Separation method and application of colonic lamina propria cells

Abstract

The invention discloses a method for separating colonic lamina propria cells of mice with colitis and application of the method. According to the method, separation of the propria neutrophile granulocyte on the colon tissue with the complete length is tried for the first time, and the total time required by cell separation is effectively shortened through multiple digestion reagents with different compositions, so that apoptosis of the neutrophile granulocyte caused by long-time exposure of an intestinal sample is effectively avoided; the problems of low cell yield, poor activity and the like are solved, and a new thought is provided for separation of the neutrophile granulocytes.

Description

Technical Field

[0001] The present invention relates to the technical field of biological cell separation, in particular to a method for separating colon lamina propria cells and application thereof. Background Art

[0002] As the body's largest mucosal immune interface, the intestine bears a unique dual immune mission: on the one hand, it must maintain immune tolerance to food antigens and commensal microorganisms; on the other hand, it must be able to rapidly identify and eliminate invading pathogens. These functions rely on a sophisticated multi-layered defense system, encompassing the coordinated actions of physical barriers, immune cell networks, and commensal microbiota. The intestinal mucosal immune system is composed of three major lymphoid compartments: the lamina propria, located beneath the basement membrane of the intestinal villi; the intraepithelial compartment, containing intraepithelial lymphocytes, located above the basement membrane between columnar epithelial cells; and lymph nodes embedded in the intestinal wall and Peyer's patches. The intestinal lamina propria is rich in immune cells, including natural killer cells, macrophages, and various T cells. These immune cells collectively participate in diverse immune responses, achieving a balance between defense and tolerance.

[0003] The intestinal mucosal immune system is the body's primary line of defense against invading pathogens such as bacteria and viruses. Long-term exposure to a complex environment allows the intestinal mucosal immune system to develop tolerance to various stimuli from food and symbiotic bacteria, while also generating immune responses to stimuli from pathogens.

[0004] Colitis is a complex disease characterized by chronic inflammation and barrier dysfunction in the colonic mucosa. Its core pathogenesis is closely related to overactivation of the mucosal immune system. As the core effector region of mucosal immunity, the intestinal lamina propria drives disease progression when immune cell imbalance, barrier disruption, and disrupted microbial interactions occur.

[0005] Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract caused by dysfunction of the innate and adaptive immune responses. Impaired innate immunity leads to the body's lack of control over the altered homeostasis of the intestinal microbial environment and activates the adaptive immune system, thereby promoting the occurrence of secondary inflammatory responses and leading to tissue damage. Neutrophils are key players in intestinal innate immunity, and neutrophil infiltration in the intestinal mucosa is an important hallmark of IBD. Studies have shown that neutrophils are a type of heterogeneous and dual-natured cell, which are believed to have dual functions in IBD, namely, both destructive and protective effects. In addition, existing studies have also emphasized the association between neutrophils and disease development and intestinal microbiota. Therefore, the isolation and analysis of immune cells in the intestinal lamina propria can provide key research tools and theoretical basis for in-depth analysis of the molecular mechanisms of imbalance in intestinal immune homeostasis, the development of disease-specific biomarkers and targeted treatment strategies.

[0006] In the prior art, there are a variety of methods for dissociating mouse intestinal components, but it is usually necessary to first cut the intestine into small segments of 0.5-1 cm, and then separate the intestinal epithelial cells by multiple oscillations, and digest for a long time with a variety of digestive enzymes to achieve cell separation. However, these methods often have problems such as overly complicated operating steps and long digestion time, which leads to low cell yield and poor activity, greatly limiting subsequent research based on isolated cells. Moreover, neutrophils themselves are sensitive and fragile, and usually begin to undergo apoptosis in 4-6 hours. Too long cell digestion and separation will seriously affect the quality of neutrophils. Therefore, when faced with large-scale samples (such as more than 20 samples), the existing methods will cause a large amount of neutrophil loss due to the long pre-treatment time. Therefore, there is an urgent need for an improved method for separating neutrophils in the colon lamina propria that is suitable for large sample experiments and is efficient. Summary of the Invention

[0007] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide a method for isolating neutrophils in the colonic lamina propria of colitis mice and its application. The present invention attempts for the first time to achieve the separation of neutrophils in the lamina propria on the complete length of colon tissue, and effectively shortens the total time required for cell separation by using a variety of digestion reagents with different compositions, thereby effectively avoiding the apoptosis of neutrophils caused by long-term exposure of intestinal samples, resulting in low cell yield and poor activity, and provides a new idea for the separation of neutrophils.

[0008] The first aspect of the present invention provides a method for isolating colon lamina propria immune cells, comprising the following steps:

[0009] (1) digesting the colon and intestine using pre-digestion reagent 1, pre-digestion reagent 2, and enzymatic digestion reagent in sequence to obtain an enzymatic digestion solution;

[0010] (2) Isolate colonic lamina propria immune cells from enzymatic digestion fluid.

[0011] In some embodiments of the invention, the colon is an animal colon.

[0012] In some embodiments of the present invention, the animals include humans and non-human mammals; the non-human mammals include but are not limited to mice (including rats, mice and guinea pigs), rabbits, dogs, horses, primates (including gorillas and monkeys).

[0013] In some embodiments of the present invention, the animal may be a disease model animal, and the disease is a colon-related disease, including but not limited to colitis.

