in the following quantities: Wnt3a 100 ng / mL, R-spondin1 200 ng / mL, Noggin 100 ng / mL, B27 additive stock solution 1 mL, N2 additive stock solution 0.5 mL, N-acetylcysteine ​​1.25 mM, epidermal growth factor (EGF) 50 ng / mL, SB202190 10 μM, Y27632 10 μM, nicotinamide 10 mM, A83-01 500 nM, 5% penicillin-streptomycin (100×), and DMEM / F12 basal solution 46 mL.

[0042] The microscopic results of primary duck intestinal organoids cultured for 1-5 days in this embodiment are as follows: Figure 6 China A~ Figure 6 As shown in Figure E, the increased volume of duck intestinal organoids and the decreased activity of duck intestinal organoids after long-term in vitro culture are evident.

[0043] Comparative Example 1 For the pre-culture process, duck eggs that have been normally incubated for 17 days are used. After the eggshell surface is disinfected by wiping with alcohol, the chicken embryo is removed and the entire duodenum to jejunum pregut segment is immediately cut off. The isolated intestinal segment is cleaned with PBS solution containing penicillin-streptomycin antibiotics at a volume concentration of 10% and then cut into 2-3 mm pieces. 3 The tissue blocks were then digested with 10 mL of 0.5 mg / mL type I collagenase preheated at 37°C for 40 min at 80 rpm in a constant temperature shaking incubator at 37°C. After digestion, the mixture was centrifuged at 200 g for 5 min at room temperature, and the digestive fluid was discarded. 10 mL of DMEM / F12 medium containing 5% (v / v) FBS was added to the tissue block pellet, and the tissue block was gently pipetted 20–30 times using a 3 mL Pasteur pipette. The suspension was first filtered through a 70 μm cell sieve to remove undissociated intestinal tissue blocks, yielding filtrate 1. Filtrate 1 was then filtered through a 40 μm cell sieve to remove scattered single cells; the cells adhering to the sieve mesh were the intestinal crypt cell clusters.

[0044] Comparative Example 2 1) The duodenum and jejunum segments of 29-year-old duck embryos were isolated under sterile conditions.

[0045] 2) Place the duodenal and jejunal segments into a 4°C pre-cooled 10% DPBS solution containing penicillin-streptomycin. Peel off the attached mesenteric and adipose tissue. Use a 1mL pipette tip to aspirate the 4°C pre-cooled 10% DPBS solution to clean the intestinal contents until the excreted fluid is clear. Then, cut open the duodenal and jejunal segments, wash them, and cut them into 1cm×3cm pieces. Collect the pieces in a 15mL centrifuge tube containing a fresh 4°C pre-cooled 10% DPBS solution containing penicillin-streptomycin. Aspirate and wash until there is no obvious turbidity. Remove the supernatant, leaving just enough 4°C pre-cooled 10% DPBS solution to cover the pretreated duodenal and jejunal segments.

[0046] Add 1g of pretreated duodenal and jejunal segments to 8mL of Gentle Cell DissociationReagent cell lysate was shaken and digested at 37°C, 50 rpm, and for 20 minutes. The number of single cells or the presence of crypt cell clusters in the supernatant after shaking and digestion were observed using an optical microscope. If the number of single cells exceeded a preset single-cell count threshold (single cells comprising 10% of the total cell structure in the field of view) or crypt cell clusters were present, the supernatant was removed, and the precipitate was retained. If the number of single cells was less than or equal to the preset single-cell count threshold or no crypt cell clusters were present, shaking and digestion was repeated at 37°C, 50 rpm, and for 20 minutes, until the number of single cells exceeded the preset single-cell count threshold or crypt cell clusters were present. The supernatant was then removed, and the precipitate was retained.

[0047] 3) Add the obtained precipitate to 7 mL of 10% DPBS solution pre-cooled at 4℃, shake once for 3 min. The solution will become cloudy instantly. Filter the supernatant through a 70 μm filter to obtain the first filtrate. Add another 7 mL of 10% DPBS solution pre-cooled at 4℃, shake once for 3 min until the surface becomes cloudy. Filter the supernatant through a filter to obtain the second filtrate. Add another 7 mL of 10% DPBS solution pre-cooled at 4℃, shake once for 3 min until the surface becomes cloudy. Filter the supernatant through a filter to obtain the third filtrate. Pipette 1 mL of the filtrate from each tube into a 6-well plate. Under an optical microscope, the primary, secondary, and tertiary filtrates were divided into turbid and clear filtrates based on the presence or absence of white flocculent matter and the density of crypts. Turbid filtrate was defined as filtrate containing white flocculent matter or filtrate without white flocculent matter but with a crypt density ≥ the preset crypt density threshold (300 crypts / well). Clear filtrate was defined as filtrate without white flocculent matter.

[0048] 4) Spread the turbid filtrate evenly in a sterile bacterial culture dish and collect the white flocculent material, which is duck small intestinal crypt stem cells. In the turbid filtrate, the crypt cell clusters can form a large water surface tension in the sterile bacterial culture dish, and will aggregate together due to the surface tension of water, so the collected white flocculent material is a relatively pure crypt cell cluster.

[0049] Separate the filtrate where the crypt density is less than the preset crypt density threshold (300 crypts / well), discard the filtrate, and do not collect the crypt cell clusters.

