Method for preparing exosomes isolated from wharton's jelly-derived mesenchymal stem cells, and cosmetic material composition comprising exosomes prepared using same

Abstract

The present application provides: a method for preparing exosomes isolated from Wharton jelly-derived mesenchymal stem cells; and a cosmetic material composition comprising exosomes prepared using same. More specifically, the present application provides a method for preparing exosomes whereby exosomes derived from a culture medium composition of Wharton's jelly-derived mesenchymal stem cells can be economically prepared by being isolated with high yield and high purity, and a cosmetic material composition comprising exosomes prepared using same.

Description

Method for preparing exosomes isolated from Wharton's jelly tissue-derived mesenchymal stem cells and cosmetic composition comprising exosomes prepared using the same

[0001] The present invention relates to a method for producing exosomes isolated from mesenchymal stem cells derived from Wharton's jelly tissue and a cosmetic composition comprising exosomes produced using the same.

[0002] Exosomes are a type of vesicle composed of a lipid bilayer that is released by cells to the outside. Possessing capabilities similar to those of stem cells, they currently act as skin boosters that effectively aid damaged skin tissue. Specifically, because they contain various substances such as proteins, lipids, and nucleic acids similar to those found inside their parent cells, they can play a crucial role in intercellular information transmission.

[0003] In particular, stem cell-derived exosomes retain the therapeutic effects of stem cells, but unlike cells, they lack the ability to self-replicate, which reduces the risk of tumor formation, and they are safe because they do not induce an immune response. For these reasons, stem cell exosomes are attracting attention as delivery vehicles for useful substances, such as next-generation drugs.

[0004] However, there are still limitations to exosome separation and purification technology with current technology. Specifically, ultracentrifugation is currently the most widely used method for manufacturing cosmetic compositions containing stem cell exosomes for economic reasons. However, using ultracentrifugation has the disadvantage of a high possibility of contamination and incorporation of substances other than exosomes, and a low yield.

[0005] In addition, if complex manufacturing processes such as chromatography are used, the yield of exosomes is low, and separation requires a lot of time and cost. Furthermore, the separation process may cause damage to the manufactured exosomes or lead to contamination. Therefore, if exosomes separated from this are used in cosmetic compositions, there is a fatal disadvantage that it may cause safety issues for the user.

[0006] As background technology of the present invention, Korean published patent No. 2022-0033182 describes a method for processing exosomes and a skin whitening cosmetic composition using the same.

[0007] The purpose of the present invention is to provide a manufacturing method capable of isolating exosomes isolated from Wharton's jelly tissue-derived mesenchymal stem cells with high yield and high purity through a simple process.

[0008] Another objective of the present invention is to provide a cosmetic composition that utilizes exosomes isolated from Wharton's jelly-derived mesenchymal stem cells to enhance the skin absorption rate of useful substances, ensure safety for the human body, and minimize side effects on the skin.

[0009] Another objective of the present invention is to provide a pharmaceutical composition comprising exosomes obtained using the manufacturing method of the present invention.

[0010] To achieve the above objective, the present invention provides a cosmetic composition for skin improvement comprising exosomes as an active ingredient, which are produced with high yield and high purity from mesenchymal stem cells derived from Wharton's jelly of the umbilical cord.

[0011]

[0012] According to one aspect, a method for producing exosomes derived from Wharton jelly with increased exosome yield is provided, comprising: I) a Wharton jelly tissue isolation step for isolating Wharton jelly tissue from an umbilical cord; II) a stem cell isolation step for isolating stem cells from the isolated Wharton jelly tissue; III) a subculture step for isolating the isolated stem cells, wherein the subculture step includes culturing in a serum-free culture medium at least once during the later subculture; IV) a centrifugation and pressurization filtration step for centrifuging and pressurizing the culture medium obtained from the subculture; and V) an exosome isolation step for isolating exosomes by concentrating and filtering the centrifuged and pressurized filtrate using tangential-flow filtration (TFF).

[0013] According to one embodiment, step II) may include treating with collagenase type I, hyaluronidase, or a combination thereof, and performing centrifugation to isolate stem cells.

[0014] According to one embodiment, the centrifugation of step II) may include performing it at least once for 5 to 10 minutes at 1,800 to 2,500 rpm.

[0015] According to one embodiment, step III) may include subculturing for 5 to 15 days at 37°C and CO2 conditions.

[0016] According to one embodiment, step III) may include adding 5 to 30 ng / ml of FGF to the serum-free medium and culturing for 24 to 48 hours.

[0017] According to one embodiment, the centrifugation of step IV) may include proceeding at 900 to 1200 g for 20 to 40 minutes.

[0018] According to one embodiment, the pressurized filtration of step IV) may include performing the filtration under vacuum using a pore filter of 0.3 to 0.6 μm.

[0019] According to one embodiment, the filtration of step V) may include pressurized filtration using a pore filter of 0.1 to 0.3 μm.

