Method for producing culture supernatant of cells

Abstract

To provide a method for producing cells or culture supernatants of cells that, even when cells cultured in a culture medium that includes cell growth factors or the like are used, contain less of components of the culture medium such as the cell growth factors.SOLUTION: A method for producing cells or culture supernatants of cells includes: a step of culturing cells in a first culture medium that does not include blood serum to acquire first cells; a step of culturing the acquired first cells in a second culture medium that has lower concentration of components of the first culture medium than the first culture medium so as to acquire second cells and a culture supernatant of the second cells; a step of removing the culture supernatant of the second cells from the acquired second cells and culture supernatant of the second cells so as to acquire the second cells; and a step of culturing the acquired second cells in a third culture medium that has lower concentration of the components of the first culture medium than the first culture medium so as to acquire third cells and a culture supernatant of the third cells and producing the third cells or the culture supernatant of the third cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a cell culture supernatant, and more specifically, to a method for producing a cell culture supernatant using cells cultured in a medium containing a cell growth factor or the like, the method comprising producing a cell culture supernatant in which the content of components contained in the medium, such as the cell growth factor, is reduced. [Background technology]

[0002] Mesenchymal stem cells are somatic stem cells present in bone marrow, synovium, fat, and umbilical cord, and have been reported to have the ability to differentiate into cartilage, bone, fat, and nerve cells (Non-Patent Document 1). Because they can be isolated from adult tissues, they are used in regenerative treatment of damaged areas of the meniscus and cartilage (Non-Patent Document 2). In addition, mesenchymal stem cells also have anti-inflammatory and immunoregulatory effects, and are therefore known to have been widely used for various diseases, such as liver disease, graft-versus-host disease, and autoimmune diseases.

[0003] In this context, it has become clear that the function of these mesenchymal stem cells transplanted into the body in cell transplantation therapy is greatly influenced by physiologically active substances such as various cytokines secreted by mesenchymal cells.

[0004] When mesenchymal stem cells are cultured in vitro, these physiologically active substances are released into the culture medium. There have been reports of successful cases in which the culture medium used for culturing mesenchymal stem cells was collected and used to regenerate tissues, since the culture medium contains a large amount of substances released from the cells (Non-Patent Documents 3, 4, and 5).

[0005] In this way, the culture supernatant produced during the culture process of mesenchymal stem cells is rich in signaling substances that play an important role in cell activity, such as growth factors and immune regulatory factors (cytokines) secreted from stem cells, including exosomes, and is thought to be useful for restoring the functions of damaged tissues and cells in the body. The exosomes, growth factors, and cytokines abundant in the supernatant act on surrounding stem cells, and are expected to have a functional recovery effect such as tissue regeneration and improved immunity by collecting cells at areas where function has been reduced due to aging or damage.

[0006] During cell culture, fetal bovine serum (FBS, FCS), human plasma, albumin, FGF2, EGF, and other components are often added as cell growth factors. It is generally believed that the medium components can be removed from cells cultured in a medium containing such components by washing with a buffer such as PBS or HBSS. It is currently believed that the safety of cells treated as regenerative medicine products or specific cell processed products, as well as exosomes and supernatants undergoing clinical research, can be improved by removing the medium components in a similar manner. Thus, there is a demand for recovering physiologically active substances derived from human stem cells while minimizing contamination by medium components.

[0007] Patent Document 1 describes an invention having the objective of providing a method for producing a culture supernatant with a reduced amount of cell growth factors, and describes culturing immortalized dental pulp-derived stem cells in DMEM containing 10% FBS, culturing the cells in a serum-free medium not containing FBS (MesenCult medium, StemPro MSC medium, BMN211 medium, StemMACS medium, MSC NutriStem medium, or Xuri MSC medium), then removing the culture medium, and culturing the cells in DMEM medium not containing FBS. Patent Document 1 also describes culturing adipose-derived stem cells in MesenCult medium containing FBS, culturing the cells in MesenCult medium not containing FBS, then removing the culture medium, and culturing the cells in DMEM medium not containing FBS. Patent Document 1 also describes culturing primary dental pulp-derived stem cells in MesenCult medium containing FBS, culturing the cells in MesenCult medium not containing FBS, then removing the culture medium, and culturing the cells in DMEM medium not containing FBS. In addition, Patent Document 1 describes culturing primary dental pulp-derived stem cells in MesenCult medium containing FBS, culturing the cells in MesenCult, DMEM, or Prime-XV medium, and then removing the culture medium and culturing the cells in DMEM medium. Patent Document 1 describes that in these cases, BSA derived from fetal bovine serum was not detected in the supernatant after the final culture.

[0008] The written opinion of Patent Document 1 asserts that the reduction of impurities contained in the culture supernatant is achieved by combining these FBS-free serum-free media, such as MesenCult medium, StemPro MSC medium, BMN211 medium, StemMACS medium, MSC NutriStem medium, or Xuri MSC medium, or serum-free media, such as MesenCult medium or Prime-XV medium, with FBS-free DMEM medium.

[0009] Patent Document 2 describes a method for culturing animal cells, which is characterized in that adhesive animal cells are cultured in a serum-containing medium until they adhere to a culture substrate, and then the cells adhered to the culture substrate are directly cultured in a serum-free medium, as a method for solving the problem that, in culturing animal cells, in order to culture cells that have been cultured in a serum-containing medium in a serum-free medium, the serum components in the medium must be gradually reduced to acclimate the cells to a serum-free environment, and that proteins cannot be produced efficiently during this period. However, Patent Document 2 does not describe or suggest performing multiple washing operations (medium exchange).

[0010] Patent Document 3 describes that a serum-free culture supernatant can be prepared by culturing dental pulp stem cells in a medium that does not contain serum (serum-free medium), and that a serum-free culture supernatant can also be obtained by performing one or more subcultures and culturing the last or penultimate subculture in a serum-free medium. However, Patent Document 3 does not describe any experimental results showing that the culture was performed multiple times in a serum-free medium, and this description lacked eligibility as a description for identifying a cited invention.

[0011] Patent Document 4 also describes that a serum-free culture supernatant can be prepared by culturing dental pulp stem cells in a medium that does not contain serum (serum-free medium), and that a serum-free culture supernatant can also be obtained by performing one or more subcultures and culturing the last or penultimate subculture in a serum-free medium. However, Patent Document 4 also does not describe experimental results showing that the culture was performed multiple times in a serum-free medium, and this description lacked eligibility as a description for identifying a cited invention.

[0012] Patent Document 5 describes that, in order to acclimate the cells to serum-free floating cells, they were subcultured in a serum-free medium supplemented with various nutrients until cell growth stabilized. However, it is envisaged here that recombinant human FSH (follicle-stimulating hormone) will be extracted from the culture supernatant of the obtained cells, and it is not envisaged that the culture supernatant itself will be used as a pharmaceutical composition, cosmetic composition, or food composition, or that it will be included in these compositions.

[0013] Patent Document 6 describes that the cells were passaged 4 to 6 times in serum-free medium to be habituated to the serum-free medium. However, it is assumed here that useful proteins are extracted from the culture supernatant of the obtained cells, and it is not assumed that the culture supernatant itself of the cells is used as a pharmaceutical composition, cosmetic composition, or food composition, or is included in these compositions. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Patent No. 6860915 [Patent Document 2] JP 2014-039503 A [Patent Document 3] International Publication No. 2011 / 118795 [Patent Document 4] International Publication No. 2014 / 126176 [Patent Document 5] JP 2009-273427 A [Patent Document 6] JP 2009-045019 A [Non-patent literature]

[0015] [Non-Patent Document 1] Colter, D. C., Sekiya, I. & Prockop, D. J. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci. 2001;98:7841-7845. [Non-Patent Document 2] Sekiya I., Muneta T., Horie M. & Koga H. Arthroscopic Transplantation of Synovial Stem Cells Improves Clinical Outcomes in Knees With Cartilage Defects. Clin Orthop Relat Res. 2015;473:2316-26. [Non-Patent Document 3] Osugi M., Katagiri, W., Yoshimi, R., Inukai, T., Hibi, H., Ueda M. Conditioned media from mesenchymal stem cells enhanced bone regeneration in rat calvarial bone defects. Tissue Engineering Part A.2012;18:1479-1489 [Non-Patent Document 4] Shohara, R., Yamamoto, A., Takikawa, S., Iwase, A., Hibi, H., Kikkawa, F., Ueda, M. Mesenchymal stromal cells of human umbilical cord Wharton's jelly accelerate wound healing by paracrine mechanisms. Cytotherapy,2012;14(10):1171-1181 [Non-Patent Document 5] Kawai, T., Katagiri, W., Osugi, M., Sugimura, Y., Hibi, H., Ueda, M. Secretomes from bone marrow-derived mesenchymal stromal cells enhance periodontal tissue regeneration. Cytotherapy,2015;17(4):369-381 Summary of the Invention [Problem to be solved by the invention]

[0016] In the examples of Patent Document 1, it is described that when the method of the invention of Patent Document 1 was used, no impurities were detected in the culture supernatant, and further reduction of impurities was not considered in the method of the invention of Patent Document 1. Also, as mentioned above, according to the opinion of Patent Document 1, the reduction of impurities in the culture supernatant in the invention of Patent Document 1 is due to the interaction of each component obtained by combining a medium containing a specific component with a medium containing a specific component, and removing the culture medium itself was not positioned as a means constituting a feature for solving the problem of the invention of Patent Document 1. Due to these circumstances, a person skilled in the art would be hindered from developing the invention of Patent Document 1 on the premise of the invention of Patent Document 1 by further repeating the process of removing the culture medium. Also, in Patent Document 1 and the like, after the culture in the serum-containing medium, the culture in the serum-free medium is performed, and there is no description of culturing in the serum-free medium after the culture in the serum-free medium.

[0017] However, the inventors have found that when producing cell culture supernatant using cells cultured in a medium containing cell growth factors, even if a process for removing the culture medium is included, cell growth factors are sometimes detected in the produced cell culture supernatant (see, for example, sample circle 2 in Figures 3 and 4 of the present application).

[0018] Therefore, an object of the present invention is to provide a method for producing cells or cell culture supernatant in which the content of components contained in a medium, such as cell growth factors, is reduced, even when cells cultured in the medium containing the cell growth factors or the like are used. [Means for solving the problem]

[0019] The present inventors considered that since various components contained in the medium exert a wide range of actions such as proliferation activity by being taken up into cells, it cannot be denied that there may be components that are transiently taken up into cells and then released back into the medium. They considered that the medium components (FBS, plasma, albumin, FGF2, etc.) used in the cell proliferation process may be taken up into cells and then released back into the medium after a certain period of time, and after extensive research, they found that the medium components confirmed under such a phenomenon can be removed by performing a washing operation (medium exchange). That is, it was common knowledge that unnecessary components such as recombinant proteins derived from the medium can be washed away by washing with PBS, etc., but in fact, those taken up into the cells are not washed away by a momentary washing operation, but are released into the medium over time, and therefore, the present inventors found that it is necessary to provide a period for release in the medium. In general, in order to culture cells, it is necessary to use a proliferation medium containing serum, recombinant proteins, antibiotics, etc., but such impurities may lead to side effects such as anaphylactic shock and hypoglycemic symptoms, and therefore a technology for removing medium-derived components is desired from the viewpoint of safety. It was previously thought that impurities could be removed by washing with PBS (phosphate buffered saline) or the like, but the present inventors discovered that these impurities are taken up into cells during culture and gradually re-released over time, and discovered a method for removing them. Impurities derived from such growth media are difficult to remove after the production of supernatants, exosomes, etc., and the method of the present invention is excellent in that it can produce highly safe cells and cell culture supernatants.

