Chick intestinal tract crypt stem cell separating medium and separating method and application thereof

Abstract

The invention relates to the technical field of organoids, and particularly discloses a chick intestinal tract crypt stem cell separation medium and a separation method and application thereof, and the technical key points are as follows: the separation medium provided by the scheme of the invention can efficiently separate chick intestinal tract crypts with complete structure, good activity and high purity under a low-temperature condition by optimizing component combination; according to the separation method provided by the scheme of the invention, the separation efficiency and the culture quality of the intestinal stem cells and the intestinal organs are remarkably improved through the matching of the separation liquid and the washing liquid. By utilizing the method disclosed by the invention, a high-quality stem cell source can be provided for culture of intestinal organs, drug screening and disease model construction, and the method has important scientific research and application values.

Description

Technical Field

[0001] This invention relates to the field of organoid technology, and more specifically, to a separation solution for chicken intestinal crypt stem cells, its separation method, and its application. Background Technology

[0002] Damage to or dysregulation of the intestinal epithelium increases intestinal permeability, leading to diseases such as inflammation and colorectal cancer. Intestinal epithelial cells originate from the crypts and are renewed on average every 3-5 days. Among them, small intestinal stem cells expressing Lgr5 in the crypts are the driving force for intestinal epithelial self-renewal, maintaining intestinal homeostasis and function. The activity of small intestinal stem cells is crucial for studying disease mechanisms and developing treatments.

[0003] Organoid technology offers advantages such as mimicking the tissue microenvironment, ease of operation, and avoidance of ethical issues, making it widely applicable in disease research, drug development, and regenerative medicine. Furthermore, it allows for the exploration of diet-microbe-host interactions, providing a better understanding of the interaction between feed nutrients and the host gut, and offering new tools for studying intestinal structure and function in livestock and poultry nutrition. Current research on crypt isolation methods in poultry intestines is relatively limited. Commonly used methods in pigs and mice include the EDTA method and the collagenase method. The EDTA method offers high isolation efficiency but causes significant damage to intestinal crypts, resulting in numerous impurities and poor crypt activity. Collagenase isolation is gentler but lacks standardized methods, is costly, and involves complex purity and sourcing of collagenase, leading to long isolation times.

[0004] The inventors of this application discovered through previous research that when the EDTA chelating agent used to isolate intestinal crypts in mice and pigs is used to isolate intestinal crypts in chickens, it will cause a certain degree of damage to the structure and activity of the chicken intestinal crypts.

[0005] To address these issues, the inventors of this application have developed a method for separating and applying a solution for separating intestinal crypt stem cells in young chickens, thereby resolving the aforementioned problems in the prior art. Summary of the Invention

[0006] The purpose of this invention is to solve the technical problems mentioned in the background art above, and to provide a separation solution for chicken intestinal crypt stem cells, its separation method and application, with the aim of improving the viability of intestinal crypt stem cells.

[0007] The above-mentioned objective of the present invention is achieved as follows: One aspect of the present invention provides a separation solution for chicken intestinal crypt stem cells, the separation solution being composed of the following components at various concentrations: pH stabilizers: 4.27 mM Na2HPO4-7H2O, 1.47 mM KH2PO4 and 10 mM HEPES; Osmotic pressure maintaining agents: 136.9 mM NaCl and 2.682 mM KCl; Nutrients: 2 mM L-Glutamine and 2.5 mM glucose; Metal ion chelating agent: 30 mM Na2EDTA·2H2O; Reducing agent: 1.5 mM DTT.

[0008] Another aspect of the present invention provides a washing solution for isolating intestinal crypt stem cells in chicks, the washing solution comprising the following components at various concentrations: pH stabilizers: 4.27 mM Na2HPO4-7H2O, 1.47 mM KH2PO4 and 10 mM HEPES; Osmotic pressure maintaining agents: 136.9 mM NaCl and 2.682 mM KCl; Nutrients: 2 mM L-Glutamine and 2.5 mM glucose.

[0009] Another aspect of the present invention provides a method for isolating intestinal crypt stem cells based on the above-mentioned separation solution and washing solution, comprising the following steps: S1. Cut 10 cm of the cleaned chicken small intestine and remove impurities by hand shaking in the washing solution under low temperature conditions; S2. Remove the washing solution, place the small intestine in the separation solution and incubate and vibrate for a period of time. Repeat step S1 to obtain intestinal crypt stem cells.

[0010] Furthermore, in step S1, the 10 cm chicken intestines need to be cut into 5 cm segments.

[0011] Furthermore, in step S1, the hand-cranking frequency is 2-3 times / s.