[0050] Separate the filtrate with a crypt density ≥ the preset crypt density threshold (300 crypts / well). Add the filtrate to a clean 50mL centrifuge tube, add 10% DPBS solution (pre-chilled at 4℃) to make a volume of 7mL, and centrifuge at 200×g for 3min at 4℃ in an ultra-low temperature centrifuge. Discard the supernatant to obtain the centrifuged precipitate. Resuspend the precipitate in 7mL of 5% F12 medium (pre-chilled at 4℃) and centrifuge to obtain the resuspension. Place 1mL of the resuspension in a 6-well plate and observe under an optical microscope. Select the resuspension with the number of intact crypt structures > the preset threshold (100 crypt structures / 10x field) and the number of contaminating cells < the preset threshold (20 contaminating cells / 10x field) and centrifuge at 290g for 3min at 4℃ in an ultra-low temperature centrifuge. The resulting precipitate is the duck small intestinal crypt stem cells.

[0051] like Figure 7 China A~ Figure 7Figure I shows a schematic diagram of the culture of duck small intestinal crypt stem cells obtained in Comparative Example 2, Example 1, and Example 2.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A duck intestinal crypt stem cell isolation method, characterized by, Includes the following steps: 1) Separate small intestine segments from ducklings aged 1-28 days; 2) Pre-treat the small intestinal segment obtained in step 1), and then perform shaking digestion 1 to 3 times. Observe the number of single cells or whether crypt cell clusters appear in the upper suspension after shaking digestion. When the number of single cells present is greater than the preset single cell number threshold or crypt cell clusters appear, remove the upper suspension and retain the precipitate. 3) Add the precipitate obtained in step 2) to a 10% DPBS solution pre-cooled to 4°C, shake by hand 1-3 times, filter, and obtain the filtrate. Based on the presence of white flocculent matter and crypt density in the filtrate, the filtrate is divided into turbid filtrate and clear filtrate. Turbid filtrate is filtrate with white flocculent matter or filtrate without white flocculent matter but with crypt density ≥ preset crypt density threshold. Clear filtrate is filtrate without white flocculent matter. 4) Spread the turbid filtrate from step 3) evenly in a culture dish and collect the white flocculent material, which is duck small intestinal crypt stem cells; separate the clear filtrate from step 3) where the crypt density is less than the preset crypt density threshold, centrifuge to remove the supernatant, and obtain a centrifuged precipitate. Resuspend the precipitate in F12 medium with a penicillin-streptomycin double antibiotic solution at 4°C and centrifuge to obtain a resuspension. Take the resuspension where the number of intact crypt structures is greater than the preset threshold for the number of intact crypt structures and the number of contaminating cells is less than the preset threshold for the number of contaminating cells, centrifuge, and the resulting precipitate is duck small intestinal crypt stem cells.

2. The duck intestinal crypt stem cell isolation method of claim 1, wherein, The small intestinal segments mentioned in step 1) are the duodenum and jejunum segments.

3. The duck intestinal crypt stem cell isolation method of claim 1, wherein, The pretreatment described in step 2) is as follows: the small intestinal segment is placed in a 10% DPBS solution of penicillin-streptomycin antibiotic solution pre-cooled at 4°C, the attached mesentery and adipose tissue are peeled off, the contents are cleaned, the small intestinal segment is then cut open, washed, shredded, and collected in a fresh 10% DPBS solution of penicillin-streptomycin antibiotic solution pre-cooled at 4°C, washed until there is no obvious turbidity, and the supernatant is removed.

4. The duck intestinal crypt stem cell isolation method of claim 1, wherein, The shaking digestion in step 2) specifically involves: shaking the pretreated small intestinal segment in cell lysis buffer for digestion, wherein the ratio of the pretreated small intestinal segment to the cell lysis buffer is 1g:8mL, the shaking digestion temperature is 37℃, the shaking digestion speed is 40~60r / min, and the shaking digestion time is 15~25min.

5. The duck intestinal crypt stem cell isolation method of claim 1, wherein, The preset single-cell number threshold mentioned in step 2) is that the single-cell number accounts for 5% to 15% of the total cellular structure in the field of view.

6. The duck intestinal crypt stem cell isolation method of claim 1, wherein, The hand-cranking time mentioned in step 3) is 2-5 minutes.

7. The duck intestinal crypt stem cell isolation method of claim 1, wherein, The preset crypt density threshold mentioned in step 3) is 200~500 crypts / pore.

8. The duck intestinal crypt stem cell isolation method of claim 1, wherein, The preset threshold for the number of complete crypt structures in step 4) is 50-150 per 10x microscope field of view; the preset threshold for the number of miscellaneous cells in step 4) is 10-40 per 10x microscope field of view.

9. A method for culturing duck small intestinal organoids using duck small intestinal crypt stem cells obtained by the method for isolating duck small intestinal crypt stem cells according to any one of claims 1 to 8, characterized in that, Includes the following steps: The duck small intestinal crypt stem cells were resuspended in duck intestinal organoid culture medium, then pre-chilled on ice was added and mixed well. The mixture was allowed to stand until the pre-chilled on ice had fully solidified, and then duck intestinal organoid culture medium was added. The duck intestinal organoid culture medium was replaced every 2-3 days and cultured continuously at 37°C with 5% CO2.

10. The method for culturing duck small intestine organoids according to claim 9, characterized in that, The duck intestine organoid culture medium comprises the following components in appropriate amounts: Wnt3a 100ng / mL, R-spondin1 200ng / mL, Noggin 100ng / mL, B27 additive stock solution 1mL, N2 additive stock solution 0.5mL, N-acetylcysteine ​​1.25mM, epidermal growth factor EGF 50ng / mL, SB202190 10μM, Y27632 10μM, nicotinamide 10mM, A83-01 500nM, 5% penicillin-streptomycin (100×), DMEM / F12 basal solution 46mL.

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