[0020] That is, the present invention provides the following. [Aspect 1] A method for producing a cell or a cell culture supernatant, comprising the steps of: Culturing the cells in a serum-free first medium to obtain a first cell; Culturing the obtained first cells in a second medium having a lower concentration of a component contained in the first medium than the first medium, and obtaining second cells and a culture supernatant of the second cells; removing the culture supernatant of the second cells from the obtained second cells and the culture supernatant of the second cells to obtain the second cells; and a step of culturing the obtained second cells in a third medium having a lower concentration of a component contained in the first medium than in the first medium, obtaining the third cells and a culture supernatant of the third cells, and producing the third cells or the culture supernatant of the third cells. A method comprising: [Aspect 2] A method for producing a cell or a cell culture supernatant, comprising the steps of: Culturing the cells in the first medium to obtain the first cells; Culturing the obtained first cells in a second medium having a lower concentration of a component contained in the first medium than the first medium, and obtaining second cells and a culture supernatant of the second cells; removing the culture supernatant of the second cells from the obtained second cells and the culture supernatant of the second cells to obtain the second cells; Culturing the obtained second cells in a third medium having a lower concentration of a component contained in the first medium than that of the first medium, and obtaining third cells and a culture supernatant of the third cells; removing the culture supernatant of the third cells from the obtained third cells and the culture supernatant of the third cells to obtain the third cells; and a step of culturing the obtained third cell in a fourth medium having a lower concentration of a component contained in the first medium than in the first medium to obtain a fourth cell and a culture supernatant of the fourth cell, thereby producing the fourth cell or the culture supernatant of the fourth cell. A method comprising: [Aspect 3] The method according to aspect 2, comprising the steps of: removing the culture supernatant of the fourth cell from the obtained fourth cell and the culture supernatant of the fourth cell to obtain the fourth cell; and culturing the obtained fourth cell in a fifth medium having a lower concentration of components contained in the first medium than in the first medium to obtain the fifth cell and the culture supernatant of the fifth cell, thereby producing the fifth cell or the culture supernatant of the fifth cell. [Aspect 4] A method according to aspect 3, comprising the steps of removing the culture supernatant of the fifth cell from the obtained fifth cell and the culture supernatant of the fifth cell to obtain the fifth cell, and culturing the obtained fifth cell in a sixth medium having a lower concentration of components contained in the first medium than in the first medium to obtain the sixth cell and the culture supernatant of the sixth cell, and producing the sixth cell or the culture supernatant of the sixth cell. [Aspect 5] The method of embodiment 2, wherein the cells are adipose tissue-derived stem cells. [Aspect 6] The method of embodiment 2, wherein the cell is a human cell. [Aspect 7] The method according to embodiment 2, wherein the component contained in the first culture medium is a cell growth factor. [Aspect 8] The method of embodiment 7, wherein the cell growth factor is a non-human protein. [Aspect 9] The method of embodiment 7, wherein the cell growth factor is serum or plasma. [Aspect 10] The method according to embodiment 7, wherein the first culture medium comprises 0.5% by volume or more of a cell growth factor. [Aspect 11] 3. The method of embodiment 2, wherein the first medium comprises 0.0000001% to 1% by volume of the recombinant protein. [Aspect 12] 11. The method of embodiment 10, wherein the first culture medium comprises 8% or more by volume of a cell growth factor. [Aspect 13] The method according to embodiment 2, wherein the second medium, in which the concentration of a component contained in the first medium is lower than that of the first medium, is a basal medium. [Aspect 14] The method according to embodiment 2, wherein the third medium, which contains a component of the first medium at a lower concentration than the first medium, is a basal medium. [Aspect 15] The method according to embodiment 2, wherein the fourth medium, in which the concentration of a component contained in the first medium is lower than that of the first medium, is a basal medium. [Aspect 16] The method according to embodiment 3, wherein the fifth medium, in which the concentration of a component contained in the first medium is lower than that of the first medium, is a basal medium. [Aspect 17] The method according to embodiment 4, wherein the sixth medium, in which the concentration of a component contained in the first medium is lower than that of the first medium, is a basal medium. [Aspect 18] The method according to aspect 2, wherein the obtained first cells are cultured in a second medium containing a component contained in the first medium at a lower concentration than that of the first medium for a period of 1 hour to 192 hours. [Aspect 19] The method according to aspect 2, wherein the obtained second cells are cultured in a third medium containing a component of the first medium at a lower concentration than that of the first medium for 1 hour to 192 hours. [Aspect 20] The method according to aspect 2, wherein the obtained third cells are cultured in a fourth medium having a component contained in the first medium at a lower concentration than that in the first medium for a period of 1 hour to 192 hours. [Aspect 21] The method according to aspect 3, wherein the obtained fourth cells are cultured in a fifth medium having a component contained in the first medium at a lower concentration than that in the first medium for a period of 1 hour to 192 hours. [Aspect 22] The method according to aspect 4, wherein the obtained fifth cells are cultured in a sixth medium having a component contained in the first medium at a lower concentration than that of the first medium for a period of 1 hour to 192 hours. [Aspect 23] The method of embodiment 2, comprising washing the first cells prior to obtaining the first cells. [Aspect 24] The method of embodiment 2, comprising washing the second cells prior to obtaining the second cells. [Aspect 25] The method of embodiment 2, further comprising washing the third cells prior to obtaining the third cells. [Aspect 26] The method of embodiment 3, comprising washing the fourth cell prior to obtaining the fourth cell. [Aspect 27] 5. The method of embodiment 4, comprising washing the fifth cell prior to obtaining the fifth cell. [Aspect 28] The method according to any one of Aspects 1 to 27, wherein the cells or a culture supernatant of the cells are intended for use as or for inclusion in a pharmaceutical composition, a cosmetic composition, a food composition, a regenerative medicine product, or a specific cell processed product. Effect of the Invention

[0021] According to the present invention, even when cells cultured in a medium containing cell growth factors and the like are used, it is possible to produce a cell culture supernatant in which the content of components contained in the medium, such as the cell growth factors, is reduced, which leads to an improvement in the safety of exosomes and supernatants that are expected to be used in regenerative medicine and new drug development. In addition, according to the present invention, washed cells can be produced, and the cells produced by the present invention can be used, for example, in cell transplantation therapy. That is, according to the present invention, it is possible to significantly remove impurities that could not be removed by conventional washing methods. The present invention is widely applicable to products that require cell culture, such as supernatants and exosomes, from regenerative medicine in general, and can become an important technology that contributes to improving safety in the future. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is a photograph showing the appearance of cells in sample circle 4, which was recovered by carrying out the method of Example 1 using cells cultured in a medium obtained by adding FBS to DMEM medium so as to contain FBS at various concentrations (numbers in the upper left corner: volume %). [Diagram 2]FIG. 2 is a graph showing the results of an ELISA test for samples circle 1 to circle 4 collected by carrying out the method of Example 1 using cells cultured in DMEM medium (not containing FBS). [Diagram 3] Figure 3 is a graph showing the results of ELISA tests on samples 1 to 4 recovered by the method of Example 1 using cells cultured in a medium obtained by adding FBS to DMEM medium so that it contained 10% FBS by volume. [Figure 4] Figure 4 is a graph showing the results of ELISA tests on samples 1 to 4 recovered by the method of Example 1 using cells cultured in a medium obtained by adding FBS to DMEM medium so that the medium contained 20% FBS by volume. [Diagram 5] FIG. 5 is a schematic diagram showing the steps of the tests carried out in Examples 2 and 3. [Figure 6] FIG. 6 is a schematic diagram showing each step of the test for sample recovery carried out in Example 4. [Figure 7] FIG. 7 is a graph showing the VEGF protein concentration in each sample obtained in Example 4. [Figure 8] FIG. 8 is a schematic diagram showing the steps of the test carried out in Example 5. [Figure 9] FIG. 9 is a graph showing the concentration of HGF in each sample obtained in Example 5. [Figure 10] FIG. 10 is a graph showing the concentration of IL-6 in each sample obtained in Example 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention relates to a method for producing a cell or a culture supernatant of a cell, the method comprising the steps of: (first step) culturing cells in a serum-free first medium to obtain first cells; (Second step) culturing the obtained first cells in a second medium having a lower concentration of a component contained in the first medium than the first medium, preferably a second medium not containing a component contained in the first medium, to obtain second cells and a culture supernatant of the second cells; (third step) removing the culture supernatant of the second cells from the obtained second cells and the culture supernatant of the second cells to obtain the second cells; and (Fourth step) A step of culturing the obtained second cells in a third medium having a lower concentration of a component contained in the first medium than the first medium, preferably a third medium not containing a component contained in the first medium, to obtain a third cell and a culture supernatant of the third cell, and to produce the third cell or the culture supernatant of the third cell. The present invention provides a method comprising:

[0024] First step: Similar to the first step described below, the cells are cultured in a first medium that does not contain serum.

[0025] Second step: This is the same as the second step described below.

[0026] Third step: This is the same as the third step described below.

[0027] Fourth step: Similar to the fourth step described below, the method for producing the culture supernatant of the cell of the present invention may include (fifth step) a step of removing the culture supernatant of the third cell from the obtained third cell and the culture supernatant of the third cell to obtain the third cell, and (sixth step) a step of culturing the obtained third cell in a fourth medium in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably not containing the components contained in the first medium, to obtain the fourth cell and the culture supernatant of the fourth cell, and to produce the fourth cell or the culture supernatant of the fourth cell. That is, the method for producing the culture supernatant of the cell of the present invention not only includes a method of using the culture supernatant of the obtained third cell as the culture supernatant of the cell to be produced, but also includes a method of removing the culture supernatant of the obtained third cell to obtain the third cell, further culturing the obtained third cell or a cell derived therefrom to obtain a culture supernatant of another cell and another cell, and using the culture supernatant of the other cell or another cell thus obtained as the culture supernatant of the cell to be produced. The method for producing the cell culture supernatant of the present invention may also include (seventh step) a step of removing the culture supernatant of the fourth cell from the obtained fourth cell and the culture supernatant of the fourth cell to obtain the fourth cell, and (eighth step) a step of culturing the obtained fourth cell in a fifth medium in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably not containing the components contained in the first medium, to obtain the fifth cell and the culture supernatant of the fifth cell, and to produce the fifth cell or the culture supernatant of the fifth cell. That is, the method for producing the cell culture supernatant of the present invention includes not only a method of using the culture supernatant of the obtained fourth cell as the culture supernatant of the producing cell, but also a method of removing the culture supernatant of the obtained fourth cell to obtain the fourth cell, further culturing the obtained fourth cell or a cell derived therefrom to obtain a culture supernatant of another cell and another cell, and using the culture supernatant of the another cell or another cell thus obtained as the culture supernatant of the producing cell or cell.Furthermore, the method for producing the culture supernatant of the cell of the present invention may include (ninth step) a step of removing the culture supernatant of the fifth cell from the obtained fifth cell and the culture supernatant of the fifth cell to obtain the fifth cell, and (tenth step) a step of culturing the obtained fifth cell in a sixth medium in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably not containing the components contained in the first medium, to obtain the sixth cell and the culture supernatant of the sixth cell, and to produce the sixth cell or the culture supernatant of the sixth cell. That is, the method for producing the culture supernatant of the cell of the present invention not only includes a method of using the culture supernatant of the obtained fifth cell as the culture supernatant of the cell to be produced, but also includes a method of removing the culture supernatant of the obtained fifth cell to obtain the fifth cell, further culturing the obtained fifth cell or a cell derived therefrom to obtain a culture supernatant of another cell and another cell, and using the culture supernatant of the another cell or another cell thus obtained as the culture supernatant of the cell to be produced. Similarly, the method for producing the culture supernatant of the cell of the present invention may include (step 11) a step of removing the culture supernatant of the sixth cell from the obtained sixth cell and the culture supernatant of the sixth cell to obtain the sixth cell, and (step 12) a step of culturing the obtained sixth cell in a seventh medium in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably not containing the components contained in the first medium, to obtain the culture supernatant of the seventh cell and the seventh cell, and to produce the seventh cell or the culture supernatant of the seventh cell. That is, the method for producing the culture supernatant of the cell of the present invention includes not only a method of using the culture supernatant of the obtained sixth cell as the culture supernatant of the cell to be produced, but also a method of removing the culture supernatant of the obtained sixth cell to obtain the sixth cell, further culturing the obtained sixth cell or a cell derived therefrom to obtain a culture supernatant of another cell and another cell, and using the culture supernatant of the another cell or another cell thus obtained as the culture supernatant of the cell to be produced. The culture supernatant of such other cells can be obtained by the same method as described above in detail.Therefore, in the method for producing a cell culture supernatant of the present invention, the cell or cell culture supernatant produced may be the culture supernatant of the obtained third cell or the third cell, the culture supernatant of the obtained fourth cell or the fourth cell, the culture supernatant of the obtained fifth cell or the fifth cell, the culture supernatant of the obtained sixth cell or the sixth cell, or the culture supernatant of the obtained seventh cell or the seventh cell, but is not limited thereto, and may also be another cell or the culture supernatant of another cell obtained by a similar method. Here, the fifth to twelfth steps are the same as the fifth to twelfth steps described below.

