Method for culturing tonsil-derived mesenchymal stem cells by using n-acetylcysteine (NAC)

Abstract

The present invention relates to a culture medium containing N-acetylcysteine (NAC) for culturing tonsil-derived mesenchymal stem cells, and a method for culturing tonsil-derived mesenchymal stem cells by using same. According to the culture medium and the culture method of the present invention, an antioxidant effect can be enhanced by effectively reducing reactive oxygen species (ROS) when tonsil-derived mesenchymal stem cells are cultured.

Description

Culturing method of tonsil-derived mesenchymal stem cells using N-acetylcysteine ​​(NAC)

[0001] The present invention relates to a tonsil-derived mesenchymal stem cell culture medium containing N-acetylcysteine ​​(NAC) and a method for culturing tonsil-derived mesenchymal stem cells using the same, and more particularly, to a method for culturing tonsil-derived mesenchymal stem cells capable of improving the antioxidant capacity of tonsil-derived mesenchymal stem cells using a culture medium containing N-acetylcysteine.

[0002] Stem cells are cells that can differentiate into various cell types that make up biological tissues. They are a general term for undifferentiated cells obtained from various tissues of embryos, fetuses, and adults. Stem cells are characterized by their ability to differentiate into specific cells in response to differentiation stimuli (environment), the ability to self-renew (produce cells identical to themselves) through cell division, and the flexibility (plasticity) to differentiate into different cell types depending on differentiation stimuli.

[0003] Stem cells can be classified into totipotent stem cells, pluripotent stem cells, and multipotent stem cells according to their differentiation ability.

[0004] A pluripotent stem cell is a cell that has the pluripotent property that can develop into a complete organism. Cells up to the 8-cell stage after fertilization of an egg and sperm have this property, and if these cells are separated and transplanted into the uterus, they can develop into a complete organism. Pluripotent stem cells are cells that can develop into various cells and tissues derived from the ectoderm, mesoderm, and endoderm. They are derived from the inner cell mass located inside the blastocyst that appears 4-5 days after fertilization. These are called embryonic stem cells and can differentiate into various other tissue cells, but cannot form new living organisms. Multipotent stem cells are stem cells that can differentiate only into cells specific to the tissues and organs in which they are contained. They are involved in the growth and development of each tissue and organ in the fetal, neonatal, and adult stages, as well as in maintaining homeostasis in adult tissues and inducing regeneration when tissues are damaged. Tissue-specific multipotent cells are collectively called mesenchymal stem cells.

[0005] Meanwhile, mesenchymal stem cells (MSCs) are non-hematopoietic, multipotent fibroblast-like cells that exhibit the ability to differentiate into mesodermal lineages, including osteocytes, adipocytes, and chondrocytes. MSCs can be readily isolated from various tissues, including bone marrow, adipose tissue, amniotic fluid, and Wharton's jelly (substantia gelatinea funiculi umbilicalis), and possess immunomodulatory properties, enabling successful allogeneic transplantation. Furthermore, MSCs can be derived from various tissue types. However, it is known that MSCs from various sources can exhibit heterogeneity due to differences in culturing methods under in vitro proliferation culture conditions, differences in stem cell donor sources, or differences in stem cell extraction sites.

[0006] However, some mesenchymal stem cells are difficult to utilize due to significant limitations in obtaining them. For example, mesenchymal stem cells derived from umbilical cord blood and adipose tissue must be obtained through invasive methods. The most non-invasive method for obtaining mesenchymal stem cells is bone marrow extraction, but bone marrow extraction requires anesthesia and causes pain, limiting its use. As an alternative, methods for obtaining cells from peripheral blood are needed to isolate patient-specific stem cells. However, the number of mesenchymal stem cells that can be isolated from adults using peripheral blood alone is too small, the isolation methods are not economical, and even if isolated, they often do not proliferate smoothly to a level usable for cell therapy. Therefore, a more practical alternative is needed. Furthermore, adult stem cells obtained from elderly patients have significantly lower proliferative capacity than those obtained from younger patients, and their ability to secrete various factors and migrate to lesions is also reduced. Therefore, there is a need to obtain cells from tissues that can be naturally isolated or discarded from younger patients. Furthermore, these cells must be readily available in sufficient quantities for experiments and maintain differentiation potential during subculture.

[0007] Meanwhile, tonsil-derived mesenchymal stem cells (TMSCs) are undifferentiated stem cells that have the ability to self-replicate and differentiate into two or more new cells, derived from the tonsils, which are located in the back of the throat and nose and primarily defend the body from invading substances such as bacteria while also functioning as lymphoepithelial immune tissue.

