Methods for co-culturing enteric glial cells with intestinal organoids, egc-organoid co-culture systems

Abstract

This invention belongs to the field of biology and discloses a method for co-culturing enteric glial cells (EGCs) and intestinal organoids. Primary cultured and purified enteric glial cells are digested and seeded onto the membrane of a Transwell chamber. Intestinal organoids are placed in individual Matrigel drops in a co-culture plate. The Transwell chambers are placed in the co-culture plate, and growth medium is added to ensure free exchange of the growth medium through the Transwell membrane. The EGC-organoid co-culture system obtained based on this method provides a model for studying diarrhea and intestinal health in large mammals. It also lays the research foundation for developing innovative intervention strategies targeting the enteric nervous system and provides a research model for further exploring the regulatory roles of nutrients or related genes in intestinal health.

Description

Technical Field

[0001] This invention relates to the field of biology, and more particularly to a method for co-culturing enteric glial cells and intestinal organoids, and an EGCs-organoid co-culture system. Background Technology

[0002] Early weaning is a critical bottleneck in large-scale pig farming. Because piglets' intestinal and other organ functions are not yet fully developed at this stage, their digestive and absorptive capacity and stress tolerance are insufficient. Faced with sudden and drastic changes in psychology, nutrition, and environment, piglets often exhibit "early weaning syndrome," characterized by reduced feed intake, digestive disorders, diarrhea, and stunted growth. This not only severely hinders production performance but also causes significant economic losses to the pig farming industry. Early weaning makes the piglet's intestine a "central organ of stress." Traditional research has focused on the epithelial barrier and immune inflammation, but has almost neglected the "gut brain"—the enteric nervous system—which independently regulates gastrointestinal function. EGCs, as the "cerebellum" of this system, are core to maintaining intestinal homeostasis. This method, for the first time, constructs a microenvironment containing "glial-epithelial" dialogue in vitro, making it possible to directly study how the enteric nervous system regulates the intestinal barrier, repair, and immunity under stress conditions. This opens a breakthrough in elucidating the specific neuro-epithelial interaction mechanism of intestinal damage caused by weaning stress. The primary significance of establishing a method for co-culturing enteric glial cells (EGCs) and intestinal organoids lies in filling a key gap in research on piglet intestinal health and providing an irreplaceable model for elucidating the core mechanisms of weaning stress. Secondly, this method lays the research foundation for developing innovative intervention strategies targeting the enteric nervous system.

[0003] In developing this platform, the Transwell membrane co-culture protocol can be referenced; Transwell membrane-based cell co-culture protocols are already relatively mature, as can be seen in the following existing technologies:

[0004] Patent application US8802432B2, entitled "Methods and Cell Culture for Promoting Organogenesis and Tissue Development," and CA2899865A1, entitled "Methods for Generating Retinal Pigment Epithelial (RPE) Cells from Induced Pluripotent Stem Cells (IPSCS)."

[0005] When this solution attempts to adopt the applicant's previously established and relatively mature culture medium usage strategies, such as the patent application with publication number CN115340960B, which describes a method for constructing a co-culture system of porcine intestinal organoids with enterotoxigenic Escherichia coli or macrophages, and publication number CN114657119B, which describes a method for isolating and primary culturing intestinal glial cells of newborn piglets, we found that the applicability of the culture medium was relatively poor.

[0006] Therefore, the technical problem that this solution needs to solve is: how to cultivate an EGCs-organoid co-culture system with better activity. Summary of the Invention

[0007] The purpose of this invention is to provide a method for co-culturing enteric glial cells and intestinal organoids. The EGCs-organoid co-culture system obtained by this method can provide a model for studying diarrhea and intestinal health in large mammals, lay the research foundation for developing innovative intervention strategies targeting the enteric nervous system, and provide a research model for in-depth exploration of the regulatory role of nutrients or related genes.

[0008] The method of the present invention is specifically as follows:

[0009] A method for co-culturing enteric glial cells and intestinal organoids involves digesting and seeding primary cultured and purified enteric glial cells onto the membrane of a Transwell chamber, placing intestinal organoids in individual Matrigel drops in a co-culture plate, placing the Transwell chamber in the co-culture plate, and adding growth medium to ensure that the growth medium can be freely exchanged through the membrane of the Transwell chamber.

