Intestinal muscle layer cell separation and extraction method and separation and extraction reagent
By using a step-down dissociation solution combination and a neutralization solution, the problem of separating the intestinal mucosa and submucosa in existing technologies has been solved, achieving high-proportion and high-activity intestinal muscle layer cell extraction, and improving separation purity and cell analysis reliability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING DRUM TOWER HOSPITAL
- Filing Date
- 2023-01-17
- Publication Date
- 2026-07-21
Smart Images
Figure CN116948943B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cell extraction, specifically relating to a method for separating and extracting intestinal muscle layer cells and a separation and extraction reagent. Background Technology
[0002] The intestines are both a digestive and immune organ. The intestinal wall consists of five layers, from the inside out: the mucus layer, the mucosa, the submucosa, the muscular layer, and the serosa. Figure 1 As shown, the muscular layer is mainly composed of muscle cells. During surgery or infection, a large number of immune cells (such as macrophages and lymphocytes) infiltrate the muscular layer, interacting with the muscle cells and leading to intestinal wall inflammation and swelling, intestinal motility disorders, and various adverse consequences such as intestinal obstruction, paralytic ileus, and enterogenic infections. Therefore, isolating and extracting intestinal muscular layer cells has significant medical value and helps to deepen our understanding of the mechanisms of intestinal disease development.
[0003] Current intestinal muscle layer separation techniques mostly involve mechanically peeling away the mucosa and submucosa to expose the muscle layer, such as in Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation. J.Vis.Exp.(67),e4086 10.3791 / 4086,DOI:10.3791 / 4086(2012).,Zou Kang. Study on the mechanism of SRC-3 inhibiting intestinal wall inflammation in mice after surgery. Master's thesis, Fujian Medical University, 2015.05. The two techniques have the following disadvantages: (1) The intestinal mucosa is distributed in a fold-like manner, which is difficult to fully spread out under in vitro conditions. It is difficult to fully peel off the mucosa in the folds by mechanical external force alone, which easily causes residue; (2) When the intestine is diseased, the intestinal wall is inflamed, edematous and adhered, and the boundaries of each layer are unclear. It is even more difficult to accurately peel off the mucosal layer and submucosa by mechanical external force; (3) The mucosal layer is rich in a large number of immune cells. In the disease state, it is attacked by a variety of pathogenic microorganisms. If the mucosal layer is not fully peeled off, it is easy to cause immune cells and microorganisms to remain, which interferes with the subsequent functional analysis and mechanism exploration of muscle layer cells.
[0004] Patent application CN112342193A discloses a method for mechanically peeling off the serosal layer from the outside to expose the muscle layer. Because the serosal layer is composed of a thin layer of flattened mesothelial cells and connective tissue, and is tightly adhered to the underlying muscle layer, it is difficult to completely peel it off from the surface of the muscle layer by mechanical force. In disease states, this adhesion is even more pronounced, easily leading to unnecessary residues. CN114540272A discloses a highly efficient and active method for separating and extracting mouse intestinal epithelial cells and immune cells. This method uses a combination of digestion and mechanical separation to separate and extract intestinal epithelial cells, interepithelial immune cells, and lamina propria immune cells, but does not involve the extraction of muscle layer cells.
[0005] To address the aforementioned issues, we have been seeking an ideal technological solution. Summary of the Invention
[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a method and reagents for separating and extracting intestinal myocytes with high cell viability and a large proportion of exposed myocytes.
[0007] The technical solution of this invention is as follows: a reagent for separating and extracting intestinal muscle layer cells, which is composed of a mucus layer dissociation solution, a mucosal layer dissociation solution, a submucosal layer dissociation solution, a muscle layer digestion solution, and a neutralization solution. The mucus layer dissociation solution, the mucosal layer dissociation solution, and the submucosal layer dissociation solution are buffer solutions containing 0.1-1.0 wt% trypsin, and the concentration of trypsin in the mucus layer dissociation solution, the mucosal layer dissociation solution, and the submucosal layer dissociation solution changes in a decreasing stepwise manner. The submucosal layer dissociation solution also contains 20-40 mU / ml elastase. The digestion solution is a buffer solution containing 0.5-0.75 wt% collagenase I / IV, 15-25 U / ml neutral protease, and 150-250 U / ml DNase I. Buffer solutions, used as solvents for dissociating and digesting enzymes, can be balanced salt solutions such as PBS, Earle's solution, D-Hank's balanced solution, and Hank's balanced solution; cell culture media such as DMEM, RPMI 1640, MEM, and DMEM / F12 can also be used; and isotonic solutions such as physiological saline or 0.278 mol·L⁻¹ can also be used. -1 Glucose solution, etc.