[0028] The present invention further provides a method for producing a cell or a cell culture supernatant, comprising the steps of: (First step) culturing cells in a first medium to obtain first cells; (Second step) culturing the obtained first cells in a second medium having a lower concentration of a component contained in the first medium than the first medium, preferably a second medium not containing a component contained in the first medium, to obtain second cells and a culture supernatant of the second cells; (third step) removing the culture supernatant of the second cells from the obtained second cells and the culture supernatant of the second cells to obtain the second cells; (fourth step) culturing the obtained second cells in a third medium having a lower concentration of a component contained in the first medium than the first medium, preferably a third medium not containing a component contained in the first medium, to obtain third cells and a culture supernatant of the third cells; (fifth step) removing the culture supernatant of the third cells from the obtained third cells and the culture supernatant of the third cells to obtain the third cells; and (Sixth step) A step of culturing the obtained third cell in a fourth medium in which the concentration of a component contained in the first medium is lower than that of the first medium, preferably in which the component is not contained in the first medium, to obtain a fourth cell and a culture supernatant of the fourth cell, and to produce the fourth cell or the culture supernatant of the fourth cell. The present invention provides a method comprising:

[0029] First step: Examples of the cells include stem cells. Examples of the stem cells include stem cells derived from tissues such as adipose tissue, umbilical cord, amniotic membrane, amniotic fluid, dental pulp, Wharton's jelly, CPJ (Cord Placenta Junction), chorion, liver, lung, spine, umbilical cord blood, placenta, peripheral blood, dermis, endometrium, breast milk, hair follicles, etc. Examples of the stem cells derived from these tissues include stem cells isolated from these tissues. Examples of the stem cells include mesenchymal stem cells (MSCs), hematopoietic stem cells, neural crest stem cells, etc. Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in multiple tissues such as umbilical cord, bone marrow, and adipose tissue. Mesenchymal stem cells (MSCs) are rare cells, but it is estimated that they are present in bone marrow at a ratio of 1 per 10,000 to 100,000 nucleated bone marrow cells. Mesenchymal stem cells (MSCs) have the ability to self-proliferate and can differentiate into various cell types such as osteoblasts (bone cells), chondrocytes, muscle cells, adipocytes, etc. In addition, examples of cells include ES cells, iPS cells, and cells induced to differentiate from these stem cells.

[0030] Examples of the cells include animal cells. Examples of the animals include vertebrates and invertebrates. Examples of the vertebrates include fish, amphibians, reptiles, birds, and mammals. Examples of the mammals include mice and humans. Examples of the invertebrates include arthropods such as insects, crustaceans, arachnids, and myriapods, as well as mollusks. Preferred animals are mammals, and preferred mammals are humans.

[0031] In the step of culturing cells in a first medium to obtain first cells, the time for culturing the cells in the first medium is, for example, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 15 hours or more, 20 hours or more, 25 hours or more, 30 hours or more, 35 hours or more, 40 hours or more, 45 hours or more, 50 hours or more, 55 hours or more, 60 hours or more, 65 hours or more, 70 hours or more, 75 hours or more, 80 hours or more, 85 hours or more, 90 hours or more, 95 hours or more, 100 hours or more, 105 hours or more, 110 hours or more, 115 hours or more to 120 hours or more, 125 hours or more to 130 hours or more, 135 hours or more to 140 hours or more, 145 hours or more to 150 hours or more, 155 hours or more to 160 hours or more, or 165 hours or more.

[0032] In the step of culturing cells in a first medium to obtain first cells, the time for culturing the cells in the first medium is, for example, 400 hours or less, 350 hours or less, 300 hours or less, 250 hours or less, 200 hours or less, 195 hours or less, 190 hours or less, 185 hours or less, 180 hours or less, 175 hours or less, 170 hours or less, 165 hours or less, 160 hours or less, 155 hours or less, 150 hours or less, 145 hours or less, 140 hours or less. The duration of the leave is 1 hour or less, 135 hours or less, 130 hours or less, 125 hours or less, 120 hours or less, 115 hours or less, 110 hours or less, 105 hours or less, 100 hours or less, 95 hours or less, 90 hours or less, 85 hours or less, 80 hours or less, 75 hours or less, 70 hours or less, 65 hours or less, 60 hours or less, 55 hours or less, 50 hours or less, 45 hours or less, 40 hours or less, 35 hours or less, 30 hours or less, 25 hours or less, or 20 hours or less.

[0033] In the step of culturing cells in a first medium to obtain first cells, the time for culturing the cells in the first medium is within a range specified by, for example, any of the above-mentioned lower limits and any of the above-mentioned upper limits.

[0034] In the step of culturing cells in a first medium to obtain first cells, the temperature at which the cells are cultured in the first medium is, for example, 20° C. to 45° C., 30° C. to 40° C., or 35° C. to 38° C. The oxygen concentration during the culture is usually 20%, but is, for example, 0% to 40%, 5% to 35%, 10% to 30%, or 15% to 25%.

[0035] Examples of the components contained in the first medium include recombinant proteins, antibiotics, and cell growth factors. The components contained in the first medium are preferably cell growth factors. The cell growth factors are, for example, proteins of organisms from which the cells cultured in the first medium are not derived. That is, when the cells cultured in the first medium are derived from humans, the cell growth factors are, for example, non-human proteins. The components contained in the first medium also include proteins derived from humans (human proteins). Human proteins derived from the medium are basically synthetic proteins, and there are safety concerns depending on the method of producing the secondary raw materials. The present invention aims to remove synthetic substances containing these human proteins and increase the composition ratio of cell-derived secretions (proteins, etc.). Examples of cell growth factors include peptides with a molecular weight of 20,000 or less that exert their effects at low concentrations by binding to a receptor.More specifically, examples of cell growth factors include epidermal growth factor (EGF) or a substance having substantially the same activity as EGF (e.g., TGFalpha, etc.), insulin or a substance having substantially the same activity as insulin (e.g., insulin, insulin-like growth factor (IGF)-1, IGF-2, etc.), fibroblast growth factor (FGF) or a substance having substantially the same assay as FGF (FGF) (e.g., acidic FGF, basic FGF, keratinocyte growth factor (KGK), FGF-10, etc.), and other cell growth factors (e.g., cytokines such as IL-2, IL-15, IL-7, IL-9, and IL-21, colony stimukating factor (CSF), erythropoietin (EPO), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), etc.). Examples of cell growth factors include serum, plasma, and albumin. Examples of serum and plasma include human serum and plasma as well as non-human serum and plasma.

[0036] When the first medium contains a recombinant protein, antibiotic, or cell growth factor, the amount of the recombinant protein, antibiotic, or cell growth factor contained in the first medium is, for example, 0.1 vol% or more, 0.2 vol% or more, 0.3 vol% or more, 0.4 vol% or more, 0.5 vol% or more, 1 vol% or more, 2 vol% or more, 3 vol% or more, 4 vol% or more, 5 vol% or more, 6 vol% or more, 7 vol% or more, 8 vol% or more, 9 vol% or more, 10 vol% or more, 11 vol% or more, 12 vol% or more, 13 vol% or more, 14 vol% or more, 15 vol% or more, 16 vol% or more, 17 vol% or more, 18 vol% or more, or 19 vol% or more. The amount of recombinant protein, antibiotic, or cell growth factor contained in the first culture medium is, for example, 0.0001 μg / ml or more, 0.001 μg / ml or more, 0.01 μg / ml or more, 0.1 μg / ml or more, 1 μg / ml or more, 10 μg / ml or more, 100 μg / ml or more, or 1000 μg / ml or more. The amount of recombinant protein contained in the first culture medium is, for example, 0.0000001 volume% or more, 0.000001 volume% or more, 0.00001 volume% or more, 0.0001 volume% or more, 0.001 volume% or more, 0.01 volume% or more, 0.1 volume% or more, or 1 volume% or more. Here, the amount of a recombinant protein, antibiotic, or cell growth factor contained in a first culture medium being equal to or greater than a predetermined numerical volume % or equal to or greater than a predetermined numerical value μg / ml may imply that the total amount of a recombinant protein, antibiotic, or cell growth factor contained in the first culture medium is equal to or greater than the predetermined numerical volume % or equal to or greater than the predetermined numerical value μg / ml, and may also imply that the total amount of a specific recombinant protein, antibiotic, or cell growth factor contained in the first culture medium is equal to or greater than the predetermined numerical volume % or equal to or greater than the predetermined numerical value μg / ml.

[0037] When the first medium contains a recombinant protein, an antibiotic, or a cell growth factor, the amount of the recombinant protein, antibiotic, or cell growth factor contained in the first medium is, for example, 35% by volume or less, 34% by volume or less, 33% by volume or less, 32% by volume or less, 31% by volume or less, 30% by volume or less, 29% by volume or less, 28% by volume or less, 27% by volume or less, 26% by volume or less, 25% by volume or less, 24% by volume or less, 23% by volume or less, 22% by volume or less, or 21% by volume or less. Also, the amount of the recombinant protein, antibiotic, or cell growth factor contained in the first medium is 1,000,000 μg / ml or less, 100,000 μg / ml or less, 10,000 μg / ml or less, or 5,000 μg / ml or less. The amount of recombinant protein contained in the first medium is, for example, 1% by volume or less, 0.1% by volume or less, 0.01% by volume or less, 0.001% by volume or less, 0.0001% by volume or less, 0.00001% by volume or less, 0.000001% by volume or less, or 0.0000001% by volume or less. Here, the amount of recombinant protein, antibiotic, or cell growth factor contained in the first medium being a predetermined numerical value vol% or less or a predetermined numerical value μg / ml or less may imply that the total amount of recombinant protein, antibiotic, or cell growth factor contained in the first medium is the predetermined numerical value vol% or less or the predetermined numerical value μg / ml or less, and may also imply that the total amount of a specific recombinant protein, antibiotic, or cell growth factor contained in the first medium is the predetermined numerical value vol% or less or the predetermined numerical value μg / ml or less.

[0038] When the first culture medium contains a recombinant protein, an antibiotic, or a cell growth factor, the amount of the recombinant protein, the antibiotic, or the cell growth factor contained in the first culture medium is, for example, within a range specified by any of the above lower limits and any of the above upper limits.

