Culture media, methods, and models for constructing polarized 3d human intestinal organoids

Abstract

The application discloses a culture medium, a method and a model for constructing polarized 3D human intestinal organoids, and relates to the field of organoid culture. The culture medium comprises 5-15 mu g / mL of Wnt3a, 30-100 mu g / mL of R-spondin-1, R-spondin-2 and / or R-spondin-3, 10-50 nmol of a gamma-secretase inhibitor, 2-10 mu mol of a Wnt agonist, 50-200 mu mol of an HDAC inhibitor, 0.5-2% of an N2 supplement, 1-4% of a B27 supplement, 5-20 mu g / mL of IGF-1, 0.1-2 mu mol of a TGF-beta receptor inhibitor, and the rest is Williams E solution. The model obtained by the application realizes long-term stable mature differentiation state and physiological polarity maintenance.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, and in particular to a culture medium, method and model for constructing polarized 3D human intestinal organoids. Background Technology

[0002] Intestinal organoids are important in vitro models that mimic the structure and function of intestinal tissue, and are widely used in drug absorption and metabolism studies, exploration of intestinal disease mechanisms, and toxicity evaluation. The polarity of intestinal epithelial cells (such as apical-basal polarity) is fundamental to their barrier function, substance transport, and signal transduction. However, intestinal organoids constructed using existing technologies often suffer from insufficient polarization, inability to maintain a long-term polarized state, or unstable polarization, leading to significant differences in function between the constructed intestinal organoids and real intestinal tissue. This limits the application of intestinal organoids in drug screening, disease mechanism research, and toxicity evaluation.

[0003] Traditional 2D intestinal cell models constructed using existing technologies involve culturing a single layer of intestinal epithelial cells in Transwell chambers, where polarization is induced by mechanical support. The drawbacks of traditional 2D intestinal cell models include the lack of intercellular interactions, the inability to form a three-dimensional structure, transepithelial electrical resistance (TEER) values ​​significantly lower than in real intestinal tissue, and short-lived polarization duration, typically ≤5 days.

[0004] Existing technologies can also construct basic 3D intestinal organoid models. Specifically, the culture media used to construct 3D intestinal organoids include growth factors such as Wnt3a, R-spondin, and EGF, which support organoid proliferation. However, existing culture media lack polarization-inducing components, leading to insufficient apical-basal polarity formation, disordered cell polarity within the organoid, and incomplete barrier function. Related technologies have also attempted polarization induction when constructing 3D intestinal organoid models, but the results are unstable, easily leading to apoptosis, and the barrier function is maintained for a short time, typically ≤7 days.

[0005] Therefore, providing a 3D intestinal organoid model with stable polarization effect and long-term barrier function maintenance is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] This invention discloses a culture medium, method, and model for constructing polarized 3D human intestinal organoids, in order to solve the technical problems of unstable polarization effect and short barrier function maintenance time in related 3D intestinal organoid models.

[0007] To solve the above problems, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a culture medium for constructing polarized 3D human intestinal organoids.

[0009] This invention provides a culture medium for constructing polarized 3D human intestinal organoids, comprising the following components:

[0010] Wnt3a at concentrations of 5–15 μg / mL

[0011] 30~100 μg / mL of R-spondin-1, R-spondin-2 and / or R-spondin-3,

[0012] 10-50 nmol of γ-secretase inhibitors,

[0013] 2~10 μmol of Wnt agonist,

[0014] 50~200 μmol of HDAC inhibitor,

[0015] 0.5-2% N2 supplement,

[0016] 1-4% B27 supplement,

[0017] 5~20 μg / mL of IGF-1,

[0018] 0.1~2 μmol of TGF-β receptor inhibitors,

[0019] The remainder is Williams E solution.

[0020] According to an optional implementation, the γ-secretase inhibitor is one or more of DAPT, LY411575, MK0752, curcumin, BMS708163, RO4929097, PF03084014, LY450139, and AZD3293; and / or, the amount of the γ-secretase inhibitor is 20 nmol.