[0010] The growth medium comprises the following components:

[0011] Basic culture medium: Advanced DMEM / F12;

[0012] L-WRN cell conditioned medium: 40-50 vol%

[0013] 1× specification Gentamicin / Amphotericin B: 1 volume;

[0014] Bismuth subsalicylate: 1 vol%

[0015] 1× specification N2: 1 volume;

[0016] 1×size B27: 1 volume;

[0017] EGF: 50 ng / mL;

[0018] GDNF: 20 ng / mL;

[0019] bFGF: 10 ng / mL;

[0020] CHIR99021: 10μM;

[0021] HEPES: 1 mM;

[0022] N-acetylcysteine: 1 mM;

[0023] 1× Glutamax: 1x volume;

[0024] Valproic acid: 1 mM.

[0025] In the above method, the membrane of the Transwell chamber is a 0.4 μm pore size polycarbonate membrane.

[0026] In the above method, the incubation time is 24 hours.

[0027] In the above method, the primary culture and purification method for enteric glial cells is as follows:

[0028] Enteric glial cells were seeded onto porous cell culture slides coated with laminin and Poly-D lysine and cultured on enteric glial cell culture medium for 2 days, followed by further culture on serum-free enteric glial cell culture medium for 3-5 days. The serum-free enteric glial cell culture medium is simply enteric glial cell culture medium with FBS removed.

[0029] The formulation of the enteric glial cell culture medium:

[0030] Basic culture medium: DMEM / F12 medium;

[0031] FBS 10%

[0032] Appropriate amount of dual antibodies;

[0033] 1× size NeuroCult™ SM1: 1x volume;

[0034] GDNF: 100 ng / mL;

[0035] CNTF: 20 ng / mL;

[0036] IGF: 20 ng / mL;

[0037] NGF: 10 ng / mL;

[0038] NT3: 10 ng / mL;

[0039] PDGF: 10 ng / mL;

[0040] 1× Glutamax specification: 1 volume.

[0041] In the above method, the intestinal organoid is prepared by separating intestinal crypts from the anterior segment of the jejunum of piglets;

[0042] Intestinal crypts resuspended in culture medium were mixed with growth-reducing factor Matrigel at a ratio of 4:6 and seeded into preheated 24-well plates at a density of 10-20 crypts per well. The mixture was then allowed to stand at 37°C for 10-20 minutes to allow Matrigel to solidify. 500 μL of the preheated growth medium at 37°C was added along the well wall.

[0043] The culture plates were placed in a 37°C, 5% CO2 incubator and the culture medium was changed every 2-3 days. Mature and structurally complete intestinal organoids could be formed after 5-7 days.

[0044] Meanwhile, this invention also discloses an EGCs-organoid co-culture system, which is prepared using any of the methods described above.

[0045] This application has at least the following beneficial effects:

[0046] ① This invention provides a method for co-culturing enteric glial cells and intestinal organoids; ② The system for co-culturing enteric glial cells and intestinal organoids using the above method can provide a model for studying diarrhea and intestinal health in large mammals, lay a research foundation for developing innovative intervention strategies targeting the enteric nervous system, and provide a research model for further exploring the regulatory role of nutrients or related genes in the process. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the cultivation process of the present invention;

[0048] Figure 2 These are microscopic images of co-culture in Example 1 of the present invention;

[0049] Figure 3 This is a microscopic photograph of the co-culture of Comparative Example 1 of the present invention;

[0050] Figure 4 These are microscopic images of the co-culture of Comparative Example 2 of the present invention;

[0051] Figure 5 This is a microscopic photograph of the co-culture of Comparative Example 3 of the present invention. Detailed Implementation

[0052] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0053] Example 1

[0054] The present invention relates to a method for co-culturing enteric glial cells and intestinal organoids as follows:

[0055] Part 1: Isolation and Culture of Primary Enteric Glial Cells from Piglets

[0056] 1. Matrix coating:

[0057] Laminin and Poly-D-lysine stored at -20°C were transferred to 4°C for slow thawing. Sterile cell slides were placed in 24-well plates. First, 100 μL of a 100 μg / mL Poly-D-lysine solution was added to each well to cover the slide, and the plate was incubated at room temperature for 1 hour. The slides were then washed with ultrapure water and dried at room temperature for 1 hour.