[0008] This invention utilizes the loose structure and easy injection of the submucosa. A neutralizing solution can be pre-injected into the submucosa to elevate both the submucosa and the mucosa, achieving physical isolation and mechanical separation from the muscle layer. After the dissociation solution gradually digests the submucosa layer, it automatically mixes with the neutralizing solution, terminating digestion and protecting the muscle layer cells from damage. The neutralizing solution can be a balanced salt solution, cell culture medium, or isotonic solution. Examples of balanced salt solutions include PBS, Earle's solution, D-Hank's balanced solution, and Hank's balanced solution. Cell culture media include DMEM, RPMI 1640, MEM, and DMEM / F12.
[0009] Based on the structural characteristics of each layer of the intestinal wall, a "step-down" dissociation solution combination was designed, with the highest concentration in the mucus layer, the middle concentration in the mucosal layer, and the lowest concentration in the submucosa. The dissociation solution is primarily composed of trypsin, which is used to degrade the cytoskeleton and intercellular connections. Because the submucosa is rich in elastin and reticular fibers, elastase is added to the dissociation solution for this layer to improve digestive efficiency. To maximize the protection of muscle layer cell activity, the muscle layer digestive solution is designed as a combination of collagenase, neutral protease, and deoxyribonuclease I (DNase I). Collagenase effectively digests the intercellular matrix with minimal cell damage, neutral protease has mild proteolytic activity without damaging cell membrane integrity and works synergistically with collagenase, and DNase I degrades free DNA released during cell separation, preventing DNA-induced cell aggregation and enhancing digestive efficiency.
[0010] As a further improvement to the technical solution, in order to enhance the dissociation efficiency, the mucosal layer dissociation solution is composed of an epithelial layer dissociation solution and a lamina propria dissociation solution, with the trypsin concentration in the epithelial layer dissociation solution being greater than that in the lamina propria dissociation solution.
[0011] As a further improvement to the technical solution, in order to enhance the dissociation efficiency, the trypsin concentrations in the mucus layer dissociation solution, the mucosal layer dissociation solution, and the submucosal layer dissociation solution are 0.5-1.0 wt%, 0.1-0.5 wt%, and 0.05-0.1 wt%, respectively.
[0012] As a further improvement to the technical solution, in order to enhance the dissociation efficiency, the trypsin concentrations in the epithelial layer dissociation solution and the lamina propria dissociation solution are 0.2-0.5 wt% and 0.1-0.3 wt%, respectively.
[0013] As a further improvement to the technical solution, in order to enhance the dissociation efficiency, the trypsin concentrations in the mucus layer dissociation solution, epithelial layer dissociation solution, lamina propria dissociation solution, and submucosal layer dissociation solution are 0.5wt%, 0.25wt%, 0.1wt%, and 0.05wt%, respectively.
[0014] As a further improvement to the technical solution, in order to enhance the dissociation efficiency, the trypsin concentrations in the mucus layer dissociation solution, epithelial layer dissociation solution, lamina propria dissociation solution, and submucosal layer dissociation solution are 1 wt%, 0.5 wt%, 0.25 wt%, and 0.1 wt%, respectively.
[0015] As a further improvement to the technical solution, in order to enhance the dissociation efficiency, the mucus layer dissociation solution, the mucosa layer dissociation solution and the submucosa layer dissociation solution each contain 20-40 mM of EDTA. EDTA acts as a chelating agent to enhance the hydrolytic function of trypsin.
[0016] As a further improvement to the technical solution, in order to reduce cytotoxicity, the neutralizing solution is DMEM containing 10% (V / V) fetal bovine serum.
[0017] A method for extracting intestinal muscle layer cells using the aforementioned separation and extraction reagent includes the following steps:
[0018] (1) Collect intestinal specimens, divide them into 3-5 equal segments, wash them with PBS solution to remove blood or dirt from the surface of the specimens, and remove any remaining fat or connective tissue from the surface of the specimens.