[0021] According to an optional embodiment, the Wnt agonist is one or more of Wnt3a, Wnt5a, CHIR99021, BIO, XAV939, WAY316606, Foxy5, SB216763, and Tideglusib; and / or, the amount of the Wnt agonist is 5 μmol.

[0022] According to an optional implementation, the HDAC inhibitor is one or more of TSA, SAHA, LBH589, sodium butyrate, VPA, MS275, RG2833, MGCD0103, FK228, Tubastatin A, and ACY1215; and / or, the amount of the HDAC inhibitor is 100 μmol.

[0023] According to one optional implementation, the volume ratio of N2 supplement to B27 supplement is 1:2; and / or, the amount of N2 supplement is 1% by volume; and the amount of B27 supplement is 2% by volume.

[0024] According to an optional implementation, the TGF-β receptor inhibitor is one or more of SB431542, A83-01, GW788388, LY2157299, RepSox, SD208, LY364947, and E-616452; and / or, the amount of the TGF-β receptor inhibitor is 0.5 μmol.

[0025] According to an optional implementation, the culture medium for constructing polarized 3D human intestinal organoids comprises components of the following formulation:

[0026] 10 μg / mL Wnt3a,

[0027] 50 μg / mL R-spondin-1,

[0028] LY411575 at 20 nmol

[0029] 5 μmol of CHIR99021,

[0030] 100 μmol sodium butyrate,

[0031] 1% N2 supplement,

[0032] 2% B27 supplement,

[0033] 10 μg / mL IGF-1,

[0034] 0.5 μmol of A83-01,

[0035] The remainder is Williams E solution.

[0036] A second aspect of the present invention provides a method for culturing polarized 3D human intestinal organoids.

[0037] This invention provides a method for culturing polarized 3D human intestinal organoids. The method involves culturing human intestinal stem cells in a culture medium, which is the culture medium for constructing polarized 3D human intestinal organoids as described in any of the technical solutions of this invention. The culturing method includes at least the following steps:

[0038] Step 100: Human intestinal stem cells are embedded in a matrix using a culture medium and then seeded into a multi-well plate;

[0039] Step 200: Add 1 mL of culture medium to each well and incubate at 37°C and 5% CO2.

[0040] Step 300: In subsequent culture, replace 40-60% of the original culture medium with fresh culture medium once a day.

[0041] A third aspect of the present invention provides a 3D human intestinal organoid model.

[0042] The 3D human intestinal organoid model of the present invention is based on human intestinal stem cells, and the intestinal cells obtained by culturing them using the culture method for constructing polarized 3D human intestinal organoids as described in any of the technical solutions of the present invention are as follows, and the polarization state and / or barrier function of the 3D human intestinal organoid model are maintained for more than 21 days.

[0043] A fourth aspect of the present invention provides an application of a 3D human intestinal organoid model.

[0044] The 3D human intestinal organoid model of the present invention can be used for one or more of the following: intestinal disease mechanism, intestinal disease drug screening, pharmacological and toxicological evaluation of candidate drugs, and candidate drug metabolism research.

[0045] The technical solution adopted in this invention can achieve the following beneficial effects:

[0046] In the first aspect, the present invention constructs a culture medium for polarized 3D human intestinal organoids, comprising 5-15 μg / mL Wnt3a, 30-100 μg / mL R-spondin-1, R-spondin-2 and / or R-spondin-3, 10-50 nmol of γ-secretase inhibitor, 2-10 μmol of Wnt agonist, 50-200 μmol of HDAC inhibitor, 0.5-2% N2 supplement, 1-4% B27 supplement, 5-20 μg / mL IGF-1, 0.1-2 μmol of TGF-β receptor inhibitor, and the balance being Williams E solution. This culture medium, through the combination of its components, can provide the necessary energy source and nutrients for the culture of human intestinal stem cells, and can also effectively promote the directed induction of human intestinal stem cells into mature differentiated cell types (such as goblet cells and absorptive cells), while inhibiting the excessive proliferation of human intestinal stem cells.

