A digestive solution for isolating gastric body stem cells and methods of use thereof

Abstract

The application discloses a kind of digestive juice for separating gastric corpus stem cell and its use method, belong to stem cell separation technical field;The digestive juice includes the following concentration components:18.5~28 nM Na2HPO4, 40~48 nM KH2PO4, 460.5~530 nM NaCl, 1~15.2 nM KCl, 200~350.55 nM D-furan fructosyl-D-glucopyranoside, 400~605 nM D-glucitol, 2~4 nM ethylenediaminetetraacetic acid disodium and 2~3 nM 1,4-dithiothreitol.The application also discloses the use method of the above digestive juice.The digestive juice for separating gastric corpus stem cell and its use method of the application can solve the problems of low cell yield, low activity, insufficient purity and poor repeatability of existing digestive juice and supporting use method, and have wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of stem cell separation technology, specifically relating to a digestive fluid for separating gastric stem cells and its method of use. Background Technology

[0002] The gastric body is the main site of gastric acid and pepsinogen secretion. Its mucosa is rich in complex acid-secreting glands, including parietal cells, chief cells, neck mucous cells, and stem cells with proliferative and differentiation capabilities. Therefore, successfully isolating and culturing fully functional gastric body stem cells is of great significance for constructing in vitro gastric body organoid models, elucidating the physiological and pathological mechanisms of the stomach, and developing new therapies.

[0003] Currently, the digestive fluids and related methods used for isolating gastric body stem cells are mostly directly transplanted from small intestinal crypts or embryonic stem cell research systems. They use collagenase, neutral protease, hyaluronidase, and trypsin as core enzyme combinations. These schemes have been optimized in rodent models over a long period of time and can stably obtain gastric epithelial cell clusters or single-cell suspensions. However, when these schemes are extended to species with different histological and physiological characteristics, such as pigs, dogs, non-human primates, and even human gastric body tissues, significant adaptability defects are exposed.

[0004] The original design of existing digestive fluids was not intended for the unique extracellular microenvironment of the gastric body mucosa. The gastric glands are deeply embedded in a connective tissue network rich in dense collagen fibers and myofibroblasts. Moreover, there are significant differences in gastric wall thickness, collagen cross-linking density, glycosaminoglycan composition, and basement membrane toughness among different species. Universal digestive fluid formulations often expose significant limitations, such as low cell yield, low activity, insufficient purity, and poor reproducibility.

[0005] Therefore, there is an urgent need in this field to develop a new digestive fluid and separation method to provide a stable, homogeneous, and highly active source of stem cells for the study of gastric physiological and pathological mechanisms and the construction of organoid in vitro models. Summary of the Invention

[0006] The problem this invention aims to solve is to provide a digestive fluid for isolating gastric stem cells and a method for using it, in order to address the issues of low cell yield, low activity, insufficient purity, and poor reproducibility in existing digestive fluids and their associated methods.

[0007] The technical solution adopted to solve its technical problem is a digestive fluid for isolating gastric stem cells, comprising the following components at the following concentrations: 18.5~28 nM Na2HPO4, 40~48 nM KH2PO4, 460.5~530 nM NaCl, 1~15.2 nM KCl, 200~350.55 nM D-fructofuranosyl-D-glucopyranoside, 400~605 nM D-glucanol, 2~4 nM disodium ethylenediaminetetraacetate, and 2~3 nM 1,4-dithiothreitol.

[0008] The beneficial effects of the above-mentioned technical solution of the present invention are as follows: In the digestive fluid used to isolate gastric stem cells, D-fructofuranosyl-D-glucopyranoside, D-glucanol and 1,4-dithiothreitol are added in proportion to provide an isotonic and stable osmotic pressure environment during digestion, so as to improve the integrity and activity of stem cells isolated from gastric tissue and provide energy for stem cells.

[0009] Preferably, the digestive fluid used for isolating gastric stem cells comprises the following components at the following concentrations: 25.2 nM Na2HPO4, 43.7 nM KH2PO4, 490.8 nM NaCl, 1 nM KCl, 320 nM D-fructofuranosyl-D-glucopyranoside, 498.3 nM D-glucanol, 2.5 nM disodium EDTA and 2.5 nM 1,4-dithiothreitol.

[0010] The present invention also provides a method for using the digestive fluid for isolating gastric stem cells described above, comprising the following steps: (1) Separate and break up the gastric body tissue, wash it, and obtain gastric body tissue fragments; (2) Incubate the gastric body tissue fragments and wash them until the supernatant is clear to obtain gastric body tissue; (3) The gastric body tissue was placed in a digestive solution for separating gastric body stem cells and digested. Then it was washed until the supernatant was clear, pressed to separate and filtered to obtain gastric body stem cells.

