WO2023113395A1 - Method for optimally culturing organoid derived from basal cells or luminal cells isolated from human salivary gland tissue - Google Patents

Abstract

The present invention relates to a method for optimally culturing an organoid derived from basal cells or luminal cells isolated from human salivary gland tissue. The present invention enables separating and culturing an organoid of basal and luminal cells of a salivary gland, and can be applied to identifying functions of stem cells derived from a salivary gland, developing progenitors that are more conducive to regeneration and exploring the mechanisms thereof, classifying a carcinoma according to the type of cells derived from salivary gland cancer, and establishing a platform for developing a therapeutic agent optimized for carcinoma.

Description

Optimal method for culturing organoids derived from basal cells or luminal cells isolated from human salivary gland tissue

The present invention relates to an optimal method for culturing organoids derived from basal cells or luminal cells isolated from human salivary gland tissue.

The salivary gland is an exocrine gland that secretes digestive juices into the mouth, and the main cells responsible for its function are acinar epithelial cells that produce serous or mucus and ductal epithelial cells that transport saliva to the oral cavity. and myoepithelial cells, which are considered to help secretion of saliva by contraction. Among them, ductal epithelial cells are cells that form a line constituting the lumen, and include luminal cells constituting the lumen and basal cells that surround the luminal cells from the outside ( basal cells).

Salivary gland dysfunction is mainly caused by radiation therapy, Sjogren's syndrome, aging, etc., and causes various complications such as chewing and swallowing difficulties, language disorders, loss of taste, and oral infections due to decreased salivary secretion, greatly reducing the quality of life. Treatment methods such as artificial saliva substitutes and sialagogues can provide symptomatic treatment, but are far from fundamental recovery of salivary gland function. To solve this problem, stem cell-based regenerative medicine has recently emerged as a fundamental treatment method.

Among the exocrine glands, in the prostate and mammary glands, separation conditions for basal cells and luminal cells have been previously established, and studies on the characteristics of each cell and progenitor ability are being conducted. However, until now, there has been no study on the separation of basal cells and luminal cells in the salivary gland, and few studies on the culture conditions of the isolated cell-derived organoids.

An object of the present invention is to provide an organoid culture composition derived from basal cells or luminal cells isolated from salivary gland tissue and an optimal method for culturing the organoids.

In order to achieve the above object, the present invention provides a basal cell-derived organoid culture solution composition isolated from salivary gland tissue containing a medium containing CHIR-99201, NRG1 and RSPO3 as an active ingredient.

In addition, the present invention provides an organoid culture composition derived from luminal cells isolated from salivary gland tissue containing a medium containing NRG1, RSPO3 and Forskolin as an active ingredient.

In addition, the present invention comprises the steps of (1) isolating salivary gland epithelial cells from salivary gland tissue; (2) separating basal cells from the separated salivary gland epithelial cells; and (3) culturing the isolated basal cells in the culture composition.

In addition, the present invention comprises the steps of (1) isolating salivary gland epithelial cells from salivary gland tissue; (2) separating luminal cells from the separated salivary gland epithelial cells; and (3) culturing the isolated luminal cells in the culture composition.

The present invention relates to an optimal method for culturing organoids derived from basal cells or luminal cells isolated from human salivary gland tissue. function of salivary gland-derived stem cells in the future, development of progenitors that are more helpful in regeneration and exploration of their mechanisms, classification of carcinomas according to cell types derived from salivary gland cancer, and It can be applied to the establishment of a treatment development platform optimized for carcinoma.

1 shows a method for separating human salivary gland-derived cells into basal cells and luminal cells.

Figure 2 shows the results of analysis of the expression pattern of the RSPO family in salivary gland tissue.

3 shows the culture patterns of basal cell-derived organoids for each condition.

4 shows the results of comparison of organoid growth of basal cell-derived organoids by culture conditions.

5 shows the results of comparing the growth of organoids of luminal cell-derived organoids according to culture medium conditions.

6 shows additional comparison results of organoid growth of luminal cell-derived organoids by culture conditions.

