Methods for producing supernatant cell cultures

Abstract

The invention proposes a method for producing cells or cell supernatants in which the content of a component such as a cell growth factor or similar substance in the medium is reduced, even when the cells are cultured in a medium containing a cell growth factor or similar substance.The method for producing cells or cell supernatant includes: firstly, culturing cells in a serum-free medium to obtain the first cells; secondly, culturing these obtained first cells in a second medium containing a lower concentration of the component in the first medium than in the first medium to obtain the second cells and the supernatant of the second cells; thirdly, removing the supernatant of the second cells from the obtained second cells and the supernatant of the second cells to obtain the second cells; and thirdly, culturing the obtained second cells in a third medium containing a lower concentration of the component in the first medium than in the first medium to obtain the third cells and the supernatant of the third cells, thus producing the third cells or the supernatant of the third cells.

Description

Method for producing cell culture supernatant

[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, wherein the content of components contained in the medium, such as the cell growth factor, is reduced.

[0002] Mesenchymal stem cells are somatic stem cells found in bone marrow, synovium, adipose tissue, 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 treatments for damaged meniscus and cartilage (Non-Patent Document 2). Furthermore, because mesenchymal stem cells also have anti-inflammatory and immunomodulatory effects, they are known to have already been widely used to treat a variety of diseases, including 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 tissue regeneration using the culture medium recovered from mesenchymal stem cell culture, which 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 mesenchymal stem cell culture process is rich in signaling substances that play an important role in cell activity, such as exosomes and other growth factors and immune regulatory factors (cytokines) secreted by stem cells, and is thought to be useful for restoring the function of damaged tissues and cells in the body. The exosomes, growth factors, and cytokines abundant in the supernatant act on surrounding stem cells, and by attracting cells to areas where function has declined due to aging or damage, it is expected to have functional recovery effects such as tissue regeneration and improved immunity.

[0006] Cell culture often involves the addition of growth factors such as fetal bovine serum (FBS, FCS), human plasma, albumin, FGF2, and EGF. It is generally believed that media components can be removed from cells cultured in media containing these components by washing with buffers such as PBS or HBSS. It is now believed that similar removal of media components can enhance safety for cells used in regenerative medicine products and specific cell processing products, as well as exosomes and supernatants undergoing clinical research. Thus, there is a need to recover physiologically active substances derived from human stem cells while minimizing contamination by media components.

[0007] Patent Document 1 describes an invention aimed at providing a method for producing a culture supernatant containing reduced amounts 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 (MesenCult medium, StemPro MSC medium, BMN211 medium, StemMACS medium, MSC NutriStem medium, or Xuri MSC medium) without FBS, subsequently removing the culture medium, and culturing the cells in DMEM without FBS. Patent Document 1 also describes culturing adipose-derived stem cells in MesenCult medium containing FBS, culturing the cells in MesenCult medium without FBS, subsequently removing the culture medium, and culturing the cells in DMEM without FBS. Patent Document 1 also describes culturing primary dental pulp-derived stem cells in MesenCult medium containing FBS, culturing the cells in MesenCult medium without FBS, subsequently removing the culture medium, and culturing the cells in DMEM without 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 also describes that in these cases, BSA derived from fetal bovine serum was not detected in the supernatant after the final culture.

[0008] The 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 involves culturing adhesive animal cells in a serum-containing medium until they adhere to a culture substrate in a serum-free medium, and then culturing the cells in a serum-free medium. This method solves the problem that, in order to culture cells that have been cultured in a serum-containing medium, the serum components in the medium must be gradually reduced to acclimate the cells to a serum-free environment, and efficient protein production cannot be achieved during this period. The method is characterized by culturing adhesive animal cells in a serum-containing medium until they adhere to a culture substrate, and then directly culturing the cells that have adhered to the culture substrate in a serum-free medium. However, Patent Document 2 does not describe or suggest performing multiple washing procedures (medium changes).

[0010] Patent Document 3 describes that a serum-free culture supernatant can be prepared by culturing dental pulp stem cells in a serum-free medium, and that a serum-free culture supernatant can also be obtained by subculturing the stem cells one or more times and culturing the stem cells in a serum-free medium for the last or penultimate subculturing. However, Patent Document 3 does not describe any experimental results showing that the stem cells were cultured in a serum-free medium multiple times, and this description was deemed inadequate for identifying the 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 serum-free medium, and that a serum-free culture supernatant can also be obtained by subculturing the stem cells one or more times and culturing the stem cells in a serum-free medium for the last or penultimate subculturing. However, Patent Document 4 also does not describe any experimental results showing that the stem cells were cultured in a serum-free medium multiple times, and this description was not eligible to be used to identify the cited invention.

[0012] Patent Document 5 describes that, in order to adapt the cells to serum-free suspension, they were subcultured in a serum-free medium supplemented with various nutrients until cell growth stabilized. However, this document envisages extracting recombinant human FSH (follicle-stimulating hormone) from the culture supernatant of the obtained cells, and does not envisage using the cell culture supernatant itself as a pharmaceutical composition, cosmetic composition, or food composition, or including it in any of these.

[0013] Patent Document 6 describes that the cells were passaged in serum-free medium 4 to 6 times to be adapted 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 will be used as or included in a pharmaceutical composition, cosmetic composition, or food composition.

[0014] Patent Publication No. 6860915, Japanese Patent Application Laid-Open No. 2014-039503, International Publication No. 2011 / 118795, International Publication No. 2014 / 126176, Japanese Patent Application Laid-Open No. 2009-273427, Japanese Patent Application Laid-Open No. 2009-045019

[0015] 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.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.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;18:1479-1489Shohara, 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):1171-1181Kawai, 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.

[0016] The examples in Patent Document 1 state 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. Furthermore, as mentioned above, the written opinion in Patent Document 1 states that the reduction of impurities in the culture supernatant in the invention of Patent Document 1 is due to the interaction of the components 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. Given these circumstances, a person skilled in the art would be prevented from further developing the invention of Patent Document 1 by further repeating the process of removing the culture medium. Furthermore, Patent Document 1 and other documents describe culturing in a serum-containing medium followed by culturing in a serum-free medium, but do not describe culturing in a medium free of the components contained in the serum-free medium after culturing 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 of 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 a medium containing the cell growth factors or the like are used.

