Mammary gland lactation type organ as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedicine, and discloses a mammary gland lactation organoid and a preparation method and application thereof. The mammary gland lactation organoid is obtained by induced culture of mammary gland globules; the mammary gland ball is obtained by inducing and culturing a cell aggregate; the cell aggregate is obtained by three-dimensional aggregation culture of MCF10A cells. Through two-stage culture of promoting cell proliferation culture and promoting lactation culture, based on Areg-mediated cell lineage directional differentiation and a PRL-driven hormone activation synergistic mechanism, the mammary gland lactation type organ model with an efficient secretion function is successfully constructed, specific differentiation and lactation function reconstruction of mammary gland cells are realized, and the mammary gland lactation type organ model with an efficient secretion function is obtained. The invention provides a good solution for improving breast milk yield and accessibility, can be used as an important experimental platform for researching breast function regulation and molecular mechanism in the lactation period, and can be widely applied to the related research field of breast lactation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a mammary gland lactogenic organoid, its preparation method, and its application. Background Technology

[0002] Current in vitro mammary organoid lactation models face multiple technical bottlenecks: Qu et al. (Qu Y. et al. Differentiation of Human Induced Pluripotent Stem Cells to Mammary-like Organoids. Stem Cell Reports, (2017)) demonstrated that while mammary organoids derived from induced pluripotent stem cells (iPSCs) can express milk proteins under prolactin-insulin-hydrocortisone complex culture, they suffer from inherent defects such as low secretion efficiency (insufficient yield of human milk-like products) and lengthy preparation cycles; while Sumbal's team (Sumbal J. et al. Primary Mammary Organoid Model of Lactation and Involution. Front Cell Dev Biol, ...) The mouse mammary organoid system developed in (2020), titled "Primary Mammary Organoid Model of Lactation and Degeneration," can simulate the dynamic process of lactation and degeneration. However, its translational application value is limited by cross-species heterogeneity, namely the fundamental differences between mouse and human mammary glands in terms of tissue structure and molecular regulation. Furthermore, while existing biomaterial construction strategies have promoted research on mammary gland development, they generally face common challenges such as insufficient organoid maturity, low differentiation efficiency, and high culture costs, which seriously hinder their application in elucidating lactation mechanisms and large-scale preparation of human milk-like products. These key technical bottlenecks not only restrict breakthroughs in basic research on mammalian biology but also limit the functional reconstruction of human breast milk components. Therefore, establishing a human mammary gland lactation model with significant lactation function and achieving specific differentiation of mammary gland cells and reconstruction of lactation function are technical problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0003] This invention provides a mammary gland lactogenic organoid, its preparation method, and its application, in order to solve the problems of insufficient maturity, low differentiation efficiency, low lactation efficiency, and high cost of existing lactogenic organoids, thereby establishing a mammary gland lactation model with significant lactation function and realizing specific differentiation of mammary gland cells and reconstruction of lactation function.

[0004] In a first aspect, the present invention provides a lactogenic organoid of a mammary gland, wherein the lactogenic organoid is obtained by inducing and culturing mammary globules using a second induction medium; the mammary globules are obtained by inducing and culturing cell aggregates using a first induction medium; and the cell aggregates are obtained by three-dimensional aggregation and culture of MCF10A cells. The first induction medium is a first basal medium supplemented with bimodal protein Areg; the second induction medium is a second basal medium supplemented with prolactin PRL.

[0005] In one alternative embodiment, the cell aggregates are obtained by three-dimensional aggregation culture of MCF10A cells using a second basal medium.

[0006] In one alternative embodiment, the concentration of the bimodal protein Areg in the first induction medium is 50-200 ng / mL.

[0007] In one alternative embodiment, the concentration of the bimodal protein Areg in the first induction medium is 100-200 ng / mL.

[0008] In one alternative embodiment, the concentration of prolactin PRL in the second induction medium is 50-200 ng / mL.

[0009] In one alternative embodiment, the concentration of prolactin PRL in the second induction medium is 100-200 ng / mL.

[0010] In one optional embodiment, the first basal culture medium comprises DMEM / F12 basal culture medium, horse serum, cholera toxin, hydrocortisone, and insulin; the horse serum has a volume fraction of 5% in the first basal culture medium; the cholera toxin has a concentration of 100 ng / mL in the first basal culture medium; the hydrocortisone has a concentration of 0.5 µg / mL in the first basal culture medium; and the insulin has a concentration of 10 µg / mL in the first basal culture medium.

[0011] In one optional embodiment, the second basal culture medium comprises DMEM / F12 basal culture medium, horse serum, epidermal growth factor (EGF), cholera toxin, hydrocortisone, and insulin; the horse serum has a volume fraction of 5% in the second basal culture medium; the EGF concentration in the second basal culture medium is 20 ng / mL; the cholera toxin concentration in the second basal culture medium is 100 ng / mL; the hydrocortisone concentration in the second basal culture medium is 0.5 µg / mL; and the insulin concentration in the second basal culture medium is 10 µg / mL.

[0012] In one alternative embodiment, the mammary lactogenic organoid has basal cells and luminal cells.

[0013] In one alternative embodiment, the mammary lactogenic organoid has mammary epithelium and is capable of producing human milk-like products.

[0014] In one alternative implementation, the mammary globules express the luminal cell marker K8.

[0015] In one alternative embodiment, the mammary lactogenic organoid expresses the basal cell marker K14.

[0016] In one alternative embodiment, the mammary gland lactogenic organoid is a human mammary gland lactogenic organoid.

[0017] In one alternative embodiment, the diameter of the cell aggregate is 150-300 μm.

[0018] In one alternative embodiment, the human milk-like product comprises human milk protein and fat; the human milk protein comprises at least one of α-lactalbumin, β-casein, and lactoferrin; and the fat comprises milk fat.