The present invention provides a basal cell-derived organoid culture composition isolated from salivary gland tissue containing CHIR-99201, NRG1 and RSPO3-containing medium as an active ingredient.

Preferably, the culture medium composition may include CHIR-99201 at a final concentration of 0.5 to 2 µM, NRG1 at 2.5 to 10 ng/mL, and RSPO3 at 0.5 to 2%, but is not limited thereto.

In addition, the present invention provides an organoid culture composition derived from luminal cells isolated from salivary gland tissue containing a medium containing NRG1, RSPO3 and Forskolin as an active ingredient.

Preferably, the culture medium composition may include NRG1 at a final concentration of 2.5 to 100 ng/mL, RSPO3 at 0.5 to 10%, and Forskolin at 2.5 to 10 μM, but is not limited thereto.

In addition, the present invention comprises the steps of (1) isolating salivary gland epithelial cells from salivary gland tissue; (2) separating basal cells from the separated salivary gland epithelial cells; and (3) culturing the isolated basal cells in the culture medium composition according to the above.

Preferably, the step of separating the basal cells may be separating CD49f ⁺ CD26 ^- cells, but is not limited thereto.

In addition, the present invention comprises the steps of (1) isolating salivary gland epithelial cells from salivary gland tissue; (2) separating luminal cells from the separated salivary gland epithelial cells; and (3) culturing the isolated luminal cells in the culture composition according to the above.

Preferably, in the step of separating the luminal cells, CD49f ⁺ CD26 ⁺ cells may be separated, but is not limited thereto.

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

“Saliva” is a mixed fluid secreted from the parotid, submandibular, sublingual, and mucous glands present in the oral mucosa. Saliva is a key component of the human body, produced by 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 homeostasis of the human body as well as oral health. For example, mucin and immunoglobulin, which are major components of saliva, play a primary defense role against external infections, and protect the oral mucosa and teeth by lubricating the oral cavity and teeth, maintaining moisture, and neutralizing pH. do. In addition, there are digestive enzymes such as amylase such as ptyalin in saliva, which are responsible for promoting digestion by decomposing starch to maltose units. In addition, the body's water metabolism and body temperature can be controlled by the secretion of saliva, and toxic substances (I, Hg, Pb, etc.) are excreted.

“Salivary gland” is an organ that produces and secretes saliva, and includes major salivary glands such as the parotid gland, the submaxillary gland, and the sublingual gland. ), and minor salivary glands distributed in various parts of the oral mucosa, such as mucous glands that exist in the mucous membrane of the oral cavity.

In the present invention, "organoid" means that it can be made into a form like 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’ in organ, and has the word ‘something similar to an organ’. Through the 3D culture method, organoids have better arrangement of cells and their functions, and have the form and function like organs with functions. Stem cell research and 3D cell culture have been developed, and organoids are attracting attention along with research on optimization of growth and differentiation factors that can be differentiated into various tissues.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<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.

Advanced DMEM/F12 (ADF12) (# 12634010, Gibco), HEPES (#15630-080, Gibco), GlutaMAX (# 35050-061, Gibco), Primocin (# ant-pm, Invivogen), B-27 Supplement (50X ), serum free (# 17504-044, Gibco), NAC (N-acetyl-cysteine; # A9165, Sigma), Nicotinamide (# N0636, Sigma), A83-01 (# 2939, Tocris), Prostaglandin E2 (# 2296 , Tocris), Wnt3a-CM (#J2-001, MBL)