[0019] The present inventors believed that because various components contained in culture media exert a wide range of effects, including proliferation activity, upon intracellular uptake, it was impossible to rule out the possibility that some components may be transiently taken up into cells and then subsequently released back into the culture medium. Considering the possibility that medium components used in the cell growth process (e.g., FBS, plasma, albumin, FGF2) may be taken up into cells and then released back into the culture medium after a certain period of time, the present inventors conducted extensive research and found that medium components observed under such a phenomenon can be removed by performing a washing procedure (medium exchange). Specifically, while it was previously believed that unwanted components derived from the culture medium, such as recombinant proteins, could be washed away with PBS or other methods, the present inventors discovered that components taken up into cells are not washed away by a quick wash but are instead released into the culture medium over time, and therefore, a period of release in the culture medium is necessary. Cell culture generally requires the use of growth media containing serum, recombinant proteins, antibiotics, etc., but these impurities may lead to side effects such as anaphylactic shock and hypoglycemia, and therefore a technology for removing medium-derived components is desired from a safety perspective. While it was previously thought that impurities could be removed by washing with PBS (phosphate buffered saline) or the like, the present inventors discovered that these impurities are taken up by cells during culture and gradually re-released over time, and have developed 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 advantageous 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 cells or a cell culture supernatant, comprising: culturing cells in a serum-free first medium to obtain first cells; culturing the obtained first cells in a second medium having components contained in the first medium at concentrations lower than those in the first medium to obtain second cells and a culture supernatant for the second cells; removing the culture supernatant for the second cells from the obtained second cells and the culture supernatant for the second cells to obtain second cells; and culturing the obtained second cells in a third medium having components contained in the first medium at concentrations lower than those in the first medium to obtain third cells and a culture supernatant for the third cells, thereby producing third cells or a culture supernatant for the third cells. [Aspect 2] A method for producing cells or a cell culture supernatant, comprising: culturing cells in a first medium to obtain first cells; culturing the obtained first cells in a second medium having a component contained in the first medium at a concentration lower than that of the first medium, thereby obtaining second cells and a culture supernatant for the second cells; removing the culture supernatant for the second cells from the obtained second cells and the culture supernatant for the second cells, thereby obtaining second cells; culturing the obtained second cells in a third medium having a component contained in the first medium at a concentration lower than that of the first medium, thereby obtaining third cells and a culture supernatant for the third cells; removing the culture supernatant for the third cells from the obtained third cells and the culture supernatant for the third cells, thereby obtaining third cells; and culturing the obtained third cells in a fourth medium having a component contained in the first medium at a concentration lower than that of the first medium, thereby obtaining fourth cells and a culture supernatant for the fourth cells, thereby producing fourth cells or a culture supernatant for the fourth cells. [Aspect 3] The method described in Aspect 2, comprising the steps 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; and culturing the obtained fourth cells in a fifth medium in which the concentrations of components contained in the first medium are lower than in the first medium to obtain the fifth cells and the culture supernatant of the fifth cells, thereby producing the fifth cells or the culture supernatant of the fifth cells.[Aspect 4] The method described in 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, thereby producing the sixth cell or the culture supernatant of the sixth cell. [Aspect 5] The method described in Aspect 2, wherein the cell is an adipose tissue-derived stem cell. [Aspect 6] The method described in Aspect 2, wherein the cell is a human cell. [Aspect 7] The method described in Aspect 2, wherein the component contained in the first medium is a recombinant protein, a cell growth factor, or a human protein. [Aspect 8] The method described in Aspect 7, wherein the recombinant protein is a human protein and the cell growth factor is a non-human protein. [Aspect 9] The method described in Aspect 7, wherein the cell growth factor is serum or plasma. [Aspect 10] The method described in Aspect 7, wherein the first medium contains 0.5% by volume or more of a cell growth factor. [Aspect 11] The method of Aspect 2, wherein the first medium comprises 0.0000001% to 1% by volume of the recombinant protein. [Aspect 12] The method of Aspect 10, wherein the first medium comprises 8% or more by volume of a cell growth factor. [Aspect 13] The method of Aspect 2, wherein the second medium, which contains a component in a lower concentration than the first medium, is a basal medium. [Aspect 14] The method of Aspect 2, wherein the third medium, which contains a component in a lower concentration than the first medium, is a basal medium. [Aspect 15] The method of Aspect 2, wherein the fourth medium, which contains a component in a lower concentration than the first medium, is a basal medium. [Aspect 16] The method of Aspect 3, wherein the fifth medium, which contains a component in a lower concentration than the first medium, is a basal medium. [Aspect 17] The method of Aspect 4, wherein the sixth medium, which contains a component in a lower concentration than the first medium, is a basal medium. [Embodiment 18] The method according to embodiment 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 1 hour to 192 hours.[Aspect 19] The method of Aspect 2, wherein the obtained second cells are cultured for 1 hour to 192 hours in a third medium, in which the concentration of a component contained in the first medium is lower than that of the first medium. [Aspect 20] The method of Aspect 2, wherein the obtained third cells are cultured for 1 hour to 192 hours in a fourth medium, in which the concentration of a component contained in the first medium is lower than that of the first medium. [Aspect 21] The method of Aspect 3, wherein the obtained fourth cells are cultured for 1 hour to 192 hours in a fifth medium, in which the concentration of a component contained in the first medium is lower than that of the first medium. [Aspect 22] The method of Aspect 4, wherein the obtained fifth cells are cultured for 1 hour to 192 hours in a sixth medium, in which the concentration of a component contained in the first medium is lower than that of the first medium. [Aspect 23] The method of Aspect 2, comprising a step of washing the first cells prior to obtaining the first cells. [Aspect 24] The method of Aspect 2, comprising a step of washing the second cells prior to obtaining the second cells. [Aspect 25] The method of Aspect 2, comprising a step of washing the third cells prior to obtaining the third cells. [Aspect 26] The method of Aspect 3, comprising a step of washing the fourth cells prior to obtaining the fourth cells. [Aspect 27] The method of Aspect 4, comprising a step of washing the fifth cells prior to obtaining the fifth cells. [Aspect 28] The method of any one of Aspects 1 to 27, wherein the cells or cell culture supernatant are for producing a pharmaceutical composition, a cosmetic composition, a food composition, a regenerative medicine product or a specific cell processed product. [Aspect 29] The method of any one of Aspects 1 to 27, wherein the cells or cell culture supernatant are 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.

[0021] According to the present invention, even when cells are cultured in a medium containing cell growth factors, etc., 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, leading to improved safety of exosomes and supernatants that are expected to be used in regenerative medicine and new drug discovery. Furthermore, 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. In other words, the present invention makes it 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 may become an important technology that will contribute to improving safety in the future.

[0022] FIG. 1 is a photograph showing the appearance of cells in sample 4, which was collected by the method of Example 1 using cells cultured in a medium obtained by adding FBS to DMEM medium to contain various concentrations (volume percentages: upper left values). FIG. 2 is a graph showing the results of an ELISA test for samples 1 to 4, which were collected by the method of Example 1 using cells cultured in DMEM medium (without FBS). FIG. 3 is a graph showing the results of an ELISA test for samples 1 to 4, which were collected by the method of Example 1 using cells cultured in a medium obtained by adding FBS to DMEM medium to contain 10% FBS by volume. FIG. 4 is a graph showing the results of an ELISA test for samples 1 to 4, which were collected by the method of Example 1 using cells cultured in a medium obtained by adding FBS to DMEM medium to contain 20% FBS by volume. FIG. 5 is a schematic diagram showing the steps of the tests conducted in Examples 2 and 3. FIG. 6 is a schematic diagram showing the steps of the test for sample collection conducted in Example 4. FIG. 7 is a graph showing the VEGF protein concentration in each sample obtained in Example 4. Figure 8 is a schematic diagram showing the steps of the test performed in Example 5. Figure 9 is a graph showing the HGF concentration in each sample obtained in Example 5. Figure 10 is a graph showing the IL-6 concentration in each sample obtained in Example 5.