[0019] In a second aspect, the present invention also provides a kit for constructing the said mammary gland lactogenic organoid, the kit comprising MCF10A cells, a first induction medium and a second induction medium; The first induction medium is a first basal medium supplemented with bimodal protein Areg; the second induction medium is a second basal medium supplemented with prolactin PRL.

[0020] In one alternative embodiment, the concentration of the bimodal protein Areg in the first induction medium is 50-200 ng / mL.

[0021] In one alternative embodiment, the concentration of the bimodal protein Areg in the first induction medium is 100-200 ng / mL.

[0022] In one alternative embodiment, the concentration of prolactin PRL in the second induction medium is 50-200 ng / mL.

[0023] In one alternative embodiment, the concentration of prolactin PRL in the second induction medium is 100-200 ng / mL.

[0024] In one alternative implementation, the kit further includes a second basal culture medium.

[0025] In one optional embodiment, the second basal culture medium comprises DMEM / F12 basal culture medium, horse serum, epidermal growth factor (EGF), cholera toxin, hydrocortisone, and insulin; the horse serum has a volume fraction of 5% in the second basal culture medium; the EGF concentration in the second basal culture medium is 20 ng / mL; the cholera toxin concentration in the second basal culture medium is 100 ng / mL; the hydrocortisone concentration in the second basal culture medium is 0.5 µg / mL; and the insulin concentration in the second basal culture medium is 10 µg / mL.

[0026] In one optional embodiment, the first basal culture medium comprises DMEM / F12 basal culture medium, horse serum, cholera toxin, hydrocortisone, and insulin; the horse serum has a volume fraction of 5% in the first basal culture medium; the cholera toxin has a concentration of 100 ng / mL in the first basal culture medium; the hydrocortisone has a concentration of 0.5 µg / mL in the first basal culture medium; and the insulin has a concentration of 10 µg / mL in the first basal culture medium.

[0027] Thirdly, the present invention also provides a method for constructing the said mammary gland lactogenic organoid, the method comprising: performing three-dimensional aggregation culture on MCF10A cells to obtain cell aggregates; performing a first-stage induction culture on the cell aggregates using a first induction medium to obtain mammary globules; and performing a second-stage induction culture on the mammary globules using a second induction medium to obtain mammary gland lactogenic organoids; The first induction medium is a first basal medium supplemented with bimodal protein Areg; the second induction medium is a second basal medium supplemented with prolactin PRL.

[0028] In one alternative embodiment, the concentration of the bimodal protein Areg in the first induction medium is 50-200 ng / mL.

[0029] In one alternative embodiment, the concentration of the bimodal protein Areg in the first induction medium is 100-200 ng / mL.

[0030] In one alternative embodiment, the concentration of prolactin PRL in the second induction medium is 50-200 ng / mL.

[0031] In one alternative embodiment, the concentration of prolactin PRL in the second induction medium is 100-200 ng / mL.

[0032] In one optional embodiment, the method includes: performing three-dimensional aggregation culture of MCF10A cells using a second basal culture medium to obtain cell aggregates; performing a first-stage induction culture of the cell aggregates using a first induction culture medium to obtain mammary globules; and performing a second-stage induction culture of the mammary globules using a second induction culture medium to obtain mammary lactogenic organoids.

[0033] In one optional embodiment, the second basal culture medium comprises DMEM / F12 basal culture medium, horse serum, epidermal growth factor (EGF), cholera toxin, hydrocortisone, and insulin; the horse serum has a volume fraction of 5% in the second basal culture medium; the EGF concentration in the second basal culture medium is 20 ng / mL; the cholera toxin concentration in the second basal culture medium is 100 ng / mL; the hydrocortisone concentration in the second basal culture medium is 0.5 µg / mL; and the insulin concentration in the second basal culture medium is 10 µg / mL.

[0034] In one optional embodiment, the first basal culture medium comprises DMEM / F12 basal culture medium, horse serum, cholera toxin, hydrocortisone, and insulin; the horse serum has a volume fraction of 5% in the first basal culture medium; the cholera toxin has a concentration of 100 ng / mL in the first basal culture medium; the hydrocortisone has a concentration of 0.5 µg / mL in the first basal culture medium; and the insulin has a concentration of 10 µg / mL in the first basal culture medium.

[0035] In one optional implementation, the method includes the following steps: Step 1: Digest adherent MCF10A cells into single cells; Step 2: Use the second basal culture medium to perform three-dimensional aggregation culture on the single cells obtained in Step 1 for 48-72 hours to obtain cell aggregates; Step 3: Use the first induction medium to induce the cell aggregates obtained in Step 2 for the first stage of induction culture for 2-4 days to obtain mammary globules; Step 4: Use the second induction medium to perform a second-stage induction culture on the mammary spheres obtained in Step 3, and continue culturing for 2 to 28 days to obtain mammary lactogenic organoids.

[0036] In one optional implementation, in step two, the three-dimensional aggregation culture is carried out using inverted drop culture in a culture dish or 96-well ultra-low adsorption U-shaped plate culture; in steps three and four, the first stage induction culture and the second stage induction culture are carried out in a low adsorption culture container; the low adsorption culture container includes an ultra-low adsorption six-well plate and a 96-well ultra-low adsorption U-shaped plate.

[0037] In one optional implementation, in step two, the three-dimensional aggregation culture is carried out using a 96-well ultra-low adsorption U-shaped plate.

[0038] In one optional embodiment, in step two, when the three-dimensional aggregation culture is carried out in a 96-well ultra-low adsorption U-shaped plate, the number of single cells seeded per well is 3.0 × 10⁻⁶. 3 indivual.