Recombinant RSPO3-Fc fusion protein conditioned medium (hRSPO3-CM) (R001, U-Protein Express BV), Noggin-Fc Fusion Protein conditioned medium (hNOG-CM) (#N002, U-Protein Express BV), Nrg1 (Neuregulin- 1;# 100-03, Peprotech), FGF2 (# 100-18B, Peprotech), FGF10 (# 100-26, Peprotech), CHIR-99021 (# 4423, Tocris), EGF (# 236-EG, R&D Systems) , Y-27632 dihydrochloride (# 1254, Tocris), Forskolin (# 1099, Tocris), Zombie Violet Fixable Viability Kit (# 423114, Biolegend), Brilliant Violet (BV) 605 anti-human CD45 Antibody (# 368524, Biolegend), FITC anti-human CD31 Antibody (# 303104, Biolegend), PE/Cy7 anti-human/mouse CD49f Antibody (# 313621, Biolegend), PE anti-human CD26 Antibody (# 302705, Biolegend), UltraComp eBead Compensation Beads (# 01222242 , Invitrogen), Human TruStain FcX (Fc Receptor Blocking Solution) (# 422302, Biolegend), 1×HBSS solution (# 14025092, Gibco), EDTA solution (# E8008, Sigma), Collagenase, type II (# 4176, Worthington) , Matrigel (#356231, Corning), TrypLE express (#12605-010, Life technologies), CellBanker 1 (Zenoaq), CellTiter-Glo 3D Cell Viability Assay (#G9681, Promega), BSA (Bovine Serum Albumin; # BSA-68700-1kg, LSP), 48 well plate for suspension culture (# 677 102, Greiner Bio-one)

2. Human Salivary Gland Tissue Digestion and Storage

(1) Human salivary gland samples were put in 1× HBSS supplemented with 1% BSA and stored at 4° C. until the experiment was performed on the same day or the next day.

(2) After mincing the sample with a blade, transfer it to a new conical tube.

(3) Add 1 mL of a digestion solution prepared by adding 5 mg/mL of collagenase type II and 10 μM of Y-27632 to Advanced DMEM/F12 per 50 mg of salivary gland sample.

(4) React for 1 hour at 200 rpm in a 37 ° C shaker.

(5) After centrifugation at 500 g for 5 minutes, the Pellet was dissolved in trypLE express 2 mL and Y-27632 10 μM, and reacted at 37℃ for 10 minutes.

(6) Filter the sample with a 100 μm strainer to remove undigested tissues, add at least 2 mL of Advanced DMEM/F12 to stop the reaction, and centrifuge at 500 g for 5 minutes to obtain only cells.

(7) After counting the cells, calculate that at least 2×10 ⁵ cells enter one cryovial (1 mL), dissolve the cell pellet in CellBanker 1, Y-27632 10 μM, and dispense 1 mL each into the cryovial. Store at -80 ° C for 2-3 days, then transfer to a nitrogen tank and store for a long period of time.

3. Isolation of luminal and basal cells from human salivary gland tissue-derived cells

buffer preparation

- Sorting buffer - 1: PBS + HEPES (1×) + EDTA (2 mM) + Y-27632 (10 μM)

- Sorting buffer - 2 : Sorting buffer - 1 + BSA 0.5%

(1) Cell stock (at least 1 × 10 ⁶ total cells) is taken out of the nitrogen tank, reacted at 37 ℃ until half melted, and then centrifuged at 500 g for 5 minutes to obtain cell pellets.

(2) Add 200 μl Sorting buffer -1 and 1 μl zombie violet, mix well, cover with aluminum foil and wait 15 minutes at RT.

(3) After adding 1 mL of Sorting buffer -2, centrifuge at 500 g for 5 minutes to obtain a pellet.

(4) Add 40 μl of Sorting buffer-2 and 2 μl of Human TruStain FcX, mix well, and wait for 10 minutes at RT.

(5) Sorting buffer - After adding a solution containing 1 μl of each of the antibodies in Table 1 below to 100 μl of Sorting buffer-2, keep it in an ice-cold state and wait for 30 minutes.

antibody BV605 anti-human CD45 Antibody FITC anti-human CD31 Antibody PE-Cy7 anti-human/mouse CD49f Antibody PE anti-human CD26 Antibody

(6) After adding 1 mL of Sorting buffer -2, mix well and centrifuge at 500 g for 5 minutes to obtain a pellet.

(7) Repeat (6) one more time.

(8) Add 1mL of sorting buffer -2 to the cell pellet, mix (vortexing), and filter the solution through a filter facs tube to obtain single cells.