[0014] In some embodiments of the present invention, the animal is a colitis mouse.

[0015] In some embodiments of the present invention, the pre-digestion reagent 1 and the pre-digestion reagent 2 do not contain Ca 2+ and Mg 2+ .

[0016] In some embodiments of the present invention, the intestinal tract of the colitis mouse is not sheared.

[0017] In the present invention, the "shearing treatment" refers to shearing the intestinal tissue to a size of 1 cm or less according to conventional methods in the art. In the present invention, the shearing treatment does not include simply dissecting the intestine longitudinally to expose the intestinal epithelial layer.

[0018] In some embodiments of the present invention, the pre-digestion reagent 1 includes a chelating agent.

[0019] In some embodiments of the present invention, the chelating agent in the pre-digestion reagent 1 includes ethylenediaminetetraacetic acid (EDTA).

[0020] In some embodiments of the present invention, the final concentration of the chelating agent in the pre-digestion reagent 1 is 2-10 mM.

[0021] In some embodiments of the present invention, the pre-digestion reagent 1 further includes at least one of a reducing agent and a stabilizer.

[0022] In some embodiments of the present invention, the reducing agent of the pre-digestion reagent 1 includes dithiothreitol (DTT).

[0023] In some embodiments of the present invention, the balanced salt of the pre-digestion reagent 1 includes HEPES.

[0024] In some embodiments of the present invention, the pre-digestion reagent 1 further comprises at least one of the following components at the following concentrations: a reducing agent at 0.5-3 mM and a stabilizer at 10-20 mM.

[0025] In some embodiments of the present invention, the pre-digestion reagent 1 further includes PBS buffer as a solvent.

[0026] In some embodiments of the present invention, the PBS buffer does not contain Ca 2+ and Mg 2+ .

[0027] In some embodiments of the present invention, the pre-digestion reagent 1 includes the following components based on the final concentration of each component in the solvent: 5-10 mM EDTA, 0.5-3 mM DTT and 10-20 mM HEPES buffer.

[0028] In some embodiments of the present invention, the pre-digestion reagent 1 includes the following components based on the final concentration of each component in the solvent: 5 mM EDTA, 1 mM DTT and 10 mM HEPES buffer.

[0029] In some embodiments of the present invention, based on the final concentration of each component in the solvent, the pre-digestion reagent 1 is a PBS buffer solution containing 5 mM EDTA, 1 mM DTT and 10 mM HEPES buffer.

[0030] In some embodiments of the present invention, the dosage of the pre-digestion reagent 1 is: 2-5 mL pre-digestion reagent 1 per colon.

[0031] In some embodiments of the present invention, the dosage of the pre-digestion reagent 1 is: 2 mL of pre-digestion reagent 1 per colon.

[0032] In some embodiments of the present invention, the pre-digestion time is 8-12 minutes.

[0033] In some embodiments of the present invention, the pre-digestion time is 10 minutes.

[0034] In some embodiments of the present invention, the pre-digestion reagent 2 includes a chelating agent.

[0035] In some embodiments of the present invention, the chelating agent in the pre-digestion reagent 2 includes EDTA.

[0036] In some embodiments of the present invention, the final concentration of the chelating agent in the pre-digestion reagent 2 is 2-10 mM.

[0037] In some embodiments of the present invention, the pre-digestion reagent 2 further includes a balanced salt.

[0038] In some embodiments of the present invention, the balanced salt of the pre-digestion reagent 2 includes HEPES.

[0039] In some embodiments of the present invention, the pre-digestion reagent 2 further comprises 10-20 mM balanced salt.

[0040] In some embodiments of the present invention, the pre-digestion reagent 2 further includes PBS buffer as a solvent.

[0041] In some embodiments of the present invention, the PBS buffer does not contain Ca 2+ and Mg 2+ .

[0042] In some embodiments of the present invention, the pre-digestion reagent 2 includes the following components based on the final concentration of each component in the solvent: 2-10 mM EDTA and 10-20 mM HEPES buffer.

[0043] In some embodiments of the present invention, the pre-digestion reagent 2 includes the following components based on the final concentration of each component in the solvent: 5 mM EDTA and 10 mM HEPES buffer.

[0044] In some embodiments of the present invention, based on the final concentration of each component in the solvent, the pre-digestion reagent 2 is a PBS buffer solution containing 5 mM EDTA and 10 mM HEPES buffer.

[0045] In some embodiments of the present invention, the dosage of the pre-digestion reagent 2 is: 2-5 mL pre-digestion reagent 2 per colon.

[0046] In some embodiments of the present invention, the dosage of the pre-digestion reagent 2 is: 2 mL of pre-digestion reagent 2 per colon.

[0047] In some embodiments of the present invention, the digestion time is 8-12 minutes.

[0048] In some embodiments of the present invention, the digestion time is 10 minutes.

[0049] In some embodiments of the present invention, the enzymatic digestion reagent includes collagenase and nuclease.

[0050] In some embodiments of the present invention, the collagenase comprises at least one of collagenase D, collagenase IV, trypsin, and neutral protease.

[0051] In some embodiments of the present invention, the collagenase is collagenase D.

[0052] In some embodiments of the present invention, the nuclease comprises at least one of DNase I, Endonuclease V and Fragmentase.