[0058] Add 100 μL of 60 μg / mL laminin solution to each well and incubate at 37°C for 1 hour. After incubation, discard the solution, wash twice with sterile PBS, and store in an incubator for later use.

[0059] 2. Tissue Acquisition and Preprocessing:

[0060] Collect the anterior segment of the jejunum from slaughtered piglets and rinse repeatedly 3-5 times with PBS containing 3% penicillin and antibiotics until the washing solution is clear.

[0061] The cleaned intestinal segments were placed in pre-cooled Kerbs buffer (components: 126 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.2 mM NaH2PO4, 1.2 mM MgCl2, pH 7.0-7.4) containing 1× Gentamicin / Amphotericin B Solution (500X) and continuously circulated with 95% O2 / 5% CO2.

[0062] 3. Separation of the serosa from the longitudinal muscle layer:

[0063] Gently insert the intestinal segment into the plunger of a 2 mL syringe and make two longitudinal incisions along the mesenteric attachment. Wipe the muscle longitudinally with a PBS-moistened cotton swab to remove the serosa and longitudinal muscle layer. Place the treated tissue in PBS at 4°C for later use.

[0064] 4. Enzymatic digestion and cell separation:

[0065] Cut the tissue into 0.3-0.5 cm pieces. 3 Add small pieces of the mixture to preheated digestion solution (PBS containing 1 mg / mL collagenase I, 1 mg / mL bovine serum albumin, 20 μg / mL DNase I, 1 mM HEPES, and 1 mM Glutamax) and digest for 1 hour at 37°C and 200 rpm in a shaker.

[0066] The digestate was filtered through a 70 μm cell sieve, and the filtrate was centrifuged at 400 g for 8 minutes at 4°C to collect the cell pellet. The cells were resuspended in 0.25% EDTA-trypsin and digested at 37°C for 7 minutes. Digestion was terminated by adding an equal volume of DMEM containing 10% FBS. The digestate was then filtered again through a 70 μm cell sieve and gently ground. The filtrate was centrifuged at 500 g for 8 minutes at 4°C to obtain the final enteric glial cell enrichment pellet.

[0067] 5. Primary culture and purification:

[0068] Cells were resuspended in a special culture medium for enteric glial cells and seeded onto pre-coated slides.

[0069] Two days after inoculation, cells were seeded in DMEM / F12 basal medium supplemented with 10% FBS, 1× Penicillin & Streptomycin, 1× NeuroCult™ SM1, and a combination of neurotrophic factors (100 ng / mL GDNF, 20 ng / mL CNTF, 20 ng / mL IGF, and 10 ng / mL NGF, NT3, PDGF), and 1× Glutamax. Two days later, the medium was replaced with the same FBS-free medium (all other components remained unchanged) to inhibit fibroblast growth. Subsequently, the medium could be switched back to maintenance medium containing a low concentration of serum, depending on the cell condition. The medium was changed every two days, and cell morphology and purity were observed regularly under a microscope. After approximately 3-5 days of culture, the cells could be used for co-culture.

[0070] Part Two: Culture of Intestinal Organoids

[0071] 1. Separation of intestinal crypts:

[0072] The anterior segment of the jejunum of piglets was harvested, washed with ice-cold PBS, and then longitudinally dissected. The serosa was peeled off, and the tissue was cut into 1-2 mm pieces. 3 Small pieces were placed in cold PBS containing 1% BSA. Impurities were removed by repeated sedimentation until the supernatant was clear. The tissue pieces were incubated with digestion solution (containing 2 mM EDTA, 10 mM DTT, and 1× antibiotic) at room temperature for 2 hours with gentle shaking at 40 rpm. After digestion, the mixture was filtered through a 100 μm cell sieve, and the filtrate was centrifuged at 300 g for 5 minutes at 4°C. The precipitate was resuspended in cold PBS containing 1% BSA and centrifuged again at 200 g for 5 minutes at 4°C. The resulting precipitate was the intestinal crypt.