[0020] According to one embodiment, the Wharton jelly-derived exosomes comprise 1×10 per 1 ml unit volume. 10 to 9×10 10 It may contain a number of exosomes.

[0021]

[0022] According to another aspect, a cosmetic composition comprising exosomes produced by a method for producing exosomes derived from Wharton's jelly that increases the yield of exosomes according to the present invention is provided.

[0023] According to one embodiment, the present invention can provide a manufacturing method that can isolate exosomes isolated from Wharton jelly tissue-derived mesenchymal stem cells with high yield and high purity by a simple process.

[0024] According to one embodiment, the present invention can provide a cosmetic composition that utilizes exosomes isolated from Wharton's jelly-derived mesenchymal stem cells to enhance the skin absorption rate of useful substances, ensure safety for the human body, and minimize side effects on the skin.

[0025] According to one embodiment, the present invention may provide a pharmaceutical composition comprising exosomes obtained using the manufacturing method of the present invention.

[0026] FIG. 1 is a schematic diagram illustrating a method for preparing exosomes isolated from mesenchymal stem cells derived from Wharton jelly tissue according to one embodiment of the present invention.

[0027] FIGS. 2a to 2e show the results of the characterization (NTA) of exosomes of Example 1 (serum-free culture medium of mesenchymal stem cells derived from Wharton jelly) and Comparative Examples 1 to 4 according to one embodiment of the present invention (Fig. 2a (Example 1), Fig. 2b (Comparative Example 1), Fig. 2c (Comparative Example 2), Fig. 2d (Comparative Example 3), and Fig. 2e (Comparative Example 4)).

[0028] Figure 3 is a photograph showing the process of separating Wharton's jelly from the umbilical cord of a mammal according to one embodiment of the present invention in sequence.

[0029] The object, specific advantages, and novel features of the present disclosure will become more apparent from the following detailed description and embodiments in conjunction with the accompanying drawings.

[0030] Prior to this, terms and words used in this specification and claims shall not be interpreted in their ordinary and dictionary meanings, but must be interpreted in a meaning and concept consistent with the technical spirit of this disclosure, based on the principle that the inventor may appropriately define the concept of the terms to best describe his invention.

[0031] In this specification, where a component, such as a layer, part, or substrate, is described as being "connected" or "coupled" to another component, this may mean that it is directly "connected" or "coupled" to another component, or that one or more other components may be interposed between the two components. In contrast, where a component is described as being "directly connected" or "directly coupled" to another component, no other components may be interposed between the two components.

[0032] The terms used herein are merely for describing specific embodiments and are not intended to limit the disclosure. The singular expression includes the plural expression unless the context clearly indicates otherwise.

[0033] In this specification, terms such as “comprising” or “having” are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0034] In this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components. Furthermore, throughout the specification, "on" means located above or below the subject part, and does not necessarily mean located on the upper side with respect to the direction of gravity.

[0035] The present disclosure is capable of various modifications and may have various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present disclosure to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present disclosure. In describing the present disclosure, if it is determined that a detailed description of related prior art may obscure the essence of the present disclosure, such detailed description is omitted.

[0036] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In describing with reference to the accompanying drawings, identical or corresponding components are given the same reference numerals, and redundant descriptions thereof will be omitted.

[0037]

[0038] FIG. 1 is a schematic diagram illustrating a method for preparing exosomes isolated from mesenchymal stem cells derived from Wharton jelly tissue according to one embodiment of the present invention.

[0039] Referring to FIG. 1, a method for producing exosomes derived from Wharton jelly that increases the yield of exosomes according to the present invention comprises: I) a Wharton jelly tissue isolation step (S10) for isolating Wharton jelly tissue from an umbilical cord; II) a stem cell isolation step (S20) for isolating stem cells from the isolated Wharton jelly tissue; III) a step of subculturing the isolated stem cells, wherein the subculturing step (S30) includes culturing in a serum-free culture medium at least once during the later subculturing; IV) a centrifugation and pressurized filtration step (S40) for centrifuging and pressurizing the culture medium obtained from the subculturing step; and V) an exosome isolation step (S50) for isolating exosomes by concentrating and filtering the centrifuged and pressurized filtrate using tangential-flow filtration (TFF).

[0040] The umbilical cord (UC) is an organ responsible for supplying oxygen and nutrients from the mother to enable the fetus to grow in the placenta, as well as for removing fetal waste. The umbilical cord is generally composed of three blood vessels (two umbilical arteries and one umbilical vein), with Wharton's jelly filling the space between the umbilical cord epithelial cell layer and the blood vessels.

[0041] Here, Wharton's jelly is a gelatinous substance composed of mucopolysaccharides (hyaluronic acid) and chondroitin sulfate found in the umbilical cord or the vitreous humor of the eye, containing a large amount of mesenchymal stem cells (MSCs) with excellent differentiation ability. In particular, the mesenchymal stem cells (MSCs) contained in Wharton's jelly are called WJ-MSCs. While general mesenchymal stem cells are known to have excellent safety in that they do not mutate into cancer, they are known to have the disadvantage of having lower efficacy compared to embryonic stem cells or induced pluripotent stem cells.