(9) After sorting, store isolated single cells in Advanced DMEM/F12.

The sorting method is the same as in FIG. 1 .

4. Luminal Cell and Basal Cell Derivation of organoids Production and culture maintenance

(1) The separated cells are centrifuged at 500 g for 5 minutes.

(2) Calculate the number of cells so that 1.0 × 10 ³ cells enter 1 well of a 48 well plate from the number of sorted cells.

(3) After mixing 20 μl of Matrigel per well with the cell pellet in a 48-well plate heated in a 37 ℃ incubator for more than 1 hour, dispense it into each well to form a dome shape.

(4) After storing in an incubator at 37 °C for 20 minutes, 250 μl of the culture medium is added to each well and cultured.

(5) The culture conditions tested at this time are shown in Table 2 for basal cell-derived organoids and Table 3 for luminal cell-derived organoids.

(6) After removing the existing culture medium at intervals of 2-3 days, replace it with a new culture medium.

Basal media screening One. cnr 2. cn 3. cr 4. nr 5. c 6. n 7. r ADF12 Glutamax 1× HEPES 1 × Primocin 1× B27vit A+ 1 × NAC 1 mM Nicotinamide 5M A83-01 5 µM PGE2 3 µM hNOG-CM 2 % hFGF2 5 ng/mL hFGF10 10 ng/mL Y-27632* 10 µM CHIR99021 1 µM 1 µM 1 µM 1 µM hRSPO3-CM One% One% One% One% hNRG1 5 ng/mL 5 ng/mL 5 ng/mL 5 ng/mL

Luminal media screening 1. cnr 2.cNRs 3. cNRe 4.cNRf 5.NRef 6.cNRef ADF12 Glutamax 1 × HEPES 1× Primocin 1 × B27vit A+ 1 × NAC 1 mM Nicotinamide 5M A83-01 5 µM PGE2 3 µM WNT3a-CM One % hNoggin 2 % hFGF2 5 ng/mL hFGF10 10 ng/mL Y-27632* 10 µM CHIR99021 1 µM 1 µM 1 µM 1 µM 1 µM hRSPO3 CM One% 5% 5% 5% 5% 5% hNRG1 5 ng/mL 50 ng/mL 50 ng/mL 50 ng/mL 50 ng/mL 50 ng/mL hEGF 50 ng/mL 50 ng/mL 50 ng/mL Forskolin 5 µM 5 µM 5 µM

* Y-27632 is removed after processing for 7-10 days after organoid production.

5. Analysis of human salivary gland stem cell-derived basal-luminal organoids

(1) 14 days after the start of organoid culture, cell viability is measured as follows.

(2) Move the CellTiter-Glo 3D reagent from -20 ℃ at least 2 hours before the experiment to 4 ℃.

(3) 30 minutes before the experiment, take out the CellTiter-Glo 3D reagent at room temperature, remove the old culture medium from the 48 well plate, add 100 μl of the new warmed culture medium to each well, and wait at room temperature for 30 minutes.

(4) After mixing the melted CellTiter-Glo 3D reagent well, add 100 μl to each well.

(5) After shaking at 150 rpm for 5 minutes, stand by at room temperature for 25 minutes.

(6) Transfer the mixed solution to a 96 well plate and analyze after measuring luminescence.

<Example>

FACS (fluorescence-activated cell sorting) was performed on basal cells and luminal cells from human salivary gland tissue samples, and the following strategies were tried for organoid culture and medium optimization, respectively.

1) Separate basal cells into CD49f ⁺ CD26 ^- and luminal cells into CD49f ⁺ CD26 ⁺ according to known isolation conditions from prostate and mammary glands.

2) Make organoid culture media based on Advanced DMEM/F12 media to screen culture conditions for organoid culture.

3) In order to find the optimal culture conditions, screening is performed with culture media under various conditions. After sorting with the above three strategies, organoids were formed using Matrigel from basal single cells and luminal single cells, and after screening for 2 weeks in each condition, the optimal conditions were selected.