[0073] 2. Organoid formation and culture:

[0074] The isolated crypts were mixed with the growth-reducing factor Matrigel at a ratio of 4:6 and seeded into preheated 24-well plates at a density of 20-30 crypts per well. The Matrigel was allowed to solidify at 37°C for 10-20 minutes. 500 μL of preheated optimized organoid growth medium was added along the well wall. The optimized organoid growth medium formulation was: Advanced DMEM / F12, supplemented with 40-50% L-WRN conditioned medium (rich in Wnt3a, R-spondins, and Noggin), 1× Gentamicin / Amphotericin B, 1% penicillin antibiotics, 1× N2, 1× B27, 50 ng / mL EGF, 20 ng / mL GDNF, 10 ng / mL bFGF, 10 μM CHIR99021, 1 mM HEPES, 1 mM N-acetylcysteine, 1× Glutamax, and 1 mM valproic acid. The culture plates were placed in a 37°C, 5% CO2 incubator and the culture medium was changed every 2-3 days. Mature, structurally complete intestinal organoids will form after about 5-7 days.

[0075] Part Three: Establishment of a Co-training System

[0076] 1. Preparations before co-cultivation:

[0077] Select primary enteric glial cells with good growth status and high purity (culture for about 3-5 days) and intestinal organoids with mature structure and uniform size (culture for about 5-7 days).

[0078] 2. Co-training model:

[0079] refer to Figure 1 Enteric glial cells were digested, resuspended, and seeded onto the membrane of a Transwell chamber (0.4 μm pore size polycarbonate membrane).

[0080] Single or small amounts of Matrigel-embedded intestinal organoids were placed in individual Matrigel drops in the lower culture plate. Optimized organoid growth medium (same composition as above) was added to the co-culture plate, ensuring free exchange of medium through the chamber membranes. This allowed glial cells and organoids to share the same medium in a 37°C, 5% CO2 incubator environment. After approximately 24 hours of culture, the tissues were observed to be in good growth condition, demonstrating paracrine signal communication.

[0081] The culture results can be used as a reference. Figure 2 (Scale bar 50μm);

[0082] pass Figure 2It is evident that the organoid activity is good in the EGCs-organoid co-culture system of the present invention.

[0083] Comparative Example 1

[0084] Generally the same as Example 1, except that:

[0085] The culture medium specifically for primary enteroglial cells of piglets is based on the applicant's prior application (CN114657119B, A method for isolating and primary culturing enteroglial cells of newborn piglets), and its formula is as follows:

[0086] DMEM / F12 medium, 10% FBS, 1 volume of Penicillin & Streptomycin solution (100X), 100 ng / ml GDNF, 10 ng / mL PDGF, 1x Glutamax (100X).

[0087] Enteric glial cells can be successfully cultured using this culture medium;

[0088] The enteric glial cells were applied to the third part of Example 1, and the culture results are shown below. Figure 3 ;

[0089] pass Figure 3 It is evident that in the EGCs-organoid co-culture system of Comparative Example 1, the organoids almost lost their activity.

[0090] Comparative Example 2

[0091] Generally the same as Example 1, except that:

[0092] Organoid growth medium formulation: Advanced DMEM / F12, supplemented with 40-50% L-WRN conditioned medium (rich in Wnt3a, R-spondins, Noggin), 1× Gentamicin / Amphotericin B, 1% penicillin antibiotics, 1× N2, 1× B27, 50 ng / mL EGF, 10 μM Y27632, 0.5 μM LY2157299, 10 μM CHIR99021, 1 mM HEPES, 1 mM N-acetylcysteine, 1× Glutamax, 1 mM valproic acid.

[0093] The enteric glial cells were applied to the third part of Example 1, and the culture results are shown below. Figure 4 ;

[0094] pass Figure 4It can be seen that in the EGCs-organoid co-culture system of Comparative Example 2, organoid activity and... Figure 2 Its activity is slightly lower compared to others.

[0095] Comparative Example 3

[0096] Generally the same as Example 1, except that:

[0097] The organoid growth medium used in the culture of intestinal organoids was Advanced DMEM / F12, supplemented with 40-50% L-WRN conditioned medium (rich in Wnt3a, R-spondins, and Noggin), 1× Gentamicin / Amphotericin B, 1% penicillin antibiotics, 1× N2, 1× B27, 50 ng / mL EGF, 10 μM Y27632, 0.5 μM LY2157299, 10 μM CHIR99021, 1 mM HEPES, 1 mM N-acetylcysteine, 1× Glutamax, and 1 mM valproic acid.