[0042] On the other hand, Wharton jelly mesenchymal stem cells (WJ-MSCs) can ensure high safety as there is no possibility of the teratoma effect, which involves transformation into a teratoma, and their efficacy is about seven times higher than that of other stem cells. In addition, they possess more stem cells than umbilical cord blood, which contains superior stem cells compared to bone marrow, thus offering advantageous benefits in fields such as regenerative medicine, tissue engineering, and immune modulation.

[0043] Meanwhile, the amount of exosomes contained in stem cells varies greatly depending on the separation method, and as described above, the method of producing exosomes according to the prior art has the disadvantage of low yield due to a high possibility of contamination and incorporation of substances other than exosomes.

[0044] In addition, due to the complex manufacturing process, the yield of exosomes is low, and separation requires a lot of time and cost. For this reason, damage or contamination may occur to the manufactured exosomes during the separation process, which may cause safety issues for users when used in cosmetic compositions.

[0045] However, the method for producing exosomes isolated from mesenchymal stem cells of Wharton's jelly tissue provided by this institution has the advantage of simplifying the manufacturing process while enabling high yield and high purity.

[0046]

[0047] Step I) above is a step (S10) of separating Wharton jelly tissue from the umbilical cord. Although not limited thereto, the middle portion of the umbilical cord can be used after disinfecting it with ethanol and cutting off both ends. When transporting the umbilical cord, a frozen transport container must be used to maintain a temperature of 4 to 10°C and minimize the transport time.

[0048] Although not limited thereto, the above-mentioned severed umbilical cord may be cut to a length of about 2 cm and the arterial and venous vessels and surface tissues removed, or the arterial and venous vessels and surface tissues removed and then cut to obtain Whaton's Jelly (WJ) tissue. The blood obtained from the Whaton's Jelly tissue may be removed with physiological saline, and the physiological saline may be Dulbecco phosphate-buffered saline (DPBS) containing 1% penicillin-streptomycin, though not limited thereto. Additionally, the Whaton's Jelly tissue may be obtained by cutting the umbilical cord longitudinally and then cutting it with a scalpel, though not limited thereto.

[0049]

[0050] Step II) above is a stem cell isolation step (S20) for isolating stem cells from the separated Wharton jelly tissue. Although not limited thereto, stem cells may be differentiated by treating the chopped Wharton jelly tissue with collagenase type I, hyaluronidase, or a combination thereof.

[0051] In this specification, "stem cell" refers to a proliferating stem cell, and said stem cell can release exosomes containing genetic information, proteins, growth factors, etc. into the culture medium. In this invention, exosomes are extracted by utilizing the culture medium discarded during stem cell subculture, making it economical and environmentally friendly. The degree of differentiation promotion of said differentiated stem cells may vary depending on the amounts of collagenase type I and hyaluronidase used. The weight ratio of hyaluronidase to collagenase for effectively differentiating said Wharton jelly-derived stem cells may be 1:0.2 to 5, but is not limited thereto. The medium containing said Wharton jelly tissue may be used after being left for 2 to 5 hours after treatment with said enzymes, followed by washing 2 to 4 times with PBS (phosphate buffered saline).

[0052] The above-mentioned differentiated stem cells can be separated by centrifugation. Furthermore, the above-mentioned differentiated stem cells can also be obtained through density differential centrifugation. Although not limited thereto, the density differential centrifugation may be suitable for 1 to 5 times at 1,800 to 2,500 rpm for 5 to 10 minutes, and 2 to 3 times may be more suitable from the perspective of economic efficiency, high yield, and obtaining high purity.

[0053]

[0054] Step III) above is a subculture step (S30) for subculture the isolated Wharton jelly-derived stem cells. The subculture may be performed until 70 to 80% confluence is reached, although this is not limited thereto. The subculture step (S30) includes culturing in a serum-free culture medium at least once during the later subculture. The serum-free culture medium may be a commonly used serum-free medium. For example, it may refer to a culture medium that does not contain fetal bovine serum (FBS), phenolsulfonphthalein (PSP), or a combination thereof, but is not limited thereto.

[0055] According to one embodiment, but not limited thereto, step III) may involve subculturing for 5 to 15 days under 37°C CO2 conditions, and 7 to 15 days may be more suitable. In the above range of conditions, a larger amount of exosomes can be obtained than when cultured under other conditions.

[0056] According to one embodiment, but not limited thereto, step III) may include adding 5 to 30 ng / ml of fibroblast growth factor (FGF) to the serum-free culture medium, for example, a medium without FBS, or adding only fibroblast growth factor and culturing for 24 to 48 hours. Growth factors refer to physiologically active substances that promote cell division, proliferation, and differentiation. Examples of growth factors that facilitate cell division, proliferation, and differentiation in the absence of FBS include GDF-11 (growth and differentiation factor 11), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF). However, fibroblast growth factor (FGF) may be more suitable for promoting division, proliferation, and differentiation from Wharton's jelly-derived stem cells.