[0039] In one optional implementation, in step two, the three-dimensional aggregation culture is carried out using an inverted hanging drop culture in a 10 cm culture dish.

[0040] In one optional implementation, in step two, when the three-dimensional aggregation culture is performed using an inverted hanging drop culture in a 10 cm culture dish, the number of MCF10A single cells per drop is controlled to be 3.0 × 10⁻⁶. 3 indivual.

[0041] In one alternative implementation, in step three, the first stage of induction culture is carried out in an ultra-low adsorption six-well plate.

[0042] In one alternative implementation, in step three, the first stage of induction culture is carried out in a 96-well ultra-low adsorption U-shaped plate.

[0043] In one alternative implementation, in step four, the second stage of induction culture is carried out in an ultra-low adsorption six-well plate.

[0044] In one alternative implementation, in step four, the second stage of induction culture is carried out in a 96-well ultra-low adsorption U-shaped plate.

[0045] In one alternative implementation, the cell aggregates are induced in a first stage using a first induction medium for 2 days to obtain mammary globules.

[0046] In one alternative implementation, the cell aggregates are induced in a first stage using a first induction medium for 4 days to obtain mammary globules.

[0047] In one alternative implementation, the mammary globules are induced in a second stage using a second induction medium for 6 days to obtain mammary lactogenic organoids.

[0048] In one alternative implementation, a second induction culture medium is used to induce a second stage of culture of mammary globules for 10 days to obtain mammary lactogenic organoids.

[0049] In one alternative implementation, the mammary globules are induced in a second stage using a second induction medium for 28 days to obtain mammary lactogenic organoids.

[0050] In one alternative implementation, the constructed mammary gland lactogenic organoids are observed using immunofluorescence staining, where K8 represents luminal cells, K14 represents basal cells, milk represents human milk protein complex, aLA represents α-lactalbumin, β-casein represents β-casein, and LTF represents lactoferrin.

[0051] Fourthly, the present invention also provides the application of the described mammary gland lactogenic organoid, the described kit, or the method thereof in any of the following: A1. Production of human milk-like products; A2. Screening for drugs that promote or inhibit lactation; A3. Safety evaluation of medication use during lactation; A4. Screen for medications to prevent and / or treat lactation-related diseases; A5. Research on the mechanisms of mammary gland development and lactation; A6. Research on the pathological mechanisms of lactation-related diseases.

[0052] In one alternative implementation, the lactation-related disease includes at least one of abnormal milk secretion, impaired milk drainage, and abnormal milk composition.

[0053] The technical solution of this invention has the following advantages: 1. This invention provides a lactogenic organoid system for the mammary gland, wherein the lactogenic organoid is obtained by inducing and culturing mammary globules using a second induction medium; the mammary globules are obtained by inducing and culturing cell aggregates using a first induction medium; the cell aggregates are obtained by three-dimensional aggregation culture of MCF10A cells; the first induction medium is a first basal medium supplemented with biregular protein Areg; the second induction medium is a second basal medium supplemented with prolactin PRL. MCF10A cells, as normal human mammary epithelial cells, express the β-casein gene (Csn2) and the α-lactalbumin gene (Lactalbumin Alpha, LALBA), possessing certain lactogenic functions. They can be three-dimensionally cultured to obtain spherical structures, actively responding to prolactin and factor stimulation and efficiently secreting human milk-like components; simultaneously, they can avoid the problems of excessively long stem cell differentiation cycles and high culture costs. This invention constructs a lactogenic organoid system based on MCF10A cells, obtaining near-natural human milk product components through dynamic stimulation, possessing safety and controllability, and providing a good solution for the standardized production of human milk-like products.

[0054] Furthermore, this invention successfully constructed a highly efficient mammary gland lactation organoid model through a two-stage culture process—cell proliferation-promoting culture and lactation-promoting culture—based on the synergistic mechanism of Areg-mediated cell lineage-directed differentiation and PRL-driven hormone activation. In the first stage, temporal stimulation by the EGF family key factor, the bimodal protein Areg, significantly promoted the proliferation of mammary ductal cells and the formation of three-dimensional mammary globules. In the second stage, the hormonal regulation of prolactin (PRL) induced functional differentiation of mammary globules and achieved continuous lactation, thus establishing a highly biomimetic in vitro mammary gland lactation model. This model not only stably and efficiently secretes human milk-like bioactive products in vitro, exhibiting rapid and high-throughput characteristics, providing an innovative solution for improving breast milk production and accessibility, but also serves as an important experimental platform for studying the regulation and molecular mechanisms of mammary gland function during lactation, with wide applications in mammary gland lactation-related research fields.

[0055] 2. This invention also provides a kit for constructing the aforementioned lactogenic organoids of the mammary gland. The kit comprises the first induction medium, the second induction medium, and the second basal medium. The first induction medium comprises a first basal medium and the bimodal protein Areg, and the second induction medium comprises a second basal medium and prolactin PRL. The addition of Areg to the first induction medium promotes the proliferation of luminal cells in cell aggregates to form mammary globules. The addition of PRL to the second induction medium promotes lactation from the mammary globules, generating classic human milk-like products such as whey protein and casein. The lactogenic organoids constructed using the kit provided by this invention can stably and efficiently secrete human milk-like bioactive products containing α-lactalbumin, β-casein, lactoferrin, and milk fat in vitro, and can continuously lactate for up to 28 days until the lactogenic organoids are depleted. Attached Figure Description