1. Isolation of basal and luminal cells through fluorescence-activated cell sorting

In human salivary gland samples, basal cells and luminal cells were isolated from prostate and mammary glands by FACS under well-known conditions (FIG. 1). As a result, it can be confirmed that luminal cells, which are the CD49f ⁺ CD26 ⁺ population, and basal cells, which are the CD49f ⁺ CD26 ^- population, are well differentiated within the epithelial cells.

2. Basal organoid culture screening

In the basal organoid, by adding CHIR-99201, NRG1, and RSPO3, respectively, it was confirmed whether organoid culture and growth from single cells were maintained under each condition. CHIR-99021 is a GSK3 inhibitor and activates Wnt/beta-catenin signaling. NRG1 is a growth factor that induces hetero-dimerization of ErbB2 and ErbB3 and is a member of the EGF family. RSPO3 is involved in Wnt pathway activation, and RSPO3 expression is the highest among RSPO1, 2, 3, and 4 in salivary glands (FIG. 2). Seven conditions were created with the above three materials and applied to the human salivary gland organoid-derived basal organoid culture medium to confirm the culture and growth of organoids.

One 2 3 4 5 6 7 cnr cn cr nr c n r

* c; chir-99021 (1 μM), n; nrg1 (5 ng/mL), r; rspo3 (1%)

After culturing in 48 wells for 2 weeks, brightfield images were obtained through a microscope. As shown in Figure 3, it was confirmed that organoid formation and maintenance were relatively well under condition 1, and to quantitatively analyze this, CellTiter-Glo 3D reagent was used to determine how many living cells were present in the basal organoid under each condition. It was confirmed whether there were many (Fig. 4). As a result, consistent with the organoid photographic images, it was confirmed that the cell viability was significantly higher in condition 1 compared to other conditions. Therefore, it was determined that condition No. 1 containing CHIR-99021, NRG1, and RSPO3 was most appropriate for the basal organoid culture medium.

3. Luminal Organoid Culture Screening

Next, optimization of the luminal organoid culture medium was performed based on the basal organoid medium. Through previous studies, it was confirmed that the optimized basal organoid medium was not suitable for maintaining the growth of luminal organoids. Therefore, it was confirmed that other factors are necessary, and the following experiments were conducted to verify this. First, WNT3a-CM used in small intestine and liver organoids, which basically consist only of luminal cells, was added to the basal organoid culture medium. After that, EGF growth factors belonging to the same family as NRG1 were tested after increasing the concentrations of NRG1 and RSPO3, which are essential for the growth of basal organoids. In addition, Forskolin for amplifying intracellular calcium signals was screened. Finally, the following six conditions were prepared and applied to human salivary gland organoid-derived luminal organoid cultures to confirm the culture and growth of organoids.

One 2 3 4 5 6 cnr cNR cNRe cNRf NRef cNRef

c; chir-99021 (1 μM), n; nrg1 (5 ng/mL), r; RSPO3 (1%), N; nrg1 (50 ng/mL), R; RSPO3 (5%), e; EGF (50 ng/mL), f; forskolin (5 μM)

The experimental results are shown in FIG. 5 . When statistical processing was performed based on condition 5, which showed the best growth, condition 5 showed a significant increase compared to all conditions except condition 4. The increase in the amount of Nrg1 or RSPO3 and the addition of EGF alone did not have a significant effect on the growth of luminal organoids (compared to conditions 1 to 3), but when Forskolin, which increases intracellular calcium signal, was added to the culture medium, a significant increase in growth was observed (when conditions 4 and 5 were compared with other conditions), and in the condition where chir-99021, EGF, and Forskolin were all contained (condition 6), growth was significantly reduced when compared to conditions 4 and 5. It was confirmed that the culture medium in which all factors were added was not the optimal culture medium. As a result, it was confirmed that condition No. 5 (NRef), which showed the most significant difference when compared with other conditions, was a culture medium composition optimized for growth of luminal cell-derived organoids among screening conditions.