[0047] Secondly, the 3D human intestinal organoid model obtained using the culture medium and culture method of the present invention possesses polarization function and physiological simulation. Specifically, this 3D human intestinal organoid model can stably maintain high cell viability (ATP content remains above 85% of the initial level) for 21 days, and Villin expression remains at a high level and exhibits typical apical-out polarization characteristics, indicating that this 3D human intestinal organoid model has achieved a long-term stable mature differentiation state and maintenance of physiological polarity.

[0048] Thirdly, the culture medium formulation and the 3D human intestinal organoid model constructed based on this invention not only maintain the differentiation state and physiological polarity of mature intestinal epithelium for a long period, but also possess good predictive sensitivity and clinical relevance for enterotoxicity, providing a reliable and efficient new tool for intestinal toxicity assessment, drug screening, and in vitro model research of intestinal diseases. Compared with traditional unpolarized or poorly differentiated organoid models, the 3D human intestinal organoid model of this invention has significant advantages in simulating clinical intestinal toxicity reactions. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 These are fluorescence staining results (40X) of human intestinal organoid model sections obtained in Example 1 and Comparative Example 1 of this application. The left side shows the detection results of Example 1, and the right side shows the detection results of Comparative Example 1.

[0051] Figure 2 This is a graph showing the changes in cell viability during 21 days of culture in Example 1 of this application;

[0052] Figure 3 This is a graph showing the changes in cell viability during 21 days of culture in Example 2 of this application;

[0053] Figure 4 This is a graph showing the changes in cell viability during 21 days of culture in Example 3 of this application;

[0054] Figure 5 This is a graph showing the changes in Villin protein expression in the model during 21 days of culture in Example 1 of this application;

[0055] Figure 6 This is a graph showing the changes in cell viability during the 21-day culture process of Comparative Example 1.

[0056] Figure 7 This is a diagram showing the cell viability test results of the model obtained in Example 1 of this application after being treated with different concentrations of abexicillin for 48 hours.

[0057] Figure 8 This is a diagram showing the cell viability test results of the model obtained in Example 2 of this application after being treated with different concentrations of abexicillin for 48 hours.

[0058] Figure 9 This is a diagram showing the cell viability test results of the model obtained in Example 3 of this application after being treated with different concentrations of abecicilline for 48 hours.

[0059] Figure 10 This is a graph showing the cell viability test results of the model obtained in Comparative Example 1 after treatment with different concentrations of abecicilline for 48 hours. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0061] The terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and not to describe a specific order or sequence. It should be understood that such use of data can be interchanged where appropriate so that embodiments of this application can be implemented in orders other than those illustrated or described herein, and the objects distinguished by "first," "second," etc., are generally of the same class and the number of objects is not limited; for example, a first object can be one or more. Furthermore, in the specification and claims, "and / or" indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0062] The following is an explanation of the terms used in this application:

[0063] Human intestinal stem cells: These are adult pluripotent cells located at the base of the human intestinal crypts, possessing unlimited self-renewal capacity and the potential to differentiate into all functional cells of the intestinal epithelium. Their differentiated progeny cells can form all functional cell types of the intestinal epithelium, maintaining the structural integrity and physiological function of the intestinal mucosa. They are also the core starting cells for in vitro culture of intestinal organoids.

[0064] 2D intestinal cell model: Intestinal cells in two dimensions. This model is usually cultured on planar carriers such as culture dishes or multi-well plates. Intestinal cells attach to the surface of the carrier and form a monolayer of cells. It can be used to evaluate drug toxicity, drug metabolism, etc. Its advantages are simple operation, low cost, and high throughput, making it suitable for basic research and initial screening experiments.

[0065] 3D intestinal cell model: A three-dimensional intestinal model, which is usually formed into a sphere or similar shape, more closely resembles the morphology and function of the real intestine. Its advantage is that it is closer to the physiological state of the in vivo intestine, with a high degree of simulation, and is suitable for precise mechanism research, drug screening and personalized medicine research.