[0012] Furthermore, the washing solution in step S1 and the separation solution in step S2 are both used in a volume of 15 mL each time.

[0013] Furthermore, in step S1, the washing is repeated 3 times, each time for 5 minutes; in step S2, the incubation-vibration cycle is repeated once, with an incubation time of 10 minutes, and step S1 is repeated 2 to 5 times, each time for 5 minutes.

[0014] Furthermore, the low temperature is 0~8℃.

[0015] The present invention also provides a method for enriching chicken intestinal crypt stem cells, which involves enriching the chicken intestinal crypts obtained by the above separation method by differential precipitation.

[0016] The present invention also provides the application of intestinal crypt stem cells obtained by the above isolation method or the above enrichment method in constructing organoids and then evaluating the effect of drug intervention on the activity of intestinal stem cells.

[0017] In the above-described scheme of the present invention, the separation solution and washing solution provided contain NaCl and KCl to maintain osmotic pressure and ion balance, preventing cell swelling or shrinkage; Na2HPO4·7H2O, KH2PO4 and HEPES to maintain stable solution pH and provide a suitable acid-base environment; L-Glutamine and glucose to provide crypt energy and maintain cell metabolism; Na2EDTA·2H2O to chelate metal ions and promote cell dissociation; and DTT to break disulfide bonds, promote cell dissociation, and protect cells from oxidative damage.

[0018] Furthermore, based on the separation liquid of the present invention, the present invention also provides the application of the above separation liquid in the separation of chicken intestinal crypts; the above solution of the present invention improves and innovates the existing method for separating chicken intestinal crypts, that is, under low temperature conditions, chicken intestinal crypts with complete structure and high activity and purity are obtained by the above separation liquid.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. Significantly improved separation efficiency and purity: By using the specific separation solution combination of this invention under low temperature conditions, combined with step-by-step washing, incubation and gentle manual shaking, impurities and villus fragments in intestinal tissue can be efficiently removed, and crypts with intact structure and high purity can be successfully obtained, providing high-quality seed cells for subsequent organoid culture. 2. Significantly improved cell viability and yield: The present invention avoids the use of complex enzyme preparations such as collagenase and adopts a gentle method mainly based on physical separation, which greatly reduces the mechanical and enzymatic damage to the crypts, thereby ensuring that the separated crypts have extremely high cell viability and stable crypt yield with good reproducibility. 3. The method is simple to operate and has obvious cost advantages: The method of this invention has simplified steps and standardized operation procedures. It does not rely on special or expensive enzyme preparations, which significantly reduces experimental costs and operational complexity, while shortening the separation time and making it easier to promote and apply in the laboratory. 4. Outstanding application value: The chicken intestinal crypts obtained using the method of this invention have high activity and complete function, providing a powerful tool for poultry intestinal physiological and pathological research, nutrient absorption, drug screening and disease model construction, and have significant scientific research and industrial application value.

[0020] In summary, the present invention overcomes the shortcomings of existing methods, such as "significant damage to intestinal crypts, numerous impurities during separation, poor crypt activity, lack of unified standards, high cost, complex purity and sources of collagenase, and long separation time," and provides a highly efficient, gentle, low-cost solution that improves the viability of intestinal crypt cells. Attached Figure Description

[0021] Figure 1 A schematic diagram of the crypt fluff shaft structure in a 14-day-old chick. Figure 2 Observation of crypt size in chicks of different ages (40×); (ah represents 16-day, 17-day, 18-day, 19-day, 3-day, 5-day, 7-day and 14-day-old chicks, respectively). Figure 3 The crypt morphology obtained by different methods (40×) (Method 1: mucosal scraping method, Method 2: hand shaking method, Method 3: shaking table method). Figure 4 The crypt morphology obtained by separation at different hand-cranking frequencies (40×) (A: hand-cranking frequency of 3~4 times / s, B: hand-cranking frequency of 2~3 times / s, C: hand-cranking frequency of 1~2 times / s). Figure 5 The crypt morphology obtained by separating different segments of the small intestine (40×) (A: small intestine sampling length of 2cm, B: 5cm, C: 10cm). Figure 6 The effect of different cell sieve pore sizes on the morphology of intestinal crypts in chicks (40×). Figure 7 The effect of different Na2EDTA-2H2O concentrations on the crypt morphology of the broiler intestine (40×). Figure 8 The effect of different amounts of separation solution on the morphology of intestinal crypts in chicks (40×). Figure 9 A schematic diagram of crypt purification and enrichment observation and organoid growth effect (40×). Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] The present invention provides a separation solution for chicken intestinal crypt stem cells, a separation method thereof, and its application, aiming to solve the problems of large damage to intestinal crypts, many separation impurities, poor crypt activity, lack of unified standards, high cost, and long separation time in the prior art, and to improve the viability of intestinal crypt stem cells.