[0023] The present invention provides a method for producing cells or a cell culture supernatant, comprising: (a first step) culturing cells in a serum-free first medium to obtain first cells; (a second step) culturing the obtained first cells in a second medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably not containing the components contained in the first medium, to obtain second cells and a culture supernatant of the second cells; (a 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 second cells; and (a fourth step) culturing the obtained second cells in a third medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably not containing the components contained in the first medium, to obtain third cells and a culture supernatant of the third cells, thereby producing third cells or a culture supernatant of the third cells.

[0024] First step: This is the same as the first step described below, but the cells are cultured in a serum-free first medium.

[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] Step 4: Similar to Step 4 described below, the method for producing a cell culture supernatant of the present invention may include (step 5) removing the culture supernatant of the third cells from the obtained third cells and the culture supernatant of the third cells to obtain third cells, and (step 6) culturing the obtained third cells in a fourth medium having a lower concentration of components contained in the first medium than the first medium, preferably not containing the components contained in the first medium, to obtain fourth cells and the culture supernatant of the fourth cells, and producing fourth cells or the culture supernatant of the fourth cells. That is, the method for producing a cell culture supernatant of the present invention not only includes a method in which the culture supernatant of the obtained third cells is used as the culture supernatant of the cells to be produced, but also a method in which the culture supernatant of the obtained third cells is removed to obtain third cells, further culturing the obtained third cells or cells derived therefrom to obtain other cells and the culture supernatant of the other cells, and using the obtained other cells or the culture supernatant of the other cells to produce the cells to be produced. Furthermore, the method for producing a cell culture supernatant of the present invention may include (a 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 (an eighth step) a step of culturing the obtained fourth cell in a fifth medium having a lower concentration of components contained in the first medium than 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 a cell culture supernatant of the present invention not only includes a method of using the culture supernatant of the obtained fourth cell as the culture supernatant of the cell to be produced, 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 another cell and the culture supernatant of the another cell, and using the obtained another cell or the culture supernatant of the another cell as the culture supernatant of the cell to be produced.Furthermore, the method for producing a cell culture supernatant of the present invention may include (a ninth step) a 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, and (a tenth step) a step of culturing the obtained fifth cells in a sixth medium having a lower concentration of components contained in the first medium than the first medium, preferably not containing the components contained in the first medium, to obtain the sixth cells and the culture supernatant of the sixth cells, and to produce the sixth cells or the culture supernatant of the sixth cells. That is, the method for producing a cell culture supernatant of the present invention not only includes a method of using the culture supernatant of the obtained fifth cells as the culture supernatant of the cells to be produced, but also a method of removing the culture supernatant of the obtained fifth cells to obtain the fifth cells, further culturing the obtained fifth cells or cells derived therefrom to obtain another cell and the culture supernatant of the another cell, and using the obtained another cell or the culture supernatant of the another cell as the culture supernatant of the cells to be produced. Similarly, the method for producing a cell culture supernatant of the present invention may include (an eleventh step) removing the culture supernatant of the sixth cells from the obtained sixth cells and the culture supernatant of the sixth cells to obtain the sixth cells, and (a twelfth step) culturing the obtained sixth cells in a seventh medium having a lower concentration of components contained in the first medium than the first medium, preferably not containing the components contained in the first medium, to obtain the seventh cells and the culture supernatant of the seventh cells, and producing the seventh cells or the culture supernatant of the seventh cells. That is, the method for producing a cell culture supernatant of the present invention not only includes a method in which the culture supernatant of the obtained sixth cells is used as the culture supernatant of the cells to be produced, but also a method in which the culture supernatant of the obtained sixth cells is removed to obtain the sixth cells, further culturing the obtained sixth cells or cells derived therefrom to obtain another cell and the culture supernatant of the another cell, and using the obtained another cell or the culture supernatant of the another cell to be used as the culture supernatant of the cells 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 of 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 to these, and may also be a culture supernatant of another cell or another cell obtained by a similar method. Note that steps 5 to 12 are the same as steps 5 to 12 described below.

[0028] Furthermore, the present invention provides a method for producing cells or a cell culture supernatant, comprising: (first step) culturing cells in a first medium to obtain first cells; (second step) culturing the obtained first cells in a second medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably not containing the components 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 second cells; (fourth step) culturing the obtained second cells in a third medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably not containing the components 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 third cells; (Sixth step) culturing the obtained third cells in a fourth medium in which the concentrations of 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, to obtain fourth cells and a culture supernatant of the fourth cells, and to produce the fourth cells or the culture supernatant of the fourth cells.

[0029] Step 1: Examples of cells include stem cells. 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, and hair follicles. Stem cells derived from these tissues include stem cells isolated from these tissues. Examples of stem cells include mesenchymal stem cells (MSCs), hematopoietic stem cells, and neural crest stem cells. Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in multiple tissues, such as the umbilical cord, bone marrow, and adipose tissue. Although mesenchymal stem cells (MSCs) are rare, they are estimated to exist in bone marrow at a rate of 1 per 10,000 to 100,000 nucleated bone marrow cells. Mesenchymal stem cells (MSCs) have the ability to self-proliferate and differentiate into various cell types, such as osteoblasts (bone cells), chondrocytes, muscle cells, and adipocytes. Examples of cells that can be used include embryonic stem cells (ES cells), iPS cells, and cells induced to differentiate from these stem cells.

[0030] Examples of cells include animal cells. Examples of animals include vertebrates as well as invertebrates. Examples of vertebrates include fish, amphibians, reptiles, birds, and mammals. Examples of mammals include mice and humans. Examples of 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 may be, 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 following are the working hours: 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 culture medium to obtain first cells, the time for culturing the cells in the first culture medium is, for example, within a range specified by 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 may be, 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. Cell growth factors are, for example, proteins from organisms other than those from which the cells cultured in the first medium are 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. Furthermore, the components contained in the first medium also include proteins derived from humans (human proteins). Human proteins derived from the medium are essentially synthetic proteins, and safety concerns exist depending on the method of producing the secondary raw materials. The purpose of the present invention is to remove synthetic substances containing these human proteins and increase the proportion 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 receptors.More specifically, examples of cell growth factors include epidermal growth factor (EGF) or substances having substantially the same activity as EGF (e.g., TGFalpha), insulin or substances having substantially the same activity as insulin (e.g., insulin, insulin-like growth factor (IGF)-1, IGF-2), fibroblast growth factor (FGF) or substances having substantially the same assay as FGF (e.g., acidic FGF, basic FGF, keratinocyte growth factor (KGK), FGF-10), and other cell growth factors (e.g., cytokines such as IL-2, IL-15, IL-7, IL-9, and IL-21, colony stimukting 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), heregulin, angiopoietin, etc.). Examples of cell growth factors include serum, plasma, albumin, etc. Examples of serum and plasma include human serum and plasma as well as non-human serum and plasma.