[0039] In the step of culturing cells in a first medium to obtain the first cells, for example, the first cells and the culture supernatant of the first cells are separated prior to obtaining the first cells. The separation of the first cells and the culture supernatant of the first cells is performed, for example, by centrifugation. The centrifugation is performed, for example, under conditions of 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The time for which the centrifugation is performed is, for example, 1 minute to 20 minutes, 2 minutes to 15 minutes, 3 minutes to 10 minutes, or 4 minutes to 7 minutes. The separation may be performed by filter filtration. The pore size of the filter is, for example, 1 μm or less, 0.9 μm or less, 0.8 μm or less, 0.7 μm or less, 0.6 μm or less, 0.5 μm or less, 0.4 μm or less, or 0.3 μm or less, and, for example, 0.05 μm or more, 0.06 μm or more, 0.07 μm or more, 0.08 μm or more, 0.09 μm or more, 0.1 μm or more, or 0.2 μm or more, with 0.22 μm, 0.45 μm, etc. being preferred. The culture supernatant of the first cells is preferably removed.

[0040] The step of culturing cells in a first medium to obtain the first cells may include a step of washing the first cells prior to obtaining the first cells. In the step of washing the first cells, for example, the first cells are washed using a solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium. Here, the solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, may be a solution in which the concentrations of all the components contained in the first medium are lower than that of the first medium, preferably does not contain all the components contained in the first medium, but is not limited thereto, and may be a solution in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium. The solution in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium, may be a solution in which the concentration of another specific component contained in the first medium is the same as or higher than that of the first medium, or a solution containing another specific component not contained in the first medium. The solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, includes, for example, a buffer, physiological saline, medium, etc. Examples of the medium include a basal medium, a low serum medium, a serum-free medium, etc. Examples of the basal medium include, for example, an IMDM medium, a Medium 199 medium, an Eagle's Minimum Essential Medium (EMEM) medium, an αMEM medium, a Dulbecco's modified Eagle's Medium (DMEM) medium, a Ham's F12 medium, a RPMI 1640 medium, a Fischer's medium, and a mixed medium thereof. In addition, the basal medium may be, for example, a medium (MEM, MEM-a, DMEM, DMEM high-glucose, PRIM, etc.) that does not contain synthetic proteins and is composed of inorganic salts, vitamins, amino acids, sugars, etc.Here, the basal medium may contain, as necessary, substances such as N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, ethanolamine, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), sodium pyruvate, vitamins, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. The solution in which the concentration of the components contained in the first medium is lower than that of the first medium, and preferably does not contain the components contained in the first medium, may be, for example, a second medium in which the concentration of the components contained in the first medium is lower than that of the first medium, and preferably does not contain the components contained in the first medium.

[0041] Second step: In the step of culturing the obtained first cells in a second medium in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, to obtain the second cells and the culture supernatant of the second cells, the second medium in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, may be a medium in which the concentration of all the components contained in the first medium is lower than that of the first medium, preferably does not contain all the components contained in the first medium, but is not limited thereto, and may be a medium in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium. The medium in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium, may be a medium in which the concentration of another specific component contained in the first medium is the same as or higher than that of the first medium, or a medium containing another specific component not contained in the first medium. Examples of the medium in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, include a basal medium, a low-serum medium, and a serum-free medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. The basal medium may also be, for example, a medium (MEM, MEM-a, DMEM, DMEM high-glucose, PRIM, etc.) that is composed of inorganic salts, vitamins, amino acids, sugars, etc. that do not contain synthetic proteins.Here, the basal medium may contain substances such as N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, ethanolamine, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), sodium pyruvate, vitamins, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts, as necessary.

[0042] The time for culturing the obtained first cells in a second medium in which the concentration of a component contained in the first medium is lower than that of the first medium, preferably which does not contain a component contained in the first medium, is, for example, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 15 hours or more, or 20 hours or more.

[0043] The time for culturing the obtained first cells in a second medium in which the concentration of a component contained in the first medium is lower than that of the first medium, preferably containing no components contained in the first medium, is, for example, 250 hours or less, 240 hours or less, 230 hours or less, 220 hours or less, 210 hours or less, 200 hours or less, 190 hours or less, 180 hours or less, 170 hours or less, 160 hours or less, 150 hours or less, 140 hours or less, 130 hours or less, 120 hours or less, 110 hours or less, 100 hours or less, 95 hours or less, 90 hours or less, 85 hours or less, 80 hours or less, 75 hours or less, 70 hours or less, 65 hours or less, 60 hours or less, 55 hours or less, 50 hours or less, 45 hours or less, 40 hours or less, 35 hours or less, 30 hours or less, or 25 hours or less.

[0044] The time for culturing the obtained first cells in a second medium having a lower concentration of a component contained in the first medium than that of the first medium, preferably not containing a component contained in the first medium, is within a range specified by, for example, any of the above-mentioned lower limits and any of the above-mentioned upper limits.

[0045] The temperature at which the obtained first cells are cultured in a second medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably in which no components contained in the first medium are contained, is, for example, 20° C. to 45° C., 30° C. to 40° C., or 35° C. to 38° C. The oxygen concentration during the culture is usually 20%, but is, for example, 0% to 40%, 5% to 35%, 10% to 30%, or 15% to 25%.

[0046] Third step: In the step of removing the culture supernatant of the second cells from the obtained second cells and the culture supernatant of the second cells to obtain the second cells, for example, the second cells and the culture supernatant of the second cells are separated from each other prior to removing the culture supernatant of the second cells. The separation of the second cells and the culture supernatant of the second cells is performed, for example, by centrifugation. The centrifugation is performed, for example, under conditions of 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The time for which the centrifugation is performed is, for example, 1 minute to 20 minutes, 2 minutes to 15 minutes, 3 minutes to 10 minutes, or 4 minutes to 7 minutes. The separation may be performed by filter filtration. The pore size of the filter is, for example, 1 μm or less, 0.9 μm or less, 0.8 μm or less, 0.7 μm or less, 0.6 μm or less, 0.5 μm or less, 0.4 μm or less, or 0.3 μm or less, and for example, 0.05 μm or more, 0.06 μm or more, 0.07 μm or more, 0.08 μm or more, 0.09 μm or more, 0.1 μm or more, or 0.2 μm or more, with 0.22 μm, 0.45 μm, etc. being preferred.

[0047] The step of removing the culture supernatant of the second cells from the obtained second cells and the culture supernatant of the second cells to obtain the second cells may include a step of washing the second cells prior to obtaining the second cells. In the step of washing the second cells, for example, the second cells are washed using a solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium. Here, the solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, may be a solution in which the concentration of all the components contained in the first medium is lower than that of the first medium, preferably does not contain all the components contained in the first medium, but is not limited thereto, and may be a solution in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium. The solution in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium, may be a solution in which the concentration of another specific component contained in the first medium is the same as or higher than that of the first medium, or a solution containing another specific component not contained in the first medium. The solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, includes, for example, a buffer, physiological saline, medium, etc. Examples of the medium include a basal medium, a low serum medium, a serum-free medium, etc. Examples of the basal medium include, for example, an IMDM medium, a Medium 199 medium, an Eagle's Minimum Essential Medium (EMEM) medium, an αMEM medium, a Dulbecco's modified Eagle's Medium (DMEM) medium, a Ham's F12 medium, a RPMI 1640 medium, a Fischer's medium, and a mixed medium thereof. In addition, the basal medium may be, for example, a medium (MEM, MEM-a, DMEM, DMEM high-glucose, PRIM, etc.) that does not contain synthetic proteins and is composed of inorganic salts, vitamins, amino acids, sugars, etc.Here, the basal medium may contain, as necessary, substances such as N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, ethanolamine, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), sodium pyruvate, vitamins, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. The solution in which the concentration of the components contained in the first medium is lower than that of the first medium, and preferably does not contain the components contained in the first medium, may be, for example, a third medium in which the concentration of the components contained in the first medium is lower than that of the first medium, and preferably does not contain the components contained in the first medium.

[0048] Fourth step: In the step of culturing the obtained second cells in a third medium in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, to obtain the third cells and the culture supernatant of the third cells, the third medium in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, may be a medium in which the concentration of all the components contained in the first medium is lower than that of the first medium, preferably does not contain all the components contained in the first medium, but is not limited thereto, and may be a medium in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium. The medium in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium, may be a medium in which the concentration of another specific component contained in the first medium is the same as or higher than that of the first medium, or a medium containing another specific component not contained in the first medium. Examples of the medium in the third medium in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, include a basal medium, a low-serum medium, and a serum-free medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. The basal medium may also be, for example, a medium (MEM, MEM-a, DMEM, DMEM high-glucose, PRIM, etc.) that is composed of inorganic salts, vitamins, amino acids, sugars, etc. that do not contain synthetic proteins.Here, the basal medium may contain substances such as N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, ethanolamine, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), sodium pyruvate, vitamins, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts, as necessary.

[0049] The time for culturing the obtained second cells in a third medium in which the concentration of a component contained in the first medium is lower than that of the first medium, preferably which does not contain a component contained in the first medium, is, for example, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 15 hours or more, or 20 hours or more.

[0050] The time for culturing the obtained second cells in a third medium having a lower concentration of a component contained in the first medium than the first medium, preferably not containing any component contained in the first medium, is, for example, 250 hours or less, 240 hours or less, 230 hours or less, 220 hours or less, 210 hours or less, 200 hours or less, 190 hours or less, 180 hours or less, 170 hours or less, 160 hours or less, 150 hours or less, 140 hours or less, 130 hours or less, 120 hours or less, 110 hours or less, 100 hours or less, 95 hours or less, 90 hours or less, 85 hours or less, 80 hours or less, 75 hours or less, 70 hours or less, 65 hours or less, 60 hours or less, 55 hours or less, 50 hours or less, 45 hours or less, 40 hours or less, 35 hours or less, 30 hours or less, or 25 hours or less.

[0051] The time for culturing the obtained second cells in a third medium having a lower concentration of a component contained in the first medium than that of the first medium, preferably a third medium containing no components contained in the first medium, is within a range specified by, for example, any of the above-mentioned lower limits and any of the above-mentioned upper limits.

[0052] The temperature at which the obtained second cells are cultured in a third medium in which the concentrations of the components contained in the first medium are lower than those in the first medium, preferably in a third medium that does not contain the components contained in the first medium, is, for example, 20° C. to 45° C., 30° C. to 40° C., or 35° C. to 38° C. The oxygen concentration during the culture is usually 20%, but is, for example, 0% to 40%, 5% to 35%, 10% to 30%, or 15% to 25%.

[0053] Fifth step: In the step of removing the culture supernatant of the third cells from the obtained third cells and the culture supernatant of the third cells to obtain the third cells, for example, the third cells and the culture supernatant of the third cells are separated from each other prior to removing the culture supernatant of the third cells. The separation of the third cells and the culture supernatant of the third cells is carried out, for example, by centrifugation. The centrifugation is carried out under conditions of, for example, 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The time for which the centrifugation is carried out is, for example, 1 minute to 20 minutes, 2 minutes to 15 minutes, 3 minutes to 10 minutes, or 4 minutes to 7 minutes. The separation may be carried out by filter filtration. The pore size of the filter is, for example, 1 μm or less, 0.9 μm or less, 0.8 μm or less, 0.7 μm or less, 0.6 μm or less, 0.5 μm or less, 0.4 μm or less, or 0.3 μm or less, and for example, 0.05 μm or more, 0.06 μm or more, 0.07 μm or more, 0.08 μm or more, 0.09 μm or more, 0.1 μm or more, or 0.2 μm or more, with 0.22 μm, 0.45 μm, etc. being preferred.