[0057]

[0058] The growth factor added to the serum-free medium stimulates the growth factor receptor signaling pathway of Wharton's jelly-derived stem cells and can increase the content of growth factors, collagen, and fibronectin within exosomes. This can affect the proliferation and motility of human fibroblasts and promote the re-secretion of other growth factors, thereby affecting the proliferation and motility of surrounding cells.

[0059]

[0060] Step IV) above is a centrifugation and pressurization filtration step (S40) in which the culture medium obtained from the subculture is centrifuged and then pressurized. Although not limited thereto, the centrifugation may be carried out at 900 to 1200 g for 20 to 40 minutes, and the centrifuged separated liquid may include, but is not limited thereto, pressurized filtration under vacuum using a pore filter of 0.3 to 0.6 μm.

[0061]

[0062] Step V) is an exosome separation step (S50) in which the centrifuged and pressurized filtered filtrate is concentrated and filtered by tangential-flow filtration (TFF) to separate exosomes. By using the tangential-flow filtration method, cells and large cell fragments can be removed and the solution concentrated. Tangential-flow filtration is an effective and economical method for removing impurities from large samples. By using tangential-flow filtration, compared to conventional filtration methods, the adsorption and clogging of exosomes on the filter can be minimized, making it useful for process scale-up. Additionally, by using tangential-flow filtration, the increase of contaminants that can clog pores can be prevented, and the centrifuged culture medium can be concentrated to about 1 / 100 of its volume.

[0063] In the present invention, a pressurized subculture obtained solution was concentrated using a tangential flow filtration method. The concentrated filtrate may further include a step of filtering using a pore filter of 0.1 to 0.3 μm, although not limited thereto. By using a filtration filter of the pore size above on the concentrated solution concentrated using the tangential flow filtration method, a final concentrated solution can be obtained, and the components most likely to cause side effects in the concentrated solution can be removed until the very end, thereby producing exosomes that minimize side effects when applied as a cosmetic composition.

[0064] The exosomes obtained above are not limited thereto, but the average value of the exosome particles may be 130 to 180 nm, the mode may be 130 to 150 nm, and the standard deviation (SD) may be 40 to 60.

[0065]

[0066] Compared to conventional exosome manufacturing methods, the purification process listed above has the advantage of producing exosomes with high yield and high purity through a simple process, while increasing safety and minimizing side effects when applied as a cosmetic composition. Specifically, by using the purification process, only active ingredients such as regeneration-related proteins, protein derivatives, and various growth factors loaded into exosomes can be included, and harmful factors can be excluded, thereby providing a composition containing high-efficiency exosomes.

[0067] The purification of the exosomes above is not limited to this, but may be performed by resuspending them in purified water to separate them, or by using a buffer composition for separating exosomes such as PEG (polyethylene glycol) and dextran.

[0068]

[0069] As described above, conventional methods for manufacturing exosomes consume excessive time and cost, have a high possibility of contamination and low stability, and have a low yield of exosomes. However, according to the present invention, by optimizing the manufacturing process (e.g., culture medium conditions, type and frequency of centrifugation, filter size and time, etc.), exosomes can be manufactured quickly and inexpensively with high yield and high purity.

[0070]

[0071] According to one embodiment, the Wharton jelly-derived exosomes comprise 1×10 per 1 ml unit volume. 10 to 9×10 10 It may contain a number of exosomes.

[0072] FIGS. 2a to 2e show the results of the NTA (Non-Tested Analysis) of exosomes of Example 1 (serum-free culture medium of mesenchymal stem cells derived from Wharton jelly) and Comparative Examples 1 to 4 according to one embodiment of the present invention (Fig. 2a (Example 1), Fig. 2b (Comparative Example 1), Fig. 2c (Comparative Example 2), Fig. 2d (Comparative Example 3), and Fig. 2e (Comparative Example 4)). Referring to FIGS. 2a to 2e, Example 1 (Fig. 2a), in which the medium was changed to serum-free at the final stage of subculture, contains the highest number concentration of exosomes within 1 ml. This represents a particle number concentration approximately 73 times higher than that of the exosome raw material of Comparative Example 4 (Fig. 2e) (see Table 2 below).

[0073]

[0074] As described above, Wharton jelly-derived stem cells possess more stem cells than umbilical cord blood and have high safety due to a very low teratoma effect. In addition, high-purity exosomes can be produced with a very high yield from the culture medium of Wharton jelly-derived stem cells by the manufacturing method of the present invention, thereby providing exosome-containing cosmetics that are economical, safe, and highly effective compared to exosome-containing cosmetics with similar efficacy.

