WO2004104187A1 - Fibroblast and method of culturing the same - Google Patents

Abstract

It is intended to provide a fibroblast which makes it possible to culture cells (in particular, fibroblasts) in a tissue cut off from a living body in a required amount while sustaining favorable biocompatibility, and a method of culturing the same.

Description

 Specification

 Fibroblast and culture method thereof

 Technical field

 The present invention relates to a portion of cells excised from a living body, particularly to fibroblasts derived from dermal tissue of skin and a method for culturing the same.

 Background art

 [0002] For example, when a depression (scratch, wrinkle, self-depression, etc.) occurs on the skin of a living body for some reason, restoring or restoring the state before the depression is formed is in the medical field, In particular, it is of great significance as a treatment mainly aimed at beauty (removing wrinkle lines, repairing dents in accidents, etc.).

 [0003] In order to perform such treatment, it is conceivable to use artificial skin (for example, Japanese Patent Application Publication No. 2001-104346).

 [0004] However, incorporation of artificial matter such as artificial skin into a living body is not preferable because rejection may occur.

 [0005] Further, techniques such as artificial skin and skin transplantation are intended to compensate for widespread damage of the skin due to burns or the like. This is to cope with the case where the maintenance of dyed water and the local immunity are lost. Therefore, it is not originally targeted to deal with a case where a defect occurs in the elastic fiber under the skin, such as a wrinkle or a recess due to an accident.

 [0006] On the other hand, in recent years, cells of the skin of a living body (patient) for the purpose of repairing a concave portion of the skin are cultured, and the cultured skin cells are filled in the concave portion as described above, and the concave portion is filled. Techniques for restoring to the same level as the surrounding area (レ, so-called “re-transplantation”) have been implemented.

 [0007] With such a technique, the problem of rejection does not occur after re-implantation, and it is convenient for dealing with the loss of elastic fibers below the skin.

[0008] Here, skin cells, particularly fibroblasts, need a three-dimensional structure (matrix) to serve as a "scaffold" for growth. In other words, without providing such a three-dimensional structure, even if only fibroblasts are injected into a patient (living body), the injected fibroblasts will not be sufficiently injected. Do not grow.

 [0009] However, it has been difficult for conventional culture techniques to culture skin cells of a living body (patient) to an extent sufficient to fill the living body (patient) and repair the recess.

[0010] At the time of culturing, small pieces (for example, skin pieces) having cells to be cultured are placed on a substance called a medium, and cultured in an environment similar to that of a living body. As this medium, various types are commercially available. For example, when culturing human skin cells, a medium called a mammalian cell culture basic medium is used.

 [0011] Here, it is difficult to make the nutrients contained in such a medium exactly the same as the nutrients supplied to the skin tissue in the living body, and therefore, a sufficient amount of skin can be obtained by conventional culture techniques. The cells could not be cultured.

 [0012] Disclosure of the Invention

 The present invention has been made in view of the above points, and is a fibroblast capable of culturing cells of a tissue excised from a living body, particularly fibroblasts, in a required amount while having biocompatibility. It is intended to provide a cell culture method and fibroblasts obtained by the culture method.

 [0013] This object is solved by the means described below.

 [0014] According to the first aspect of the present invention, the culture method of the present invention includes at least a part of the same blood to be collected in the incubator having fibrin deposited on the culture surface. Filling the fibroblasts into the incubator filled with the liquid medium; and culturing the introduced fibroblasts. Features. It is preferable that a sheet made of a polymer material derived from a living body (for example, fibrin) is spread.

 [0015] By using the culture method of the present invention, the cultured cells do not penetrate the polymer material sheet (for example, fibrin sheet) and adhere to the bottom of the casing (the Petri dish 20). It is very easily recovered from the member for use.

[0016] In the method for forming a sheet with fibrin and laying the sheet on the casing (a Petri dish 20), blood (autologous blood) is collected from a living body having skin cells (fibroblasts) to be cultured. And the step of coagulating the blood (autologous blood) Collecting blood (eg, by centrifugation) and crushing the collected (coagulated) blood (autologous blood) (eg, physically, by aspirating it into a syringe and pushing it out with a needle) It is preferable that the collected (coagulated) blood (for example, blood extruded from an injection needle) be spread over the bottom of a casing (a Petri dish 20) of a culture device.