[0066] Polarity of intestinal epithelial cells: As typical polar epithelial cells, intestinal epithelial cells have their cell membranes, cytoplasmic components, and organelles distributed asymmetrically in space, forming structural and functional polarization. They also form two distinct functional domains, the apical membrane and the basolateral membrane, along the intestinal lumen-basement membrane axis. At the same time, they form a polarized monolayer epithelial barrier through intercellular junctions. This polar distribution is the structural basis for the intestinal epithelium to complete core physiological functions such as nutrient absorption, substance transport, and barrier defense.

[0067] The polarization state of intestinal epithelial cells refers to the final functional state of intestinal epithelial cells after differentiation and maturation from non-polar undifferentiated cells, exhibiting apical-basolateral membrane separation, polar distribution of protein molecules, and functional orientation. It is the core marker of intestinal epithelial cell differentiation and maturation. Unpolarized cells can only complete basic proliferation and cannot perform specific functions such as nutrient absorption and barrier defense.

[0068] Drug candidates are compounds or molecules that are screened out during the drug development process and have the potential to treat specific diseases. Drug candidates must undergo a series of rigorous laboratory and clinical trials to determine their safety, efficacy, and side effects.

[0069] In drug screening and disease mechanism research, the use of reliable cell models for drug testing is crucial. One related technology utilizes intestinal stem cells to induce differentiation and obtain intestinal cell models. However, the inventors have discovered that existing intestinal cell models suffer from defects such as loss of barrier function, low polarization efficiency, and unstable polarization states, limiting their application in drug screening and disease mechanism research.

[0070] While 3D intestinal organoid models can be obtained through related technologies, the barrier function of these models is maintained for a short period, typically ≤7 days, failing to simulate the physiological characteristics of real intestinal tissue. Specifically, the expression of polarization markers (such as ZO-1 and E-cadherin) decreases rapidly over time, indicating that the function of 3D intestinal organoid models is not maintained for a long time.

[0071] In this regard, this application provides a culture medium and culture method for constructing polarized 3D human intestinal organoids. Through this culture medium and culture method, the obtained 3D human intestinal organoid model can stably maintain high cell viability (ATP content is maintained at more than 85% of the initial level) for 21 days, and Villin expression remains at a high level and exhibits typical apical-out polarization characteristics, indicating that the 3D human intestinal organoid model has achieved a long-term stable mature differentiation state and maintenance of physiological polarity.

[0072] The culture medium, method, and model for constructing polarized 3D human intestinal organoids provided in this application will be described in detail below with reference to the accompanying drawings, through specific embodiments and application scenarios.

[0073] This embodiment describes the culture medium for constructing polarized 3D human intestinal organoids, which includes the following formulation components:

[0074] Wnt3a at concentrations of 5–15 μg / mL

[0075] 30~100 μg / mL of R-spondin-1, R-spondin-2 and / or R-spondin-3,

[0076] 10-50 nmol of γ-secretase inhibitors,

[0077] 2~10 μmol of Wnt agonist,

[0078] 50~200 μmol of HDAC inhibitor,

[0079] 0.5-2% N2 supplement,

[0080] 1-4% B27 supplement,

[0081] 5~20 μg / mL of IGF-1,

[0082] 0.1~2 μmol of TGF-β receptor inhibitors,

[0083] The remainder is Williams E solution.

[0084] Preferably, the γ-secretase inhibitor is one or more of DAPT, LY411575, MK0752, curcumin, BMS708163, RO4929097, PF03084014, LY450139, and AZD3293. More preferably, the amount of γ-secretase inhibitor used is 20 nmol.

[0085] Preferably, the Wnt agonist is one or more of Wnt3a, Wnt5a, CHIR99021, BIO, XAV939, WAY316606, Foxy5, SB216763, and Tideglusib. More preferably, the amount of Wnt agonist used is 5 μmol.

[0086] Preferably, the HDAC inhibitor is one or more selected from TSA, SAHA, LBH589, sodium butyrate, VPA, MS275, RG2833, MGCD0103, FK228, Tubastatin A, and ACY1215. More preferably, the amount of HDAC inhibitor used is 100 μmol.

