KR20220050548A - Culture compositions for growth and inducing differentiation of salivary organoids from human salivary glandular stem cells and method for organoid maturation using the same - Google Patents

Abstract

The present invention relates to a culture fluid composition for inducing growth and differentiation of human salivary gland stem cell-derived salivary gland organoid and a method for maturing organoid using the same. The present invention establishes the culture fluid composition to which neuregulin-1 (Nrg1) and retinyl acetate (RA) necessary for growth and maintenance of human salivary gland organoid are added. The present invention establishes organoid of a shape similar to tissue through 3D culture in vitro with the culture fluid composition to which a notch inhibitor (DAPT) is added for differentiation induction of the grown organoid therethrough. The organoid can be used for disease modeling, pathology research, drug screening, toxicity assessment, and gene manipulation.

Description

Culture compositions for growth and inducing differentiation of salivary organoids from human salivary glandular stem cells and method for organoid maturation using the same

The present invention relates to a culture medium composition for inducing growth and differentiation of salivary gland organoids derived from human salivary gland stem cells and a method for maturation of the organoids using the same.

"Organoid" means that cells derived from tissues or embryonic stem cells can be cultured in 3D form to form an artificial organ. Organoid is a suffix that has the same meaning as ‘organ’ of an organ, and has the word ‘organ-like’. Organoids have a more well-arranged cell and cell function through a three-dimensional culture method, and have a functional organ-like form and function. Organoids are receiving attention along with research on optimization of growth and differentiation factors that can differentiate into various tissues, with stem cell research and 3D cell culture being developed.

Growing cells and cell-derived organoids longer in vitro, increasing the number of passages, and making them biomimetic can obtain more cells from single cells or from cells in the starting stage compared to conventional methods. means to make it easier for research. For example, it can be used in various fields such as screening for pharmacological substances, embryology research, and functional recovery through transplantation.

Growing cells in a gland expansion medium can proliferate while maintaining the properties of their stem cells or progenitor cells. In this case, the organoid is composed of stem cells or progenitor cells. Therefore, growing in a growth medium has the advantage of obtaining an increased amount of stem cells or progenitor cells and organoids containing these cells.

However, when the salivary gland organoids are applied to actual research, it can be confirmed that the salivary gland morphology is simulated and the unique markers disappear easily and are not expressed. This is a phenomenon caused by tube-shaped organs along with complex cell signaling steps unique to the salivary gland, and development of an effective culture medium for inducing growth and differentiation of salivary gland organoids and an effective organoid maturation method using the same is required.

Korean Patent Registration No. 10-1953978 (Registered on February 25, 2019)

It is an object of the present invention to provide a culture medium composition for growth or maintenance of salivary gland organoids derived from salivary gland stem cells comprising Neuregulin-1 (Nrg1) and vitamin A (Vitamin A) as active ingredients.

In addition, another object of the present invention is to provide a culture solution composition for inducing differentiation of salivary gland organoids derived from salivary gland stem cells comprising Nrg1 and DAPT, a Notch inhibitor, as active ingredients.

Another object of the present invention is to provide a method for culturing salivary gland organoids derived from salivary gland stem cells and a method for differentiating salivary gland organoids using the composition.

In order to achieve the above object, the present invention provides a culture solution composition for growth or maintenance of salivary gland organoids derived from salivary gland stem cells comprising Nrg1 and vitamin A as active ingredients.

In addition, the present invention provides a culture medium composition for inducing differentiation of salivary gland organoids derived from salivary gland stem cells comprising Nrg1 and DAPT, a Notch inhibitor, as active ingredients.

In addition, the present invention comprises the steps of (1) isolating salivary gland stem cells from salivary gland tissue; (2) contacting the isolated salivary gland stem cells with an extracellular matrix (ECM); and (3) culturing the salivary gland stem cells in contact with the ECM in the culture solution composition for growth or maintenance of the salivary gland stem cell-derived salivary gland organoids, and growing them into salivary gland organoids. provides

In addition, the present invention comprises the steps of (1) culturing salivary gland organoids in a culture solution composition containing Nrg1; And (2) it provides a salivary gland organoid differentiation method comprising the step of further culturing by adding DAPT, a Notch inhibitor, to the salivary gland organoid culture medium.