[0056] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0057] Figure 1 This is a technical roadmap for constructing human mammary gland lactogenic organoids based on MCF10A cells according to the present invention; Figure 2 This is a morphological observation diagram of human mammary gland lactogenic organoids constructed at different differentiation times according to Example 1 of the present invention; scale bar, 500 µm; Figure 3 This is a morphological observation diagram of human mammary gland lactogenic organoids constructed at different differentiation times according to Embodiment 2 of the present invention; scale bar, 500 µm; Figure 4 This is a morphological observation diagram of human mammary gland lactogenic organoids constructed at different differentiation times in Example 3 of the present invention; scale bar, 500 µm; Figure 5 This is a morphological observation diagram of human mammary gland lactogenic organoids constructed at different differentiation times in Example 4 of the present invention; scale bar, 500 µm; Figure 6 This is an immunofluorescence image of the human mammary gland lactogenic organoid constructed in Example 1 of the present invention; in the immunofluorescence image, K8 represents luminal cells, K14 represents basal cells, milk represents human milk protein complex, aLA represents α-lactalbumin, β-casein represents β-casein, and LTF represents lactoferrin. Figure 7 This is an immunofluorescence image of the human mammary gland lactating organoid constructed in Example 2 of the present invention; in the immunofluorescence image, K8 represents luminal cells, K14 represents basal cells, milk represents human milk protein complex, aLA represents α-lactalbumin, β-casein represents β-casein, and LTF represents lactoferrin. Figure 8 This is an immunofluorescence image of the human mammary gland lactating organoid constructed in Example 3 of the present invention; in the immunofluorescence image, K8 represents luminal cells, K14 represents basal cells, milk represents human milk protein complex, aLA represents α-lactalbumin, β-casein represents β-casein, and LTF represents lactoferrin. Figure 9 This is an immunofluorescence image of the human mammary gland lactating organoid constructed in Example 4 of the present invention; in the immunofluorescence image, K8 represents luminal cells, K14 represents basal cells, milk represents human milk protein complex, aLA represents α-lactalbumin, β-casein represents β-casein, and LTF represents lactoferrin. Figure 10 This is an immunofluorescence image of the human mammary gland lactogenic organoid constructed in Comparative Example 1 of this invention; in the immunofluorescence image, K8 represents luminal cells, K14 represents basal cells, milk represents human milk protein complex, aLA represents α-lactalbumin, β-casein represents β-casein, and LTF represents lactoferrin. Figure 11 This is an immunofluorescence image of the human mammary gland lactogenic organoid constructed in Comparative Example 2 of this invention; in the immunofluorescence image, K8 represents luminal cells, K14 represents basal cells, milk represents human milk protein complex, aLA represents α-lactalbumin, β-casein represents β-casein, and LTF represents lactoferrin. Figure 12This is an immunofluorescence image of the human mammary gland lactating organoid constructed in Comparative Example 3 of this invention; in the immunofluorescence image, K8 represents luminal cells, K14 represents basal cells, milk represents human milk protein complex, aLA represents α-lactalbumin, β-casein represents β-casein, and LTF represents lactoferrin. Figure 13 These are Oil Red staining results of human mammary gland lactogenic organoids constructed in Example 1 and Comparative Examples 1 to 3 of this invention. Detailed Implementation

[0058] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0059] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0060] First basal culture medium: DMEM / F12 medium (source culture, catalog number L340KJ), 5% (v / v) horse serum (Gibco, catalog number 16050122), 100 ng / mL cholera toxin (Sigma, catalog number C8052), 0.5 µg / mL hydrocortisone (Sigma, catalog number H0888), 10 µg / mL insulin (Solepro, catalog number I8830). Second basal culture medium: DMEM / F12 medium (source culture, catalog number L340KJ), 5% (v / v) horse serum (Gibco, catalog number 16050122), 20 ng / mL EGF (peprotech), 100 ng / mL cholera toxin (Sigma, catalog number C8052), 0.5 µg / mL hydrocortisone (Sigma, catalog number H0888), 10 µg / mL insulin (Solepro, catalog number I8830). Cells: MCF10A cells were purchased from ATCC; Reagents: Areg was purchased from MCE; PRL was purchased from Sino Biological. Culture containers: 10 cm petri dish (Jet Biotech, catalog number TCD010100), ultra-low adsorption six-well plate (Corning, catalog number 3471), 96-well ultra-low adsorption U-shaped plate (Corning, catalog number 7007). Antibodies: K8 (Biolegend, catalog number 904804), K14 (Biolegend, catalog number 906004), human milk protein (Nordic Mubio, catalog number RAHu / TM), β-casein (Invitrogen, PA5-118998), α-lactalbumin aLA (Cloud clone, catalog number PAB018Hu01), lactoferrin LTF (Cloud clone, catalog number PAA077Hu01), Cy3 goat anti-mouse secondary antibody (Jackson, catalog number 115-165-146), Goat anti-Chicken IgY (H+L) secondary antibody, Alexa Fluor TM 488 (Invitrogen, Cat. No. A-11039), Donkeyanti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, AlexaFluor TM 647 (Invitrogen, catalog number A-31573).

[0061] Example 1: A method for constructing human mammary gland lactogenic organoids This embodiment provides a human mammary gland lactogenic organoid, which is obtained by inducing and culturing mammary globules using a second induction medium; the mammary globules are obtained by inducing and culturing cell aggregates using a first induction medium; the cell aggregates are obtained by three-dimensional aggregation culture of MCF10A cells. The construction method of the human mammary gland lactogenic organoid is as follows (the technical route for constructing human mammary gland lactogenic organoids based on MCF10A cells is as follows). Figure 1 (as shown) 1. Culture of MCF10A cells MCF10A cells are a human non-tumorigenic epithelial cell line with strong adherence. Cell digestion requires a relatively long time; therefore, double the volume of 0.25% (g / 100 mL) EDTA trypsin is used for digestion, with the time controlled within 15 minutes. Cells grow rapidly, with a routine passage ratio of 1:5 to 1:10, and the culture medium is changed every two days. Cells are cultured at 37 ℃ and 5% (v / v) CO2 in a secondary basal medium. The specific steps are as follows: (1) Take the second basal culture medium and preheat it to room temperature (25℃). Culture MCF10A cells in a six-well plate at a seeding density of 3×10⁻⁶. 4 ~5×10 4 One hole / hole.