[0019] (2) Cut the intestine along the axis, lay it flat to fully expose the inner surface, then fix the edge of the intestine and wash away the contents and impurities of the intestinal cavity;
[0020] (3) Use a syringe to inject the neutralizing solution into the submucosa until the intestinal mucosa is lifted and bulged.
[0021] (4) Add mucus layer dissociation solution, place in a constant temperature shaker, and incubate at 37°C and 5% (V / V) CO2 until the mucus layer on the intestinal wall disappears. Then remove the mucus layer dissociation solution and wash the intestinal tract with PBS solution.
[0022] (5) Add mucosal layer dissociation solution, place in a constant temperature shaker, and incubate at 37°C and 5% (V / V) CO2 until the intestinal wall mucosal epithelial layer disappears. Then remove the mucosal layer dissociation solution and wash the intestinal tract with PBS solution.
[0023] (6) Add submucosal layer dissociation solution, place in a constant temperature shaker, and incubate at 37°C and 5% (V / V) CO2 until the submucosal layer of the intestinal wall disappears. Use PBS solution to wash the intestinal tract with the submucosal layer dissociation solution.
[0024] (7) Add digestive solution, place in a constant temperature shaker, and incubate at 37°C and 5% (V / V) CO2 for 25-40 minutes. Collect the cell suspension, filter, centrifuge, and collect the precipitate to obtain intestinal muscle layer cells.
[0025] As a further improvement to the technical solution, step (5) involves adding epithelial layer dissociation solution C, placing it in a constant temperature shaker, and incubating it at 37°C and 5% (V / V) CO2 until the intestinal wall mucosal epithelial layer disappears. Then, the epithelial layer dissociation solution is removed, and the intestinal tract is washed with PBS solution. Next, lamina propria dissociation solution is added, placed in a constant temperature shaker, and incubated at 37°C and 5% (V / V) CO2 until the lamina propria disappears. Then, the lamina propria dissociation solution is removed, and the intestinal tract is washed with PBS solution.
[0026] This invention possesses significant substantive features and substantial advancements compared to existing technologies. Specifically, the muscle layer cell extraction reagent of this invention exhibits a high dissociation rate of cells from the outer surface of the intestinal muscle layer. Furthermore, the extraction method of this invention utilizes a neutralization solution to pre-achieve physical isolation and mechanical separation between the outer tissue of the intestinal muscle layer and the muscle layer itself, minimizing damage to muscle layer cells from the dissociation solution. The separation and extraction method of this invention offers the advantages of high cell viability and a large exposure rate of muscle layer cells. Attached Figure Description
[0027] Figure 1 This is a schematic diagram of the intestinal wall structure.
[0028] Figure 2 Electron micrographs of intestinal muscle layer cells before and after separation and extraction. Detailed Implementation
[0029] The technical solution of the present invention will be further described in detail below through specific embodiments. The reagents and their sources used in the examples and comparative examples are: trypsin: ThermoFisher#27250018, elastase: Sigma#E7885, collagenase I: Sigma#C0130, collagenase IV: Sigma#C5138, neutral protease: Roche#04942086001, EDTA: Sigma#324506, DNase I: ThermoFisher#EN0521. Other reagents not specified are commercially available or general-purpose reagents.
[0030] The experimental method in the example is as follows
[0031] Three C57BL / 6 mice, aged 12–16 weeks, were sacrificed by cervical dislocation.
[0032] 1. Take intestinal specimens and divide them into 3-5 equal segments. Wash them with PBS solution to remove blood or dirt from the surface of the specimens and remove any remaining fat or connective tissue.
[0033] 2. Use sterile scissors to cut the intestinal tube along the axial direction, place it on a grid plate, spread the intestinal tube flat so that the inner surface is fully exposed, fix the edges of the intestinal tube with fixation pins, immerse it in PBS solution, and wash away the contents and impurities of the intestinal lumen.
[0034] 3. Use a syringe to inject an appropriate amount of neutralizing solution into the submucosa, causing the intestinal mucosa to be raised and bulging.