[0087] Preferably, the volume ratio of N2 supplement to B27 supplement is 1:2. Preferably, the dosage of N2 supplement is 1% by volume; the dosage of B27 supplement is 2% by volume.

[0088] Preferably, the TGF-β receptor inhibitor is one or more of SB431542, A83-01, GW788388, LY2157299, RepSox, SD208, LY364947, and E-616452. More preferably, the amount of TGF-β receptor inhibitor used is 0.5 μmol.

[0089] Preferably, the culture medium for constructing polarized 3D human intestinal organoids comprises the following components: 10 μg / mL Wnt3a, 50 μg / mL R-spondin-1, 20 nmol LY411575, 5 μmol CHIR99021, 100 μmol sodium butyrate, 1% N2 supplement, 2% B27 supplement, 10 μg / mL IGF-1, 0.5 μmol A83-01, and the balance being Williams E solution.

[0090] In the above formulation, Wnt3a is used to maintain stem cell proliferation; R-spondin-1, R-spondin-2, and / or R-spondin-3 are used to synergistically activate the Wnt signaling pathway; γ-secretase inhibitors inhibit the Notch signaling pathway and induce differentiation and polarization of intestinal stem cells; Wnt agonists enhance the expression of polarity-related proteins; HDAC inhibitors promote the differentiation of intestinal epithelial cells and the expression of tight junction proteins to enhance barrier integrity and stabilize cell polarization phenotype; N2 supplements support neuroepithelial differentiation; B27 supplements provide lipids and antioxidants; IGF-1 inhibits apoptosis; TGF-β receptor inhibitors reduce fibroblast contamination; and Williams E solution provides the necessary energy source for cell culture to maintain cell growth and function. On the other hand, the combined use of γ-secretase inhibitors (such as LY411575) and HDAC inhibitors (sodium butyrate) can enhance the blocking strength of Notch signaling and synergistically induce intestinal stem cells to differentiate into secretory cells. Thirdly, the complementary effects of N2 and B27 supplements can significantly improve cell survival and reduce oxidative stress.

[0091] The above-mentioned culture medium formula, through the combination of its components, can provide the energy source and nutrients required for the culture of human intestinal stem cells, and can also effectively promote the directional induction of human intestinal stem cells into mature differentiated cell types (such as goblet cells and absorptive cells), while inhibiting the excessive proliferation of human intestinal stem cells.

[0092] The method for constructing polarized 3D human intestinal organoids in this embodiment involves culturing human intestinal stem cells in a culture medium, wherein the culture medium is the culture medium for constructing polarized 3D human intestinal organoids as described in any of the technical solutions in this embodiment.

[0093] The method for constructing polarized 3D human intestinal organoids in this embodiment includes at least the following steps:

[0094] Step 100: Human intestinal stem cells are embedded in a substrate using a culture medium and then seeded into a multi-well plate. Exemplarily, the embedding substrate is Matrigel, Collagen I, hyaluronic acid-based hydrogel, or a synthetic hydrogel (such as PEG-based). Exemplarily, the multi-well plate is a 24-well plate, a 48-well plate, a 96-well plate, or a microfluidic chip.

[0095] Step 200: Add 1 mL of culture medium to each well and incubate at 37°C and 5% CO2.

[0096] Step 300: In subsequent cultures, replace 40-60% of the original culture medium with fresh culture medium once a day. For example, replace 50% of the original culture medium with fresh culture medium daily to ensure the activity of the culture medium components.

[0097] The 3D human intestinal organoid model obtained using the culture medium and culture method of this embodiment possesses polarization function and physiological simulation. Specifically, this 3D human intestinal organoid model can stably maintain high cell viability (ATP content remains above 85% of the initial level) for 21 days, and Villin expression remains at a high level and exhibits typical apical-out polarization characteristics, indicating that this 3D human intestinal organoid model has achieved a long-term stable mature differentiation state and maintenance of physiological polarity.

[0098] The 3D human intestinal organoid model of this embodiment is based on human intestinal stem cells. The intestinal cells are obtained by culturing them using any of the technical solutions in this embodiment to construct polarized 3D human intestinal organoids. The polarization state and / or barrier function of the 3D human intestinal organoid model are maintained for more than 21 days.