[0011] Preferably, steps (1) to (2) are performed at 2 to 15°C; the size of the gastric body tissue fragments is (0.8 to 1.2) × 3 cm. 2 .

[0012] Preferably, incubation and washing in step (2) are both performed in PBS buffer.

[0013] Preferably, in step (2), the incubation speed is 90~110 r / min, the time is 6~8 min, and it is repeated 7~10 times; the cleaning oscillation frequency is 40~50 times / min, the time is 1~5 min, and it is repeated 2~4 times.

[0014] More preferably, in step (2), the incubation speed is 100 r / min, the time is 6 min, and it is repeated 10 times; the cleaning oscillation frequency is 40 times / min, the time is 1 min, and it is repeated 2 times.

[0015] Preferably, in step (3), washing is performed in PBS buffer; pressing is performed using a glass slide; and filtration is performed using a 100 μm cell sieve.

[0016] Preferably, in step (3), the digestion speed is 50~60 r / min and the time is 30~45 min; the washing oscillation frequency is 20~40 times / min and the time is 1~3 min, repeated 1~3 times.

[0017] The beneficial effects of the above-mentioned technical solution are as follows: the digestion and cleansing are carried out under relatively mild and gentle conditions, which aims to ensure the integrity of gastric stem cells and prevent the stem cell structure from being damaged.

[0018] More preferably, in step (3), the digestion speed is 60 r / min and the time is 35 min; the washing oscillation frequency is 20 times / min and the time is 1 min, repeated 3 times.

[0019] Preferably, the suitable animals for digestive fluids used to isolate gastric stem cells include mammals.

[0020] More preferably, the digestive fluid used to isolate gastric stem cells is suitable for pigs.

[0021] More preferably, the pig is a 14-day-old piglet.

[0022] The beneficial effects of the above-mentioned technical solution of the present invention are as follows: the gastric tissue of piglets of different ages develops differently, and screening piglets of appropriate age can achieve the purpose of optimal isolation of gastric stem cells.

[0023] The present invention has the following beneficial effects: The digestive fluid of this invention for isolating gastric stem cells can efficiently dissociate tissues based on the histological and physiological characteristics of animal gastric bodies, maximize the protection of stem cell activity and function, effectively enrich the target cell population, and provide standardized supporting methods. This invention can overcome the problems of low cell yield, low activity, insufficient purity, and poor reproducibility of existing digestive fluids and supporting methods, and provide reliable, uniform, and high-quality seed cells for gastric body biological research and related translational applications. Attached Figure Description

[0024] Figure 1 The images show the morphological images of gastric body stem cells isolated in Example 1; where (a) is a morphological image of gastric body stem cells at 4×; and (b) is a morphological image of gastric body stem cells at 10×. Figure 2 The images show immunofluorescence staining of gastric body stem cells isolated in Example 1; where (a) is an immunofluorescence staining image of RUNX1, a marker of gastric body stem cells; and (b) is an immunofluorescence staining image of STMN1, a marker of gastric body stem cells. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0026] Example 1 A digestive fluid for isolating gastric stem cells comprises the following components at concentrations: 25.2 nM Na2HPO4, 43.7 nM KH2PO4, 490.8 nM NaCl, 1 nM KCl, 320 nM D-fructofuranosyl-D-glucopyranoside, 498.3 nM M-glucanol, 2.5 nM disodium EDTA and 2.5 nM 1,4-dithiothreitol.

[0027] This embodiment also provides a method for using digestive fluid for isolating gastric stem cells, including the following steps: (1) At 6℃, fresh gastric tissue from 14-day-old piglets was placed in a culture dish containing PBS buffer. The muscle layer was removed with scissors, and the tissue was then cut and broken up. Excess muscle and fat fragments of the gastric tissue were washed away to obtain a size of 1×3 cm. 2 Fragments of gastric tissue; (2) At 6℃, transfer the gastric body tissue fragments to a new 50 mL centrifuge tube containing PBS buffer. Incubate the gastric body tissue fragments at 100 r / min for 6 min, discard the supernatant, add new PBS buffer and continue incubation. Repeat 10 times. Then transfer the incubated gastric body tissue to a new 50 mL centrifuge tube containing PBS buffer and shake by hand for 1 min at a shaking frequency of 40 times / min. Repeat twice until the supernatant is clear to obtain gastric body tissue. (3) Place the gastric body tissue in a new 50 mL centrifuge tube, add the digestion solution for separating gastric body stem cells, and digest it at a shaking speed of 60 r / min for 35 min. After digestion, transfer it to a sterile centrifuge tube, add PBS buffer, and perform a second hand-shaking wash at a shaking frequency of 20 times / min for 1 min. Repeat 3 times until the supernatant is clear. Then transfer the precipitate to a new culture dish, gently press it with a glass slide to separate the gastric body stem cell structure that has not detached, suspend it in PBS buffer, and then use an enzyme-free pipette tip to aspirate the suspension and filter it through a 100 μm cell sieve. Put the filtered suspension back into a new centrifuge tube to obtain gastric body stem cells.