[0054] The step of removing the culture supernatant of the third cells from the obtained third cells and the culture supernatant of the third cells to obtain the third cells may include a step of washing the third cells prior to obtaining the third cells. In the step of washing the third cells, for example, the third cells are washed using a solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium. Here, the solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, may be a solution in which the concentration of all the components contained in the first medium is lower than that of the first medium, preferably does not contain all the components contained in the first medium, but is not limited thereto, and may be a solution in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium. The solution in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium, may be a solution in which the concentration of another specific component contained in the first medium is the same as or higher than that of the first medium, or a solution containing another specific component not contained in the first medium. The solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, includes, for example, a buffer, physiological saline, medium, etc. Examples of the medium include a basal medium, a low serum medium, a serum-free medium, etc. Examples of the basal medium include, for example, an IMDM medium, a Medium 199 medium, an Eagle's Minimum Essential Medium (EMEM) medium, an αMEM medium, a Dulbecco's modified Eagle's Medium (DMEM) medium, a Ham's F12 medium, a RPMI 1640 medium, a Fischer's medium, and a mixed medium thereof. In addition, the basal medium may be, for example, a medium (MEM, MEM-a, DMEM, DMEM high-glucose, PRIM, etc.) that does not contain synthetic proteins and is composed of inorganic salts, vitamins, amino acids, sugars, etc.Here, the basal medium may contain, as necessary, substances such as N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, ethanolamine, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), sodium pyruvate, vitamins, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. The solution in which the concentration of the components contained in the first medium is lower than that of the first medium, and preferably does not contain the components contained in the first medium, may be, for example, a fourth medium in which the concentration of the components contained in the first medium is lower than that of the first medium, and preferably does not contain the components contained in the first medium.

[0055] Sixth step: In the step of culturing the obtained third cell in a fourth medium in which the concentration of the component contained in the first medium is lower than that of the first medium, preferably not containing the component contained in the first medium, to obtain a fourth cell and a culture supernatant of the fourth cell, and to produce a fourth cell or a culture supernatant of the fourth cell, the fourth medium in which the concentration of the component contained in the first medium is lower than that of the first medium, preferably not containing the component contained in the first medium, may be a medium in which the concentration of all the components contained in the first medium is lower than that of the first medium, preferably not containing all the components contained in the first medium, but is not limited thereto, and may be a medium in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably not containing the specific component contained in the first medium. The medium in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably not containing the specific component contained in the first medium, may be a medium in which the concentration of another specific component contained in the first medium is the same as or higher than that of the first medium, or a medium containing another specific component not contained in the first medium. The fourth medium, which has a lower concentration of the components contained in the first medium than the first medium, preferably does not contain the components contained in the first medium, may be a basal medium, a low serum medium, a serum-free medium, etc. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. The basal medium may also be, for example, a medium (MEM, MEM-a, DMEM, DMEM high-glucose, PRIM, etc.) that does not contain synthetic proteins and is composed of inorganic salts, vitamins, amino acids, sugars, etc.Here, the basal medium may contain substances such as N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, ethanolamine, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), sodium pyruvate, vitamins, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts, as necessary.

[0056] The time for culturing the obtained third cells in a fourth medium in which the concentration of a component contained in the first medium is lower than that of the first medium, preferably which does not contain any component contained in the first medium, is, for example, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 15 hours or more, or 20 hours or more.

[0057] The time for culturing the obtained third cells in a fourth medium in which the concentration of a component contained in the first medium is lower than that of the first medium, preferably not containing any component contained in the first medium, is, for example, 250 hours or less, 240 hours or less, 230 hours or less, 220 hours or less, 210 hours or less, 200 hours or less, 190 hours or less, 180 hours or less, 170 hours or less, 160 hours or less, 150 hours or less, 140 hours or less, 130 hours or less, 120 hours or less, 110 hours or less, 100 hours or less, 95 hours or less, 90 hours or less, 85 hours or less, 80 hours or less, 75 hours or less, 70 hours or less, 65 hours or less, 60 hours or less, 55 hours or less, 50 hours or less, 45 hours or less, 40 hours or less, 35 hours or less, 30 hours or less, or 25 hours or less.

[0058] The time for culturing the obtained third cells in a fourth culture medium in which the concentration of a component contained in the first culture medium is lower than that of the first culture medium, preferably in which no component contained in the first culture medium is contained, is within a range specified by, for example, any of the above-mentioned lower limits and any of the above-mentioned upper limits.

[0059] The temperature at which the obtained third cells are cultured in a fourth medium in which the concentrations of the components contained in the first medium are lower than those in the first medium, preferably in a fourth medium that does not contain the components contained in the first medium, is, for example, 20° C. to 45° C., 30° C. to 40° C., or 35° C. to 38° C. The oxygen concentration during the culture is usually 20%, but is, for example, 0% to 40%, 5% to 35%, 10% to 30%, or 15% to 25%.

[0060] In obtaining the fourth cell and the culture supernatant of the fourth cell, and producing the fourth cell or the culture supernatant of the fourth cell, for example, prior to producing the fourth cell or the culture supernatant of the fourth cell, the fourth cell and the culture supernatant of the fourth cell are separated. The separation of the fourth cell and the culture supernatant of the fourth cell is performed, for example, by centrifugation. The centrifugation is performed, for example, under conditions of 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The time for which the centrifugation is performed is, for example, 1 minute to 20 minutes, 2 minutes to 15 minutes, 3 minutes to 10 minutes, or 4 minutes to 7 minutes. The separation may be performed by filter filtration. The pore size of the filter is, for example, 1 μm or less, 0.9 μm or less, 0.8 μm or less, 0.7 μm or less, 0.6 μm or less, 0.5 μm or less, 0.4 μm or less, or 0.3 μm or less, and for example, 0.05 μm or more, 0.06 μm or more, 0.07 μm or more, 0.08 μm or more, 0.09 μm or more, 0.1 μm or more, or 0.2 μm or more, with 0.22 μm, 0.45 μm, etc. being preferred.

[0061] The method for producing the cell culture supernatant of the present invention may include (seventh step) a step of removing the culture supernatant of the fourth cell from the obtained fourth cell and the culture supernatant of the fourth cell to obtain the fourth cell, and (eighth step) a step of culturing the obtained fourth cell in a fifth medium in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably not containing the components contained in the first medium, to obtain the fifth cell and the culture supernatant of the fifth cell, and to produce the fifth cell or the culture supernatant of the fifth cell. That is, the method for producing the cell culture supernatant of the present invention includes not only a method of using the culture supernatant of the obtained fourth cell as the culture supernatant of the producing cell, but also a method of removing the culture supernatant of the obtained fourth cell to obtain the fourth cell, further culturing the obtained fourth cell or a cell derived therefrom to obtain a culture supernatant of another cell and another cell, and using the culture supernatant of the another cell or another cell thus obtained as the culture supernatant of the producing cell or cell.

[0062] Seventh step: In the step of removing the culture supernatant of the fourth cell from the obtained fourth cell and the culture supernatant of the fourth cell to obtain the fourth cell, for example, the fourth cell and the culture supernatant of the fourth cell are separated from each other prior to removing the culture supernatant of the fourth cell. The separation of the fourth cell and the culture supernatant of the fourth cell is performed, for example, by centrifugation. The centrifugation is performed, for example, under conditions of 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The time for which the centrifugation is performed is, for example, 1 minute to 20 minutes, 2 minutes to 15 minutes, 3 minutes to 10 minutes, or 4 minutes to 7 minutes. The separation may be performed by filter filtration. The pore size of the filter is, for example, 1 μm or less, 0.9 μm or less, 0.8 μm or less, 0.7 μm or less, 0.6 μm or less, 0.5 μm or less, 0.4 μm or less, or 0.3 μm or less, and for example, 0.05 μm or more, 0.06 μm or more, 0.07 μm or more, 0.08 μm or more, 0.09 μm or more, 0.1 μm or more, or 0.2 μm or more, with 0.22 μm, 0.45 μm, etc. being preferred.

[0063] The step of removing the culture supernatant of the fourth cells from the obtained fourth cells and the culture supernatant of the fourth cells to obtain the fourth cells may include a step of washing the fourth cells prior to obtaining the fourth cells. In the step of washing the fourth cells, for example, the fourth cells are washed using a solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium. Here, the solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, may be a solution in which the concentration of all the components contained in the first medium is lower than that of the first medium, preferably does not contain all the components contained in the first medium, but is not limited thereto, and may be a solution in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium. The solution in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium, may be a solution in which the concentration of another specific component contained in the first medium is the same as or higher than that of the first medium, or a solution containing another specific component not contained in the first medium. The solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, includes, for example, a buffer, physiological saline, medium, etc. Examples of the medium include a basal medium, a low serum medium, a serum-free medium, etc. Examples of the basal medium include, for example, an IMDM medium, a Medium 199 medium, an Eagle's Minimum Essential Medium (EMEM) medium, an αMEM medium, a Dulbecco's modified Eagle's Medium (DMEM) medium, a Ham's F12 medium, a RPMI 1640 medium, a Fischer's medium, and a mixed medium thereof. In addition, the basal medium may be, for example, a medium (MEM, MEM-a, DMEM, DMEM high-glucose, PRIM, etc.) that does not contain synthetic proteins and is composed of inorganic salts, vitamins, amino acids, sugars, etc.Here, the basal medium may contain, as necessary, substances such as N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, ethanolamine, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), sodium pyruvate, vitamins, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. The solution in which the concentration of the components contained in the first medium is lower than that of the first medium, and preferably does not contain the components contained in the first medium, may be, for example, a fifth medium in which the concentration of the components contained in the first medium is lower than that of the first medium, and preferably does not contain the components contained in the first medium.

[0064] Eighth step: In the step of culturing the obtained fourth cell in a fifth medium in which the concentration of a component contained in the first medium is lower than that of the first medium, preferably not containing the component contained in the first medium, to obtain a fifth cell and a culture supernatant of the fifth cell, and to produce a fifth cell or a culture supernatant of the fifth cell, the fifth medium in which the concentration of a component contained in the first medium is lower than that of the first medium, preferably not containing the component contained in the first medium may be a medium in which the concentration of all components contained in the first medium is lower than that of the first medium, preferably not containing all components contained in the first medium, but is not limited thereto, and may be a medium in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably not containing the specific component contained in the first medium. The medium in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably not containing the specific component contained in the first medium, may be a medium in which the concentration of another specific component contained in the first medium is the same as or higher than that of the first medium, or a medium containing another specific component not contained in the first medium. The fifth medium, which has a lower concentration of the components contained in the first medium than the first medium, preferably does not contain the components contained in the first medium, may be a basal medium, a low serum medium, a serum-free medium, etc. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. The basal medium may also be, for example, a medium (MEM, MEM-a, DMEM, DMEM high-glucose, PRIM, etc.) that does not contain synthetic proteins and is composed of inorganic salts, vitamins, amino acids, sugars, etc.Here, the basal medium may contain substances such as N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, ethanolamine, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), sodium pyruvate, vitamins, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts, as necessary.

[0065] The time for culturing the obtained fourth cells in a fifth medium in which the concentration of components contained in the first medium is lower than that of the first medium, preferably not containing any components contained in the first medium, is, for example, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 15 hours or more, or 20 hours or more.