[0099] This embodiment applies the 3D human intestinal organoid model, which is used for one or more of the following: mechanisms of intestinal diseases, drug screening for intestinal diseases, pharmacological and toxicological evaluation of candidate drugs, and metabolic studies of candidate drugs.

[0100] The culture medium formulation and the 3D human intestinal organoid model constructed based on this formulation provided in this embodiment not only maintain the differentiation state and physiological polarity of mature intestinal epithelium for a long period of time, but also have good predictive sensitivity and clinical relevance for enterotoxicity. This provides a reliable and efficient new tool for intestinal toxicity assessment, drug screening, and in vitro model research of intestinal diseases. Compared with traditional unpolarized or poorly differentiated organoid models, the 3D human intestinal organoid model in this embodiment has significant advantages in simulating clinical intestinal toxicity responses.

[0101] The culture medium and culture method for constructing polarized 3D human intestinal organoids according to this application will be described in detail below with specific embodiments.

[0102] Example 1

[0103] This embodiment describes the culture medium for constructing polarized 3D human intestinal organoids, comprising the following components: 10 μg / mL Wnt3a, 50 μg / mL R-spondin-1, 20 nmol LY411575, 5 μmol CHIR99021, 100 μmol sodium butyrate, 1% N2 supplement, 2% B27 supplement, 10 μg / mL IGF-1, 0.5 μmol A83-01, with the balance being Williams E solution.

[0104] The method for culturing human intestinal stem cells using the above-mentioned culture medium to obtain 3D human intestinal organoids includes the following steps:

[0105] Step 100: Using the above culture medium, 500 human intestinal stem cells were embedded in Matrigel matrix and then seeded into 24-well plates.

[0106] Step 200: Add 1 mL of culture medium to each well and incubate at 37°C and 5% CO2.

[0107] Step 300: In subsequent cultures, replace 50% of the original culture medium with fresh medium daily to ensure the activity of the medium components. Culture continuously for 21 days.

[0108] Example 2

[0109] This embodiment describes the culture medium for constructing polarized 3D human intestinal organoids, comprising the following components: 5 μg / mL Wnt3a, 30 μg / mL R-spondin-2, 10 nmol DAPT, 10 μmol SB216763, 50 μmol sodium butyrate, 2% N2 supplement, 4% B27 supplement, 5 μg / mL IGF-1, 0.1 μmol A83-01, with the balance being Williams E solution.

[0110] The method for culturing human intestinal stem cells and obtaining 3D human intestinal organoids using the above-mentioned culture medium is the same as in Example 1, and will not be repeated here.

[0111] Example 3

[0112] This embodiment describes the culture medium for constructing polarized 3D human intestinal organoids, comprising the following components: 15 μg / mL Wnt3a, 100 μg / mL R-spondin-3, 50 nmol RO4929097, 2 μmol Tideglusib, 200 μmol sodium butyrate, 0.5% N2 supplement, 1% B27 supplement, 20 μg / mL IGF-1, 2 μmol A83-01, with the balance being Williams E solution.

[0113] The method for culturing human intestinal stem cells and obtaining 3D human intestinal organoids using the above-mentioned culture medium is the same as in Example 1, and will not be repeated here.

[0114] Comparative Example 1

[0115] The culture medium used to construct the comparative intestinal organoid model consisted of the following components: 50 ng / mL EGF, 500 ng / mL R-spondin-1, 100 ng / mL Noggin, 10 μg / mL Wnt3a, 1% N2 supplement, 0.5 μM SB431542, and the remainder being Advanced DMEM / F12 solution.

[0116] The method for culturing human intestinal stem cells and obtaining intestinal organoid models using the above-mentioned culture medium includes the following steps:

[0117] Step 100: Using the above culture medium, 500 human intestinal stem cells were embedded in Matrigel matrix and then seeded into 24-well plates.

[0118] Step 200: Add 1 mL of culture medium to each well and incubate at 37°C and 5% CO2.