[0062] (2) When the confluence of MCF10A cells reaches 80%~90%, the cells are rinsed with phosphate buffer (PBS buffer), and 0.4 mL of 0.25% (g / 100 mL) EDTA trypsin is added to each well for 10~15 min. The digested cells are then blown twice with a P1000 pipette tip to disperse the cell colonies into single cells. The digestion reaction is neutralized by adding an equal volume of DMEM medium (source culture, catalog number L110KJ) to the trypsin. The cells are then centrifuged at 800 rpm at room temperature (25℃) for 3 min and the supernatant is discarded.

[0063] (3) Resuspend the cell pellet obtained by centrifugation in step (2) using the second basic culture medium, and then passage it at a ratio of 1:5 to 1:10 under conditions of 37 °C and 5% (v / v) CO2. Change the culture medium every two days after passage.

[0064] 2. Preparation of cell aggregates When the confluence of MCF10A cells cultured in step 1 reaches 80%–90%, rinse the cells twice with PBS buffer, add 0.4 mL of 0.25% (g / 100 mL) EDTA trypsin to digest the cells for 10–15 min, add DMEM medium to stop digestion, collect the cell suspension into a centrifuge tube, centrifuge at 800 rpm at room temperature (25℃) for 3 min, and discard the supernatant. Resuspend the cells in the second basal medium to obtain a single-cell suspension, and then adjust the cell density of the suspension to ensure that each drop of the inverted hanging drop culture in a 10 cm culture dish contains 3 × 10-10 cells. 3 Cell aggregates were obtained by culturing single cells at 37 °C and 5% (v / v) CO2 for 48 h.

[0065] 3. Preparation of the first-stage mammary gland globules The cell aggregates obtained in step 2 were collected and centrifuged at 300 rpm for 3 min at room temperature (25℃). After discarding the supernatant, 2 mL of the first induction medium was added, and the mixture was transferred to a six-well plate with ultra-low adsorption, controlling 180 cell aggregates per well. The plates were cultured at 37℃ and 5% (v / v) CO2 for 4 days, with the medium changed every two days, to promote luminal cell proliferation and obtain the first-stage mammary spheres. The first induction medium was a first basal medium supplemented with 100 ng / mL Areg.

[0066] 4. Preparation of second-stage human mammary gland lactogenic organoids The mammary globules obtained in step 3 were collected and centrifuged at 300 rpm for 1 min at room temperature (25℃). After discarding the supernatant, 2 mL of the second induction medium was added, and the mixture was transferred to an ultra-low adsorption six-well plate, with 180 mammary globules per well. The plates were cultured at 37℃ and 5% (v / v) CO2 for 10 days. Lactation was stimulated in the mammary globules using prolactin (PRL), yielding the second-stage human mammary gland lactogenic organoids. The second induction medium was a second basal medium supplemented with 100 ng / mL PRL.

[0067] Example 2: A method for constructing human mammary gland lactogenic organoids This embodiment provides a human mammary gland lactogenic organoid. The construction method of the human mammary gland lactogenic organoid differs from that of Embodiment 1 in that steps 2 to 4 are all performed in a 96-well ultra-low adsorption U-shaped plate; wherein, when preparing cell aggregates using a 96-well ultra-low adsorption U-shaped plate, the number of single cells seeded per well is 3.0 × 10⁻⁶. 3 When preparing the first-stage mammary spheres and the second-stage human mammary lactating organoids using 96-well ultra-low adsorption U-shaped plates, each well contained one cell aggregate or one mammary sphere. The mammary spheres were induced and cultured in the second induction medium for 6 days. After 6 days of induction culture, the second-stage human mammary lactating organoids were obtained. The second induction medium was a second basal medium supplemented with 200 ng / mL PRL. Other experimental procedures were the same as in Example 1.

[0068] Example 3: A method for constructing human mammary gland lactogenic organoids This embodiment provides a human mammary gland lactating organoid. The construction method of the human mammary gland lactating organoid differs from that of Embodiment 1 in that: the induction culture time using the second induction medium is 28 days, and the mammary gland spheres are induced and cultured in an ultra-low adsorption six-well plate for 28 days to obtain the second-stage human mammary gland lactating organoid; the second induction medium is a second basal medium with an additional 200 ng / mL PRL; other experimental procedures are the same as in Embodiment 1.

[0069] Example 4: A method for constructing human mammary gland lactogenic organoids This embodiment provides a human mammary gland lactating organoid. The construction method of the human mammary gland lactating organoid differs from that of Embodiment 1 in that: the first induction medium is used for 2 days of culture, and the cell aggregates are cultured in an ultra-low adsorption six-well plate for 2 days to obtain the first stage mammary spheres; the second induction medium is used for 6 days of induction culture, and the mammary spheres are cultured in an ultra-low adsorption six-well plate for 6 days to obtain the second stage human mammary gland lactating organoid; the first induction medium is a first basal medium with an additional 200 ng / mL Areg; other experimental procedures are the same as in Embodiment 1.