In addition, the present invention comprises the steps of (1) isolating salivary gland stem cells from salivary gland tissue; (2) contacting the isolated salivary gland stem cells with an extracellular matrix (ECM); (3) culturing the salivary gland stem cells in contact with the ECM in a culture solution composition for growth or maintenance of the salivary gland stem cell-derived salivary gland organoids, and growing them into salivary gland organoids; (4) culturing the grown salivary gland organoids in a culture solution containing Nrg1; and (5) adding DAPT, a Notch inhibitor, to the salivary gland organoid culture medium of step (4) and culturing the salivary gland stem cell-derived salivary gland organoids.

The present invention relates to a culture medium composition for inducing growth and differentiation of human salivary gland stem cell-derived salivary gland organoids and a method for maturation of the organoid using the same, and more particularly, to Neuregulin-1 essential for growth and maintenance of human salivary gland organoids. 1; Nrg1) and Vitamin A (Retinyl acetate, RA) were added to establish a culture medium composition, and through this, a culture medium containing Notch inhibitor (DAPT), etc. was added to induce differentiation of grown organoids. Nodes were established through three-dimensional culture in vitro, and the organoids can be utilized for disease modeling, pathology research, drug screening, toxicity evaluation, and genetic manipulation.

1 shows a photomicrograph of human salivary gland organoids cultured in an organoid culture medium containing IGF-1 growth factor.
2 shows the results of confirming the expression of markers in human salivary gland organoids cultured in an organoid culture medium containing IGF-1 growth factor.
3 shows a photomicrograph of human salivary gland organoids cultured in an organoid culture medium containing Nrg-1 growth factor.
4 shows gene expression and micrographs of human salivary gland organoids cultured in an organoid growth culture medium containing RA.
5 shows the staining results of human salivary gland organoids cultured in an organoid growth culture medium containing Nrg-1 growth factor.
6 shows a photomicrograph of human salivary gland organoids cultured in an organoid differentiation culture medium containing Nrg-1 and DAPT.
7 shows micrographs and staining results of human salivary gland organoids cultured in a differentiated culture medium containing Nrg-1 and DAPT.

The present invention provides a culture solution composition for growth or maintenance of salivary gland organoids derived from salivary gland stem cells, comprising Nrg1 and vitamin A as active ingredients.

In addition, the present invention provides a culture medium composition for inducing differentiation of salivary gland organoids derived from salivary gland stem cells comprising Nrg1 and DAPT, a Notch inhibitor, as active ingredients.

Preferably, the culture solution composition may include a final concentration of 7.5 to 150 ng/mL of Nrg1, but is not limited thereto.

Preferably, the salivary gland stem cell-derived salivary gland organoid growth or maintenance culture solution composition may consist of the components shown in Table 1, but is not limited thereto.

Preferably, the salivary gland stem cell-derived salivary gland organoid differentiation induction culture solution composition may consist of the components shown in Table 2, but is not limited thereto.

In this specification, "vitamin A (Vitamin A)" has been described as synonymous with "retinyl acetate (Retinyl acetate; RA)".

Meanwhile, as in the present invention, culturing the cells in a gland expansion medium maintains the properties of their stem cells or progenitor cells, and can be proliferated. In this case, the organoid is composed of stem cells or progenitor cells. Therefore, growing in a growth medium has the advantage of obtaining an increased amount of stem cells or progenitor cells and organoids containing these cells.

Neuregulin-1 (Nrg1) of the present invention is an endogenous Wnt secretion agonist, and the intrinsic Wnt secretion pathway is downstream of hetero-dimerization of ErbB2 and ErbB3 in salivary gland epithelial stem cells occurs due to the activation of

In addition, the culture medium composition of the present invention includes a Wnt agonist (e.g. R-spondin), a TGF-beta inhibitor (e.g. A83-01), a cAMP pathway activator (e.g. VIP, Forskolin, Prostagladin E2), a BMP inhibitor (e.g. Noggin, Gremilin 1) ) or fibroblast growth factor (FGF) may be added.

In the above, the Wnt agonist may be any agent capable of activating the Wnt pathway. for example, one of R-spondin 1, 2, 3, 4, which is an agonist of Lgr4, 5, 6. For R-spondin 1, concentrations of 50, 100, 250, 500, and 1000 ng/mL are preferred, and for R-spondin 3, concentrations of 25, 50, 100, 250, and 500 ng/mL are preferred.