[0075]

[0076] According to another aspect, a cosmetic composition comprising exosomes produced by a method for producing exosomes derived from Wharton's jelly that increases the yield of exosomes according to the present invention is provided.

[0077] The above cosmetic composition includes exosomes produced from a culture medium of Wharton jelly-derived stem cells produced by the exosome manufacturing method of the present invention, thereby enhancing the absorption rate of the cosmetic into the skin, minimizing side effects, and increasing safety, while also having efficacy in improving skin aging, regenerating wound sites, improving sensitive skin, improving skin pigmentation, skin whitening, wrinkle improvement, and strengthening skin elasticity.

[0078]

[0079] The cosmetic composition according to the present invention may be selected from formulations such as skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, facial cleanser, treatment, beauty liquid, beauty pack, ointment, gel, liniment, liquid, patch, spray, etc.

[0080] In addition, additives may also be added to each formulation as general additives in the field of cosmetics. Examples of general additives in the field of cosmetics, though not limited thereto, may be selected from antibiotics, binders, disintegrants, diluents, lubricants, stabilizers, preservatives, fragrances, oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, colorants, and preservatives.

[0081]

[0082] The above cosmetic composition may be used in the general usage amount in the cosmetic field. The above cosmetic composition is not limited thereto. For example, it may be used 1 to 5 times a day, and the usage amount per use may be 1 ml to 10 ml, but is not limited thereto.

[0083]

[0084] According to another aspect, the exosomes produced by the method of producing Wharton jelly-derived exosomes that increases the yield of exosomes according to the present invention may be used as a pharmaceutical composition for the prevention or alleviation of inflammatory skin diseases, comprising exosomes as an active ingredient. Although not limited thereto, the inflammatory skin diseases may be selected from atopic and / or allergic dermatitis, contact dermatitis, acne, seborrheic dermatitis, urticaria, psoriasis, eczema, lupus, hair loss, etc.

[0085] The above pharmaceutical composition may include, as an active ingredient, exosomes isolated from Wharton jelly-derived stem cells in an amount of 1 to 98 parts by weight based on the total weight of the composition, and it may be suitable to include 5 to 75 parts by weight.

[0086] The above pharmaceutical composition may include additives commonly used according to conventional methods. However, it may be selected from, for example, stabilizers, surfactants, lubricants, solubilizers, buffers, sweeteners, bases, adsorbents, binders, binders, suspending agents, curing agents, antioxidants, glossers, flavoring agents, taste agents, pigments, coating agents, wetting agents, wetting regulators, fillers, defoaming agents, cooling agents, chewing agents, antistatic agents, coloring agents, sugaring agents, isotonic agents, softeners, emulsifiers, adhesives, thickeners, foaming agents, pH adjusters, excipients, dispersants, disintegrants, waterproofing agents, preservatives, preservatives, solubilizing agents, solvents, fluidizing agents, and combinations thereof.

[0087] The above pharmaceutical composition may be applied to the skin, but is not limited thereto, and its formulation may be a topical skin formulation. The above formulation may be selected from ointments, lotions, sprays, patches, creams, powders, suspensions, patches, or gels, but is not limited thereto.

[0088]

[0089] The present invention will be explained in more detail below through examples.

[0090]

[0091] Examples

[0092] Preparation Example 1. Isolation and culture of Wharton's Jelly-derived Mesenchymal Stem Cells (WJ-MSC)

[0093] The umbilical cord (UC) was collected from a full-term infant. The umbilical cord tissue was cut into lengths of approximately 2 to 3 cm and washed several times with Dulbecco's phosphate buffered saline (DPBS) containing 1% penicillin-streptomycin. After removing the two arteries, vein, and surface tissues, the Wharton tissue was finely cut with micro-scissors and washed 3 to 4 times with DPBS (Belame Shivakumar et al., 2019). Blood was removed during this process.

[0094] The above Wharton tissue was longitudinally sliced ​​and minced with a scalpel. The small pieces of minced tissue were transferred in a thin layer to a culture dish pre-coated with MEM Alpha culture medium and left in an incubator until the tissue attached to the dish. After attaching the ex-planted tissue to the surface of the plate, Advanced Dulbecco's Modified Eagle's Medium (ADMEM) was added to the culture dish. The culture dish was kept in the incubator for 3 to 4 days, and the medium was cultured. Cells with an MSC phenotype were generated from the tissue after approximately 7 to 10 days. When the isolated cells reached 70–80% confluency, the ex-planted tissue was carefully removed, and the isolated cells were trypsinized using a 0.25% trypsin-ethylenediamine-tetraacetic acid (trypsin-EDTA) solution and centrifuged at 300g for 5 minutes. Subsequently, the cell pellet was collected for further processing. Passage 3 WJ-MSC prepared as described above was used in all experiments.

[0095]

[0096] Preparation Example 2. Treatment with serum-free medium and separation / concentration

[0097] At the final stage of cell culture, the culture medium was replaced with one that did not contain FBS and phenolsulfonphthalein (PSP), and 5 to 39 ng / ml of FGF was added to the culture medium.