[0053] In some embodiments of the invention, the nuclease is DNase I.

[0054] In some embodiments of the present invention, the enzymatic digestion reagent includes the following components, based on the final concentration of each component in the solvent: 0.5-1.5 mg / mL collagenase and 20-80 U / mL nuclease.

[0055] In some embodiments of the present invention, the enzymatic digestion reagent further comprises a protease inhibitor.

[0056] In some embodiments of the invention, the protease inhibitor comprises at least one of FBS and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF).

[0057] In some embodiments of the present invention, the protease inhibitor is FBS.

[0058] In some embodiments of the present invention, the enzymatic digestion reagent includes 5-15% (v / v) of protease inhibitors based on the final concentration of each component in the solvent.

[0059] In some embodiments of the present invention, the enzymatic digestion reagent includes cell culture medium as a solvent.

[0060] In some embodiments of the present invention, the cell culture medium includes RPMI-1640 medium or the like containing Ca 2+ and Mg 2+ of cell culture medium.

[0061] In some embodiments of the present invention, the cell culture medium is RPMI-1640 medium.

[0062] In some embodiments of the present invention, the enzymatic digestion reagent includes the following components, based on the final concentration of each component in the solvent: 5-15% (v / v) protease inhibitor, 0.5-1.5 mg / mL collagenase, and 20-80 U / mL nuclease.

[0063] In some embodiments of the present invention, the enzymatic digestion reagent includes the following components, based on the final concentration of each component in the solvent: 10% (v / v) protease inhibitor, 1 mg / mL collagenase, and 40 U / mL nuclease.

[0064] In some embodiments of the present invention, the enzymatic digestion reagent is: RPMI-1640 culture medium containing 10% (v / v) FBS, 1 mg / mL collagenase D and 40 U / mL DNase I, based on the final concentration of each component in the solvent.

[0065] In some embodiments of the present invention, the amount of the enzymatic digestion reagent is: 2-5 mL of enzymatic digestion reagent per colon.

[0066] In some embodiments of the present invention, the amount of the enzymatic digestion reagent is: 2 mL of enzymatic digestion reagent per colon.

[0067] In some embodiments of the present invention, the enzymatic digestion time is 30-45 min.

[0068] In some embodiments of the present invention, the enzymatic digestion time is 40 min.

[0069] In some embodiments of the present invention, a wash buffer is used to remove the pre-digestion reagent in step (1), and the wash buffer comprises serum or a serum replacement.

[0070] In some embodiments of the present invention, the serum comprises at least one of FBS and CS.

[0071] In some embodiments of the invention, the serum is FBS.

[0072] In some embodiments of the present invention, the serum substitute comprises at least one of bovine serum albumin (BSA), polyethylene glycol (PEG), and a serum-free stop buffer.

[0073] In some embodiments of the present invention, the solvent comprises a buffer, including but not limited to PBS buffer.

[0074] In some embodiments of the present invention, the colon intestine is cut open along the extending direction of the intestine.

[0075] In some embodiments of the present invention, the length of the dissected colon is greater than 1 cm.

[0076] In some embodiments of the present invention, the separation in step (2) includes: removing impurities from the enzymatic digestion solution and then centrifuging to obtain a cell pellet, resuspending the cell pellet using a first Percoll reagent and then adding it dropwise to a second Percoll reagent, performing density gradient centrifugation, and then aspirating the cell layer at the interface of the two liquid phases to obtain immune cells in the colon propria of colitis mice.

[0077] In some embodiments of the present invention, the method specifically comprises:

[0078] (1) using pre-digestion reagent 1 to pre-digest the colon, washing to remove epithelial cells, and then adding pre-digestion reagent 2 for digestion, removing the pre-digestion reagent, and adding enzymatic digestion reagent for enzymatic digestion to obtain an enzymatic digestion solution;

[0079] (2) The enzymatic digestion solution was removed from impurities and centrifuged to obtain a cell pellet. The cell pellet was resuspended in the first Percoll reagent and then added dropwise to the second Percoll reagent. After density gradient centrifugation, the cell layer at the interface of the two liquid phases was aspirated to obtain the immune cells of the colon lamina propria of the colitis mouse.

[0080] In some embodiments of the present invention, the specific steps of the method are:

[0081] (1) Using pre-digestion reagent 1, the intestinal tract of a colitis mouse that has not been sheared is pre-digested for 8-12 minutes, washing to remove epithelial cells, and then adding pre-digestion reagent 2 for digestion for 8-12 minutes. Washing buffer is added to remove the pre-digestion reagent, and enzymatic digestion reagent is added for enzymatic digestion for 30-45 minutes to obtain an enzymatic digestion solution;

[0082] (2) The enzymatic digestion solution was filtered to remove impurities and centrifuged to obtain a cell pellet. The cell pellet was resuspended in the first Percoll reagent and then added dropwise to the second Percoll reagent. Density gradient centrifugation was performed at 700-900g for 15-22 minutes. The cell layer at the interface of the two liquid phases was aspirated to obtain immune cells in the colonic lamina propria of colitis mice.

[0083] In some embodiments of the present invention, the cleaning in step (2) comprises filtering to remove intestinal debris.

[0084] In some embodiments of the present invention, the filtering comprises filtering using a filter screen having a filtration pore size of 40-70 μm. In some embodiments of the present invention, the filtration pore size of the filter screen is 70 μm.