[0024] The separation solution for efficiently separating intestinal crypts provided in the embodiments of the present invention is composed of the following components at various concentrations: 136.9 mM NaCl, 2.682 mM KCl, 4.27 mM Na2HPO4·7H2O, 1.47 mM KH2PO4, 2 mM L-Glutamine, 30 mM Na2EDTA-2H2O, 1.5 mM DTT, 2.5 mM glucose, and 10 mM HEPES.

[0025] The provided highly efficient washing solution for separating intestinal crypts consists of the following components at the following concentrations: 136.9 mM acl, 2.682 mM Kcl, 4.27 mM Na2HPO4·7H2O, 1.47 mM KH2PO4, 2 mM L-Glutamine, 2.5 mM glucose, and 10 mM HEPES.

[0026] The above method for isolating chicken intestinal crypt stem cells includes the following steps: Step 1: Take about 10 cm of jejunum from a 14-day-old chick, cut it into 5 cm sections, put it in a pre-cooled washing solution at 4°C, remove the mesentery and fat on the outside of the intestine as much as possible, cut open the intestine, and rinse the contents clean. Step 2: Transfer the jejunum to a 50 mL centrifuge tube and add 15 mL of ice-cold washing solution. Shake by hand for 5 minutes at a frequency of about 2-3 times / s. Replace with fresh washing solution and repeat the shaking 3 times. At this point, the main impurities separated are villi and fragments of intestinal contents. Step 3: Remove the washing solution, place the jejunum into a new 50 mL centrifuge tube and add 15 mL of separation solution, and incubate while manually shaking at a frequency of about 2 to 3 times / s for 10 minutes. Step 4: Remove the separation fluid, retain the jejunum, add ice-cold washing solution, and shake by hand for 5 minutes at a frequency of about 2-3 times / s. Step 5: Repeat step 4 2-5 times, collect the supernatant into a 50 mL centrifuge tube, let it stand at 4℃ for 5 min. At this time, the precipitated cell clusters are mostly crypts. Discard the supernatant and collect the crypts.

[0027] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0028] Reference Figures 1-9 The image shown is a preferred embodiment of the present invention.

[0029] Example 1: Observation of crypt-villi axis structure Dissecting 14-day-old chicks, the jejunum was removed and longitudinally dissected. The intestinal epithelium and contents were thoroughly rinsed away with cold washing solution. Under a dissecting microscope, the muscle layer and epithelium of the jejunum were separated with forceps. A small amount of chicken intestinal epithelium was placed on a glass slide to form a compact, which was then observed under a microscope. The results are as follows: Figure 1 As shown.

[0030] Example 2: Selecting chicks of suitable age Chickens at embryonic ages of 16, 17, 18, and 19 days, and at 1, 3, 5, 7, 10, and 14 days of age, were selected, and their jejunum was collected. The intestinal crypts were then separated using the following method: (1) Take about 10 cm of jejunum, dissect the intestine longitudinally, and rinse the intestine with cold washing solution; (2) Transfer the jejunum to a 50 mL centrifuge tube, add 15 mL of ice-cold washing solution, shake manually for 5 min at a frequency of about 2 to 3 times / s, replace with new separation solution, and repeat the operation 3 times. (3) Remove the washing solution, retain the jejunum, transfer it to a new 50 mL centrifuge tube, add 15 mL of separation solution, and manually shake at a frequency of about 2 to 3 times / s for 10 min; (4) Remove the separation fluid, retain the jejunum, transfer it to a new 50 mL centrifuge tube, add 15 mL of ice-cold washing fluid, manually shake at a frequency of about 2 to 3 times / s for 5 min, replace with new washing fluid, repeat the operation 2 to 5 times, and collect the liquid containing a large number of crypts.

[0031] Experimental results are as follows Figure 2 As shown, from Figure 2 It can be seen that when the chicks are 16-19 days old, only intestinal fragments are obtained after chelation with the separation solution, and no crypts are observed. As the chicks get older, the number and length of crypts increase. The best results are observed in crypt separation in 14-day-old chicks. Therefore, 14-day-old chicks are the most suitable for crypt separation.

[0032] Example 3: Method for isolating chicken intestinal crypts Select 14-day-old chicks, remove the jejunum, clean the contents with cold detergent, and then separate the crypts using the following three different methods.