[0098] After culturing the above medium for 24 to 48 hours, centrifugation was performed, and vacuum pressure filtration was performed using a 0.45 μm pore filter.

[0099] The above culture medium was concentrated using tangential-flow filtration (TFF), and the concentrate was filtered using a 0.2 μm pore filter. The tangential-flow filtration system used was the KR2i TFF System.

[0100]

[0101] Experimental Example 1. Analysis of the size, size distribution, and concentration (number) of exosomes in Example 1 and Comparative Examples 1 to 4

[0102] To analyze the size, size distribution, and number of the Wharton jelly exosomes (WJ) of the present invention generated through Preparation Examples 1 and 2 above, Nanoparticle Tracking Analysis (NTA), which is considered the technically best measurement and analysis method at present, was selected and applied based on the contents of the Korea Food and Drug Administration’s “Guidelines for Quality, Non-clinical and Clinical Evaluation of Extracellular Vesicles” (December 2018) and the MISEV2018 (Minimal Information for Studies of Extracellular Vesicles 2018) documents proposed by the International Society of Extracellular Vesicles (ISEV). The experiment was conducted following measurement procedures verified through three inter-laboratory comparison tests and proficiency tests participated in through the EU H2020 project, the ACE nano project. The comparative examples used are as follows.

[0103]

[0104]

[0105] result

[0106] For the samples of Example 1 and Comparative Examples 1 to 4 listed in Table 2 below, the mode was observed to range from 128.2 to 230.5 nm, the mean from 172.9 to 351.5 nm, and the width of the size distribution (standard deviation, SD) from 55.3 to 227.6 nm. Additionally, the measured number concentration of the samples was 2.70 x 10⁻⁶. 7 - 3.54 X 10 10The concentrations were measured within the range of particles / mL. Among the samples, it was confirmed that the sample of Example 1 consisted of the smallest particles ([WJ] average value: 172.9 nm), while the sample of Comparative Example 2 consisted of the largest particles ([PDRN] average value: 351.5 nm). Furthermore, the sample of Example 1 was identified as the most uniform and highest concentration sample ([WJ] particle standard deviation: 55.3 nm and particle number concentration: 3.54 X 10⁶). 10 particles / mL).

[0107]

[0108] Quality Check Criteria: Pass ( Number of Valid Track > 500 ), Fail ( Number of Valid Track ≤ 500

[0109]

[0110] Experimental Example 2. BrdU:ELISA local lymph node test using female CBA / J mice of Examples 1 to 3

[0111] The following test was conducted to evaluate skin sensitization by measuring lymphocyte proliferation within the lymph nodes after the application of the test substance. Examples 1 to 3 were used as the test substances, an excipient was used as the negative control, and HCA (α-Hexylcinnamaldehyde) was used as the positive control. BrdU (5-bromo-2'-deoxyuridine) was used as the labeling substance. Examples 1 to 3 were conducted at different concentrations of the test substance: 25% (Example 3), 50% (Example 2), and 100% (Example 1).

[0112] To evaluate skin sensitization, changes in cell proliferation, ear thickness, ear and lymph node weight, and skin reaction were measured.

[0113] The composition of the test group and the setting of dosages are shown in Table 3 below.

[0114]

[0115] The administration method of the above test group was as shown in Table 4 below.

[0116]

[0117] The observation results were recorded for each individual by observing general symptoms as shown in Table 5 below. In addition, body weight and ear thickness were measured. To evaluate skin irritation, evaluation criteria were established as shown in Table 6 below, and the results were observed.

[0118]

[0119]

[0120] The results of the experiment are expressed as the mean stimulation index (mean SI), and the mean SI value of the negative control group was set to 1.

[0121]

[0122] The BrdU labeling index of the test group was defined as follows.

[0123]

[0124]

[0125] Evaluation results

[0126] - Measurement of changes in cell proliferation

[0127] The average absorbance was measured as 0.174, 0.151, 0.152, and 0.197, in order of the negative control group and the administration concentrations of the test substance: 25% (Example 3), 50% (Example 2), and 100% (Example 1). The average absorbance of the negative control group was in the range of 0.1 to 0.2. The average irritation index was 0.87, 0.88, and 1.13, in order of the administration concentrations of the test substance: 25% (Example 3), 50% (Example 2), and 100% (Example 1), indicating no skin sensitization. In contrast, the average absorbance and irritation index in the positive control group were 0.599 and 3.45, respectively, indicating skin sensitization.

[0128]

[0129] - Measurement of ear thickness, ear and lymph node weight

[0130] No statistically significant changes were observed in ear thickness, ear weight, and lymph node weight across all treatment groups. Meanwhile, in the positive control group, lymph node weight showed a statistically significant increase compared to the negative control group (P<0.01).