[0119] Step 300: In subsequent cultures, replace 50% of the original culture medium with fresh medium daily to ensure the activity of the medium components. Culture continuously for 21 days.

[0120] A 3D human intestinal organoid model with polarization function was constructed on day 21 in Example 1, and an intestinal organoid model constructed according to existing technology in Comparative Example 1 was characterized using slide immunofluorescence. The expression of LGR5, MUC2, Villin, and Ki67 in the models was detected. The results are as follows: Figure 1 As shown.

[0121] Figure 1 In the image, the left side shows the detection results of the 3D human intestinal organoid model obtained in Example 1, and the right side shows the detection results of the intestinal organoid model obtained in Comparative Example 1. Figure 1 As shown, the 3D human intestinal organoid model obtained in Example 1 expressed low levels of Ki67 and LGR5, but high levels of MUC2 and Villin; while the intestinal organoid model obtained in Comparative Example 1 expressed relatively high levels of Ki67 and LGR5, but low levels of Villin.

[0122] MUC2 and Villin were used to characterize goblet cells and villous cells, respectively. This result also shows that the culture medium and culture method of Example 1 can effectively promote the directed induction of human intestinal stem cells into mature differentiated cell types (such as goblet cells and absorptive cells), while inhibiting the excessive proliferation of stem cells, thus realizing the polarization function and physiological simulation of the human intestinal organoid model.

[0123] The changes in cell viability and dynamic changes in Villin expression in the 3D human intestinal organoid models obtained in Examples 1-3 were further examined during the 21-day culture period. The changes in cell viability in the intestinal organoid model obtained in Comparative Example 1 were also examined. The results are as follows: Figures 2-6 As shown.

[0124] like Figures 2-4 As shown, the 3D human intestinal organoid models obtained using the culture media and methods described in Examples 1-3 were able to stably maintain high cell viability (ATP content remained above 85% of the initial level) for 21 days. Figure 5 As shown, the 3D human intestinal organoid model obtained using the culture medium and method of Example 1 exhibits persistently high levels of Villin expression and displays typical apical-out polarization characteristics, indicating that this 3D human intestinal organoid model has achieved a long-term stable state of mature differentiation and maintenance of physiological polarity. Figure 6 As shown, the intestinal organoid model obtained in Comparative Example 1 experienced a rapid decline in cell viability during the corresponding culture process, with a decrease of more than 50% on day 21.

[0125] It should be noted that, Figure 2 The data presented are relative values ​​based on the test results from day 1. In the test results from days 7, 14, and 21, cell viability slightly exceeded 100%, which is considered normal experimental fluctuation. As long as cell viability does not fall below 85% of the initial level, the 3D human intestinal organoid model is considered to have stably maintained high cell viability. Further... Figure 3 , Figure 4 , Figures 6-10 Similar situations exist in both cases, where the relative values ​​are based on the test results of day 1.

[0126] To verify the application value of the polarized 3D human intestinal organoid models constructed in Examples 1-3 in predicting enterotoxicity, abemaciclib (a CDK4 / 6 inhibitor known to cause diarrhea, intestinal mucosal damage, and other gastrointestinal toxicities), a clinically known drug with proven enterotoxicity, was selected as a representative test compound. After treatment with different concentration gradients (1 μM, 10 μM, 20 μM) for 48 h, changes in cell viability were detected, and the results are as follows: Figures 7-9 As shown.

[0127] like Figures 7-9As shown, for the polarized 3D human intestinal organoid models constructed in Examples 1 to 3, abecicilline significantly reduced cell viability in a concentration-dependent manner (at a concentration of 20 μM, cell viability decreased to about 60%, which was statistically significant compared with the 0.1% DMSO control group, p<0.01). This damaging effect is highly consistent with the intestinal toxicity observed clinically.

[0128] like Figure 10 As shown, for the human intestinal organoid model constructed in Comparative Example 1, the cell viability of the organoid model did not decrease significantly with the increase of abexicillin concentration (at a concentration of 20 μM, cell viability decreased to about 80%, and the difference was not statistically significant compared with the 0.1% DMSO control group, p<0.01). This damaging effect is different from the intestinal toxicity observed in clinical practice.