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

[0037] 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, 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 culture 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 culture medium being a predetermined volume % or less or a predetermined μg / ml or less may imply that the total amount of recombinant protein, antibiotic, or cell growth factor contained in the first culture medium is the predetermined volume % or less or the predetermined 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 culture medium is the predetermined volume % or less or the predetermined 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-mentioned lower limits and any of the above-mentioned upper limits.

[0039] In the step of culturing cells in a first medium to obtain first cells, the first cells and their culture supernatant are separated, for example, prior to obtaining the first cells. Separation of the first cells and their culture supernatant is carried out, for example, by centrifugation. Centrifugation is carried out, for example, at 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The centrifugation time 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. The culture supernatant of the first cells is preferably removed.

[0040] The step of culturing cells in a first medium to obtain 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 with a solution in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably, the solution is free of components contained in the first medium. Here, the solution in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably, the solution is free of components contained in the first medium. The solution in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably, the solution is free of all components contained in the first medium, but is not limited thereto. The solution in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably, the solution is free of specific components contained in the first medium. The solution in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably, the solution is free of specific components contained in the first medium, may be a solution in which the concentrations of another specific component contained in the first medium are the same as or higher than those in the first medium, or a solution containing another specific component not contained in the first medium. Examples of the solution containing a lower concentration of components contained in the first medium than in the first medium, preferably a solution containing no components contained in the first medium, include buffer, physiological saline, and medium. Examples of the medium include basal medium, low-serum medium, and serum-free medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may also be, for example, a medium containing inorganic salts, vitamins, amino acids, sugars, and the like, without synthetic proteins (e.g., MEM, MEM-α, DMEM, DMEM high-glucose, PRIM, etc.).Here, the basal medium may optionally 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, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. A solution containing components contained in the first medium at lower concentrations than the first medium, preferably free of the components contained in the first medium, may be, for example, a second medium containing components contained in the first medium at lower concentrations than the first medium, preferably free of the components contained in the first medium.

[0041] Second step: In the step of culturing the obtained first cells in a second medium containing components contained in the first medium at lower concentrations than the first medium, preferably containing no components contained in the first medium, to obtain second cells and a culture supernatant of the second cells, the second medium containing components contained in the first medium at lower concentrations than the first medium, preferably containing no components contained in the first medium, may be a medium containing all components contained in the first medium at lower concentrations than the first medium, preferably containing no components contained in the first medium, but is not limited thereto. It may also be a medium containing a specific component contained in the first medium at lower concentrations than the first medium, preferably containing no specific component contained in the first medium. The first medium containing a specific component contained in the first medium at lower concentrations than the first medium, preferably containing no specific component contained in the first medium, may contain another specific component contained in the first medium at the same or higher concentration than the first medium, or may contain another specific component not contained in the first medium. Examples of the second medium containing a component contained in the first medium at lower concentrations than the first medium, preferably containing no components contained in the first medium, include basal medium, low-serum medium, and serum-free medium. Examples of basal media 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 mixtures thereof. The basal medium may also be, for example, a medium (MEM, MEM-α, 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 optionally 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.

[0042] The time for culturing the obtained first cells in a second medium in which the concentration of components contained in the first medium is lower than that of the first medium, preferably in which the components contained in the first medium are not contained, 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 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, which does not contain any 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 which the obtained first cells are cultured 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 in which the second medium does not contain 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.

[0045] The temperature at which the obtained first cells are cultured in a second medium containing components contained in the first medium at lower concentrations than the first medium, preferably containing no 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 may be, for example, 0% to 40%, 5% to 35%, 10% to 30%, or 15% to 25%.

[0046] Third step: In the step of obtaining second cells by removing the second cell culture supernatant from the obtained second cells and the second cell culture supernatant, the second cells and the second cell culture supernatant are separated, for example, prior to removing the second cell culture supernatant. Separation of the second cells and the second cell culture supernatant is carried out, for example, by centrifugation. Centrifugation is carried out, for example, at a speed of 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The centrifugation time 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.

[0047] The step of obtaining the second cells by removing the second cell culture supernatant from the obtained second cells and the second cell culture supernatant 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 with a solution in which the concentrations of components contained in the first medium are lower than those of the first medium, preferably, the solution is free of components contained in the first medium. Here, the solution in which the concentrations of components contained in the first medium are lower than those of the first medium, preferably, the solution is free of components contained in the first medium. The solution in which the concentrations of components contained in the first medium are lower than those of the first medium, preferably, the solution is free of all components contained in the first medium, but is not limited thereto. The solution in which the concentrations of specific components contained in the first medium are lower than those of the first medium, preferably, the solution is free of specific components contained in the first medium. The solution in which the concentrations of specific components contained in the first medium are lower than those of the first medium, preferably, the solution is free of specific components contained in the first medium, may be a solution in which the concentrations of another specific component contained in the first medium are the same as or higher than those of the first medium, or a solution containing another specific component not contained in the first medium. Examples of the solution containing a lower concentration of components contained in the first medium than in the first medium, preferably a solution containing no components contained in the first medium, include buffer, physiological saline, and medium. Examples of the medium include basal medium, low-serum medium, and serum-free medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may also be, for example, a medium containing inorganic salts, vitamins, amino acids, sugars, and the like, without synthetic proteins (e.g., MEM, MEM-α, DMEM, DMEM high-glucose, PRIM, etc.).Here, the basal medium may optionally 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, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. A solution containing components contained in the first medium at lower concentrations than the first medium, preferably free of the components contained in the first medium, may be, for example, a third medium containing components contained in the first medium at lower concentrations than the first medium, preferably free of 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 concentrations of components contained in the first medium are lower than those in the first medium, preferably in which the components contained in the first medium are not contained, to obtain third cells and a culture supernatant of the third cells, the third medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably in which the components contained in the first medium are not contained, may be a medium in which the concentrations of all components contained in the first medium are lower than those in the first medium, preferably in which all components contained in the first medium are not contained, but is not limited thereto. It may also be a medium in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably in which the specific components contained in the first medium are not contained. The medium in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably in which the specific components contained in the first medium are not contained, may be a medium in which the concentrations of other specific components contained in the first medium are the same as or higher than those in the first medium, or a medium in which other specific components not contained in the first medium are contained. Examples of the third medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably in which the components contained in the first medium are not contained, include basal medium, low-serum medium, and serum-free medium. Examples of basal media 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 mixtures thereof. The basal medium may also be, for example, a medium (MEM, MEM-α, 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 optionally 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.

[0049] The obtained second cells are cultured in a third medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably in which the components contained in the first medium are not contained, 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 which the obtained second cells are cultured in a third 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, 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 which the obtained second cells are cultured in a third medium in which the concentration of a component contained in the first medium is lower than that in the first medium, preferably in which the third medium does not contain 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.

[0052] The temperature at which the obtained second cells are cultured in a third medium containing components contained in the first medium at lower concentrations than the first medium, preferably containing no 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 may be, for example, 0% to 40%, 5% to 35%, 10% to 30%, or 15% to 25%.