[0131]

[0132] - Weight

[0133] As a result of comparing body weights between the groups, no statistically significant changes were observed in any of the test substance administration groups.

[0134]

[0135] - Skin irritation assessment and general symptoms

[0136] No skin irritation or general symptoms were observed in any of the test substance administration groups.

[0137]

[0138] To evaluate skin sensitization, changes in cell proliferation, ear thickness, ear and lymph node weight, and skin reaction were measured. As a result, the mean stimulation index (mean SI) was 0.87, 0.88, and 1.13 for the test substances administered at 25% (Example 3), 50% (Example 2), and 100% (Example 1), respectively, confirming that no skin sensitization occurred in any of the test substance administration groups.

[0139] In addition, there were no statistically significant changes in ear thickness, ear weight, and lymph node weight in all test substance administration groups compared to the negative control group.

[0140] The positive control substance showed clear skin sensitization with an average irritation index of 3.45, whereas the skin irritation evaluation score in all test substance administration groups was 0, and no erythema was observed.

[0141] Therefore, it is determined that the test substances in Examples 1 to 3 do not exhibit skin sensitization under the test conditions.

[0142]

[0143] Experimental Example 3. Skin irritation test using the human skin model of Example 1

[0144] To evaluate the skin irritation of the test substance of Example 1, the following test was conducted.

[0145] A commercially available RhE tissue model (EpiDerm™ Skin Irritation Test, MatTek Corporation) was used for the test. After applying 30 μL of the test substance from Example 1 to the tissue and culturing it, the tissue viability was determined by the absorbance at 570 nm using MTT formazan extract.

[0146] The negative control group was treated with Dulbecco's Phosphate-Buffered Saline (DPBS), and the positive control group was treated with 5% Sodium Dodecyl Sulfate (5% SDS) in the same manner.

[0147] As a result of the test, the average tissue survival rate of the test substance treatment group of Example 1 was approximately 105.6% compared to the negative control group. The tissue survival rate of the positive control group was approximately 4.4%, confirming it as an irritant.

[0148] Therefore, it is determined that the serum-free culture extract of human umbilical cord-derived mesenchymal stem cells of Example 1 does not cause skin irritation in an RhE tissue model under the conditions of this test.

[0149]

[0150] Experimental Example 4. Eye irritation test using the human cornea-like epithelial model of Example 1

[0151] To evaluate the eye irritation of the test substance of Example 1, the following test was conducted.

[0152] A commercially available RhCE tissue model (EpiOcular™ Eye Irritation Test, MatTek Corporation) was used for the test. Tissue viability in cultured tissues after applying 50 μL of the test substance from Example 1 was determined by extracting MTT formazan and checking the absorbance at OD 570 nm. Ultrapure distilled water was used for the negative control group, and methyl acetate was used for the positive control group in the same manner.

[0153]

[0154] - Confirmation test of color development and MTT reduction

[0155] When the test substance of Example 1 was mixed with water for injection and observed visually, there was no color development, and when mixed with isopropanol, the change in absorbance value did not exceed 0.08, and when mixed with MTT solution, it did not turn purple or blue.

[0156]

[0157] - Average absorbance and tissue viability

[0158] The average absorbance of the test substance treatment group in Example 1 was 2.011, and the average tissue viability was approximately 111.9% compared to the negative control group. The average absorbances of the negative control and positive control groups were 1.796 and 0.310, respectively, and the average tissue viability of the positive control group was approximately 17.3% compared to the negative control group. The deviations in average tissue viability among the negative control group, the positive control group, and the test substance treatment group in Example 1 were each less than 20%. As a result of the test, the average tissue viability of the test substance treatment group in Example 1 was approximately 111.9% compared to the negative control group. The tissue viability of the positive control group was approximately 17.3%, confirming it as an irritant.

[0159] Therefore, Example 1 is determined not to cause eye irritation to the RhCE tissue model under the test conditions.

[0160]

[0161] Experimental Example 5. Phototoxicity test using cultured 3T3 cells from Example 1

[0162] To evaluate the phototoxicity (toxicity induced after exposure to ultraviolet rays in 3T3 cells) of the test substance of Example 1, the following test was conducted.

[0163] The test substance of Example 1 was prepared by dissolving it in Hank's Balanced Salt Solution (HBSS) and then treated, and the positive control substance, Chloropromazine hydrochloride (CPZ), was prepared by dissolving it in DMSO and then diluted in HBSS and treated. The treatment concentration of the test substance of Example 1 was set as shown in Table 7 below.

[0164]

[0165] Take actively proliferating cells and place 1 x 10⁶ cells per well in a 96-well plate. 4 Canine cells were seeded and cultured for about 20 to 24 hours, after which the test substance of Example 1 was treated. One hour after treatment with the test substance of Example 1, UV (UVA, 365 nm) was irradiated using a UV irradiation device, and the cells were cultured for about 18 to 22 hours after the UV irradiation ended. Cell viability in the cultured cells was determined by absorbance at 540 nm using the neutral red staining method.