[0129] The above results fully demonstrate that the culture medium formulation provided in this application and the polarized 3D human intestinal organoid model constructed based on this formulation can not only maintain the differentiation state and physiological polarity of mature intestinal epithelium for a long time, but also have good predictive sensitivity and clinical relevance for enterotoxicity, providing a reliable and efficient new tool for intestinal toxicity assessment, drug screening, and in vitro model research of intestinal diseases. Compared with traditional unpolarized or poorly differentiated organoid models, the model in this embodiment has significant advantages in simulating clinical intestinal toxicity reactions.

[0130] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0131] Furthermore, it should be noted that the scope of the methods and apparatus in the embodiments of this application is not limited to performing functions in the order shown or discussed, but may also include performing functions substantially simultaneously or in the reverse order, depending on the functions involved. For example, the described methods may be performed in a different order than described, and various steps may be added, omitted, or combined. In addition, features described with reference to certain examples may be combined in other examples.

[0132] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A culture medium for constructing polarized 3D human intestinal organoids, characterized in that, The formula includes the following components: Wnt3a at concentrations of 5–15 μg / mL 30~100 μg / mL of R-spondin-1, R-spondin-2 and / or R-spondin-3, 10-50 nmol of γ-secretase inhibitors, such as DAPT, LY411575, or RO4929097. 2–10 μmol of a Wnt agonist, such as CHIR99021, SB216763, or Tideglusib. 50~200 μmol sodium butyrate, N2 supplement at a volume ratio of 0.5~2%, A 1-4% volume ratio of B27 supplements The volume ratio of N2 supplement to B27 supplement is 1:

2. 5~20 μg / mL of IGF-1, 0.1–2 μmol of a TGF-β receptor inhibitor, the TGF-β receptor inhibitor being A83-01. The remainder is Williams E solution.

2. The culture medium for constructing polarized 3D human intestinal organoids according to claim 1, characterized in that, The dosage of LY411575 is 20 nmol.

3. The culture medium for constructing polarized 3D human intestinal organoids according to claim 1, characterized in that, The dosage of CHIR99021 is 5 μmol.

4. The culture medium for constructing polarized 3D human intestinal organoids according to claim 1, characterized in that, The amount of sodium butyrate used is 100 μmol.

5. The culture medium for constructing polarized 3D human intestinal organoids according to claim 1, characterized in that, The dosage of N2 supplement is 1% by volume; the dosage of B27 supplement is 2% by volume.

6. The culture medium for constructing polarized 3D human intestinal organoids according to claim 1, characterized in that, The dosage of A83-01 is 0.5 μmol.

7. The culture medium for constructing polarized 3D human intestinal organoids according to claim 1, characterized in that, The formula includes the following components: 10 μg / mL Wnt3a, 50 μg / mL R-spondin-1, LY411575 at 20 nmol 5 μmol of CHIR99021, 100 μmol sodium butyrate, 1% N2 supplement by volume 2% by volume B27 supplement 10 μg / mL IGF-1, 0.5 μmol of A83-01, The remainder is Williams E solution.

8. A method for constructing polarized 3D human intestinal organoids through culture, characterized in that, The culture method uses a culture medium to culture human intestinal stem cells, wherein the culture medium is the culture medium for constructing polarized 3D human intestinal organoids as described in any one of claims 1 to 7, and the culture method includes at least the following steps: Step 100: Human intestinal stem cells are embedded in a matrix using a culture medium and then seeded into a multi-well plate; Step 200: Add 1 mL of culture medium to each well and incubate at 37°C and 5% CO2. Step 300: In subsequent culture, replace 40-60% of the original culture medium with fresh culture medium once a day.

9. A 3D human intestinal organoid model, characterized in that, The 3D human intestinal organoid model is based on human intestinal stem cells, and the intestinal cells are obtained by culturing them using the culture method for constructing polarized 3D human intestinal organoids as described in claim 8. The polarization state and / or barrier function of the 3D human intestinal organoid model are maintained for more than 21 days.

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