[0053] Fifth step: In the step of obtaining third cells by removing the culture supernatant of the third cells from the obtained third cells and the culture supernatant of the third cells, the third cells and the culture supernatant of the third cells are separated, for example, prior to removing the culture supernatant of the third cells. Separation of the third cells and the culture supernatant of the third cells is carried out, for example, by centrifugation. Centrifugation is carried out, for example, at 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The centrifugation time 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 third cell culture supernatant from the obtained third cells and the third cell culture supernatant 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 with a solution in which the concentrations of components contained in the first medium are lower than those of the first medium, preferably, the solution is free of components contained in the first medium. Here, the solution in which the concentrations of components contained in the first medium are lower than those of the first medium, preferably, the solution is free of components contained in the first medium. The solution in which the concentrations of components contained in the first medium are lower than those of the first medium, preferably, the solution is free of all components contained in the first medium, but is not limited thereto. The solution in which the concentrations of specific components contained in the first medium are lower than those of the first medium, preferably, the solution is free of specific components contained in the first medium. The solution in which the concentrations of specific components contained in the first medium are lower than those of the first medium, preferably, the solution is free of specific components contained in the first medium, may be a solution in which the concentrations of another specific component contained in the first medium are the same as or higher than those of the first medium, or a solution containing another specific component not contained in the first medium. Examples of the solution containing a lower concentration of components contained in the first medium than in the first medium, preferably a solution containing no components contained in the first medium, include buffer, physiological saline, and medium. Examples of the medium include basal medium, low-serum medium, and serum-free medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may also be, for example, a medium containing inorganic salts, vitamins, amino acids, sugars, and the like, without synthetic proteins (e.g., MEM, MEM-α, DMEM, DMEM high-glucose, PRIM, etc.).Here, the basal medium may optionally 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, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. A solution containing components contained in the first medium at lower concentrations than the first medium, preferably free of the components contained in the first medium, may be, for example, a fourth medium containing components contained in the first medium at lower concentrations than the first medium, preferably free of the components contained in the first medium.

[0055] Sixth step: In the step of producing fourth cells or a culture supernatant of the fourth cells, the obtained third cells are cultured in a fourth medium in which the concentrations of 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, to obtain fourth cells and a culture supernatant of the fourth cells. The fourth medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably in which the components contained in the first medium are not contained, may be a medium in which the concentrations of all components contained in the first medium are lower than those in the first medium, preferably in which all components contained in the first medium are not contained, but is not limited thereto, and may also be a medium in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably in which the specific components contained in the first medium are not contained. The medium in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably in which the specific components contained in the first medium are not contained, may be a medium in which the concentrations of another specific component contained in the first medium are the same as or higher than those in the first medium, or a medium containing another specific component not contained in the first medium. The fourth medium contains components contained in the first medium at lower concentrations than the first medium, preferably does not contain any of the components contained in the first medium. Examples of the fourth medium include basal media, low-serum media, and serum-free media. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may also be, for example, a medium containing inorganic salts, vitamins, amino acids, sugars, and the like, but without synthetic proteins (e.g., MEM, MEM-α, DMEM, DMEM high-glucose, PRIM, etc.).Here, the basal medium may optionally 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.

[0056] The obtained third cells are cultured in a fourth medium in which the concentration of components contained in the first medium is lower than that of the first medium, preferably in which the fourth medium does not contain any components contained in the first medium, 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 which the obtained third cells are cultured in a fourth medium in which the concentrations of components contained in the first medium are lower than those in 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.

[0058] The time for which the obtained third cells are cultured 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 the component contained in the first culture medium is not 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 containing components contained in the first medium at lower concentrations than the first medium, preferably containing no 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 may be, for example, 0% to 40%, 5% to 35%, 10% to 30%, or 15% to 25%.

[0060] When 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, the fourth cell and the culture supernatant of the fourth cell are separated prior to producing the fourth cell or the culture supernatant of the fourth cell. Separation of the fourth cell and the culture supernatant of the fourth cell is carried out, for example, by centrifugation. Centrifugation is carried out, for example, at 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The centrifugation time 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.

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

[0062] Seventh step: In the step of obtaining fourth cells by removing the fourth cell culture supernatant from the obtained fourth cells and the fourth cell culture supernatant, the fourth cells and the fourth cell culture supernatant are separated, for example, prior to removing the fourth cell culture supernatant. Separation of the fourth cells and the fourth cell culture supernatant is performed, for example, by centrifugation. Centrifugation is performed, for example, at 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The centrifugation time 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 fourth cell culture supernatant from the obtained fourth cells and the fourth cell culture supernatant 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 with a solution in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably, the solution is free of components contained in the first medium. Here, the solution in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably, the solution is free of components contained in the first medium. The solution in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably, the solution is free of all components contained in the first medium, but is not limited thereto. The solution in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably, the solution is free of specific components contained in the first medium. The solution in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably, the solution is free of specific components contained in the first medium, may be a solution in which the concentrations of another specific component contained in the first medium are the same as or higher than those in the first medium, or a solution containing another specific component not contained in the first medium. Examples of the solution containing a lower concentration of components contained in the first medium than in the first medium, preferably a solution containing no components contained in the first medium, include buffer, physiological saline, and medium. Examples of the medium include basal medium, low-serum medium, and serum-free medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may also be, for example, a medium containing inorganic salts, vitamins, amino acids, sugars, and the like, without synthetic proteins (e.g., MEM, MEM-α, DMEM, DMEM high-glucose, PRIM, etc.).Here, the basal medium may optionally 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, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. A solution containing components contained in the first medium at lower concentrations than the first medium, preferably free of the components contained in the first medium, may be, for example, a fifth medium containing components contained in the first medium at lower concentrations than the first medium, preferably free of the components contained in the first medium.

[0064] Eighth step: In the step of culturing the obtained fourth cells in a fifth medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably in a fifth medium that does not contain the components contained in the first medium, to obtain fifth cells and a culture supernatant of the fifth cells, and producing fifth cells or a culture supernatant of the fifth cells, the fifth medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably in which the components contained in the first medium are not contained, may be a medium in which the concentrations of all components contained in the first medium are lower than those in the first medium, preferably in which all components contained in the first medium are not contained, but is not limited thereto, and may also be a medium in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably in which the specific components contained in the first medium are not contained. The medium in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably in which the specific components contained in the first medium are not contained, may be a medium in which the concentrations of another specific component contained in the first medium are the same as or higher than those in the first medium, or a medium containing another specific component not contained in the first medium. The fifth medium contains components contained in the first medium at lower concentrations than the first medium, preferably does not contain any of the components contained in the first medium. Examples of the fifth medium include basal media, low-serum media, and serum-free media. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may also be, for example, a medium containing inorganic salts, vitamins, amino acids, sugars, and the like, but without synthetic proteins (e.g., MEM, MEM-α, DMEM, DMEM high-glucose, PRIM, etc.).Here, the basal medium may optionally 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.