[0008] Tonsil-derived mesenchymal stem cells have the advantage of being able to secure stem cells without additional pain for the donor, unlike mesenchymal stem cells derived from other tissues, because they are extracted from tissue discarded as medical waste after tonsillectomy. In addition, a large quantity can be obtained compared to stem cells that can be obtained by extracting them from bone marrow for 2 hours, and the cells are known to have excellent proliferation ability, resulting in a very high stem cell productivity.

[0009] Tonsil-derived mesenchymal stem cells (TMSCs) are emerging as good MSC candidates for clinical cell therapy because they are superior to other MSCs in terms of proliferation rate, differentiation potency, and immunomodulatory capacity, and successfully maintain MSC-specific properties during freezing and thawing and up to passage 15 during long-term in vitro cell culture.

[0010] Mesenchymal stem cells can differentiate into osteocytes, chondrocytes, and adipocytes, and have anti-inflammatory, immunomodulatory, anti-fibrotic, and damaged skin regenerative properties. However, while most mesenchymal stem cells can only differentiate within a limited range, tonsil-derived mesenchymal stem cells have been reported to be capable of differentiating not only into mesodermal cells such as muscle, but also into endodermal cells such as parathyroid hormone-secreting cells and insulin-secreting cells, and ectodermal cells such as motor neurons and Schwann cells. In addition, tonsil-derived mesenchymal stem cells have been reported to have therapeutic efficacy for skin inflammatory diseases and chronic inflammatory bowel diseases.

[0011] Meanwhile, reactive oxygen species (ROS) are highly reactive chemicals formed from molecular oxygen (O2). In biology, ROS are byproducts of normal oxygen metabolism. Reduction of molecular oxygen produces superoxide, and dismutation of superoxide produces hydrogen peroxide (H2O2). Hydrogen peroxide can be partially reduced to form hydroxide ions and hydroxyl radicals (·OH), or completely reduced to water.

[0012] ROS are essential for cellular function and exist at low, quiescent levels in normal cells. However, ROS levels can increase dramatically during environmental stress. Increased ROS can cause irreversible damage to DNA by oxidizing and modifying certain cellular components and interfering with their normal functions. This can lead to serious damage to cellular structures. This process, called oxidative stress, causes oxidative DNA damage, which in turn upregulates p53, leading to intrinsic mitochondrial apoptosis.

[0013] N-acetylcysteine ​​(NAC) is a precursor to cysteine, a sulfur-containing amino acid used by cells to synthesize glutathione. It is known as an antioxidant molecule that acts by increasing the synthesis of intracellular glutathione (GSH). GSH's reducing effect is attributed to its ability to directly scavenge reactive oxygen species and to recycle previously used antioxidants.

[0014] In this regard, Korean Patent Registration No. 10-1211913 discloses a medium composition for culturing amniotic membrane-derived mesenchymal stem cells containing N-acetyl-L-cysteine ​​and a method for culturing amniotic membrane-derived mesenchymal stem cells using the same, Korean Patent Publication No. 10-2016-0079390 discloses a medium composition for culturing stem cells containing N-acetyl-L-cysteine, and PCT Publication No. 2017 / 032811 discloses that a culture medium for embryos, gametes, or stem cells may additionally contain acetyl-cysteine.

[0015] Although the use of N-acetylcysteine ​​in stem cell culture is known, there has not yet been a specific example of applying N-acetylcysteine ​​in the culture of tonsil-derived mesenchymal stem cells. In order to obtain such mesenchymal stem cells in large quantities while maintaining their stem cell performance, the culture method is important, and the present inventors have confirmed that when N-acetylcysteine ​​is treated at a specific concentration, the antioxidant effect is excellent in the culture of tonsil-derived mesenchymal stem cells, and this has led to the completion of the present invention.

[0016] (Patent Document 0001) Republic of Korea Patent No. 10-1211913

[0017] (Patent Document 0002) Republic of Korea Publication Patent No. 10-2016-0079390

[0018] (Patent Document 0003) PCT Publication No. 2017 / 032811

[0019] The present invention provides a tonsil-derived mesenchymal stem cell culture solution containing N-acetylcysteine ​​and a method for culturing tonsil-derived mesenchymal stem cells using the same.

[0020] The present invention provides a culture solution of tonsil-derived mesenchymal stem cells containing N-acetylcysteine ​​(NAC) as an effective ingredient.

[0021] In addition, the present invention provides a method for culturing tonsil-derived mesenchymal stem cells, comprising a step of culturing tonsil-derived mesenchymal stem cells in a stem cell culture composition containing N-acetylcysteine ​​(NAC) as an effective ingredient.

[0022] According to the culture solution and culture method of the present invention, there is an effect that antioxidant efficacy can be increased by effectively reducing reactive oxygen compounds (ROS) during the culture of tonsil-derived mesenchymal stem cells.

[0023] Figure 1 shows the results of measuring cell viability by treating tonsil-derived human mesenchymal stem cells with NAC at concentrations of 2, 4, 6, 8, 10, 15, and 100 mM for 24 hours.