[0035] 4. Remove the PBS solution, add the mucus layer dissociation solution, and incubate in a constant temperature shaker (37℃, 5% CO2) for 10 minutes. Observe under a stereomicroscope that the intestinal wall mucus layer has disappeared.
[0036] 5. Remove the mucus layer dissociation solution and gently wash repeatedly with PBS solution 3 times.
[0037] 6. Remove the PBS solution, add the epithelial dissociation solution, and incubate in a constant temperature shaker (37℃, 5% CO2) for 15 minutes. Observe under a stereomicroscope that the intestinal wall mucosal epithelium has disappeared.
[0038] 7. Remove the epithelial layer dissociation solution, replace with PBS solution, and gently wash three times.
[0039] 8. Remove the PBS solution, add the lamina propria dissociation solution, and incubate in a constant temperature shaker (37℃, 5% CO2) for 20 minutes. Observe under a stereomicroscope that the lamina propria of the intestinal wall mucosa has disappeared.
[0040] 9. Remove the intrinsic layer dissociation solution, replace with PBS solution, and gently wash three times.
[0041] 10. Remove the PBS solution, add the submucosal layer dissociation solution, and incubate in a constant temperature shaker (37℃, 5% CO2) for 15 minutes. Observe under a stereomicroscope that the submucosal layer of the intestinal wall has disappeared.
[0042] 11. Remove the submucosal layer dissociation solution, replace with PBS solution, and gently wash three times.
[0043] 12. Remove the PBS solution, add the muscle layer digestion solution, and incubate in a constant temperature shaker (37℃, 5% CO2) for 30 minutes. Gently pipette the surface of the intestinal segment several times to collect the cell suspension. Filter the suspension through a 100um filter, centrifuge at 3000 rpm for 3 minutes at room temperature, discard the supernatant, wash with PBS solution, centrifuge again, and collect the precipitate, which is the intestinal muscle layer cells.
[0044] Example 1
[0045] Reagent composition table 1
[0046]
[0047] The buffer solution is PBS.
[0048] Figure 1 This is a schematic diagram of the intestinal wall structure.
[0049] Example 1: Comparison of effects before and after muscle layer extraction (see Example 1) Figure 2 , Figure 2 Images were taken using a LEICA DM2000 microscope and Leica Application Suite X software. Figure 2 A represents the effect before separation and extraction. Figure 2 In the diagram, B represents the effect after separation and extraction; a represents circular muscle, and b represents longitudinal muscle.
[0050] Example 2
[0051] Reagent composition table 2
[0052]
[0053]
[0054] The buffer solution is PBS.
[0055] Example 3
[0056] Reagent composition table 3
[0057]
[0058] The buffer solution is PBS.
[0059] Comparative Example 1
[0060] The difference between this comparative example and Example 1 is that no neutralizing solution was pre-injected.
[0061] Comparative Example 2
[0062] The difference between this comparative example and the previous examples lies in the fact that a single digestive solution was used during the digestion of each component of the intestinal wall, without stepwise washing with PBS. The single digestive solution consisted of PBS containing trypsin (1 wt%) and EDTA (30 mM).
[0063] The dissociation and digestion results of the examples and comparative examples are shown in the table below:
[0064]
[0065]
[0066] The method for calculating the muscle layer exposure ratio is as follows: After step 10, randomly observe 20 low-power microscope fields of 40x magnification and calculate the number of fields of view where the muscle layer exposure area exceeds 80%.
[0067] The cell viability was measured by staining the extracted muscle layer cells with Trypan Blue and calculating the proportion of cells that were not stained with blue.
[0068] The digestion results of the examples and comparative examples show that the pre-injection of neutralizing solution before extraction can improve cell activity.
[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.
Claims
1. A reagent for isolating and extracting intestinal muscle layer cells, characterized in that, It is composed of mucus layer dissociation solution, mucosal layer dissociation solution, submucosal layer dissociation solution, muscle layer digestion solution, and neutralization solution. The mucus layer dissociation solution, mucosal layer dissociation solution, and submucosal layer dissociation solution each contain 0.1-1.0 wt% trypsin buffer solution. The concentration of trypsin in the mucus layer dissociation solution, mucosal layer dissociation solution, and submucosal layer dissociation solution changes in a decreasing stepwise manner. The submucosal layer dissociation solution also contains 20-40 mU / ml elastase. The digestion solution is a buffer solution containing 0.5-0.75 wt% collagenase I / IV, 15-25 U / ml neutral protease, and 150-250 U / ml DNase I. The trypsin concentrations in the mucus layer dissociation fluid, mucosal layer dissociation fluid, and submucosal layer dissociation fluid were 0.5-1.0 wt%, 0.1-0.5 wt%, and 0.05-0.1 wt%, respectively.