[0066] The time for culturing the obtained fourth cells in a fifth medium in which the concentration of a component contained in the first medium is lower than that of the first medium, preferably containing no components contained in the first medium, is, for example, 250 hours or less, 240 hours or less, 230 hours or less, 220 hours or less, 210 hours or less, 200 hours or less, 190 hours or less, 180 hours or less, 170 hours or less, 160 hours or less, 150 hours or less, 140 hours or less, 130 hours or less, 120 hours or less, 110 hours or less, 100 hours or less, 95 hours or less, 90 hours or less, 85 hours or less, 80 hours or less, 75 hours or less, 70 hours or less, 65 hours or less, 60 hours or less, 55 hours or less, 50 hours or less, 45 hours or less, 40 hours or less, 35 hours or less, 30 hours or less, or 25 hours or less.

[0067] The time for culturing the obtained fourth cells in a fifth medium having a lower concentration of a component contained in the first medium than the first medium, preferably not containing any component contained in the first medium, is within a range specified by, for example, any of the above-mentioned lower limits and any of the above-mentioned upper limits.

[0068] The temperature at which the obtained fourth cells are cultured in a fifth medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably which does not contain any components contained in the first medium, is, for example, 20° C. to 45° C., 30° C. to 40° C., or 35° C. to 38° C. The oxygen concentration during the culture is usually 20%, but is, for example, 0% to 40%, 5% to 35%, 10% to 30%, or 15% to 25%.

[0069] In obtaining the fifth cell and the culture supernatant of the fifth cell, and producing the fifth cell or the culture supernatant of the fifth cell, for example, prior to producing the fifth cell or the culture supernatant of the fifth cell, the fifth cell and the culture supernatant of the fifth cell are separated. The separation of the fifth cell and the culture supernatant of the fifth cell is performed, for example, by centrifugation. The centrifugation is performed, for example, under conditions of 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The time for which the centrifugation is performed is, for example, 1 minute to 20 minutes, 2 minutes to 15 minutes, 3 minutes to 10 minutes, or 4 minutes to 7 minutes. The separation may be performed by filter filtration. The pore size of the filter is, for example, 1 μm or less, 0.9 μm or less, 0.8 μm or less, 0.7 μm or less, 0.6 μm or less, 0.5 μm or less, 0.4 μm or less, or 0.3 μm or less, and for example, 0.05 μm or more, 0.06 μm or more, 0.07 μm or more, 0.08 μm or more, 0.09 μm or more, 0.1 μm or more, or 0.2 μm or more, with 0.22 μm, 0.45 μm, etc. being preferred.

[0070] The method for producing the culture supernatant of the cell of the present invention may include (ninth step) a step of removing the culture supernatant of the fifth cell from the obtained fifth cell and the culture supernatant of the fifth cell to obtain the fifth cell, and (tenth step) a step of culturing the obtained fifth cell in a sixth medium in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably not containing the components contained in the first medium, to obtain the sixth cell and the culture supernatant of the sixth cell, and to produce the sixth cell or the culture supernatant of the sixth cell. That is, the method for producing the culture supernatant of the cell of the present invention includes not only a method of using the culture supernatant of the obtained fifth cell as the culture supernatant of the producing cell, but also a method of removing the culture supernatant of the obtained fifth cell to obtain the fifth cell, further culturing the obtained fifth cell or a cell derived therefrom to obtain a culture supernatant of another cell and another cell, and using the culture supernatant of the another cell or another cell thus obtained as the culture supernatant of the producing cell or cell.

[0071] Ninth step: In the step of removing the culture supernatant of the fifth cell from the obtained fifth cell and the culture supernatant of the fifth cell to obtain the fifth cell, for example, the fifth cell and the culture supernatant of the fifth cell are separated from each other prior to removing the culture supernatant of the fifth cell. The separation of the fifth cell and the culture supernatant of the fifth cell is performed, for example, by centrifugation. The centrifugation is performed, for example, under conditions of 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The time for which the centrifugation is performed is, for example, 1 minute to 20 minutes, 2 minutes to 15 minutes, 3 minutes to 10 minutes, or 4 minutes to 7 minutes. The separation may be performed by filter filtration. The pore size of the filter is, for example, 1 μm or less, 0.9 μm or less, 0.8 μm or less, 0.7 μm or less, 0.6 μm or less, 0.5 μm or less, 0.4 μm or less, or 0.3 μm or less, and for example, 0.05 μm or more, 0.06 μm or more, 0.07 μm or more, 0.08 μm or more, 0.09 μm or more, 0.1 μm or more, or 0.2 μm or more, with 0.22 μm, 0.45 μm, etc. being preferred.

[0072] The step of removing the culture supernatant of the fifth cells from the obtained fifth cells and the culture supernatant of the fifth cells to obtain the fifth cells may include a step of washing the fifth cells prior to obtaining the fifth cells. In the step of washing the fifth cells, for example, the fifth cells are washed using a solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium. Here, the solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, may be a solution in which the concentration of all the components contained in the first medium is lower than that of the first medium, preferably does not contain all the components contained in the first medium, but is not limited thereto, and may be a solution in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium. The solution in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably does not contain the specific component contained in the first medium, may be a solution in which the concentration of another specific component contained in the first medium is the same as or higher than that of the first medium, or a solution containing another specific component not contained in the first medium. The solution in which the concentration of the components contained in the first medium is lower than that of the first medium, preferably does not contain the components contained in the first medium, includes, for example, a buffer, physiological saline, medium, etc. Examples of the medium include a basal medium, a low serum medium, a serum-free medium, etc. Examples of the basal medium include, for example, an IMDM medium, a Medium 199 medium, an Eagle's Minimum Essential Medium (EMEM) medium, an αMEM medium, a Dulbecco's modified Eagle's Medium (DMEM) medium, a Ham's F12 medium, a RPMI 1640 medium, a Fischer's medium, and a mixed medium thereof. In addition, the basal medium may be, for example, a medium (MEM, MEM-a, DMEM, DMEM high-glucose, PRIM, etc.) that does not contain synthetic proteins and is composed of inorganic salts, vitamins, amino acids, sugars, etc.Here, the basal medium may contain, as necessary, substances such as N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, sodium selenite, ethanolamine, collagen precursor, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipid, amino acid, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acid (NEAA), sodium pyruvate, vitamin, low molecular weight compound, antibiotic, antioxidant, pyruvic acid, buffer, inorganic salt, etc. The solution in which the concentration of the component contained in the first medium is lower than that of the first medium, preferably does not contain the component contained in the first medium, may be, for example, a medium in which the concentration of the component contained in the first medium is lower than that of the first medium, preferably does not contain the component contained in the first medium, and is used for the next culture.

[0073] Tenth step: In the step of culturing the obtained fifth cell in a sixth medium in which the concentration of the component contained in the first medium is lower than that of the first medium, preferably not containing the component contained in the first medium, to obtain the sixth cell and the culture supernatant of the sixth cell, and to produce the sixth cell or the culture supernatant of the sixth cell, the sixth medium in which the concentration of the component contained in the first medium is lower than that of the first medium, preferably not containing the component contained in the first medium may be a medium in which the concentration of all the components contained in the first medium is lower than that of the first medium, preferably not containing all the components contained in the first medium, but is not limited thereto, and may be a medium in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably not containing the specific component contained in the first medium. The medium in which the concentration of a specific component contained in the first medium is lower than that of the first medium, preferably not containing the specific component contained in the first medium, may be a medium in which the concentration of another specific component contained in the first medium is the same as or higher than that of the first medium, or a medium containing another specific component not contained in the first medium. The sixth medium, which has a lower concentration of the components contained in the first medium than the first medium, preferably does not contain the components contained in the first medium, may be a basal medium, a low serum medium, a serum-free medium, etc. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. The basal medium may also be, for example, a medium (MEM, MEM-a, DMEM, DMEM high-glucose, PRIM, etc.) that does not contain synthetic proteins and is composed of inorganic salts, vitamins, amino acids, sugars, etc.Here, the basal medium may contain substances such as N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, ethanolamine, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), sodium pyruvate, vitamins, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts, as necessary.

[0074] The time for culturing the obtained fifth cells in a sixth medium in which the concentration of components contained in the first medium is lower than that of the first medium, preferably not containing any components contained in the first medium, is, for example, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 15 hours or more, or 20 hours or more.

[0075] The time for culturing the obtained fifth cells in a sixth medium having a lower concentration of a component contained in the first medium than the first medium, preferably containing no components contained in the first medium, is, for example, 250 hours or less, 240 hours or less, 230 hours or less, 220 hours or less, 210 hours or less, 200 hours or less, 190 hours or less, 180 hours or less, 170 hours or less, 160 hours or less, 150 hours or less, 140 hours or less, 130 hours or less, 120 hours or less, 110 hours or less, 100 hours or less, 95 hours or less, 90 hours or less, 85 hours or less, 80 hours or less, 75 hours or less, 70 hours or less, 65 hours or less, 60 hours or less, 55 hours or less, 50 hours or less, 45 hours or less, 40 hours or less, 35 hours or less, 30 hours or less, or 25 hours or less.

[0076] The time for culturing the obtained fifth cells in a sixth medium having a lower concentration of a component contained in the first medium than the first medium, preferably a sixth medium not containing any component contained in the first medium, is within a range specified by, for example, any of the above-mentioned lower limits and any of the above-mentioned upper limits.

[0077] The temperature at which the obtained fifth cells are cultured in a sixth medium in which the concentrations of the components contained in the first medium are lower than those in the first medium, preferably which does not contain the components contained in the first medium, is, for example, 20°C to 45°C, 30°C to 40°C, or 35°C to 38°C.

[0078] In obtaining the sixth cell and the culture supernatant of the sixth cell, and producing the sixth cell or the culture supernatant of the sixth cell, for example, prior to producing the sixth cell or the culture supernatant of the sixth cell, the sixth cell and the culture supernatant of the sixth cell are separated. The sixth cell and the culture supernatant of the sixth cell are separated, for example, by centrifugation. The centrifugation is performed under conditions of, for example, 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The time for which the centrifugation is performed is, for example, 1 minute to 20 minutes, 2 minutes to 15 minutes, 3 minutes to 10 minutes, or 4 minutes to 7 minutes. The separation may be performed by filter filtration. The pore size of the filter is, for example, 1 μm or less, 0.9 μm or less, 0.8 μm or less, 0.7 μm or less, 0.6 μm or less, 0.5 μm or less, 0.4 μm or less, or 0.3 μm or less, and for example, 0.05 μm or more, 0.06 μm or more, 0.07 μm or more, 0.08 μm or more, 0.09 μm or more, 0.1 μm or more, or 0.2 μm or more, with 0.22 μm, 0.45 μm, etc. being preferred.

[0079] The method for producing the cell culture supernatant of the present invention may include (11th step) a step of removing the culture supernatant of the sixth cell from the obtained sixth cell and the culture supernatant of the sixth cell to obtain the sixth cell, and (12th step) a step of culturing the obtained sixth cell in a seventh medium having a lower concentration of the components contained in the first medium than the first medium, preferably a seventh medium not containing the components contained in the first medium, to obtain the seventh cell and the culture supernatant of the seventh cell, and to produce the seventh cell or the culture supernatant of the seventh cell. That is, the method for producing the cell culture supernatant of the present invention includes not only a method of using the culture supernatant of the obtained sixth cell as the culture supernatant of the producing cell, but also a method of removing the culture supernatant of the obtained sixth cell to obtain the sixth cell, further culturing the obtained sixth cell or a cell derived therefrom to obtain a culture supernatant of another cell and another cell, and using the obtained culture supernatant of the another cell or another cell to obtain the producing cell or cell culture supernatant. Such a culture supernatant of another cell can be obtained by the same method as the method described above in detail. Therefore, in the method of producing a cell or a culture supernatant of a cell of the present invention, the cell or the culture supernatant of a cell to be produced may be the culture supernatant of the obtained fourth cell or the culture supernatant of the fourth cell, the culture supernatant of the obtained fifth cell or the culture supernatant of the fifth cell, the culture supernatant of the obtained sixth cell or the culture supernatant of the sixth cell, or the culture supernatant of the obtained seventh cell or the culture supernatant of the seventh cell, but is not limited thereto, and may also be the culture supernatant of another cell obtained by a similar method.