In the above, it is advantageous to add the TGF-beta inhibitor to the medium because it increases the efficiency of human organoid formation. TGF-beta signaling is involved in many cellular functions, including cell growth, cell fate and apoptosis. Signal transduction typically begins with the binding of TGF-beta superfamily substrates to type 1 receptors. Type 1 receptors then phosphorylate SMAD, acting as a transcription factor in the nucleus and regulating target gene expression. For example, for A83-01, concentrations of 50, 500, 1000, 2000, 5000 nM are preferred.

In the above, the BMP inhibitor means to inhibit BMP activity by sticking to a BMP molecule, or to interfere with binding to BMP by sticking to a BMP receptor. Examples of BMP inhibitors include Noggin and Gremlin. These diffusive proteins bind to the BMP substrate and inhibit their binding to their receptors. The addition of these BMP inhibitors helps to maintain stem cells. The preferred BMP inhibitor is Noggin, which was added at 10, 25, 50 or 100 ng/mL to the growth medium. The preferred concentration is 100 ng/mL.

In the above, FGF can be selected to act on FGF receptor 1 and FGF receptor 2, mainly, FGF1, FGF2, FGF7, FGF10 was used. For FGF1, concentrations of 20, 40, 100 ng/mL, for FGF2, concentrations of 5, 20, 40, and 100 ng/mL, for FGF7, concentrations of 20, 40, and 100 ng/mL, FGF10 , concentrations of 10, 20, 40 and 100 ng/mL are preferred. Sometimes concentrations of 250 or 500 ng/mL may be used.

In addition, the present invention comprises the steps of (1) isolating salivary gland stem cells from salivary gland tissue; (2) contacting the isolated salivary gland stem cells with an extracellular matrix (ECM); and (3) culturing the salivary gland stem cells in contact with the ECM in the culture solution composition for growth or maintenance of the salivary gland stem cell-derived salivary gland organoids, and growing them into salivary gland organoids. provides

Preferably, the cultured salivary gland organoids express ductal markers CK5 and CK7, acinar cell markers SLC12A2 and AQP5, myoepithelial cell markers ACTA2, and Ki67 and LGR5 may be, but is not limited thereto.

In addition, the present invention comprises the steps of (1) culturing salivary gland organoids in a culture solution composition containing Nrg1; And (2) it provides a salivary gland organoid differentiation method comprising the step of further culturing by adding DAPT, a Notch inhibitor, to the salivary gland organoid culture medium.

In addition, the present invention comprises the steps of (1) isolating salivary gland stem cells from salivary gland tissue; (2) contacting the isolated salivary gland stem cells with an extracellular matrix (ECM); (3) culturing the salivary gland stem cells in contact with the ECM in a culture solution composition for growth or maintenance of the salivary gland stem cell-derived salivary gland organoids, and growing them into salivary gland organoids; (4) culturing the grown salivary gland organoids in a culture solution containing Nrg1; and (5) adding DAPT, a Notch inhibitor, to the salivary gland organoid culture medium of step (4) and culturing the salivary gland stem cell-derived salivary gland organoids.

In the present invention, the method for culturing salivary gland stem cells includes culturing the salivary gland stem cells by contacting them with an extracellular matrix (ECM). Any suitable extracellular matrix may be used. The isolated salivary gland stem cells are preferably cultured in an environment that partially mimics the cellular microenvironment in which the salivary gland stem cells naturally exist. This cell niche can be mimicked by culturing the stem cells in the presence of a biomaterial such as an extracellular matrix that provides key regulatory signals that control stem cell fate. The cell niche is determined in part by the extracellular matrix produced by stem cells and surrounding cells and cells in the niche. In a preferred method of the present invention, the salivary gland stem cells are cultured in contact with an extracellular matrix. "Contact" means physical or mechanical or chemical contact, meaning that force needs to be used to separate the resulting population of salivary gland organoids or salivary gland stem cells from the extracellular matrix. Preferably, the salivary gland stem cells are embedded in the ECM. The culture medium of the present invention can be diffused into the extracellular matrix.

Definitions of terms used in the present invention are as follows.

“Saliva” is a mixed solution secreted from the parotid, submandibular, sublingual and mucous glands in the oral mucosa. Saliva is a key component of the human body, produced in the salivary glands and discharged into the oral cavity. Saliva is an essential component of the human body and contains bioactive proteins, digestive enzymes, mucus, immunoglobulins, and various salts.