[0065] The obtained fourth cells are cultured in a fifth medium in which the concentration of components contained in the first medium is lower than that of the first medium, preferably in which the fifth medium does not contain any components contained in the first medium, 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 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 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 which the obtained fourth cells are cultured in a fifth medium in which the concentration of a component contained in the first medium is lower than that in the first medium, preferably in which the fifth medium does not contain 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 containing no 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 may be, for example, 0% to 40%, 5% to 35%, 10% to 30%, or 15% to 25%.

[0069] When 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, the fifth cell and the culture supernatant of the fifth cell are separated prior to producing the fifth cell or the culture supernatant of the fifth cell. Separation of the fifth cell and the culture supernatant of the fifth cell is carried out, for example, by centrifugation. Centrifugation is carried out, for example, at 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The centrifugation time 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.

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

[0071] Ninth step: In the step of obtaining fifth cells by removing the culture supernatant of the fifth cells from the obtained fifth cells and the culture supernatant of the fifth cells, the fifth cells and the culture supernatant of the fifth cells are separated, for example, prior to removing the culture supernatant of the fifth cells. Separation of the fifth cells and the culture supernatant of the fifth cells is carried out, for example, by centrifugation. Centrifugation is carried out, for example, at 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The centrifugation time 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.

[0072] The step of removing the fifth cell culture supernatant from the obtained fifth cells and the fifth cell culture supernatant 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 with a solution in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably, the solution does not contain the components contained in the first medium. Here, the solution in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably, the solution does not contain the components contained in the first medium, may be a solution in which the concentrations of all components contained in the first medium are lower than those in the first medium, preferably, the solution does not contain all components contained in the first medium, but is not limited thereto. It may also be a solution in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably, the solution does not contain the specific component contained in the first medium. The solution in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably, the solution does not contain the specific component contained in the first medium may be a solution in which the concentrations of another specific component contained in the first medium are the same as or higher than those in the first medium, or a solution containing another specific component not contained in the first medium. Examples of the solution containing a lower concentration of components contained in the first medium than in the first medium, preferably a solution containing no components contained in the first medium, include buffer, physiological saline, and medium. Examples of the medium include basal medium, low-serum medium, and serum-free medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may also be, for example, a medium containing inorganic salts, vitamins, amino acids, sugars, and the like, without synthetic proteins (e.g., MEM, MEM-α, DMEM, DMEM high-glucose, PRIM, etc.).Here, the basal medium may optionally 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, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. The solution containing components contained in the first medium at lower concentrations than the first medium, preferably free of the components contained in the first medium, may be, for example, a medium containing components contained in the first medium at lower concentrations than the first medium, preferably free of the components contained in the first medium, and used for the next culture.

[0073] Tenth step: In the step of culturing the obtained fifth cells in a sixth medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably in a sixth medium that does not contain the components contained in the first medium, to obtain sixth cells and a culture supernatant of the sixth cells, and producing sixth cells or a culture supernatant of the sixth cells, the sixth medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably in which the components contained in the first medium are not contained, may be a medium in which the concentrations of all components contained in the first medium are lower than those in the first medium, preferably in which all components contained in the first medium are not contained, but is not limited thereto, and may also be a medium in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably in which the specific components contained in the first medium are not contained. The medium in which the concentrations of specific components contained in the first medium are lower than those in the first medium, preferably in which the specific components contained in the first medium are not contained, may be a medium in which the concentrations of another specific component contained in the first medium are the same as or higher than those in the first medium, or a medium containing another specific component not contained in the first medium. The sixth medium contains components contained in the first medium at lower concentrations than the first medium, preferably does not contain any of the components contained in the first medium. Examples of the sixth medium include basal media, low-serum media, and serum-free media. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may also be, for example, a medium containing inorganic salts, vitamins, amino acids, sugars, and the like, but without synthetic proteins (e.g., MEM, MEM-α, DMEM, DMEM high-glucose, PRIM, etc.).Here, the basal medium may optionally 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.

[0074] The obtained fifth cells are cultured in a sixth medium in which the concentrations of components contained in the first medium are lower than those in the first medium, preferably in which the sixth medium does not contain any components contained in the first medium, 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 which the obtained fifth cells are cultured in a sixth medium in which the concentrations of components contained in the first medium are lower than those in 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 which the obtained fifth cells are cultured in a sixth medium in which the concentration of a component contained in the first medium is lower than that in the first medium, preferably in which the sixth medium does not contain 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 in which the components contained in the first medium are not contained, is, for example, 20°C to 45°C, 30°C to 40°C, or 35°C to 38°C.

[0078] When obtaining the sixth cells and the culture supernatant of the sixth cells and producing the sixth cells or the culture supernatant of the sixth cells, for example, the sixth cells and the culture supernatant of the sixth cells are separated prior to producing the sixth cells or the culture supernatant of the sixth cells. The separation of the sixth cells and the culture supernatant of the sixth cells is carried out, for example, by centrifugation. The centrifugation is carried out, for example, at 500 rpm to 5,000 rpm, 750 rpm to 3,000 rpm, or 1,000 rpm to 2,000 rpm. The centrifugation time 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.

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

[0080] Uses of Cells or Cell Culture Supernatant: The cells or cell culture supernatant produced by the method of producing cells or cell culture supernatant of the present invention can be used, for example, to produce pharmaceutical compositions, cosmetic compositions, food compositions, regenerative medicine products, or specific cell processed products. Furthermore, the cells or cell culture supernatant produced by the method of producing cells or cell culture supernatant of the present invention can be used, for example, as pharmaceutical compositions, cosmetic compositions, food compositions, regenerative medicine products, or specific cell processed products, or can be used as part of these. That is, the cells or cell culture supernatant produced by the method of producing cells or cell culture supernatant of the present invention are intended for producing pharmaceutical compositions, cosmetic compositions, food compositions, regenerative medicine products, or specific cell processed products. Furthermore, the cells or cell culture supernatant produced by the method of producing cells or cell culture supernatant of the present invention are intended for use, for example, as pharmaceutical compositions, cosmetic compositions, food compositions, regenerative medicine products, or specific cell processed products. Furthermore, the cells or cell culture supernatants produced by the method for producing cells or cell culture supernatants of the present invention are intended for inclusion in, for example, pharmaceutical compositions, cosmetic compositions, food compositions, regenerative medicine products, or specific cell processed products. Examples of pharmaceutical compositions include compositions for treating or preventing cancer, demyelinating diseases, eye diseases, eye disorders, etc. Examples of cosmetic compositions include compositions for use in skin care, such as compositions for improving skin wrinkles, blemishes, and firmness, and compositions for promoting skin regeneration.