[0085] In some embodiments of the present invention, the concentration of Percoll in the first Percoll reagent in step (2) is lower than that in the second Percoll reagent.

[0086] In some embodiments of the present invention, the concentration of Percoll in the first Percoll reagent is less than or equal to 40%.

[0087] In some embodiments of the present invention, the concentration of Percoll in the first Percoll reagent is 40%.

[0088] In some embodiments of the present invention, the concentration of Percoll in the second Percoll reagent is greater than or equal to 70%.

[0089] In some embodiments of the present invention, the concentration of Percoll in the second Percoll reagent is 80%.

[0090] In some embodiments of the present invention, the density gradient centrifugation conditions are: 700-900 g centrifugation for 15-22 min (the centrifuge is set to 3 accelerations and 1 deceleration). The 3 accelerations and 1 deceleration means that the speed is increased by 3 accelerations during acceleration and decelerated by 1 acceleration during deceleration.

[0091] A second aspect of the present invention provides a method for isolating neutrophils from the colonic lamina propria, comprising the following steps:

[0092] The colonic lamina propria neutrophils are screened out from the colonic lamina propria immune cells separated by the separation method described above.

[0093] In some embodiments of the present invention, the screening methods include: flow cytometry, immunomagnetic bead sorting, microfluidics and density gradient centrifugation.

[0094] In some embodiments of the invention, the screening method is flow cytometry.

[0095] In some embodiments of the present invention, an Fc receptor binding inhibitor is added to the colon lamina propria immune cells before performing flow cytometry.

[0096] In some embodiments of the present invention, the Fc receptor binding inhibitors include but are not limited to TruStainFcX TM Antibody (Biolengend).

[0097] In some embodiments of the present invention, when flow cytometry is used for screening, at least one neutrophil marker-specific antibody gating strategy is used for screening.

[0098] In some embodiments of the present invention, the neutrophil marker includes at least one of CD45, Ly6G and CD11b.

[0099] In some embodiments of the present invention, when flow cytometry is used for screening, antibodies against at least one of the above-mentioned neutrophil-specific markers are used for screening.

[0100] In some embodiments of the present invention, neutrophil-specific antibodies include CD45 antibody, Ly6G antibody, and CD11b antibody.

[0101] In some embodiments of the present invention, the neutrophil marker-specific antibodies are in different fluorescence channels, including at least one of APC-A700, APC, and V610 channels.

[0102] In some embodiments of the present invention, the flow cytometry screening is achieved using a neutrophil-specific antibody based on a gating strategy.

[0103] In some embodiments of the present invention, flow cytometry screening of neutrophils is performed using CD45 antibody on the APC-A700 channel, Ly6G antibody on the APC channel, and CD11b antibody on the V610 channel.

[0104] In some embodiments of the present invention, the specific steps of the method are:

[0105] (1) Using pre-digestion reagent 1, the intestinal tract of a colitis mouse that has not been sheared is pre-digested for 8-12 minutes, washing to remove epithelial cells, and then adding pre-digestion reagent 2 for digestion for 8-12 minutes. Washing buffer is added to remove the pre-digestion reagent, and enzymatic digestion reagent is added for enzymatic digestion for 30-45 minutes to obtain an enzymatic digestion solution;

[0106] (2) The enzymatic digestion solution was filtered to remove impurities and centrifuged to obtain a cell pellet. The cell pellet was resuspended in a first Percoll reagent and then added dropwise to a second Percoll reagent. Density gradient centrifugation was performed at 700-900g for 15-22 minutes. The cell layer at the interface between the two liquid phases was aspirated to obtain immune cells in the colonic lamina propria of the colitis mouse;

[0107] (3) Fc receptor binding inhibitors were added to the immune cells of the colonic lamina propria of colitis mice, and then neutrophils were screened from the immune cells of the colonic lamina propria of colitis mice using flow cytometry based on CD45 antibodies on the APC-A700 channel, Ly6G antibodies on the APC channel, and CD11b antibodies on the V610 channel.

[0108] A third aspect of the present invention provides a cell separation kit or set for isolating colon lamina propria immune cells and / or colon lamina propria neutrophils.

[0109] In some embodiments of the present invention, the cell separation kit or set comprises the pre-digestion reagent 1, pre-digestion reagent 2 and enzymatic digestion reagent as described in the above aspects.

[0110] In some embodiments of the present invention, the cell separation kit or set further comprises the washing buffer and the first Percoll reagent and the second Percoll reagent as described above.

[0111] A fourth aspect of the present invention provides use of the cell separation kit or set described in the above aspects for isolating colon lamina propria immune cells and / or colon lamina propria neutrophils.

[0112] In some embodiments of the present invention, the use does not involve the diagnosis and treatment of a disease.

[0113] The beneficial effects of the present invention are:

[0114] The present invention proposes an efficient method for isolating lamina propria immune cells. During the process of clearing intestinal epithelial fragments, the present invention maintains the long shape of the colon and allows a large area of ​​intestinal tissue to contact the pre-digestion fluid, effectively exposing the intestinal lamina propria, simplifying the steps of clearing epithelial fragments, and greatly accelerating the speed of isolating immune cells. The operation steps are simpler than existing methods, and can effectively avoid the apoptosis of neutrophils caused by long-term exposure of intestinal samples, and the problems of low cell yield and poor activity. It is particularly suitable for conducting large-sample experiments, saving time and cost for the efficient development of subsequent research.