Saliva plays a very important role in maintaining the homeostasis of the human body as well as oral health. For example, the main components of saliva, mucin and immunoglobulin, play a primary defense role against external infection, and protect the oral mucosa and teeth through lubrication and moisture maintenance of the oral cavity and teeth, and neutralizing pH. do. In addition, saliva contains digestive enzymes such as amylase, such as ptyalin, which promotes digestion by decomposing starch to maltose units. In addition, the body's water metabolism and body temperature control can be achieved by the secretion of saliva, and toxic substances (I, Hg, Pb, etc.) are excreted.

The “salivary gland” is an organ that produces and secretes saliva. ) and minor salivary glands distributed in various parts of the oral mucosa, such as mucous glands existing in the mucous membrane of oral cavity, such as mucous glands.

In the present invention, "organoid" means that it can be made in the form of an artificial organ by culturing it in a 3D form using cells derived from tissues or embryonic stem cells. Organoid is a suffix that has the same meaning as ‘organ’ of an organ, and has the word ‘organ-like’. Organoids have a more well-arranged cell and cell function through a three-dimensional culture method, and have a functional organ-like form and function. Organoids are receiving attention along with research on optimization of growth and differentiation factors that can differentiate into various tissues, with stem cell research and 3D cell culture being developed.

Hereinafter, the present invention will be described in more detail through the following examples. However, the present invention is not limited by these examples.

< Experimental example >

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Reagents

The following reagents were used in the present invention.

Bovine Serum Albumin (BSA), 1×HBSS solution (# 14025092, Gibco), Collagenase, type II (# 4176, Worthington), Hyaluronidase (# H3506, Sigma), Y-27632 dihydrochloride (# 1254, Tocris) ), 48 well plate for suspension culture (# 677102, Greiner Bio-one), Reduced Growth Factor Basement Membrane Matrix, Type 2 (BME 2) (# 3533-001-02, Trevigen), Advanced DMEM/F12 (# 12634010, Gibco), Primocin (#anti-pm, Invivogen), HEPES (#15630-080, Gibco), GlutaMAX (#35050-061, Gibco), B27 (#17504-044, Gibco), B27, minus vitamin A (# 12587-010, Gibco), NAC (N-acetyl-cysteine; #A9165, Sigma), Nicotinamide (#N0636, Sigma), A83-01 (#2939, Tocris), Noggin (#6057-NG, R&D), Nrg1 (Neuregulin-1; #100-03, Peprotech), FGF2 (#100-18B, Peprotech), FGF7 (#100-19, Peprotech), FGF10 (#100-26, Peprotech), RSPO1 (R-spondin 1, #120-38, Peprotech), Afamin/Wnt3a (#J-ORMW301R, MBL), TrypLE express (#12605-010, Life technologies), CellBanker 1 (Zenoaq).

2. Derived from human salivary gland stem cells organoid produce

(1) After collecting a human salivary gland sample, put it in a preservative solution (1× HBSS with 1% BSA) and store it in a refrigerator at 4℃ until the next day or the next day.

(2) Collagenase type II 0.5 mg/ml, hyaluronidase 0.5 mg/ml, Y-27632 10 mM is added to the preservation solution with the salivary gland sample to obtain a digestion solution After making a total of 20 ml, it is stored in a stirred incubator at 37°C at 200 rpm for 30 minutes.

(3) Filter the reacted salivary gland sample with a 70 μm strainer to remove undissolved tissue, and then centrifuge the filtered solution at 300 g for 5 minutes to obtain only cells.

(4) After counting the number of cells, count the number of cells to contain 1.0 × 10 ⁴ cells based on a 48-well plate, and centrifuge again under the same conditions.

(5) Take out the 48-well plate heated at 37° C. for at least 1 hour in advance, mix 20 ul of BME 2 matrix per well with the cell pellet, and then dispense into each well to make a dome shape. Thereafter, after storage at 37° C. for 20 minutes, the culture medium is put into each well of 250 ul and cultured.

(6) At this time, the composition of the culture medium is shown in Table 1.

(7) Remove the existing culture medium every 2-3 days during culture, and then replace it with a new one.