[0098] The organoid growth medium used to establish the co-culture system in Part III was formulated as follows: Advanced DMEM / F12, supplemented with 40-50% L-WRN conditioned medium (rich in Wnt3a, R-spondins, and Noggin), 1× Gentamicin / Amphotericin B, 1% penicillin antibiotics, 1× N2, 1× B27, 50 ng / mL EGF, 0.5 ng / mL GDNF, 0.01 ng / mL bFGF, 10 μM CHIR99021, 1 mM HEPES, 1 mM N-acetylcysteine, 1× Glutamax, and 1 mM valproic acid.

[0099] The enteric glial cells were applied to the third part of Example 1, and the culture results are shown below. Figure 5 ;

[0100] pass Figure 5 It can be seen that in the EGCs-organoid co-culture system of Comparative Example 3, organoids and Figure 2 Its activity is relatively poor.

[0101] Summarize:

[0102] As can be seen from the above Examples 1, 1, 2, and 3, the EGCs-organoid co-culture system based on Transwell membranes is closely related to the culture medium associated with intestinal organoids, the culture medium for enteric glial cells, and the co-culture medium. Successfully culturing enteric glial cells and organoids is only the basic goal. Optimization of the culture medium for the co-culture system is also closely related to the final co-culture state.

[0103] The significance of this study lies in the fact that the co-culture system of enteric glial cells and intestinal organoids using the above methods can provide a model for studying diarrhea and intestinal health in large mammals, lay the research foundation for developing innovative intervention strategies targeting the enteric nervous system, and provide a research model for further exploring the regulatory role of nutrients or related genes.

Claims

1. A method for co-culturing enteric glial cells and intestinal organoids, characterized in that, Primary cultured and purified enteric glial cells were digested and seeded onto the membrane of a Transwell chamber. Intestinal organoids were placed in individual Matrigel drops in a co-culture plate. Transwell chambers were placed in the co-culture plate and growth medium was added to ensure that the growth medium could be freely exchanged through the membrane of the Transwell chamber. The growth medium comprises the following components: Basic culture medium: Advanced DMEM / F12; L-WRN cell conditioned medium: 40-50 vol% 1× specification Gentamicin / Amphotericin B: 1 volume; Bismuth subsalicylate: 1 vol% 1× specification N2: 1 volume; 1×size B27: 1 volume; EGF: 50 ng / mL; GDNF: 20 ng / mL; bFGF: 10 ng / mL; CHIR99021: 10μM; HEPES: 1 mM; N-acetylcysteine: 1 mM; 1× Glutamax: 1x volume; Valproic acid: 1 mM.

2. The method according to claim 1, characterized in that, The membrane of the Transwell chamber is a 0.4 μm pore size polycarbonate membrane.

3. The method according to claim 1, characterized in that, The incubation time is 24 hours.

4. The method according to claim 1, characterized in that, The method for primary culture and purification of the enteric glial cells is as follows: Enteric glial cells were seeded onto porous cell culture slides coated with laminin and Poly-D lysine and cultured on enteric glial cell culture medium for 2 days, followed by further culture on serum-free enteric glial cell culture medium for 3-5 days. The serum-free enteric glial cell culture medium is simply enteric glial cell culture medium with FBS removed. The formulation of the enteric glial cell culture medium: Basic culture medium: DMEM / F12 medium; FBS 10% Appropriate amount of dual antibodies; 1× size NeuroCult™ SM1: 1x volume; GDNF: 100 ng / mL; CNTF: 20 ng / mL; IGF: 20 ng / mL; NGF: 10 ng / mL; NT3: 10 ng / mL; PDGF: 10 ng / mL; 1× Glutamax specification: 1 volume.

5. The method according to claim 1, characterized in that, The method for preparing the intestinal organoids is as follows: intestinal crypts are obtained by isolating the anterior segment of the jejunum of piglets; The intestinal crypts resuspended in the culture medium were mixed with the growth factor Matrigel at a ratio of 4:6 and seeded into a preheated 24-well plate at a density of 20-30 crypts per well. The plate was then incubated at 37°C for 10-20 minutes to allow the Matrigel to solidify. 500 μL of the preheated growth medium was added along the well wall. The culture plates were placed in a 37°C, 5% CO2 incubator and the culture medium was changed every 2-3 days. Mature and structurally complete intestinal organoids could be formed after 5-7 days.

6. An EGCs-organoid co-culture system, characterized in that, It is prepared by the method described in any one of claims 1 to 5.

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