[0115] The present invention also provides a method for isolating neutrophils from the colonic lamina propria, which uses a flow cytometry sorting method to sort the immune cell suspension. The isolated colonic lamina propria immune cells are clearly grouped, the neutrophil markers are obvious, the yield is high, and the activity is high, and the cells can be further applied to related research.

[0116] In addition, the present invention also provides a set of isolation kits and packages for lamina propria immune cells or neutrophils, which contain a variety of different digestion reagents. By combining specific processing temperature, time, centrifugation conditions, etc., high-quality separation effects can be stably maintained. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 1 is a diagram of the colorectal anatomy of a colitis mouse in an embodiment of the present invention.

[0118] Figure 2 This is a light microscopic image of the immune cells in the colon lamina propria obtained in an example of the present invention.

[0119] Figure 3 This is a flow cytometry result diagram of obtaining colon lamina propria immune cells in an embodiment of the present invention.

[0120] Figure 4 This is a diagram showing the flow cytometry results of neutrophils in the colonic lamina propria of the present invention. DETAILED DESCRIPTION

[0121] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0122] Example 1

[0123] In this embodiment, an efficient method for isolating neutrophils from the colonic lamina propria of colitis mice is provided, which specifically comprises the following steps:

[0124] (1) Colitis mouse model:

[0125] In this example, according to conventional procedures in the art, dextran sodium sulfate (DSS) was used to induce a colitis model in 6 experimental mice, and the induction was continued through drinking water for 5 consecutive days. After the induction was completed, the colitis mice were obtained.

[0126] (2) Obtaining and pre-processing the colon of colitis mice:

[0127] Colitis mice were killed by cervical dislocation, and the abdominal wall and peritoneum were longitudinally opened with scissors to expose the abdominal viscera. The colon of the mice was then removed with scissors (from the cecum to the anus). Figure 1 and stored temporarily in a culture dish containing pre-cooled PBS.

[0128] The dissected mouse colon was cleaned with pre-chilled PBS, and the mesentery, adipose tissue, and Peyer's patches were removed. The intestine was then cut open longitudinally and, without further fragmentation, transferred directly to a 5 mL centrifuge tube while maintaining its long, strip-like shape. The intestinal contents were then rinsed with pre-chilled PBS until all feces and other contents were completely removed. Then, 3 mL of pre-chilled PBS was added to the rinsed colon of the colitis mouse, shaken upside down, and rinsed again for 1 minute.

[0129] (3) Predigestion:

[0130] The cleaned colon of the colitis mouse obtained in step (2) was taken and transferred into 2 mL of PBS containing 5 mM EDTA, 1 mM dithiothreitol (DTT) and 10 mM HEPES buffer at a final concentration, and pre-digested on a shaker at 37°C and 200 rpm for 10 min.

[0131] In this embodiment, PBS containing EDTA is Ca-free. 2+ Mg 2+ PBS.

[0132] The pre-digested colon of the colitis mouse was picked up with forceps, rinsed thoroughly with PBS, drained of the digestion fluid, and freed of intestinal epithelial cell debris, and then transferred to a 5 mL centrifuge tube.

[0133] The rinse solution and the remaining cell fluid were collected and used to calculate the efficiency of epithelial digestion in the pre-digestion step.

[0134] 2 mL of PBS containing 5 mM EDTA and 10 mM HEPES buffer was added to the centrifuge tube, and pre-digested again on a shaker at 37°C and 200 rpm for 10 min.

[0135] In this embodiment, PBS containing EDTA is Ca-free. 2+ Mg 2+PBS.

[0136] After digestion, the colon was removed and rinsed with RPMI-1640 medium containing 2% (v / v) fetal bovine serum (FBS) by shaking up and down to completely remove residual EDTA. The water on the rinsed colon was then removed and transferred to a new 5 mL centrifuge tube.

[0137] The rinse solution and the remaining cell solution were collected and used to calculate the epithelial digestion efficiency in this digestion step.

[0138] (4) Enzymatic digestion:

[0139] The colon in the centrifuge tube was completely minced (less than or equal to 0.5 cm), and 2 mL of RPMI-1640 medium containing a final concentration of 10% FBS, 1 mg / mL collagenase D (purchased from Roche), and 40 U / mL DNase I was added as an enzymatic digestion solution. The colon was then digested on a shaker at 37°C and 100 rpm for 40 min. After the digestion was completed, the colon was shaken upside down for 1 min.

[0140] In this embodiment, the enzymatic digestion solution needs to be freshly prepared, and collagenase D needs to be stored at -20°C before use. Repeated freezing and thawing should be avoided to avoid affecting the separation effect.

[0141] (5) Cell separation:

[0142] The digested solution was filtered through a 70 μm filter to remove intestinal debris, and then washed with PBS containing 2% FBS. The washing solution was collected and centrifuged at 500 g for 10 min at 4°C. The supernatant was removed and the precipitate was collected.

[0143] Add 5 mL of 40% Percoll (purchased from YEASEN) to the precipitate and resuspend it, then gently add the suspension dropwise onto 5 mL of 80% Percoll (to allow it to be layered with 80% Percoll, and the suspension will be above 80% Percoll), avoiding mixing the two.