[0070] Comparative Example 1: A method for constructing human mammary gland lactogenic organoids This comparative example provides a human mammary gland lactogenic organoid. The construction method of this human mammary gland lactogenic organoid differs from that of Example 1 in that it lacks the induction of prolactin (PRL). The specific construction method is as follows: (1) The culture of MCF10A cells is carried out in the same manner as in Example 1; (2) Preparation of cell aggregates, the specific process is the same as in Example 1; (3) Preparation of the first-stage mammary globules, the specific process is the same as in Example 1; (4) Preparation of the second-stage human mammary gland lactating organoids: Mammary gland globules were cultured for 10 days using the first induction medium, and other experimental procedures were the same as in Example 1.

[0071] Comparative Example 2: A method for constructing human mammary gland lactogenic organoids This comparative example provides a human mammary gland lactogenic organoid. The construction method of this human mammary gland lactogenic organoid differs from that of Example 1 in that it lacks the induction of the bimodal protein Areg. The specific construction method is as follows: (1) The culture of MCF10A cells is carried out in the same manner as in Example 1; (2) Preparation of cell aggregates, the specific process is the same as in Example 1; (3) Collect the cell aggregates prepared in step (2), centrifuge at 300 rpm and room temperature (25℃) for 3 min, discard the supernatant and add 2 mL of the second induction medium, transfer to an ultra-low adsorption six-well plate, control 180 cell aggregates per well, and culture at 37 ℃ and 5% (v / v) CO2 for 10 days to stimulate lactation with prolactin PRL; the second induction medium is a second basal medium with an additional 100 ng / mL PRL.

[0072] Comparative Example 3: A method for constructing human mammary gland lactogenic organoids This comparative example provides a human mammary gland lactogenic organoid. The construction method of this human mammary gland lactogenic organoid differs from that of Example 1 in that it lacks the induction of bimodal protein Areg and prolactin PRL. The specific construction method is as follows: (1) The culture of MCF10A cells is carried out in the same manner as in Example 1; (2) Preparation of cell aggregates, the specific process is the same as in Example 1; (3) Collect the cell aggregates prepared in step (2), centrifuge at 300 rpm and room temperature (25℃) for 3 min, discard the supernatant, add 2 mL of the second basic culture medium, transfer to an ultra-low adsorption six-well plate, control 180 cell aggregates per well, and continue to culture for 10 days at 37 ℃ and 5% (v / v) CO2.

[0073] Experiment Example 1: Morphological observation of various stages of human mammary gland lactogenic organoid development using an inverted microscope. This experiment demonstrates the morphological observation of various stages of development of human mammary gland lactogenic organoids constructed in Examples 1 to 4. The specific experimental procedure is as follows: Human mammary gland lactogenic organoids prepared in Examples 1 to 4 were placed under an inverted microscope to observe their growth at each stage. The results are as follows: Figures 2-4 As shown.

[0074] Figure 2 The results showed that in the culture system of Example 1, the human mammary gland lactating organoids grew normally. After entering the lactation period, cavities began to form inside the organoids, and the cell structure became more compact.

[0075] Figure 3 The results showed that the human mammary gland lactogenic organoids constructed in Example 2 grew normally, and the growth rate of the lactogenic organoids remained normal as the growth cycle lengthened.

[0076] Figure 4 The results showed that the human mammary gland lactation organoid constructed in Example 3 grew normally. As the culture time was extended, a distinct cavity structure developed inside the organoid. The cavity continued to expand, while the cells remained tightly packed.

[0077] Figure 5 The results showed that the human mammary gland lactogenic organoid constructed in Example 4 grew normally, and the lactogenic organoid continued to grow normally as the growth cycle lengthened.

[0078] The above results demonstrate that the human mammary gland lactogenic organoids constructed by the methods shown in Examples 1 to 4 of this invention have a fast growth rate and short cycle, and can quickly enter and maintain a long-term, stable lactation state; at the same time, the regular cavity structure they develop lays a solid morphological foundation for the efficient synthesis and secretion of milk.

[0079] Experiment Example 2: Identification of human mammary gland lactogenic organoids by immunofluorescence staining In this experiment, the human breast lactogenic organoids constructed in Examples 1-4 and Comparative Examples 1-3 were subjected to immunofluorescence detection of frozen sections to identify their lactation function. The specific experimental procedure is as follows: (1) Use a P1000 pipette to aspirate the culture medium from the well plate and collect the lactating organoids into a 15 mL centrifuge tube. Centrifuge at 300 rpm and room temperature (25℃) for 3 min, and discard the supernatant to obtain the sample. Immerse the sample in 4% (g / 100 mL) paraformaldehyde fixation buffer and let it stand at room temperature (25℃) for 15 min to fix the organoid culture. After fixation, discard the paraformaldehyde fixation buffer and gently wash the sample with PBS for 5 min each time, washing 2-3 times.

[0080] (2) After discarding the PBS, transfer the sample to a clean and sterile 1.5 mL EP tube, and embed the sample with 3% (g / 100 mL) agarose solution at 40℃ (the agarose solution is prepared by agarose powder (Sangon Biotech, catalog number BS081) and PBS (Yuanpei, catalog number B310KJ)). After the agarose block solidifies, take out the sample block and place it in a clean and sterile 2 mL EP tube, and add 1~2 mL of 30% (g / 100 mL) sucrose solution to cover the sample. Place the sample vertically in a 4℃ refrigerator to dehydrate overnight.

[0081] (3) After wiping the liquid off the sample block surface on the second day, embed the sample with OCT embedding agent (SAKURA, catalog number 4583) and perform frozen sectioning. The section thickness is 7~10 µm.

[0082] (4) Before staining, place the frozen sections at room temperature (25°C) for 10-15 min to ensure that the samples recover to room temperature (25°C), and then soak and wash the sections with distilled water for 3 min to remove excess OCT embedding agent.

[0083] (5) 200 µL of blocking solution (composed of 90% (v / v) of 0.1% (v / v) PBST solution and 10% (v / v) goat serum (Beyotime, catalog number C0265)) was added to each sample and allowed to permeate the lactating organoids at room temperature (25°C) for 2 h.