[0028] Example 2 A digestive fluid for isolating gastric stem cells comprises the following components at concentrations: 18.5 nM Na2HPO4, 40 nM KH2PO4, 460.5 nM NaCl, 1 nM KCl, 200 nM D-fructofuranosyl-D-glucopyranoside, 400 nM D-glucanol, 2 nM disodium ethylenediaminetetraacetate, and 2 nM 1,4-dithiothreitol.

[0029] This embodiment also provides a method for using digestive fluid for isolating gastric stem cells, including the following steps: (1) At 2℃, fresh gastric tissue from 14-day-old piglets was placed in a culture dish containing PBS buffer. The muscle layer was removed with scissors, and the tissue was then cut and broken up. Excess muscle and fat fragments of the gastric tissue were washed away, yielding a size of 0.8 × 3 cm. 2 Fragments of gastric tissue; (2) At 2℃, the gastric body tissue fragments were transferred to a new 50 mL centrifuge tube containing PBS buffer. The gastric body tissue fragments were incubated at 90 r / min for 8 min. The supernatant was discarded, and new PBS buffer was added and incubation was continued. This was repeated 7 times. Then, the incubated gastric body tissue was transferred to a new 50 mL centrifuge tube containing PBS buffer and hand-washed at a shaking frequency of 40 times / min for 1 min. This was repeated 4 times until the supernatant was clear, and the gastric body tissue was obtained. (3) Place the gastric body tissue in a new 50 mL centrifuge tube, add the digestion solution for separating gastric body stem cells, and digest it at a shaking speed of 50 r / min for 45 min. After digestion, transfer it to a sterile centrifuge tube, add PBS buffer, and perform a second hand-shaking wash at a shaking frequency of 30 times / min for 2 min. Repeat once until the supernatant is clear. Then transfer the precipitate to a new culture dish, gently press it with a glass slide, separate the gastric body stem cell structure that has not detached, suspend it in PBS buffer, and use an enzyme-free pipette tip to aspirate the suspension and filter it through a 100 μm cell sieve. Put the filtered suspension back into a new centrifuge tube to obtain gastric body stem cells.

[0030] Example 3 A digestive fluid for isolating gastric stem cells comprises the following components at concentrations: 28 nM Na2HPO4, 48 nM KH2PO4, 530 nM NaCl, 15.2 nM KCl, 350.55 nM D-fructofuranosyl-D-glucopyranoside, 605 nM D-glucanol, 4 nM disodium EDTA and 3 nM 1,4-dithiothreitol.

[0031] This embodiment also provides a method for using digestive fluid for isolating gastric stem cells, including the following steps: (1) At 15℃, fresh gastric tissue from 14-day-old piglets was placed in a culture dish containing PBS buffer. The muscle layer was removed with scissors, and the tissue was then cut and broken up. Excess muscle and fat fragments in the gastric tissue were washed away, yielding a size of 1.2×3cm. 2 Fragments of gastric tissue; (2) At 15°C, transfer the gastric body tissue fragments to a new 50 mL centrifuge tube containing PBS buffer. Incubate the gastric body tissue fragments at 110 r / min for 6 min, discard the supernatant, add new PBS buffer and continue incubation. Repeat 10 times. Then transfer the incubated gastric body tissue to a new 50 mL centrifuge tube containing PBS buffer and shake by hand at a shaking frequency of 50 times / min for 5 min. Repeat twice until the supernatant is clear to obtain gastric body tissue. (3) Place the gastric body tissue in a new 50 mL centrifuge tube, add the digestion solution for separating gastric body stem cells, and digest it at a shaking speed of 60 r / min for 30 min. After digestion, transfer it to a sterile centrifuge tube, add PBS buffer, and perform a second hand-shaking wash at a shaking frequency of 40 times / min for 1 min. Repeat 3 times until the supernatant is clear. Then transfer the precipitate to a new culture dish, gently press it with a glass slide, separate the gastric body stem cell structure that has not detached, suspend it in PBS buffer, and use an enzyme-free pipette tip to aspirate the suspension and filter it through a 100 μm cell sieve. Put the filtered suspension back into a new centrifuge tube to obtain gastric body stem cells.