[0081] As shown in the Examples below, the method of the present invention unexpectedly produces a cell culture supernatant containing a high concentration of the cytokine HGF (Hepatocyte Growth Factor). Therefore, the cell culture supernatant produced by the method of the present invention can be used, for example, to treat HGF deficiency in a subject. Examples of subjects include vertebrates. Examples of vertebrates include mammals such as mice, rats, rabbits, pigs, cows, monkeys, and humans. The mammal is preferably human. Subjects can be of any age, including infants, young children, adolescents, adults, and the elderly. As an advantage of increased HGF, intrathecal administration of rhHGF has been shown to slow the progression of ALS in rat models (J Neuropathol Exp Neurol. 2007 Nov;66(11):1037-44). Furthermore, administration of HGF plasmids has been shown 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 injury has been shown to promote functional recovery in the injured area (PLoS One. 2011;6(11):e27706). Furthermore, HGF administration has been shown to alleviate insulin resistance and lipid accumulation in mice fed a high-fat diet (J Diabetes Investig. 2019 Mar;10(2):251-260). Therefore, the cell culture supernatant produced by the method of producing cell culture supernatant of the present invention can be used to treat diseases such as ALS, diabetes, peripheral vascular disorders, spinal cord injury, insulin resistance, and lipid accumulation. Examples of treatment include therapy and prevention. Examples of treatment include complete recovery and symptom alleviation.

[0082] The cells or cell culture supernatant produced by the method of producing cells or cell culture supernatant of the present invention may be administered by any administration method appropriate 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 of producing 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 of producing 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.

[0083] Example 1: Cell culture was performed using a medium containing FBS, human albumin, FGF2, and other growth-activating factors commonly used in cell culture. This example shows experimental data for FBS. Human adipose tissue-derived mesenchymal stem cells (MSCs) (P2-1) were cultured in FBS-containing medium (DMEM) for several days until confluence was reached. The medium was then completely removed (sample circle 1), washed with PBS, and then cultured in a growth factor-free basal medium (DMEM medium). After 24 hours, the medium was removed (sample circle 2), washed again with PBS, and cultured in a basal medium (DMEM medium) and recovered 24 hours later (sample circle 3). The same recovery process using basal medium (DMEM medium) was repeated again (sample circle 4). MSCs were cultured at 37°C. The oxygen concentration during cell culture was 20%.

[0084] By performing the above procedure, we were able to obtain samples with the following conditions: Sample 1: Medium containing FBS (positive control); Sample 2: Basal medium after the first wash (basal medium does not contain FBS); Sample 3: Basal medium after the second wash (basal medium does not contain FBS); Sample 4: Basal medium after the third wash (basal medium does not contain FBS). These samples were subjected to ELISA analysis for bovine albumin (not cross-reactive with human albumin) contained in FBS to determine whether bovine albumin could be detected in samples 2 through 4, thereby determining up to which sample the bovine albumin could be detected. Note that because human MSCs were used in this example, albumin (human type) secreted by the original cells themselves was not detected by the ELISA kit (which specifically reacts with bovine type).

[0085] The steps of the above experiment are described in more detail below. First, human adipose tissue-derived MSCs (passage 3) were prepared. The dish was filled with 10,000 cells / cm. 2Cells were seeded in DMEM medium at 100°C. Next, FBS was added to DMEM medium as needed to achieve a total of six FBS concentrations: 0 vol%, 1 vol%, 3 vol%, 5 vol%, 10 vol%, or 20 vol%. The resulting medium (to which L-glutamine was added to a final concentration of 1% by mass) was cultured. The medium was replaced with the same medium on day 3. On day 7, cells reached confluence under FBS-containing conditions. (1) The medium was completely removed from the 6-well plate. A portion was collected and designated Sample 1. (2) PBS was added to thoroughly wash the cells in the dish. (3) The PBS was completely removed. (4) New DMEM medium (to which L-glutamine was added 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 Sample 2. After 24 hours, steps (1) to (4) above were repeated. A portion was collected and designated Sample 3. After 24 hours, steps (1) to (4) above were repeated. A portion was collected and designated 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. Each sample was quantified using an 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 will not be observed after extraction in the first place. In that case, no cells will remain, and the operation of collecting samples four times in total would be meaningless.

[0087] The results of the ELISA test after sample collection are shown in Figures 2 to 4. In samples 1 and 2 in Figure 3 and samples 1 and 2 in Figure 4, the bovine albumin concentration was listed as 1500 ng / mL, but more accurately, these were concentrations that exceeded the measurement range of the ELISA kit and were 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] Furthermore, as shown in Figures 3 and 4, a certain amount of bovine albumin tended to be detected even at the sample circle 2 stage. This is thought to be due to bovine albumin once taken up by the cells being released back into the medium. When cells were 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 observed to be lower than when cells were cultured in a medium containing 10 vol% or 20 vol% FBS, but a similar trend was observed.

[0090] As shown in Figures 3 and 4, bovine albumin was not detected in samples 3 and 4 containing 10% or more by volume of FBS, suggesting that the amount of bovine albumin in samples 3 and 4 decreased with repeated washing (medium changes). Furthermore, when samples 3 and 4 were cultured in medium containing 5% or less by volume of FBS, a similar trend was observed, although a certain amount of bovine albumin was detected. This suggests that adding a low concentration of FBS to the medium allows bovine albumin to be efficiently taken up into the cells, resulting in the relatively long-term release (sample 4).

[0091] Examples 2 and 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 plates used were 6-well plates, and ADSC-4 medium (2 ml / well) was used for seeding. The culture conditions were as follows: medium replacement with proliferation medium (ADSC-4 + human recombinant albumin (2,000 μg / ml) + FGF2 (50 ng / ml) + 0.5% penicillin / streptomycin) was performed on days 1 and 4 after seeding. Sampling began on day 7 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 the medium was completely removed from the pre-extraction sample, the sample was washed with PBS, and DMEM with high glucose + LGln medium (2 ml / well) was added. After one day had passed, the sample was sampled to obtain a day 1 sample (day 1). After the medium was completely removed from the day 1 sample, DMEM with high glucose + LGln medium (2 ml / well) was added. After one day had passed, the sample was sampled to obtain a day 2 sample (day 2). After the medium was completely removed from the day 2 sample, DMEM with high glucose + LGln medium (2 ml / well) was added. After one day had passed, the sample was sampled to obtain a day 3 sample (day 3).

[0093] The results for umbilical cord stem cells are shown in Table 1 below. Before extraction, the existing medium was completely removed, followed by washing with PBS and then adding DMEM high-glucose medium. During extraction, DMEM was completely removed at each collection time 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. Before extraction, the existing medium was completely removed, followed by washing with PBS and then adding DMEM high-glucose medium. During extraction, DMEM was completely removed at each collection time 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. After 2 days had passed, DMEM with high glucose + LGln medium (2 ml / well) was added. This was then sampled to obtain a day 2 sample (day 2). After the medium was completely removed from the day 1 sample, DMEM with high glucose + LGln medium (2 ml / well) was added. After 2 days had passed, this was sampled to obtain a day 4 sample (day 4). After the medium was completely removed from the day 2 sample, DMEM with high glucose + LGln medium (2 ml / well) was added. After 2 days had passed, this was sampled to obtain a day 6 sample (day 6).

[0096] The results for umbilical cord stem cells are shown in Table 3 below. Before extraction, the existing medium was completely removed, followed by washing with PBS and then adding DMEM high-glucose medium. During extraction, DMEM was completely removed at each collection time from day 2 onwards, and then new DMEM high-glucose medium was added.