[0084] (6) Remove the blocking solution and add 100 μL of primary antibody diluted in the blocking solution (lumen cell marker K8, basal cell marker K14, human milk protein milk, α-lactalbumin aLA, β-casein and lactoferrin LTF, the proportions are as per the antibody instructions). Place the lactating organoids in a dark box at 4°C and incubate overnight at a horizontal level.

[0085] (7) After the primary antibody incubation is completed on the second day, wash the sample three times with 0.1% (v / v) PBST solution, 10 min each time. After washing, aspirate the supernatant, add 100 μL of the prepared secondary antibody (refer to the antibody instructions for the ratio), and incubate at room temperature (25℃) in the dark for 2 h. After the secondary antibody incubation is completed, wash the sample three to five times with 0.1% (v / v) PBST solution, 10 min each time. After washing, aspirate the supernatant as thoroughly as possible, and drop 20 μL of mounting medium containing DAPI (Sigma, catalog number F6057) onto the sample, ensuring that the sample is completely covered. Then carefully place the glass slide on the mounting medium and mount at room temperature (25℃) for at least 30 min before taking pictures.

[0086] Cell markers and secreted products were observed using immunofluorescence microscopy, and the results are as follows: Figures 6-12 As shown.

[0087] Figure 6 The results showed that the organoid constructed in Example 1 had a strong ability to synthesize and secrete milk proteins, specifically manifested in a significant accumulation of human milk protein milk, whey protein aLA, β-casein and lactoferrin LTF.

[0088] Figure 7 The results showed that the organoids constructed in Example 2 had strong milk protein synthesis and secretion capabilities, and significant accumulation of human milk protein milk and whey protein aLA.

[0089] Figure 8 The results showed that the organoids constructed in Example 3 had strong milk protein synthesis and secretion capabilities, and the production of human milk protein milk, whey protein aLA, β-casein and lactoferrin LTF showed significant accumulation.

[0090] Figure 9 The results showed that the organoids constructed in Example 4 had strong milk protein synthesis and secretion capabilities, and the production of human milk protein milk, whey protein aLA, β-casein and lactoferrin LTF showed significant accumulation.

[0091] Figure 10 The results showed that no significant expression of lactoferrin LTF and whey protein aLA was detected in the milk-like products of the lactogenic organoids constructed in Comparative Example 1, indicating that the key lactation function was lost under this comparative condition.

[0092] Figure 11 The results showed that no significant expression of whey protein aLA, β-casein and lactoferrin LTF was detected in the lactogenic organoids constructed in Comparative Example 2, indicating that the key lactation function was lost under this comparative condition.

[0093] Figure 12 The results showed that no significant expression of whey protein aLA and lactoferrin LTF was detected in the lactogenic organoids constructed in Comparative Example 3, indicating that the key lactation function was lost under this comparative condition.

[0094] In conclusion, Figures 6-12 The results show that the advantage of the human mammary gland lactogenic organoids constructed by the methods described in Examples 1 to 4 of the present invention is that they significantly improve lactation capacity, specifically reflected in the significantly higher levels of human milk protein milk, whey protein aLA, β-casein and lactoferrin LTF in the secreted milk.

[0095] Experiment Example 3: Identification of human mammary gland lactogenic organoids by Oil Red staining In this experiment, frozen sections of human mammary gland lactogenic organoids constructed in Example 1 and Comparative Examples 1-3 were stained with Oil Red chromatographs to detect their ability to secrete fatty substances. The specific experimental procedure is as follows: (1) Use a P1000 pipette to aspirate the culture medium from the well plate and collect the lactating organoids into a 15 mL centrifuge tube. Centrifuge at 300 rpm and room temperature (25℃) for 3 min, and discard the supernatant to obtain the sample. Immerse the sample in 4% (g / 100 mL) paraformaldehyde fixation buffer and let it stand at room temperature (25℃) for 15 min to fix the organoid culture. After fixation, discard the paraformaldehyde fixation buffer and gently wash the sample with PBS for 5 min each time, washing 2-3 times.

[0096] (2) After discarding the PBS, transfer the sample to a clean and sterile 1.5 mL EP tube, embed the sample with 3% (g / 100 mL) agarose solution at 40℃, and after the agarose block solidifies, take out the sample block and place it in a clean and sterile 2 mL EP tube, and add 1~2 mL of 30% (g / 100 mL) sucrose solution to cover the sample. Place the sample vertically in a 4℃ refrigerator to dehydrate overnight.

[0097] (3) After wiping the liquid off the sample block on the second day, embed the sample with OCT embedding agent and perform frozen sectioning. The section thickness is 7~10 µm.

[0098] (4) Before staining, place the frozen sections at room temperature (25°C) for 10-15 min to ensure that the samples recover to room temperature (25°C).

[0099] (5) Immerse the sections in 60% (v / v) isopropanol for 3 s, incubate in Oil Red O working solution (Sigma, catalog number O0625) at room temperature (25℃) in the dark for 30 min, discard the Oil Red O working solution, rinse once with 60% (v / v) isopropanol to remove excess dye, and then wash the sections with PBS 3 times for 5 min each time.

[0100] (6) Immerse the slides in hematoxylin staining solution (purchased from Sangon Biotech (Shanghai) Co., Ltd.) for 2 min to stain the cell nuclei; discard the staining solution, wash the slides with distilled water 3 times, 5 min each time; wipe the surface of the slides dry and then mount them with glycerol resin and take images.

[0101] The results are as follows Figure 13 As shown, the results indicate that the human mammary gland lactogenic organoid constructed in Example 1 can generate substances such as fat, indicating that it can generate complex human milk-like products and has a relatively complete lactation function.