(8) Perform subculture after 2 weeks.

growth medium final concentration Advanced DMEM/F12 Primocin (500 ×) 1 × HEPES (100 ×) 1 × Glutamax (100 ×) 1 × B27 (50 ×) plus vitamin A 1 × NAC 1 mM Nicotinamide 10 mM A83-01 0.5 μM Afamin/Wnt3a One% Rspo1 100 ng/ml Noggin 100 ng/ml Nrg1 50 ng /ml FGF7 20 ng/ml FGF2 20 ng/ml Y-27632 5 μM

** Afamin/Wnt3a is added to the culture medium only for the first 3 days after culture.

** B27 plus vitamin A contains 300 nM of retinyl acetate.

3. Derived from human salivary gland stem cells organoid differentiation

(1) Replace the culture medium with the culture medium composition shown in Table 2 in the organoids cultured for 10 days after passage as described above.

(2) Change the culture medium every 2-3 days, and after 5 days, treat the culture medium with DAPT (Notch inhibitor, 10 μM) and incubate for 2 more days.

(3) The organoid sample is harvested and used for the experiment.

Differentiation culture final concentration Advanced DMEM/F12 Primocin 1 × HEPES 1 × Glutamax 1 × B27 (without vitamin A) 1 × NAC 1 mM A83-01 0.5 μM Rspo1 100 ng/ml Nrg1 50 ng /ml FGF7 20 ng/ml FGF2 20 ng/ml FGF10 50 ng/ml DAPT** 10 μM

** DAPT is added to the culture medium only for the last 2-3 days.

4. Derived from human salivary gland stem cells organoid passage

(1) After removing the culture medium, 500 ul of TrypLE Express (10 mM Y-27632 added) per 1 well of a 48-well plate is pipetted, followed by reaction in a 37°C water bath for 10 minutes.

(2) After adding 1 ml of Advanced DMEM/F12, pipetting vigorously more than 20 times, and then centrifuging at 300 g for 5 minutes.

(3) Filter the cell pellet with a 40 μm strainer to filter out unremoved organoids.

(4) The filtered cell suspension is counted and the above organoid preparation method is repeated.

< Example >

So far, well-known culture conditions for human tissue-derived organoids have been used and applied to human salivary gland organoid cultures. Among them, organoid culture was attempted using a culture medium containing IGF-1, a growth factor used in human intestinal tissue organoid culture medium. When human salivary gland organoids were planted in Matrigel and cultured in a culture medium containing IGF-1 growth factors for 2 weeks, it was confirmed on a micrograph that the growth of the organoids was well maintained (FIG. 1, left). However, when the type of cells constituting the organoid was confirmed by H&E staining, a clear cell type in which the cytoplasm was not stained inside the organoid was observed (FIG. 1, right).

This is one of the representative phenotypes of squamous cell metaplasia, and since it is not observed in normal salivary gland tissue, it was interpreted that the characteristics of organoids representing the properties of the tissue were not well reflected. Consistent with this, when the expression of various marker genes found in salivary gland tissue was confirmed in organoids by immunostaining technique, AQP5, an acinar cell marker, CK7, a luminal cell marker, and myoepithelium Since ACTA2, a myoepithelial cell marker, was not well expressed and CK5 expressed in basal cells was mainly expressed, it was confirmed that organoids in which basal cells were mainly distributed were formed. In addition, the expression of SLC12A2 and CNN1 was observed from the inside, confirming that metaplasia into squamous cells occurred inside the organoid (FIG. 2). When the cells dividing by Ki67 were confirmed, it was confirmed that growth was mainly performed outside the organoid, and it was confirmed that LGR5 was expressed around these cells, confirming that the WNT pathway plays an important role in the growth of the organoid. can

In order to compensate for the problem that the properties of normal salivary gland tissue are not well implemented in organoids, various other factors used in the existing organoid culture medium were screened, and among them, Neuregulin-1 (Neuregulin-1; Nrg1) was added to the culture medium instead of EGF and IGF-1. As a result, in organoid culture conditions that showed a similar growth compared to the existing conditions (Fig. 3, left), cleft formation, an phenotypic trait related to salivary gland differentiation, was observed in some organoids (Fig. 3, right).

When the gene expression level according to retinyl acetate (RA) was comparatively analyzed under the above conditions, it was confirmed that Krt7 , a luminal cell marker, increased according to RA, but it was confirmed that Aqp5 , an acinar cell marker, was decreased. Rather, it was confirmed that the inhibition was (Fig. 4, left). In addition, when checking the growth degree of organoids according to the presence or absence of RA, it was confirmed that the growth of organoids was much enhanced when RA was added to the culture medium (Fig. 4, right). , the conditions were determined to be excluded from the differentiation culture medium.