[0144] The solution was subjected to density gradient centrifugation at 800g for 20 minutes at 20°C (centrifuge setting: 3 up, 1 down). After centrifugation, the white cell layer at the interface between the two liquid phases was aspirated and transferred to a new centrifuge tube. The cells were washed with PBS and resuspended to obtain immune cells from the colonic lamina propria of the colitis mouse.

[0145] After obtaining immune cells from the colonic lamina propria of colitis mice, trypan blue staining can be used to count cells to determine the cell number.

[0146] (6) Neutrophil screening:

[0147] Three samples were randomly drawn from the immune cells of the colonic lamina propria of the colitis mouse obtained in step (5), and about 1×10 6 Immune cells from the colonic lamina propria of a colitis mouse were collected. Part of the supernatant was removed by centrifugation and quantified to 50 μL. Then, Fc receptor binding inhibitor TruStain FcX was added at a volume ratio of 100:1. TM Antibody (Biolengend) was added to prevent the occurrence of nonspecific binding mediated by Fc receptors, and the cells were incubated on ice for 10 min, and then washed with PBS.

[0148] The washed cells were re-quantified to 50 μL, and the cells were stained and labeled with antibodies in sequence. Specifically, the cells were stained using eFluor TM 780 dye was used to label dead cells. Neutrophils were labeled and sorted using antibodies for CD45 in the APC-A700 channel, Ly6G in the APC channel, and CD11b in the V610 channel. For antibody-labeled flow cytometry screening, cells were re-quantified to 300 μL and transferred to a flow cytometer. Neutrophils were then sorted using a flow cytometer to obtain colonic lamina propria neutrophils from colitis mice.

[0149] The epithelial cell fluid collected from all the above steps (i.e., the rinse fluid and cell fluid from the pre-digestion and digestion steps) was pooled and filtered through a 70 μm filter to remove tissue debris. The filtrate was centrifuged at 500 g for 10 min at 4°C. The supernatant was removed and the precipitate was collected to obtain the epithelial cells. The cells were then resuspended in PBS containing 2% FBS and stained with trypan blue to count the epithelial cells, which represents the efficiency of epithelial digestion.

[0150] Comparative Example 1

[0151] In this comparative example, a conventional separation method was used to separate neutrophils from the colonic lamina propria, which specifically included the following steps:

[0152] According to conventional procedures in the art, dextran sodium sulfate (DSS) was used to induce a colitis model in 6 experimental mice, and colitis mice were obtained after the induction was completed.

[0153] Colitis mice were killed by cervical dislocation. The abdominal wall and peritoneum were longitudinally opened with scissors to expose the abdominal viscera. The mouse colon was then cut with scissors (from the cecum to the anus) and temporarily stored in a culture dish containing pre-cooled PBS.

[0154] The dissected mouse colon was cleaned with pre-cooled PBS, and the mesentery, adipose tissue and Peyer's patches were removed. The contents in the intestine were rinsed with pre-cooled PBS until the feces and other contents were completely rinsed out. Then the colon was cut into small segments of 0.5-1 cm with scissors.

[0155] 2 mL of PBS containing 5 mM EDTA, 1 mM dithiothreitol (DTT), and 10 mM HEPES buffer was added to the minced colon, and then digested on a shaker at 37° C. and 200 rpm for 10 min.

[0156] 2 mL of PBS containing a final concentration of 5 mM EDTA and 10 mM HEPES buffer was added to the minced colon as an epithelial digestion solution for digestion, and then digested on a shaker at 37°C and 200 rpm for 10 minutes.

[0157] The digested solution was filtered through a 70 μm filter to retain the digested colon tissue. The filtrate was recovered for statistical analysis of epithelial digestion efficiency. The retained colon tissue was rinsed with RMPI-1640 medium containing 2% FBS and transferred to a new 5 mL centrifuge tube.

[0158] The colon in the centrifuge tube was completely minced, and 2 mL of RPMI-1640 medium containing a final concentration of 10% FBS, 1 mg / mL collagenase D (purchased from Roche), and 40 U / mL DNase I (purchased from Roche) was added as an enzymatic digestion solution. The cells were then digested on a shaker at 37°C and 100 rpm for 40 min. After digestion, the cells were shaken upside down for 1 min.

[0159] The digested solution was filtered through a 70 μm filter to remove intestinal debris, and then washed with PBS containing 2% FBS. The washing solution was collected and centrifuged at 500 g for 10 min at 4°C. The supernatant was removed and the precipitate was collected.

[0160] After obtaining immune cells from the colonic lamina propria of colitis mice, trypan blue staining can be used to count cells to determine the cell number.

[0161] Then, according to the steps in Example 1, three samples were randomly drawn from the immune cells of the colonic lamina propria of the colitis mice, and about 1×10 6 The immune cells in the colonic lamina propria of colitis mice were stained and labeled with antibodies, and the neutrophils in the colonic lamina propria were sorted using a flow cytometer.

[0162] The epithelial digestion efficiency was calculated according to the method in Example 1.

[0163] Comparative Example 2

[0164] In this comparative example, a conventional separation method was used to separate neutrophils from the colonic lamina propria, which specifically included the following steps:

[0165] Colitis mice were obtained according to the method in Example 1, and the colons of the colitis mice were obtained therefrom.