[0102] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A lactogenic organoid of the mammary gland, characterized in that, The lactogenic organoids were obtained by inducing and culturing mammary globules using a second induction medium; the mammary globules were obtained by inducing and culturing cell aggregates using a first induction medium; the cell aggregates were obtained by three-dimensional aggregation culture of MCF10A cells; The first induction medium is a first basal medium supplemented with bimodal protein Areg; the second induction medium is a second basal medium supplemented with prolactin PRL.

2. The mammary gland lactogenic organoid according to claim 1, characterized in that, The cell aggregates were obtained by three-dimensional aggregation culture of MCF10A cells using a second basal medium.

3. The lactogenic organoid of the mammary gland according to claim 1, characterized in that, The concentration of the bimodal protein Areg in the first induction medium was 50-200 ng / mL.

4. The mammary gland lactogenic organoid according to claim 1, characterized in that, The concentration of prolactin (PRL) in the second induction medium was 50-200 ng / mL.

5. The mammary gland lactogenic organoid according to any one of claims 1-4, characterized in that, The first basal culture medium comprises DMEM / F12 basal culture medium, horse serum, cholera toxin, hydrocortisone, and insulin; the horse serum has a volume fraction of 5% in the first basal culture medium; the cholera toxin has a concentration of 100 ng / mL in the first basal culture medium; the hydrocortisone has a concentration of 0.5 µg / mL in the first basal culture medium; and the insulin has a concentration of 10 µg / mL in the first basal culture medium.

6. The mammary gland lactogenic organoid according to any one of claims 1-4, characterized in that, The second basal culture medium comprises DMEM / F12 basal culture medium, horse serum, epidermal growth factor (EGF), cholera toxin, hydrocortisone, and insulin; the horse serum has a volume fraction of 5% in the second basal culture medium; the EGF concentration in the second basal culture medium is 20 ng / mL; the cholera toxin concentration in the second basal culture medium is 100 ng / mL; the hydrocortisone concentration in the second basal culture medium is 0.5 µg / mL; and the insulin concentration in the second basal culture medium is 10 µg / mL.

7. The mammary gland lactogenic organoid according to any one of claims 1-4, characterized in that, The lactogenic organoids possess mammary epithelium and are capable of producing human milk-like products; the mammary globules express the luminal cell marker K8; and the lactogenic organoids express the basal cell marker K14.

8. The mammary gland lactogenic organoid according to claim 7, characterized in that, The human milk-like product includes human milk proteins and fats; the human milk proteins include at least one of α-lactalbumin, β-casein, and lactoferrin; the fats include milk fat.

9. A kit for constructing a mammary gland lactogenic organoid according to any one of claims 1-8, characterized in that, The kit contains MCF10A cells, a first induction medium, and a second induction medium; The first induction medium is a first basal medium supplemented with bimodal protein Areg; the second induction medium is a second basal medium supplemented with prolactin PRL.

10. A method for constructing a mammary gland lactogenic organoid according to any one of claims 1-8, characterized in that, The method includes: performing three-dimensional aggregation culture on MCF10A cells to obtain cell aggregates; performing a first-stage induction culture on the cell aggregates using a first induction medium to obtain mammary globules; and performing a second-stage induction culture on the mammary globules using a second induction medium to obtain mammary lactogenic organoids. The first induction medium is a first basal medium supplemented with bimodal protein Areg; the second induction medium is a second basal medium supplemented with prolactin PRL.

11. The method according to claim 10, characterized in that, The method includes: performing three-dimensional aggregation culture of MCF10A cells using a second basal culture medium to obtain cell aggregates; performing a first-stage induction culture of the cell aggregates using a first induction culture medium to obtain mammary globules; and performing a second-stage induction culture of the mammary globules using a second induction culture medium to obtain mammary lactogenic organoids.

12. The method according to claim 10 or 11, characterized in that, The method includes the following steps: Step 1: Digest adherent MCF10A cells into single cells; Step 2: Use the second basal culture medium to perform three-dimensional aggregation culture on the single cells obtained in Step 1 for 48-72 hours to obtain cell aggregates; Step 3: Use the first induction medium to induce the cell aggregates obtained in Step 2 for the first stage of induction culture for 2-4 days to obtain mammary globules; Step 4: Use the second induction medium to perform a second-stage induction culture on the mammary spheres obtained in Step 3, and continue culturing for 2 to 28 days to obtain mammary lactogenic organoids.

13. The method according to claim 12, characterized in that, In step two, the three-dimensional aggregation culture is carried out using inverted hanging drop culture in a culture dish or 96-well ultra-low adsorption U-shaped plate culture; in steps three and four, the first stage induction culture and the second stage induction culture are carried out in a low adsorption culture container; the low adsorption culture container includes an ultra-low adsorption six-well plate and a 96-well ultra-low adsorption U-shaped plate.

14. The method according to claim 13, characterized in that, When the three-dimensional aggregation culture was carried out in a 96-well ultra-low adsorption U-shaped plate, the number of single cells seeded per well was 3.0 × 10⁶. 3 indivual.

15. The use of the mammary lactogenic organoid according to any one of claims 1-8, the kit according to claim 9, or the method according to any one of claims 10-14 in any of the following: A1. Production of human milk-like products; A2. Screening for drugs that promote or inhibit lactation; A3. Safety evaluation of medication use during lactation; A4. Screen for medications to prevent and / or treat lactation-related diseases; A5. Research on the mechanisms of mammary gland development and lactation; A6. Research on the pathological mechanisms of lactation-related diseases; The lactation-related diseases include at least one of abnormal milk secretion, milk drainage disorders, and abnormal milk composition.

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