[0097] The results for adipose stem cells are shown in Table 4 below. Before extraction, the existing medium was completely removed, followed by washing with PBS and then adding DMEM high-glucose medium. During extraction, DMEM was completely removed at each collection time from day 2 onwards, and then new DMEM high-glucose medium was added.

[0098] As shown in Tables 1 to 4, recombinant albumin was present in the culture medium at high concentrations before extraction, exceeding the detection limit in ELISA assays. Furthermore, when the culture medium was switched to basal medium (DMEM) after PBS washing, high albumin concentrations were detected again the next day or two days later. Furthermore, when the basal medium was replaced with new medium and analyzed the next day or two days later, approximately 5–20% albumin was still detected. After the third extraction, the residual albumin ranged from 85–319 ng / ml. This concentration range, as shown in Fig. 2A of a previous paper (Biotechnology Journal, DOI: 10.1002 / biot.202100096), is close to the albumin secreted by human MSCs during culture (approximately 80 ng / ml) and can be considered the background endogenous albumin.

[0099] These results indicate that recombinant proteins are taken up to a certain extent by cultured cells during cell culture, and although they are apparently washed out by washing with PBS or other solutions, the proteins taken up once are re-released into the medium with continued culture. To keep the re-release of recombinant proteins taken up by cells to less than 5%, it is necessary to set re-release conditions in a medium that does not contain the recombinant protein at least once.

[0100] Example 4: Tests were carried out under the following conditions, as shown in the steps circled 1 to 3 in Figure 6, and samples were collected. Cell seeding density: 10,000 cells / cm 2 Plate: 6-well plate Culture conditions: Cell proliferation culture was performed using ADSC-4 medium (2 ml / well). (Note: As shown in Figure 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, for conditions 1 to 3, samples were collected as follows. Condition 1: When the cells reached confluence in the expansion culture using ADSC-4 medium (the same number of days for the expansion culture period in conditions 1 to 3), the entire medium was removed and the cells were washed once with PBS. The entire PBS was then removed, and the extraction medium (medium specified in the extraction conditions above) was added. After two days, the supernatant was collected. The supernatant was centrifuged (1,000 rpm, 3 minutes) to remove contaminants such as cell debris, and the supernatant alone was collected as a sample (condition 1). Condition 2: When the cells reached confluence in the expansion culture using ADSC-4 medium supplemented with recombinant VEGF protein (the same number of days for the expansion culture period in conditions 1 to 3), the entire medium was removed and the cells were washed once with PBS. The entire PBS was then removed, and the extraction medium (medium specified in the extraction conditions above) was added. After two days, the supernatant was collected. To remove contaminants such as cell debris from the supernatant, the cells were centrifuged (1,000 rpm, 3 minutes), and the supernatant was collected as a sample (Condition 2). Condition 3: When cells reached confluence in growth culture using ADSC-4 medium supplemented with recombinant VEGF protein (the same number of days for conditions 1 to 3), the entire medium was removed and washed once with PBS. The entire PBS was then removed, and the extraction medium (medium specified in the extraction conditions above) was added, and the entire medium was immediately collected (Sample A). The extraction medium (medium specified in the extraction conditions above) was then added again, and the supernatant was collected two days later (Sample B). To remove contaminants such as cell debris from the supernatant, Samples A and B were centrifuged (1,000 rpm, 3 minutes), and the supernatant was collected as a sample (Condition 3, immediately after) and (Condition 3, 2 days after), respectively.

[0102] The VEGF protein concentrations in these samples were measured. The results are shown in Figure 7. Condition 1 in Figure 7 indicates that there is almost no VEGF protein secreted by the cells. Condition 2 in Figure 7 indicates that the use of recombinant VEGF protein is a prerequisite for the protein to be introduced into the supernatant. Furthermore, Condition 3 in Figure 7 indicates that no VEGF protein was detected when DMEM medium was added immediately after PBS washing (condition 3, immediately after), whereas high concentrations of VEGF protein were detected after 2 days (condition 3, 2 days after). These results indicate that unwanted components, such as recombinant VEGF protein, are difficult to remove even with PBS washing once they are internalized within the cells and are re-released into the medium after a certain period of culture. While it was previously believed that unwanted components, such as recombinant proteins, could be washed away with PBS, the above example demonstrates that substances internalized within the cells are not washed away by a quick wash but are released into the medium over time, and therefore require a period of release in the medium. 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 test steps shown in Figure 8 were carried out 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 cultured and expanded 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 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 supernatant were measured. The results are shown in Figures 9 and 10. Surprisingly, as shown in Figure 9, the patented method (the method of the present invention) resulted in higher HGF (hepatocyte growth factor) secretion. Specifically, a comparison of the conventional method (supernatant extracted for two days after PBS washing) with the method of the present invention (a method that adds a one-day exposure to basal medium step to the conventional washing step) revealed that the supernatant contained an increased concentration of the cytokine HGF. As shown in Figure 10, the patented method (the method of the present invention) produced the same amount of IL-6 secretion, suggesting that the number of cells targeted in the preliminary step did not change. This indicates that the increase in HGF secretion is due to the method of the present invention, and not to a change in the number of cells targeted in the preliminary step. In other words, if cell proliferation were to proceed during the washing period in the method of the present invention, the total cell number would increase at the time of extraction, and generally, 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 that extreme cell proliferation did not occur during the washing period. Therefore, it has become clear that the increase in HGF secretion is not due to cell proliferation during the washing period, but is due to the method of the present invention.

Claims

1. A method for producing a cell or a culture supernatant of a cell, comprising: culturing cells in a first medium containing no serum to obtain a first cell; culturing the obtained first cell in a second medium having a lower concentration of a component contained in the first medium than the first medium to obtain a second cell and a culture supernatant of the second cell; removing the culture supernatant of the second cell from the obtained second cell and the culture supernatant of the second cell to obtain a second cell; and culturing the obtained second cell in a third medium having a lower concentration of a component contained in the first medium than the first medium to obtain a third cell and a culture supernatant of the third cell, thereby producing a third cell or a culture supernatant of the third cell.

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

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, and 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, and 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 of claim 2, wherein the component contained in the first culture medium is a recombinant protein, a cell growth factor, or a human protein.

8. The method of claim 7, wherein the recombinant protein is a human protein and 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. 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, which has a lower concentration of a component contained in the first medium than the first medium, is a basal medium.

17. The method according to claim 4, wherein the sixth medium, which has a lower concentration of a component contained in the first medium than 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 lower concentration of a component contained in the first medium than in 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 lower concentration of a component contained in the first medium than in 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 lower concentration of a component contained in the first medium than 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 fifth cells are cultured in a sixth 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.

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 washing the third cells prior to obtaining the third cells.

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

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

28. The method according to any one of claims 1 to 27, wherein the cells or cell culture supernatant are for producing a pharmaceutical composition, a cosmetic composition, a food composition, a regenerative medicine product, or a specific cell processed product.

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