[0166] The dissected mouse colon was cleaned with pre-chilled PBS, and the mesentery, adipose tissue, and Peyer's patches were removed. The intestine was then cut open longitudinally and, without further fragmentation, transferred directly to a 5 mL centrifuge tube while maintaining its long, strip-like shape. The intestinal contents were then rinsed with pre-chilled PBS until all feces and other contents were completely removed. Then, 3 mL of pre-chilled PBS was added to the rinsed colon of the colitis mouse, shaken upside down, and rinsed again for 1 minute.

[0167] 2 mL of PBS containing 5 mM EDTA, 1 mM dithiothreitol (DTT), and 10 mM HEPES buffer was added to the minced colon, and then digested on a shaker at 37° C. and 200 rpm for 10 min.

[0168] 2 mL of PBS containing a final concentration of 5 mM EDTA and 10 mM HEPES buffer was added to the minced colon as an epithelial digestion solution for digestion, and then digested on a shaker at 37°C and 200 rpm for 10 minutes.

[0169] The digested colon of the colitis mouse was picked up with forceps, rinsed thoroughly with PBS to drain the digestion fluid, remove intestinal epithelial cell debris, and then transferred to a 5 mL centrifuge tube.

[0170] The rinse solution and the remaining cell solution were collected and used to calculate the epithelial digestion efficiency in this digestion step.

[0171] Without further mincing the colon in the centrifuge tube, 2 mL of RPMI-1640 medium containing a final concentration of 10% FBS, 1 mg / mL collagenase D (purchased from Roche), and 40 U / mL DNase I was directly added as the enzymatic digestion solution, and then digested on a shaker at 37°C and 100 rpm for 40 min. After digestion, the tube was shaken upside down for 1 min.

[0172] The digested solution was filtered through a 70 μm filter to remove intestinal debris, and then washed with PBS containing 2% FBS. The washing solution was collected and centrifuged at 500 g for 10 min at 4°C. The supernatant was removed and the precipitate was collected.

[0173] After obtaining immune cells from the colonic lamina propria of colitis mice, trypan blue staining can be used to count cells to determine the cell number.

[0174] The epithelial digestion efficiency was calculated according to the method in Example 1.

[0175] Test Example 1

[0176] The cell separation time, epithelial digestion efficiency, lamina propria cell number and cell viability (cell viability = (total number of cells - number of dead cells) / total number of cells) in the above examples and comparative examples were statistically analyzed.

[0177] The results are shown in Tables 1 and 2.

[0178] Table 1 Comparison of the processing time between the methods in the embodiment and the comparative example

[0179]

[0180] Table 2 Comparison of separation effects between the methods in the embodiment and the comparative example

[0181] Group Epithelial digestion efficiency (epithelial cell number) Number of lamina propria cells Cell viability (%) Example 1 <![CDATA[1.206×10 7 ]]> <![CDATA[3.06×10 6 ]]> 98.89 Comparative Example 1 <![CDATA[1.107×10 7 ]]> <![CDATA[1.92×10 6 ]]> 92.90 Comparative Example 2 <![CDATA[1.230×10 7 ]]> <![CDATA[1.32×10 6 ]]> 95.38

[0182] Among them, Table 2 shows the representative treatment results of a single mouse.

[0183] From the above results, it can be seen that the method in the embodiment of the present invention, compared with the conventional method of separating epithelial cells by pre-digesting the intestine after cutting the intestine (Comparative Example 1), can shorten the experimental operation time when processing multiple samples and complete the processing of intestinal samples more quickly. The number of immune cells isolated from the lamina propria in Example 1 was 3.06×10 6 , the cell viability is 98.89%, compared with Comparative Example 2, reflecting that the method of the present invention has higher colon lamina propria immune cell separation efficiency and cell viability. Moreover, in terms of epithelial digestion efficiency, the number of cells obtained by the method in the embodiment of the present invention is almost the same as that in Comparative Example 2, indicating that the digestion steps adopted therein can indeed effectively digest and process intestinal epithelial tissue. Based on the above advantages, the method in the embodiment of the present invention can be applied to large sample experimental operations, and can quickly and efficiently complete the separation of intestinal immune cells in a shorter time, greatly reducing the exposure time of intestinal tissue, reducing the loss of related cells, and providing the possibility of maintaining the high efficiency of large sample experiments and the high activity of separated cells.

[0184] The immune cells of the colonic lamina propria of the colitis mice obtained in the above example were further observed using an optical microscope. Figure 2 shown.

[0185] It can be found that different types of immune cells can be found in the immune cells of the colon propria of colitis mice, indicating that this method can obtain all types of immune cells without exclusion of specific immune cells.

[0186] Further based on the flow screening results in the above examples (such as Figure 3 and Figure 4 As shown), it can be found that the colon propria immune cells separated by the method of the present invention are clearly clustered, and the cell viability can reach 98.89% ( Figure 3 ), and the use of CD45 antibodies of the APC-A700 channel, Ly6G antibodies of the APC channel, and CD11b antibodies of the V610 channel can separate neutrophils from the digested colonic lamina propria immune cells. The isolated colonic lamina propria immune cells of colitis mice are clearly divided into groups, and neutrophils account for 15.23% of the immune cells ( Figure 4 ).