[0166]

[0167] - Inhibitory concentration (IC50), photostimulation index, and average photoeffect

[0168] Experimental results showed that IC50 was not confirmed regardless of the presence or absence of UV irradiation. The Photo Irritation Factor was 1.000, and the Mean Photo Effect was 0.012.

[0169] The IC50 of the positive control, Chloropromazine hydrochloride, was 33.46 μg / mL without UV irradiation (-Irr) and 1.136 μg / mL with UV irradiation (+Irr). The photostimulation index and mean photoeffect were 29.488 and 0.533, respectively.

[0170]

[0171] - Cell viability

[0172] The cell viability at the test substance treatment concentrations of Example 1 was 93.50–105.30% and 95.34–102.95%, respectively, in the order of non-UV irradiation and irradiation. The cell viability of the positive control, Chloropromazine hydrochloride, was 4.01–110.68% and 2.94–102.06%, in the order of non-UV irradiation and irradiation. The average absorbance of each negative control group not irradiated with UV light was 0.4 or higher, and the deviation of the absorbance values ​​of the negative control group across all plates did not exceed 0.15 (15%).

[0173]

[0174] - Cell line sensitivity test

[0175] The sensitivity of the cell lines used in this study was 5 and 9 J / cm² compared to the non-irradiated group upon UV irradiation. 2 The percentages were 94.08% and 91.89%, respectively (PN=83).

[0176] Consequently, when the Photo Irritation Factor (PIF) and Mean Photo Effect (MPE) were checked, Example 1 showed no phototoxicity. Meanwhile, the positive control, Chloropromazine hydrochloride, showed clear positive results in both the Photo Irritation Factor and the Mean Photo Effect.

[0177] Therefore, the serum-free culture extract of human umbilical cord-derived mesenchymal stem cells of Example 1 is determined to be a substance that does not exhibit phototoxicity to cultured BALB / 3T3 clone A31 cell lines under the conditions of this test.

[0178]

[0179] Although the present disclosure has been described in detail through specific embodiments, this is for the purpose of specifically explaining the present disclosure and is not limited thereto. It is evident that modifications or improvements can be made by those skilled in the art within the technical scope of the present disclosure. All simple modifications or alterations of the present disclosure fall within the scope of the present disclosure, and the specific scope of protection of the present disclosure will be clarified by the appended claims.

Claims

1. I) Wharton jelly tissue separation step for separating Wharton jelly tissue from the umbilical cord; II) A stem cell isolation step for isolating stem cells from the above-described isolated Wharton jelly tissue; III) A step of subculture of isolated stem cells, comprising a subculture step including culturing in a serum-free culture medium at least once during the later subculture; IV) A centrifugation and pressurization filtration step of centrifuging and pressurizing the culture medium obtained from the above subculture; and V) an exosome separation step of separating exosomes by concentrating and filtering the centrifuged and pressurized filtered liquid using tangential-flow filtration (TFF); comprising a method for producing Wharton jelly-derived exosomes with increased exosome yield.

2. In Paragraph 1, II) A method for producing Wharton jelly-derived exosomes with increased exosome yield, comprising treating with collagenase type I, hyaluronidase, or a combination thereof, and isolating stem cells by performing centrifugation.

3. In Paragraph 2, II) A method for producing Wharton jelly-derived exosomes with increased exosome yield, comprising performing centrifugation in step II at 1,800 to 2,500 rpm for 5 to 10 minutes at least once.

4. In Paragraph 1, III) A method for producing Wharton jelly-derived exosomes with increased exosome yield, comprising subculturing for 5 to 15 days at 37℃ under CO2 conditions.

5. In Paragraph 1, III) A method for producing Wharton jelly-derived exosomes with increased exosome yield, comprising adding 5 to 30 ng / ml of FGF to the serum-free culture medium and culturing for 24 to 48 hours.

6. In Paragraph 1, IV) A method for producing Wharton jelly-derived exosomes with increased exosome yield, comprising centrifuging in step IV at 900 to 1200 g for 20 to 40 minutes.

7. In Paragraph 1, IV) A method for producing Wharton jelly-derived exosomes with increased exosome yield, comprising performing the pressurized filtration in step IV under vacuum using a pore filter of 0.3 to 0.6 μm.

8. In Paragraph 1, V) A method for producing Wharton jelly-derived exosomes with increased exosome yield, wherein the filtration in step V is performed by pressurized filtration using a pore filter of 0.1 to 0.3 μm.

9. In Paragraph 1, The above Wharton jelly-derived exosomes contain 1×10 per 1 ml unit volume. 10 to 9×10 10 A method for producing Wharton jelly-derived exosomes that increase the yield of exosomes, containing a number of exosomes.

10. A cosmetic composition comprising exosomes produced by a method for producing Wharton jelly-derived exosomes that increases the yield of exosomes according to any one of claims 1 to 9.

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