[0024] Figure 2 shows the results of measuring absorbance in one embodiment of the present invention (Experimental Example 1).

[0025] Figure 3 shows the results of measuring cell viability by pretreating tonsil-derived human mesenchymal stem cells with NAC in one embodiment (Experimental Example 1) of the present invention.

[0026] Figure 4 shows the results of measuring absorbance in one embodiment of the present invention (Experimental Example 2).

[0027] Figure 5 shows the results of measuring ROS by pretreating tonsil-derived human mesenchymal stem cells with NAC in one embodiment (Experimental Example 2) of the present invention.

[0028] Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the invention. These embodiments of the present invention are provided to more fully explain the invention to those of ordinary skill in the art. Therefore, the embodiments of the present invention may be modified in various ways, and the scope of the present invention is not limited to the embodiments described below.

[0029] Throughout the specification of the present invention, when a part is said to "include" a certain component, this means that it may further include other components, rather than excluding other components, unless specifically stated otherwise.

[0030] One embodiment of the present invention provides a culture solution of tonsil-derived mesenchymal stem cells containing N-acetylcysteine ​​(NAC) as an active ingredient.

[0031] In the present invention, N-acetylcysteine, also known as 2-acetamido-3-sulfanylpropanoic acid or NAC, has the following chemical formula:

[0032]

[0033]

[0034] In the present invention, the tonsil-derived stem cell refers to an undifferentiated stem cell having the ability to self-replicate and differentiate into two or more new cells, derived from the tonsil, which is a tissue located in the back of the throat and nose and primarily defends the body from substances such as bacteria invading from the outside, and at the same time functions as a lymphoepithelial immune tissue.

[0035] Tonsil-derived mesenchymal stem cells can be extracted from tonsils using methods known in the art. For example, tonsils can be extracted using methods described in Korean Patent No. 10-1508413. The tonsil-derived mesenchymal stem cell culture medium may be a culture medium, culture supernatant, a concentrate thereof, or a lyophilized product thereof obtained after culturing tonsil-derived mesenchymal stem cells and removing the cells.

[0036] In the present invention, the culture medium means a composition containing nutrients necessary to maintain cell growth and survival in vitro, and tonsil-derived mesenchymal stem cells can be conventionally cultured using a stem cell culture medium.

[0037] Tonsil-derived mesenchymal stem cell culture solution is obtained by subculturing tonsil-derived mesenchymal stem cells obtained from tonsil tissue in serum medium and then subculturing them in serum-free medium, and can be used as is or reduced by centrifugation or filtration using a filter.

[0038] The supernatant obtained after removing the cells and macromolecules can be used. In addition, the obtained supernatant can be used as is or as a concentrate obtained by concentration.

[0039] Culture media and culture conditions for culturing tonsil-derived mesenchymal stem cells are well known in the technical field to which the present invention pertains, and can be appropriately selected or modified and used by a person skilled in the art.

[0040] The above serum medium is a medium suitable for maintaining and storing the same cell type as tonsil-derived mesenchymal stem cells, and may be a DMEM medium supplemented with serum. The serum may be, but is not limited to, fetal bovine serum (FBS), and may be 1 to 10 wt% based on the total weight of the serum medium. If necessary, antibiotics, antifungal agents, and mycoplasma inhibitors may be included, and these may be 1 wt% based on the total weight of the medium. Antibiotics include antibiotics commonly used in cell culture, such as penicillin-streptomycin, antifungal agents include amphotericin-B (fungizone), and mycoplasma inhibitors include, but are not limited to, gentamicin, ciprofloxacin, tylosin, and the like.

[0041] Culturing in the above serum-free medium can be performed after removing the culture medium from the serum medium and washing the cells with a phosphate buffer solution.

[0042] The NAC may be included at a concentration of 1 to 100 mM. Specifically, it may be 1 to 90 mM, 1 to 80 mM, 1 to 70 mM, 1 to 60 mM, 1 to 50 mM, 1 to 40 mM, 1 to 30 mM, more specifically 5 to 30 mM, 5 to 25 mM, 5 to 20 mM, and even more specifically 5 to 19 mM, 5 to 18 mM, 5 to 17 mM, 5 to 16 mM, 5 to 15 mM, 6 to 15 mM, 7 to 15 mM, 8 to 15 mM, 9 to 15 mM, 9 to 14 mM, 9 to 13 mM, 9 to 12 mM, 9 to 11 mM, 10 mM, but is not limited thereto.

[0043] The above stem cell culture solution is characterized by enhanced antioxidant efficacy, and is particularly characterized by exhibiting antioxidant efficacy by reducing reactive oxygen species (ROS), but is not limited thereto.