Additional experiments were performed with the following strategies to further optimize the culture conditions for luminal cell-derived organoids. Based on condition 5, which had the best survivability, the following changes were made. 1) Based on human salivary gland tissue single cell RNA-sequencing data, RSPO3 and the receptor LGR4 are expressed at the same level in basal cells and luminal cells, so it was judged appropriate to treat RSPO3 at the same concentration as that of basal cells. concentration was lowered. Although high concentration treatment does not significantly affect the results, economic aspects were considered. On the other hand, since NRG1 is less expressed in luminal cells than in basal cells, conditions of high concentration treatment were maintained. And, in order to clearly determine whether EGF helps luminal cell survival, Nrf was set as a condition excluding EGF in condition 5. In addition, the concentration of FGF (fibroblast growth factor), which boosts the ability of stem cells as a growth factor, was increased. A83-01 is an ALK4,5,7 inhibitor, and when treated at a high concentration, it has the potential to inhibit cell growth, so the concentration was lowered. SB202190 is a p38 inhibitor that prevents downregulation of EGF and maintains EGF signal. The second screening was performed by making 5 conditions with the above materials.

One 2 3 4 5 Nrf Nref Nref + high Fgf NRef + low A Nref + SB202190

N; Nrg1 (50 ng/mL), r; RSPO3 (1%), e; EGF (50 ng/mL), f; forskolin (5 μM), high Fgf; FGF2 (10 ng/mL), FGF10 (100 ng/mL), low A;A83-01 (0.5 μM), SB202190 (0.5 μM)

The experimental results are shown in FIG. 6 . When statistically processed based on condition 1, all conditions showed a significant increase. It was confirmed that the condition in which EGF was added was not conducive to the growth of luminal organoids (comparison of the first and second conditions). Looking at the results of screening other substances based on condition No. 2 (comparison of conditions 2 to 5), there was no significant difference between each condition. As a result, it was confirmed that the various conditions additionally tried (high concentration of FGF, low concentration of A83-01, SB202190) did not significantly increase the growth of luminal cell-derived organoids when actually added to the culture medium, but rather small amounts of growth factors. was also able to induce optimal luminal cell organoid growth. In conclusion, when the NRef condition, which showed the most significant growth in the first culture screening, was additionally optimized, condition 1, Nrf, excluding EGF, was determined to be the optimal culture composition for the growth of luminal cell-derived organoids.

Having described specific parts of the present invention in detail above, it will be clear to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

An organoid culture composition derived from basal cells isolated from salivary gland tissue containing a medium containing CHIR-99201, NRG1 and RSPO3 as an active ingredient. The culture medium composition according to claim 1, wherein the culture medium composition comprises CHIR-99201 at a final concentration of 0.5 to 2 μM, NRG1 at 2.5 to 10 ng/mL, and RSPO3 at 0.5 to 2%. An organoid culture composition derived from luminal cells isolated from salivary gland tissue containing a medium containing NRG1, RSPO3 and Forskolin as an active ingredient. [Claim 4] The culture medium composition according to claim 3, wherein the culture medium composition comprises NRG1 at a final concentration of 2.5 to 100 ng/mL, RSPO3 at 0.5 to 10%, and Forskolin at 2.5 to 10 µM. (1) isolating salivary gland epithelial cells from salivary gland tissue; (2) separating basal cells from the separated salivary gland epithelial cells; and (3) A method for culturing organoids derived from basal cells isolated from salivary gland tissue, comprising culturing the isolated basal cells in the culture composition according to claim 1 or 2. The organoid culture method according to claim 5, wherein the isolating the basal cells is separating CD49f ⁺ CD26 ^- cells. (1) isolating salivary gland epithelial cells from salivary gland tissue; (2) separating luminal cells from the separated salivary gland epithelial cells; and (3) A method for culturing organoids derived from luminal cells isolated from salivary gland tissue, comprising culturing the isolated luminal cells in the culture composition according to claim 1 or 2. The organoid culture method according to claim 7, wherein the separating the luminal cells is separating the CD49f ⁺ CD26 ⁺ cells.

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