[0187] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for isolating colon lamina propria immune cells, comprising the following steps: (1) digesting the colon and intestine using pre-digestion reagent 1, pre-digestion reagent 2, and enzymatic digestion reagent in sequence to obtain an enzymatic digestion solution; (2) Isolate colonic lamina propria immune cells from enzymatic digestion fluid.

2. The separation method according to claim 1, wherein The pre-digestion reagent 1 includes a chelating agent; Optionally, the chelating agent in the pre-digestion reagent 1 includes ethylenediaminetetraacetic acid (EDTA); Optionally, the final concentration of the chelating agent in the pre-digestion reagent 1 is 2-10 mM; Optionally, the pre-digestion reagent 1 further comprises at least one of a reducing agent and a pH regulator: Optionally, the reducing agent of the pre-digestion reagent 1 comprises dithiothreitol (DTT); Optionally, the pH regulator of the pre-digestion reagent 1 includes HEPES; Optionally, the pre-digestion reagent 1 further comprises at least one of the following components at the following concentrations: 0.5-3 mM reducing agent and 10-20 mM stabilizer; Optionally, the pre-digestion reagent 1 further comprises a solvent; Optionally, the solvent in the pre-digestion reagent 1 includes PBS buffer.

3. The separation method according to claim 1, characterized in that The pre-digestion reagent 2 includes the following components: a chelating agent; Optionally, the chelating agent in the pre-digestion reagent 2 includes EDTA; Optionally, the final concentration of the chelating agent in the pre-digestion reagent 2 is 2-10 mM; Optionally, the pre-digestion reagent 2 further comprises a pH regulator; Optionally, the pH regulator of the pre-digestion reagent 2 includes HEPES; Optionally, the pre-digestion reagent 2 further comprises 10-20 mM pH regulator; Optionally, the pre-digestion reagent 2 further comprises a solvent; Optionally, the solvent in the pre-digestion reagent 2 includes PBS buffer.

4. The separation method according to claim 1, wherein The enzymatic digestion reagent includes collagenase and nuclease; Optionally, the collagenase comprises at least one of collagenase D, collagenase IV, trypsin and neutral protease; Optionally, the nuclease comprises at least one of DNase I, Endonuclease V and Fragmentase; Optionally, the enzymatic digestion reagent includes the following components, based on the final concentration of each component in the solvent: 0.5-1.5 mg / mL collagenase and 20-80 U / mL nuclease; Optionally, the enzymatic digestion reagent further comprises a protease inhibitor; Optionally, the protease inhibitor comprises at least one of fetal bovine serum (FBS) and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF); Optionally, the enzymatic digestion reagent includes 5-15% (v / v) of a protease inhibitor based on the final concentration of each component in the solvent.

5. The separation method according to claim 1, characterized in that The colon intestine is a dissected intestine; Optionally, the colon intestine is cut open along the extending direction of the intestine; Optionally, the colon intestine is a dissected intestine and is not shredded; Optionally, the length of the dissected colon is greater than 1 cm.

6. The separation method according to claim 1, characterized in that In step (1), the pre-digestion reagent is removed using a wash buffer, wherein the wash buffer comprises serum or a serum substitute; Optionally, the serum comprises at least one of FBS and calf serum (CS); Optionally, the serum substitute comprises at least one of bovine serum albumin (BSA), polyethylene glycol (PEG), and a serum-free stop buffer.

7. The separation method according to claim 1, characterized in that The colon is an animal colon; Optionally, the animals include: mice, rabbits, dogs, horses and primates; Optionally, the animal is a disease model animal; Optionally, the disease comprises a colon-related disease including: colitis; Optionally, the animal is a colitis mouse.

8. A method for isolating neutrophils from the colonic lamina propria, comprising the following steps: sieving out colonic lamina propria neutrophils from the colonic lamina propria immune cells separated by the separation method according to any one of claims 1 to 7; Optionally, the screening method comprises: Flow cytometry, immunomagnetic bead sorting, microfluidics, and density gradient centrifugation; optionally, when using flow cytometry for screening, at least one neutrophil marker-specific antibody is used for screening; Optionally, the neutrophil marker includes at least one of CD45, Ly6G and CD11b; Preferably, the neutrophil marker-specific antibodies are in different fluorescence channels, including at least one of APC-A700, APC and V610 channels.

9. A cell separation kit or set, characterized in that: The cell separation kit or set is used to separate colon lamina propria immune cells and / or colon lamina propria neutrophils; The cell separation kit or set includes a pre-digestion reagent 1, a pre-digestion reagent 2 and an enzymatic digestion reagent; Optionally, the pre-digestion reagents 1 and 2 include a chelating agent; Optionally, the chelating agent in the pre-digestion reagents 1 and 2 comprises ethylenediaminetetraacetic acid (EDTA); Optionally, the final concentration of the chelating agent in the pre-digestion reagents 1 and 2 is 2-10 mM; Optionally, the enzymatic digestion reagent includes collagenase and nuclease; Optionally, the collagenase comprises at least one of collagenase D, collagenase IV, trypsin and neutral protease; Optionally, the nuclease comprises at least one of DNase I, Endonuclease V and Fragmentase.

10. Use of the cell separation kit or set according to claim 9 for isolating colon lamina propria immune cells and / or colon lamina propria neutrophils.

Images