[0032] Experimental Example 1 20 μL of the gastric body stem cells prepared in Example 1 was evenly spread onto a glass slide and observed under a fluorescence microscope. The results are as follows: Figure 1 As shown, Figure 1 The scale bars in the middle figures (a) and (b) are 100 μm and 500 μm, respectively.

[0033] Microscopic observation revealed that the gastric body stem cells were elongated and similar in structure to intestinal stem cells, indicating that the gastric body contained stem cells. Microscopic observation of the isolated gastric body stem cells showed that they were elongated and thin, resembling chromosomes, similar in structure to porcine intestinal stem cells.

[0034] To verify whether the isolated elongated cells were gastric body stem cells, immunofluorescence staining with gastric body stem cell markers RUNX1 and STMN1 was performed. The specific steps were as follows: The digestive fluid was aspirated, and the cells were gently rinsed 2-3 times with pre-cooled PBS to remove residual digestive fluid. 0.1%-0.3% Triton X-100 was added, and the cells were incubated at room temperature for 10 min, followed by 3 washes with PBS for 5 min each. Blocking buffer (5% BSA) was added, and the cells were incubated at room temperature for 2 h. The blocking buffer was aspirated, and primary antibody dilution buffer was added, and the cells were incubated overnight at 4°C. The cells were then washed 3 times with PBS for 5 min each. Secondary fluorescent antibody was added, and the cells were incubated at room temperature in the dark for 2 h, followed by 3 washes with PBS for 5 min each. One drop of DAPI was added to the stem cells, and the cells were incubated for 10 min. After 3 washes with PBS, the cells were mounted and observed and photographed under a fluorescence microscope. The results are as follows: Figure 2 As shown, Figure 2 The scale of each small icon is 100 μm.

[0035] from Figure 2 As can be seen, RUNX1 and STMN1 are specifically expressed in the isolated gastric body stem cells, indicating that the isolated cells are gastric body stem cells.

[0036] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the scope of the present invention.

Claims

1. A digestive fluid for isolating gastric stem cells, characterized in that, The components include the following concentrations: 18.5–28 nM Na₂HPO₄, 40–48 nM KH₂PO₄, 460.5–530 nM NaCl, 1–15.2 nM KCl, 200–350.55 nM D-fructofuranosyl-D-glucopyranoside, 400–605 nM D-glucanol, 2–4 nM disodium ethylenediaminetetraacetate, and 2–3 nM 1,4-dithiothreitol.

2. The digestive fluid for isolating gastric stem cells as described in claim 1, characterized in that, The components include the following concentrations: 25.2 nM Na2HPO4, 43.7 nM KH2PO4, 490.8 nM NaCl, 1 nM KCl, 320 nM D-fructofuranosyl-D-glucopyranoside, 498.3 nM D-glucanol, 2.5 nM disodium EDTA and 2.5 nM 1,4-dithiothreitol.

3. The method of using the digestive fluid for isolating gastric stem cells as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Separate and break up the gastric body tissue, wash it, and obtain gastric body tissue fragments; (2) Incubate the gastric body tissue fragments and wash them until the supernatant is clear to obtain gastric body tissue; (3) The gastric body tissue was digested in a digestive solution used to separate gastric body stem cells, then washed until the supernatant was clear, pressed to separate and filtered to obtain gastric body stem cells.

4. The method of using the digestive fluid for isolating gastric stem cells as described in claim 3, characterized in that, Steps (1) and (2) are all performed at 2-15°C; the size of the gastric tissue fragments is (0.8-1.2) × 3 cm. 2 .

5. The method of using the digestive fluid for isolating gastric stem cells as described in claim 3, characterized in that, In step (2), the incubation speed is 90~110 r / min, the time is 6~8 min, and it is repeated 7~10 times; the cleaning oscillation frequency is 40~50 times / min, the time is 1~5 min, and it is repeated 2~4 times.

6. The method of using the digestive fluid for isolating gastric stem cells as described in claim 5, characterized in that, In step (2), the incubation speed is 100 r / min, the time is 6 min, and it is repeated 10 times; the cleaning oscillation frequency is 40 times / min, the time is 1 min, and it is repeated 2 times.

7. The method of using the digestive fluid for isolating gastric stem cells as described in claim 3, characterized in that, In step (3), the digestion speed is 50-60 r / min and the time is 30-45 min; the cleaning oscillation frequency is 20-40 times / min and the time is 1-3 min, repeated 1-3 times.

8. The method of using the digestive fluid for isolating gastric stem cells as described in claim 7, characterized in that, In step (3), the digestion speed is 60 r / min and the time is 35 min; the cleaning oscillation frequency is 20 times / min and the time is 1 min, repeated 3 times.

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