[0080] Uses of cells or cell culture supernatants: The cells or cell culture supernatant produced by the method for producing the cell or cell culture supernatant of the present invention can be used as, for example, a pharmaceutical composition, a cosmetic composition, a food composition, a regenerative medicine product, or a specific cell processed product, or can be included in these. That is, the cells or cell culture supernatant produced by the method for producing the cell or cell culture supernatant of the present invention is intended for use as, for example, a pharmaceutical composition, a cosmetic composition, a food composition, a regenerative medicine product, or a specific cell processed product. In addition, the cells or cell culture supernatant produced by the method for producing the cell or cell culture supernatant of the present invention is intended for inclusion in, for example, a pharmaceutical composition, a cosmetic composition, a food composition, a regenerative medicine product, or a specific cell processed product. Examples of pharmaceutical compositions include compositions for treating or preventing cancer, demyelinating diseases, eye diseases, eye disorders, and the like. Examples of cosmetic compositions include compositions for use in skin care, such as compositions for improving wrinkles, age spots, firmness, and the like of the skin, and compositions for promoting skin regeneration.

[0081] As shown in the examples below, the method of the present invention can unexpectedly produce a culture supernatant containing a high concentration of a cytokine called HGF (Hepatocyte Growth Factor). Therefore, the cell culture supernatant produced by the method of the present invention for producing a cell culture supernatant can be used, for example, to treat HGF deficiency in a subject. Here, the subject can be, for example, a vertebrate. The vertebrate can be, for example, a mammal such as a mouse, a rat, a rabbit, a pig, a cow, a monkey, or a human. The mammal is preferably a human. The subject can be of any age, including infants, young people, adolescents, adults, and the elderly. As an advantage of increasing HGF, it is known that intrathecal administration of rhHGF delays the progression of the disease in ALS model rats (J Neuropathol Exp Neurol. 2007 Nov;66(11):1037-44.). In addition, administration of HGF plasmid is known to improve peripheral vascular disorders in diabetic rat models (Circulation. 2001 Nov 6;104(19):2344-50.). Intrathecal administration of rhHGF to common marmosets with spinal cord injuries is known to promote functional recovery in the injured area (PLoS One. 2011;6(11):e27706.). Furthermore, it is known that administration of HGF alleviates insulin resistance and lipid accumulation in high-fat diet-fed mice (J Diabetes Investig. 2019 Mar;10(2):251-260.). Therefore, the cell culture supernatant produced by the method of producing a cell culture supernatant of the present invention can be used to treat diseases such as ALS, diabetes, peripheral vascular disease, spinal cord injury, insulin resistance, and lipid accumulation. Here, examples of treatment include treatment and prevention. Examples of treatment include complete recovery as well as alleviation of symptoms.

[0082] The cells or cell culture supernatant produced by the method for producing the cells or cell culture supernatant of the present invention may be administered by any administration method suitable for the dosage form, for example, oral administration, intravenous administration, subcutaneous administration, transdermal administration, intramuscular administration, intra-articular administration, nasal administration, intraperitoneal administration, direct injection into target tissue, inhalation administration, enteral administration, enema administration, tube feeding, etc. The dosage of the cells or cell culture supernatant produced by the method for producing the cells or cell culture supernatant of the present invention may vary depending on the age, weight, health condition, etc. of the subject. The cells or cell culture supernatant produced by the method for producing the cells or cell culture supernatant of the present invention may be administered once per day or multiple times per day, and the administration frequency can be, for example, 1 to 100 times per month. EXAMPLES

[0083] Example 1: We prepared a medium containing FBS, human albumin, FGF2, etc., which are common growth activating factors used in cell culture, and actually cultured cells. This example shows experimental data regarding FBS. Human adipose tissue-derived MSCs (mesenchymal stem cells) (P2-1) were cultured for several days in a medium (DMEM) containing FBS until they reached confluence. After that, the medium was completely collected (sample circle 1), washed with PBS, and then cultured in a basal medium (DMEM medium) that did not contain growth factors. After 24 hours, the medium was collected (sample circle 2), washed again with PBS, cultured in a basal medium (DMEM medium), and collected 24 hours later (sample circle 3). Similarly, the collection process using a basal medium (DMEM medium) was repeated again (sample circle 4). The MSCs were cultured at 37°C. The oxygen concentration during cell culture was 20%.

[0084] By carrying out the above operations, we were able to obtain samples with the following conditions. Sample circle 1: Medium containing FBS (positive control) Sample circle 2: Basal medium after the first wash (basal medium does not contain FBS) Sample circle 3: Basal medium after the second wash (basal medium does not contain FBS) Sample circle 4: Basal medium after the third wash (basal medium does not contain FBS). These samples were subjected to ELISA analysis for bovine albumin (no cross-reactivity with human albumin) contained in FBS, and by examining whether bovine albumin could be detected in samples 2 to 4, it was examined how many samples bovine albumin could be detected in. Note that since human MSCs were used in this example, albumin (human type) secreted from the original cells themselves was not detected by the ELISA kit (which reacts specifically with bovine type).

[0085] The above experimental steps are described in more detail below. First, human adipose tissue-derived MSCs (passage number 3) were prepared. The dish contained 10,000 cells / cm 2 The cells were seeded at 100°C for 24 hours. Next, FBS was added to the DMEM medium as necessary to give a total of six FBS concentrations: 0 vol%, 1 vol%, 3 vol%, 5 vol%, 10 vol%, or 20 vol%, and the cells were cultured in the resulting medium (L-glutamine was added to a final concentration of 1% by mass). On the third day, the medium was replaced with the same medium. Cells reached confluence on day 7 in FBS-containing conditions. (1) The medium was completely removed from the 6-well plate. A portion was collected and designated as sample 1. (2) PBS was added to thoroughly wash the cells in the dish. (3) The PBS was completely removed. (4) Fresh DMEM medium (containing L-glutamine to a final concentration of 1% by mass) was added. After 24 hours, steps (1) to (4) above were repeated. A portion was collected and designated as sample circle 2. After 24 hours, steps (1) to (4) above were repeated. A portion was collected and designated as sample circle 3. After 24 hours, steps (1) to (4) above were repeated. A portion was collected and designated as sample 4. After collection, samples 1 to 4 were centrifuged (1,300 rpm, 5 min), and only the upper layer was collected and stored at -80°C. Various samples were quantified by ELISA test.

[0086] Figure 1 shows an image of the cultured cells at the time of collection of sample circle 4. As shown in Figure 1, it can be seen that the cells remained viable under all conditions. However, if the extraction method induces cell death through excessive stress or heat, there is a possibility that cell survival after extraction will not be observed in the first place. In that case, there will be no cells remaining, and the operation of collecting samples four times in total would be meaningless.

[0087] The results of the ELISA test after sample recovery are shown in Figures 2 to 4. In samples circle 1 and circle 2 in Figure 3 and samples circle 1 and circle 2 in Figure 4, the bovine albumin concentration is indicated as 1500 ng / mL, but more precisely, these are high concentrations that exceed the measurement range of the ELISA kit and are above the detection limit.

[0088] As shown in Figures 3 and 4, a high concentration of bovine albumin was detected in sample 1 obtained from cells cultured in a medium containing FBS.

[0089] As shown in Figures 3 and 4, a certain amount of bovine albumin also tended to be detected at the sample circle 2 stage. This is believed to be due to the bovine albumin once taken up by the cells being released back into the medium. When cultured in a medium containing 1 vol%, 3 vol%, or 5 vol% FBS, the amount of bovine albumin at the sample circle 2 stage was found to be reduced compared to when cultured in a medium containing 10 vol% or 20 vol% FBS, but a similar tendency was observed.

[0090] As shown in Figures 3 and 4, bovine albumin was not detected in samples 3 and 4 when FBS was contained at 10% by volume or more, and it was suggested that the amount of bovine albumin in samples 3 and 4 was reduced by repeated washing (medium replacement). It should be noted that when samples 3 and 4 were cultured in a medium containing 5% by volume or less of FBS, a similar tendency was observed, although a certain amount of bovine albumin was detected. From this, it is considered that bovine albumin is efficiently taken up into the cells by adding a low concentration of FBS to the medium, and as a result, release over a relatively long period of time (sample 4) was confirmed.

[0091] Examples 2-3: Next, Examples 2 and 3 were carried out according to the procedure shown in Figure 5. In Examples 2 and 3, the cell seeding density was 10,000 cells / cm 2 The plate used was a 6-well plate, and ADSC-4 medium (2 ml / well) was used as the seeding condition. The culture conditions were as follows: medium replacement with proliferation medium (ADSC-4 + human recombinant albumin (2,000 ug / ml) + FGF2 (50 ng / ml) + 0.5% Penicilling / Streptomycin) was performed on the 1st and 4th days after seeding. Sampling was started on the 7th day after seeding. The oxygen concentration during cell culture was 20%. The temperature during cell culture was 37°C.

[0092] Example 2: The culture medium on day 7 was sampled to obtain a pre-extraction sample (before extraction). After completely removing the medium from the pre-extraction sample, the sample was washed with PBS and DMEM with-high glucose + LGln medium (2ml / well) was added and left for one day. This was then sampled to obtain a day 1 sample (day 1). After completely removing the medium from the day 1 sample, DMEM with-high glucose + LGln medium (2ml / well) was added and left for one day. This was then sampled to obtain a day 2 sample (day 2). After completely removing the medium from the day 2 sample, DMEM with-high glucose + LGln medium (2ml / well) was added and left for one day. This was then sampled to obtain a day 3 sample (day 3).

[0093] The results for the umbilical cord stem cells are shown in Table 1 below. [Table 1] Before extraction, the existing medium was completely removed, followed by washing with PBS, and then DMEM high-glucose medium was added. During extraction, DMEM was completely removed at each collection timing from day 1 onwards, and then new DMEM high-glucose medium was added.

[0094] The results for adipose stem cells are shown in Table 2 below. [Table 2] Before extraction, the existing medium was completely removed, followed by washing with PBS, and then DMEM high-glucose medium was added. During extraction, DMEM was completely removed at each collection timing from day 1 onwards, and then new DMEM high-glucose medium was added.

[0095] Example 3: The culture medium on day 7 was sampled to obtain a pre-extraction sample (before extraction). After the medium was completely removed from the pre-extraction sample, the sample was washed with PBS, and DMEM with-high glucose + LGln medium (2ml / well) was added and two days later, a sample was obtained for the day 2 sample (day 2). After the medium was completely removed from the day 1 sample, DMEM with-high glucose + LGln medium (2ml / well) was added and two days later, a sample was obtained for the day 4 sample (day 4). After the medium was completely removed from the day 2 sample, DMEM with-high glucose + LGln medium (2ml / well) was added and two days later, a sample was obtained for the day 6 sample (day 6).

[0096] The results for umbilical cord stem cells are shown in Table 3 below. [Table 3] Before extraction, the existing medium was completely removed, followed by washing with PBS, and then DMEM high-glucose medium was added. During extraction, DMEM was completely removed at each collection timing from the second day onwards, and then new DMEM high-glucose medium was added.

[0097] The adipose stem cell results are shown in Table 4 below. [Table 4] Before extraction, the existing medium was completely removed, followed by washing with PBS, and then DMEM high-glucose medium was added. During extraction, DMEM was completely removed at each collection timing from the second day onwards, and then new DMEM high-glucose medium was added.