[0033] Method 1: Mucosal scraping Approximately 10 cm of jejunum was harvested, longitudinally dissected, and its contents were thoroughly cleaned with a cold washing solution. Then, the intestinal epithelium was gently scraped off layer by layer with a scalpel and resuspended in the washing solution. Microscopic observation revealed that the villi were scraped off first, and the crypts were scraped off last.

[0034] Method 2: Hand-cranking method Select 14-day-old chicks and follow the same procedures as in Example 2.

[0035] Method 3: Shaking Bed Method (1) Take about 10 cm of jejunum, dissect the intestine longitudinally, and rinse the intestine with cold washing solution; (2) Transfer the jejunum to a 50 mL centrifuge tube, add 15 mL of ice-cold washing solution, shake at 4°C and 100 r / min for 5 min, replace with fresh separation solution, and repeat the operation 3 times. (3) Remove the washing solution, retain the jejunum, transfer it to a new 50 mL centrifuge tube, add 15 mL of separation solution, and incubate at 4℃ and 100 r / min for 10 min; (4) Remove the separation fluid, retain the jejunum, transfer it to a new 50 mL centrifuge tube, add 15 mL of ice-cold washing fluid, shake at 4℃ and 100 r / min for 5 min, replace with new washing fluid, repeat the operation 2 to 5 times, and the crypts can be separated.

[0036] The intestinal crypts of chicks obtained by the above three separation methods are as follows: Figure 3 As shown, Method 1 produces fragmented crypt structures and yields fewer complete crypts; Method 2 yields more structurally complete and active crypts; Method 3 fails to fully separate crypts, some of the separated crypts remain unseparated, and more intestinal fragments are obtained; In conclusion, Method 2 is more suitable for separating intestinal crypts in chicks.

[0037] Example 4: Selecting a suitable separation frequency Select 14-day-old chicks, take about 10 cm of jejunum, clean the contents with cold washing solution, and separate the intestinal crypts by hand-cranking at three different frequencies, which are about 1-2 times / s, 2-3 times / s, and 3-4 times / s, respectively.

[0038] Except for the different separation frequency, the operation steps are the same as in Example 2.

[0039] The intestinal crypts of chicks obtained by the above three different hand-cranking frequencies are as follows: Figure 4 As shown, shaking at a frequency of about 3-4 times / s yields more fragmented crypts than complete ones; shaking at a frequency of about 2-3 times / s yields more crypts with more complete structures compared to the other two frequencies; shaking at a frequency of about 1-2 times / s also yields relatively complete crypts, but in smaller numbers; therefore, a shaking frequency of about 2-3 times / s is more suitable for separating crypts in the intestines of chicks.

[0040] Example 5: Screening for the length of small intestinal fragments Select 14-day-old chicks, take about 10 cm of jejunum, clean the contents with cold detergent, and then cut it into small intestine segments of three different lengths of 2, 5 and 10 cm to separate the intestinal crypts.

[0041] Except for the difference in the length of the small intestine fragment, the operation steps are the same as in Example 2.

[0042] The intestinal crypts of chicks obtained by the above three different intestinal sampling lengths are as follows: Figure 5 All three sampling lengths could separate crypts, but the number of crypts separated after cutting into 5 cm segments was greater than that after cutting into 2 cm segments, and there were fewer impurities than after cutting into 10 cm segments, resulting in better separation of intestinal crypts.

[0043] Example 6: Screening for suitable cell sieve pore sizes Select 14-day-old chicks, remove the jejunum, clean the contents with ice-cold washing solution, and then filter the crypts through 100 μm, 70 μm, and 40 μm cell sieves respectively.

[0044] Except for the different cell sieve pore size, the operation steps are the same as in Example 2.

[0045] The crypts obtained from the chick intestinal crypts in Example 2, after being filtered through the three cell sieves described above, are as follows: Figure 6 As shown, the crypt structure is disrupted by growth as the pore size of the cell sieve decreases; however, the integrity of the cell sieve crypts is relatively good and their number is large; in summary, the crypts separated using this separation solution do not need to be filtered through the cell sieve.

[0046] Example 7: Screening for suitable chelating agent concentrations In this example, crypts in the intestines of chicks were isolated using Na2EDTA-2H2O concentrations of 5, 10, 30, 50, and 70 mM. Except for the different Na2EDTA-2H2O concentrations, the operation steps were the same as in Example 2.