[0044] In addition, another embodiment of the present invention provides a method for culturing tonsil-derived mesenchymal stem cells, comprising a step of culturing tonsil-derived mesenchymal stem cells in a stem cell culture composition containing N-acetylcysteine ​​(NAC) as an active ingredient.

[0045] In the case of the above culture method, the antioxidant effect is increased, and in particular, the antioxidant effect is exhibited by reducing reactive oxygen species (ROS), but is not limited thereto.

[0046] Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the following examples and experimental examples are intended to aid understanding of the present invention and are not intended to limit the scope of the present invention.

[0047] <Example 1> Preparation of NAC

[0048] To pretreat tonsil-derived human MSCs, 163.19 mg of N-acetyl-L-cysteine ​​powder (molecular weight 163.19 g / mol) was added to 10 ml of PBS, mixed, and then NaOH was added to adjust the pH to 7.4.

[0049] <Experimental Example 1> Confirmation of cell viability

[0050] 1-1. Determination of NAC pretreatment concentration

[0051] To determine the NAC pretreatment concentration for tonsil-derived human MSCs, tonsil-derived human MSCs were pretreated with different concentrations of 2, 4, 6, 8, 10, 15, and 100 mM of NAC prepared in Example 1 for 24 hours, and then cell viability was confirmed.

[0052] As a result, as shown in Fig. 1, NAC was found to be non-cytotoxic at a concentration of 10 mM after 24 hours of pretreatment.

[0053] Therefore, the concentration of NAC to be used in subsequent experiments was determined to be 10 mM.

[0054] 1-2. Confirmation of cell viability by NAC treatment in tonsil-derived human MSCs.

[0055] To determine cell viability by NAC treatment, 1x10 of tonsil-derived human MSCs (hMSC-T, ps.6) were added to the medium. 3 After inoculating at a concentration of cells / well, the following day, NAC was treated at concentrations of 10 mM and 20 mM for 24 hours. After washing with PBS and replacing the medium, PYBR was treated at concentrations of 5, 10, and 20 μg / ml. After 24 hours, the cells were washed three times with PBS, transferred to fresh medium (100 μl + 10 μl (WST solution), and incubated at 37°C for 2 hours under light protection. Afterwards, the absorbance was measured at 450 nm to confirm cell viability (Fig. 2). The positive control group was not treated with NAC.

[0056] As a result, as shown in Fig. 3, when PYBR was treated, cell viability was found to decrease by 20-30%, and when NAC was pretreated, cell viability was found to be significantly restored.

[0057] Therefore, we confirmed that cell viability could be improved when NAC was treated in tonsil-derived human MSCs.

[0058] <Experimental Example 2> Confirmation of antioxidant activity (ROS assay)

[0059] To confirm the antioxidant activity by NAC treatment, 1x10 of tonsil-derived human MSCs (hMSC-T, ps.6) were added to the medium. 4 After inoculation at a concentration of cells / well, NAC was treated at concentrations of 10 mM and 20 mM for 24 hours the following day. The cells were then washed with PBS, the medium was replaced, and PYBR was treated at concentrations of 5, 10, and 20 μg / ml. After 24 hours, the cells were washed three times with PBS and incubated in 100 μl of ROS working solution at 37°C for 30 minutes, protected from light. The absorbance was then measured at 488 / 525 nm (Fig. 4). The positive control group was not treated with NAC.

[0060] As a result, as shown in Fig. 5, ROS was found to increase when PYRB was treated, and ROS was found to significantly decrease when NAC was pretreated.

[0061] Therefore, it was confirmed that when NAC was treated on tonsil-derived human MSCs, it had an excellent antioxidant effect by reducing ROS.

[0062] According to the culture solution and culture method of the present invention, antioxidant efficacy can be increased by effectively reducing reactive oxygen compounds (ROS) during the culture of tonsil-derived mesenchymal stem cells.

Claims

1. A culture medium of tonsil-derived mesenchymal stem cells containing N-acetylcysteine ​​(NAC) as an active ingredient.

2. In paragraph 1, A culture solution of tonsil-derived mesenchymal stem cells, characterized in that the above NAC is contained at a concentration of 1 to 100 mM.

3. In paragraph 1, The above stem cell culture solution is a tonsil-derived mesenchymal stem cell culture solution characterized by enhanced antioxidant efficacy.

4. A method for culturing tonsil-derived mesenchymal stem cells, comprising a step of culturing tonsil-derived mesenchymal stem cells in a stem cell culture composition containing N-acetylcysteine ​​(NAC) as an active ingredient.

5. In paragraph 4, A method for culturing tonsil-derived mesenchymal stem cells, characterized in that the antioxidant efficacy is increased when the above culturing method is used.

6. In paragraph 4, A method for culturing tonsil-derived mesenchymal stem cells, wherein N-acetylcysteine ​​(NAC) is contained at a concentration of 1 to 100 mM.

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