2. The separation and extraction reagent according to claim 1, characterized in that, The mucosal layer dissociation solution is composed of epithelial layer dissociation solution and lamina propria dissociation solution. The concentration of trypsin in the epithelial layer dissociation solution is greater than that in the lamina propria dissociation solution.
3. The separation and extraction reagent according to claim 1 or 2, characterized in that, The trypsin concentrations in the epithelial layer dissociation solution and the lamina propria dissociation solution were 0.2-0.5 wt% and 0.1-0.3 wt%, respectively.
4. The separation and extraction reagent according to claim 1 or 2, characterized in that, The trypsin concentrations in the mucus layer dissociation fluid, epithelial layer dissociation fluid, lamina propria dissociation fluid, and submucosal layer dissociation fluid were 0.5 wt%, 0.25 wt%, 0.1 wt%, and 0.05 wt%, respectively.
5. The separation and extraction reagent according to claim 4, characterized in that, The trypsin concentrations in the mucus layer dissociation fluid, epithelial layer dissociation fluid, lamina propria dissociation fluid, and submucosal layer dissociation fluid were 1 wt%, 0.5 wt%, 0.25 wt%, and 0.1 wt%, respectively.
6. The separation and extraction reagent according to claim 1, characterized in that, The mucus layer dissociation solution, mucosa layer dissociation solution, and submucosa layer dissociation solution each contain 20-40 mM of EDTA.
7. The separation and extraction reagent according to claim 1, characterized in that, The neutralizing solution is DMEM containing 10% (V / V) fetal bovine serum.
8. A method for extracting intestinal muscle layer cells using the separation and extraction reagent according to any one of claims 1-7, comprising the following steps: (1) Collect intestinal specimens, divide them into 3-5 equal segments, wash them with PBS solution to remove blood or dirt from the surface of the specimens, and remove any remaining fat or connective tissue from the surface of the specimens. (2) Cut the intestine along the axis, spread the intestine flat to fully expose the inner surface, then fix the edge of the intestine and wash away the contents and impurities of the intestinal lumen; (3) Use a syringe to inject the neutralizing solution into the submucosa until the intestinal mucosa is raised and bulging; (4) Add mucus layer dissociation solution, place in a constant temperature shaker, and incubate at 37°C and 5% (V / V) CO2 until the mucus layer on the intestinal wall disappears. Then remove the mucus layer dissociation solution and wash the intestinal tract with PBS solution. (5) Add mucosal layer dissociation solution, place in a constant temperature shaker, and incubate at 37°C and 5% (V / V) CO2 until the intestinal wall mucosal epithelium disappears. Then remove the mucosal layer dissociation solution and wash the intestinal tract with PBS solution. (6) Add submucosal layer dissociation solution, place in a constant temperature shaker, and incubate at 37°C and 5% (V / V) CO2 until the submucosal layer of the intestinal wall disappears. Use the submucosal layer dissociation solution to wash the intestinal tract with PBS solution. (7) Add digestive solution, place in a constant temperature shaker, incubate at 37℃ and 5% (V / V) CO2 for 25-40 minutes, collect cell suspension, filter, centrifuge, and collect the precipitate as intestinal muscle layer cells.
9. The method according to claim 8, characterized in that, Step (5) involves adding epithelial layer dissociation solution C, placing it in a constant temperature shaker, and incubating it at 37°C and 5% (V / V) CO2 until the intestinal wall mucosal epithelial layer disappears. Then, the epithelial layer dissociation solution is removed, and the intestinal tract is washed with PBS. Next, lamina propria dissociation solution is added, and the intestinal tract is placed in a constant temperature shaker and incubated at 37°C and 5% (V / V) CO2 until the lamina propria disappears. Then, the lamina propria dissociation solution is removed, and the intestinal tract is washed with PBS.