[0047] The effects of different Na2EDTA-2H2O concentrations on intestinal crypts in chicks, such as Figure 7 As shown, at concentrations of 5 mM and 10 mM, the isolated jejunal crypts of chicks were mostly fragments; at a concentration of 30 mM, more jejunal crypts could be separated, and at 50 mM, although the number of crypts obtained was small, their structure was intact; at a concentration of 70 mM, only the first separation resulted in a large number of intact crypts falling off, and compared with other concentrations, the most crypts were collected, but the crypt activity decreased rapidly; in summary, the final results indicate that a Na2EDTA-2H2O concentration of 30 mM is most suitable for separating intestinal crypts of chicks.

[0048] Example 8: Screening for a suitable amount of separation solution In this example, 15 mL and 30 mL of separation solution were used to separate the intestinal crypts of chicks. The amount of separation solution used was different, but the other operation steps were the same as in Example 2.

[0049] The effects of different amounts of separation solution on the intestinal crypts of chicks, such as Figure 8 As shown, the results revealed that 15 mL of separation solution resulted in more crypts being obtained than 30 mL of separation solution due to the sufficient impact of the intestine within the liquid; thus, 15 mL of separation solution is more suitable for separating jejunal crypts in chicks.

[0050] Example 9: Crypt purification and enrichment Select 14-day-old chicks, remove the jejunum, clean the contents with ice-cold washing solution, and then separate the crypts according to method two in Example 3. The crypts can be further purified and enriched by differential precipitation, as follows: (1) Transfer the supernatant containing a mixture of crypts and a small amount of villi to a new 50 mL centrifuge tube and let it stand at 4°C for 5 min. Larger cell clumps will settle to the bottom of the tube. (2) Transfer the supernatant to a new 50 mL centrifuge tube and let it stand at 4°C for 5 min. At this time, the precipitated cell clusters are mostly crypts. Discard the supernatant and collect the crypts.

[0051] (3) Repeat steps (1) to (2) above to obtain crypts with a density of over 90%. Inoculate and observe the culture effect. The results are as follows: Figure 9 As shown.

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A solution for separating intestinal crypt stem cells from young chickens, characterized in that, The separation solution consists of the following components at the following concentrations: pH stabilizers: 4.27 mM Na2HPO4-7H2O, 1.47 mM KH2PO4 and 10 mM HEPES; Osmotic pressure maintaining agents: 136.9 mM NaCl and 2.682 mM KCl; Nutrients: 2 mM L-Glutamine and 2.5 mM glucose; Metal ion chelating agent: 30 mM Na2EDTA·2H2O; Reducing agent: 1.5 mM DTT.

2. A washing solution for isolating intestinal crypt stem cells in chicks, characterized in that, The washing solution is composed of the following components at the following concentrations: pH stabilizers: 4.27 mM Na2HPO4-7H2O, 1.47 mM KH2PO4 and 10 mM HEPES; Osmotic pressure maintaining agents: 136.9 mM NaCl and 2.682 mM KCl; Nutrients: 2 mM L-Glutamine and 2.5 mM glucose.

3. A method for isolating intestinal crypt stem cells based on the separation solution of claim 1 and the washing solution of claim 2, characterized in that, Includes the following steps: S1. Cut 10 cm of the cleaned chicken small intestine and remove impurities by hand shaking in the washing solution under low temperature conditions; S2. Remove the washing solution, place the small intestine in the separation solution and incubate and vibrate for a period of time. Repeat step S1 to obtain intestinal crypt stem cells.

4. The separation method according to claim 3, characterized in that, In step S1, the 10 cm chicken intestines need to be cut into 5 cm sections.

5. The separation method according to claim 3, characterized in that, In step S1, the hand-cranking frequency is 2-3 times / s.

6. The separation method according to claim 3, characterized in that, The washing solution in step S1 and the separation solution in step S2 are both used in 15 mL each time.

7. The separation method according to claim 3, characterized in that, In step S1, washing is repeated 3 times, each time for 5 minutes; in step S2, incubation-vibration is performed once, with an incubation time of 10 minutes. Step S1 is repeated 2 to 5 times, each time for 5 minutes.

8. The separation method according to claim 3, characterized in that, The low temperature is 0~8℃.

9. A method for enriching intestinal crypt stem cells in young chickens, characterized in that, The intestinal crypts of chicks obtained by the separation method according to any one of claims 3 to 8 are rapidly and with high quality enriched by differential sedimentation.

10. The application of intestinal crypt stem cells obtained by the isolation method according to any one of claims 3-8 or intestinal crypt stem cells obtained by the enrichment method according to claim 9 in constructing organoids and then evaluating the effect of drug interventions on the activity of intestinal stem cells.

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