As a result of staining with the H&E staining technique, a small lumen was observed. When examining the expression of various genes, ductal markers CK5 and CK7, acinar cell markers NKCC1, AQP5, and It was confirmed that ACTA2, a myoepithelial cell marker, was expressed ( FIG. 5 ). It was confirmed that the corresponding organoid was actively growing through Ki67 expression, and it was confirmed that the WNT pathway was activated in the organoid through LGR5 expression. However, since MIST1, a differentiation marker of acinar cells, is not expressed, it can be confirmed that the corresponding organoid is an organoid that has not progressed until final differentiation.

In order to determine the composition of the differentiation culture medium, previous studies on other organs have examined how various compositions related to organoid differentiation affect the differentiation of salivary gland organoids. As a result, it was confirmed that budding and cleft formation, one of the characteristics appearing in the salivary gland tissue, appeared in the culture medium containing the Notch inhibitor DAPT (FIG. 6). Among the tried conditions, the culture medium containing DAPT and Rspo1 showed the highest organoid yield and differentiation characteristics, so an additional experiment was performed by selecting the culture medium containing the above two as the composition of the differentiation medium.

Therefore, as described above, after differentiation of human salivary gland organoids for 1 week, when the shape and gene expression were confirmed, it was confirmed under a microscope that acinus was formed and branched, and as a result of H&E staining, the lumen (lumen) was enlarged and it was confirmed that it was composed of several types of cells (FIG. 7). In addition, when AQP5, an acinar cell marker, and ACTA2, a myoepithelial cell marker, were confirmed by IF staining, AQP5 was expressed near the lumen as observed in existing tissues, ACTA2 was confirmed to be expressed from the outside. Through this, it was confirmed that the expression of the marker after differentiation was more similar to that of the tissue than the expression in the existing growth culture conditions.

Claims (9)

A culture medium composition for growth or maintenance of salivary gland organoids derived from salivary gland stem cells comprising Neuregulin-1 (Nrg1) and vitamin A (Vitamin A) as active ingredients. A culture solution composition for inducing differentiation of salivary gland organoids derived from salivary gland stem cells comprising Nrg1 and DAPT, a Notch inhibitor, as active ingredients. [Claim 3] The culture solution composition according to claim 1 or 2, wherein the culture solution composition comprises Nrg1 at a final concentration of 7.5 to 150 ng/mL. According to claim 1, wherein the salivary gland stem cell-derived salivary gland organoid growth or maintenance culture solution composition, characterized in that consisting of the components shown in Table 1. The culture solution composition according to claim 2, wherein the salivary gland stem cell-derived salivary gland organoid differentiation induction culture solution composition consists of the components shown in Table 2. (1) isolating the salivary gland stem cells from the salivary gland tissue;
(2) contacting the isolated salivary gland stem cells with an extracellular matrix (ECM); and
(3) A salivary gland stem cell-derived salivary gland organoid culture method comprising the step of culturing the salivary gland stem cells in contact with the ECM in the culture solution composition according to claim 1, and growing them into salivary gland organoids. 7. The method of claim 6, wherein the cultured salivary gland organoids are ductal markers CK5 and CK7, acinar cell markers SLC12A2 and AQP5, myoepithelial cell markers ACTA2, and Ki67 and LGR5 Salivary gland stem cell-derived salivary gland organoid culture method, characterized in that the expression. (1) culturing salivary gland organoids in a culture solution composition containing Nrg1; and
(2) A salivary gland organoid differentiation method comprising the step of further culturing by adding DAPT, a Notch inhibitor, to the salivary gland organoid culture medium. (1) isolating the salivary gland stem cells from the salivary gland tissue;
(2) contacting the isolated salivary gland stem cells with an extracellular matrix (ECM);
(3) culturing the salivary gland stem cells in contact with the ECM in the culture solution composition according to claim 1, and growing them into salivary gland organoids;
(4) culturing the grown salivary gland organoids in a culture solution containing Nrg1; and
(5) A method for growth and differentiation of salivary gland stem cell-derived salivary gland organoids comprising the step of further culturing by adding DAPT, a Notch inhibitor, to the salivary gland organoid culture medium of step (4).

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