[0098] As shown in the results of Tables 1 to 4 above, the recombinant albumin contained in the culture medium was at a high concentration before the start of extraction, so the value was above the detection limit in the evaluation by the ELISA method. In addition, when the medium was switched to basal medium (DMEM) after PBS washing, high concentrations of albumin were detected again the next day or two days later. Furthermore, when the basal medium was replaced and analyzed the next day or two days later, about 5-20% albumin was detected. After that, in the third extraction, the remaining amount of albumin was in the range of 85-319 ng / ml, and this concentration range is close to the albumin (about 80 ng / ml) secreted from cells during human MSC culture, referring to the previously published paper Fig. 2A (Biotechnology Journal, DOI: 10.1002 / biot.202100096), and can be considered as the background of endogenous albumin.

[0099] From the above, it was shown that recombinant proteins, etc., are taken up to a certain extent by cultured cells during cell culture, and although they are apparently washed out by washing procedures such as PBS, the proteins taken up once are re-released into the medium with continued culture. In order to suppress the re-release of recombinant proteins taken up by cells to less than 5%, it is necessary to set re-release conditions at least once in a medium that does not contain the recombinant protein.

[0100] Example 4: Under the following conditions, tests were carried out in the steps shown in conditions 1 to 3 in FIG. 6, and samples were collected. Cell seeding density: 10,000 cells / cm 2 Plate: 6 well plate Culture conditions: Cell proliferation was performed using ADSC-4 medium (2 ml / well). (Note that, as shown in FIG. 6, recombinant VEGF protein was added at 1000 pg / ml under some conditions.) Extraction conditions: DMEM with high-glucose + L-Gln (2 ml / well)

[0101] More specifically, under conditions 1 to 3, samples were collected as follows. Condition 1: When the cells reached confluence in the growth culture using ADSC-4 medium (the same number of days of culture period for conditions 1 to 3), the entire medium was removed and the cells were washed once with PBS. Then, the entire PBS was removed and the extraction medium (medium shown in the above extraction conditions) was added. After 2 days, the supernatant was collected. To remove impurities such as cell debris from the supernatant, the cells were centrifuged (1,000 rpm, 3 minutes), and only the supernatant was collected as a sample to obtain the sample (condition 1). Condition 2: When the cells reached confluence in the growth culture using ADSC-4 medium supplemented with recombinant VEGF protein (the same number of days of culture period for conditions 1 to 3), the entire medium was removed and the cells were washed once with PBS. Then, the entire PBS was removed and the extraction medium (medium shown in the extraction conditions above) was added. After 2 days, the supernatant was collected. To remove impurities such as cell debris from the supernatant, the cells were centrifuged (1,000 rpm, 3 minutes), and only the supernatant was collected as a sample to obtain the sample (condition 2). Condition 3: When the growth culture using ADSC-4 medium supplemented with recombinant VEGF protein reached confluence (the same number of days of culture period for conditions 1 to 3), the entire medium was removed and washed once with PBS. Then, the entire PBS was removed, the extraction medium (medium shown in the above extraction conditions) was added, and the entire medium was immediately collected (sample A). Then, the extraction medium (medium shown in the above extraction conditions) was added again, and the supernatant was collected after 2 days (sample B). In order to remove impurities such as cell debris in the supernatant, samples A and B were centrifuged (1,000 rpm, 3 minutes), and only the supernatant was collected as a sample, and sample (condition 3, immediately after) and sample (condition 3, 2 days later) were obtained, respectively.

[0102] The VEGF protein concentration in these resulting samples was measured, and the results are shown in FIG. Condition circle 1 in Figure 7 indicates that there is almost no VEGF protein secreted by the cells themselves, and condition circle 2 in Figure 7 indicates that the use of recombinant VEGF protein is a prerequisite for bringing the protein into the supernatant. Furthermore, condition circle 3 in Figure 7 shows that even when DMEM medium was added immediately after PBS washing, no VEGF protein was detected (condition circle 3, immediately after), whereas high concentrations of VEGF protein were detected after two days (condition circle 3, two days later). These results indicate that unnecessary components such as recombinant VEGF protein are difficult to remove even by washing with PBS after being taken up into cells, and are released back into the medium after a certain period of culture. It was previously believed that unwanted components such as recombinant proteins derived from the culture medium could be washed away by washing with PBS or the like. However, the above examples demonstrated that substances taken up into cells are not washed away by a momentary washing operation, but are actually released into the culture medium over time, and therefore it was found that a period of release in the culture medium is required. As described above, the method of the present invention removes substances that have been taken up by cells and then re-released; these substances have not been removed by conventional removal methods such as washing with PBS, and the method of the present invention has a completely different technical concept from conventional removal methods such as washing with PBS.

[0103] Example 5: The process shown in FIG. 8 was tested under the following conditions, and samples were collected. Cell seeding density: 10,000 cells / cm 2 Plate: 6 well plate Culture conditions: Adipose or umbilical cord MSCs were expanded and cultured using Ajinomoto Kohjin Bio's StemFit for MSC (2 ml / well). The difference between the patented method (the method of the present invention) and the conventional method is the presence or absence of a 24-hour washing step using a basal medium (DMEM with high-glucose + L-Gln). The extraction period for the supernatant was performed under the same conditions.

[0104] The concentrations of HGF (Hepatocyte Growth Factor) and IL-6 in each of the resulting supernatants were measured, and the results are shown in Figures 9 and 10. Unexpectedly, it was found that the amount of HGF (Hepatocyte Growth Factor) secreted increased when the patented method (the method of the present invention) was used, as shown in Figure 9. In other words, when comparing the conventional method (supernatant extracted for two days after washing with PBS) with the method of the present invention (a method in which a step of exposing to basal medium for one day is added to the conventional washing step), it was found that the concentration of the cytokine HGF in the supernatant increased. As shown in Figure 10, even when the patented method (the method of the present invention) was used, the amount of IL-6 secreted was the same, and it is believed that there was no change in the number of cells targeted in the preliminary step, so it was clear that the increase in the amount of HGF secreted was not due to a change in the number of cells targeted in the preliminary step, but due to the method of the present invention. In other words, if cell proliferation is considered to progress during the washing period of the method of the present invention, the total number of cells at the time of the extraction operation would increase, and generally, the IL-6 concentration would also increase. However, based on the above verification results, it is believed that the basal medium such as DMEM used for cell washing does not have cell proliferation activity, and extreme cell proliferation does not occur during the washing operation period, so it was clear that the increase in the amount of HGF secreted was not due to cell proliferation during the washing operation period, but due to the method of the present invention.

Claims

1. 1. A method for producing a cell or a cell culture supernatant, comprising: Culturing the cells in a serum-free first medium to obtain first cells; Culturing the obtained first cells in a second medium having a lower concentration of a component contained in the first medium than the first medium, and obtaining second cells and a culture supernatant of the second cells; removing the culture supernatant of the second cells from the obtained second cells and the culture supernatant of the second cells to obtain the second cells; and a step of culturing the obtained second cells in a third medium having a lower concentration of a component contained in the first medium than in the first medium, obtaining the third cells and a culture supernatant of the third cells, and producing the third cells or the culture supernatant of the third cells. A method comprising:

2. 1. A method for producing a cell or a cell culture supernatant, comprising: Culturing the cells in the first medium to obtain the first cells; Culturing the obtained first cells in a second medium having a lower concentration of a component contained in the first medium than the first medium, and obtaining second cells and a culture supernatant of the second cells; removing the culture supernatant of the second cells from the obtained second cells and the culture supernatant of the second cells to obtain the second cells; Culturing the obtained second cells in a third medium having a lower concentration of a component contained in the first medium than that of the first medium, and obtaining third cells and a culture supernatant of the third cells; removing the culture supernatant of the third cells from the obtained third cells and the culture supernatant of the third cells to obtain the third cells; and a step of culturing the obtained third cell in a fourth medium having a lower concentration of a component contained in the first medium than in the first medium to obtain a fourth cell and a culture supernatant of the fourth cell, thereby producing the fourth cell or the culture supernatant of the fourth cell. A method comprising:

3. The method according to claim 2, comprising the steps of: removing the culture supernatant of the fourth cell from the obtained fourth cell and the culture supernatant of the fourth cell to obtain the fourth cell; and culturing the obtained fourth cell in a fifth medium having a lower concentration of components contained in the first medium than in the first medium to obtain the fifth cell and the culture supernatant of the fifth cell, thereby producing the fifth cell or the culture supernatant of the fifth cell.

4. The method according to claim 3, comprising the steps of: removing the culture supernatant of the fifth cell from the obtained fifth cell and the culture supernatant of the fifth cell to obtain the fifth cell; and culturing the obtained fifth cell in a sixth medium having a lower concentration of components contained in the first medium than in the first medium to obtain the sixth cell and the culture supernatant of the sixth cell, thereby producing the sixth cell or the culture supernatant of the sixth cell.

5. The method of claim 2, wherein the cells are adipose tissue-derived stem cells.

6. The method of claim 2, wherein the cell is a human cell.

7. The method according to claim 2, wherein the component contained in the first culture medium is a cell growth factor.

8. The method of claim 7, wherein the cell growth factor is a non-human protein.

9. The method according to claim 7, wherein the cell growth factor is serum or plasma.

10. The method according to claim 7, wherein the first culture medium contains 0.5% by volume or more of a cell growth factor.

11. 3. The method of claim 2, wherein the first medium comprises 0.0000001% to 1% by volume of the recombinant protein.

12. The method according to claim 10, wherein the first culture medium contains 8% or more by volume of a cell growth factor.

13. The method according to claim 2, wherein the second medium, which has a lower concentration of a component contained in the first medium than the first medium, is a basal medium.

14. The method according to claim 2, wherein the third medium, which has a lower concentration of a component contained in the first medium than the first medium, is a basal medium.

15. The method according to claim 2, wherein the fourth medium, which has a lower concentration of a component contained in the first medium than the first medium, is a basal medium.

16. The method according to claim 3, wherein the fifth medium, in which the concentration of a component contained in the first medium is lower than that of the first medium, is a basal medium.

17. The method according to claim 4, wherein the sixth medium, in which the concentration of a component contained in the first medium is lower than that of the first medium, is a basal medium.

18. The method according to claim 2, wherein the time for culturing the obtained first cells in a second medium having a component contained in the first medium at a lower concentration than that of the first medium is 1 hour to 192 hours.

19. The method according to claim 2, wherein the time for culturing the obtained second cells in a third medium having a component contained in the first medium at a lower concentration than that of the first medium is 1 hour to 192 hours.

20. The method according to claim 2, wherein the obtained third cells are cultured in a fourth medium having a component contained in the first medium at a lower concentration than that in the first medium for a period of 1 hour to 192 hours.

21. The method according to claim 3, wherein the obtained fourth cells are cultured in a fifth medium having a lower concentration of a component contained in the first medium than in the first medium for a period of 1 hour to 192 hours.

22. The method according to claim 4, wherein the obtained fifth cells are cultured in a sixth medium having a component contained in the first medium at a lower concentration than that of the first medium for a period of 1 hour to 192 hours.

23. The method of claim 2, further comprising washing the first cells prior to obtaining the first cells.

24. The method of claim 2, further comprising washing the second cells prior to obtaining the second cells.

25. The method of claim 2, further comprising the step of washing the third cells prior to obtaining the third cells.

26. The method of claim 3, further comprising washing the fourth cells prior to obtaining the fourth cells.

27. The method of claim 4, further comprising washing the fifth cell prior to obtaining the fifth cell.

28. The method according to claims 1 to 27, wherein the cells or cell culture supernatant are intended for use as or inclusion in a pharmaceutical composition, a cosmetic composition, a food composition, a regenerative medicine product, or a specific cell processed product.

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