 According to a first alternative aspect of the present invention, the culturing method of the present invention relates to a method of culturing a blood sample collected from the same collection target as described above in an incubator having a biocompatible polymer mounted on the culturing surface. Filling a liquid culture medium containing at least a part thereof; a step of introducing the fibroblasts into the incubator filled with the liquid medium; and a step of culturing the introduced fibroblasts. ; Characterized by having; Further, the biocompatible polymer is preferably a low-temperature sensitive polymer.

 [0018] When skin cells, especially fibroblasts, are cultured and proliferated, they extend below the medium, reach the bottom of the casing (dish 20), and are difficult to collect from the culture device of the present invention. There is a possibility that it will be difficult.

 With this configuration, it is possible to extremely easily collect cells from the culture surface of the culture member.

 [0020] The medium used in the first aspect of the present invention preferably contains serum collected from the same collection target. This makes it possible to obtain fibroblasts that are more compatible with the same collection target.

According to the first aspect of the present invention, it is possible to obtain more biocompatible cells by culturing and proliferating while being in contact with the extremely biocompatible material used for cell culture. .

 According to the second aspect of the present invention, the fibroblast according to the present invention is obtained by the same collection method as described above, wherein the fibroblast is cultured using a liquid medium containing at least a part of blood collected from a collection target. Derived from the subject. This “at least a part” is preferably the same collected serum as the fibroblasts derived from the collected subject.

[0023] As the liquid medium or medium in the present invention, a basic medium for culturing a mammalian cell can be used, and for example, a force including DMEM, RPMI1640, HANKS, Fl2, and the like is not limited thereto. Not.

 [0024] If the amount of the component (for example, autologous blood 26) collected from a living body with respect to the medium (24) is too small, the osmotic pressure of blood or a culture solution including proteins such as growth factors such as FGF contained in blood. Insufficient components (albumin) to maintain On the other hand, if the amount of blood (autologous blood) collected from the living body relative to the medium (24) is too large, the amount of the medium (24) becomes relatively small, which is necessary for culturing skin cells contained in the medium. Some nutrients, such as liquid nutrients such as amino acids and carbohydrates, are deficient.

 [0025] For such a reason, the above-described ratio (4% 50%) of the blood (autologous blood 26) to the medium (24) is preferable.

 [0026] By using these culture solutions and culture systems, target cells, particularly fibroblasts, that do not cause rejection can be proliferated, and can be easily recovered from the culture systems.

 [0027] According to the configuration, by adding blood collected from a living body having skin cells (fibroblasts) to be cultured to a culture medium, the blood can be added to a conventional culture medium. The protein contained and the components (albumin) required to maintain the osmotic pressure of blood and culture solution are provided in the culture device.

 [0028] Accordingly, among the skin cells, fibroblasts having particularly strong regenerative power grow and grow very well.

 [0029] Here, proteins contained in blood and components (albumin) necessary for maintaining the osmotic pressure of blood and culture solution are supplied by blood collected from other living bodies, bovine serum, or the like. It may be possible to do so. However, in this case, the proliferated cells are contaminated by contaminants that may cause toxicity to the proliferated cells, such as blood collected from other organisms or pathogens contained in bovine serum. There is a risk that it will.

 [0030] Such a situation must be prevented, especially when the cells are transplanted again.

[0031] According to the method of the present invention, blood collected from a living organism having skin cells (fibroblasts) to be cultured (autologous blood) is added to a culture medium, so that blood of another living organism is added. And the possibility of transmission through the blood of other animals (eg, cattle) is completely excluded. [0032] In the present invention, there is provided a culture case for containing the above-mentioned casing filled with a culture medium and autologous blood, wherein the culture case has an internal temperature of about 37 ° C and about 5% of carbon dioxide. The atmosphere is preferably configured to have an atmosphere containing.

[0033] If such a configuration is adopted, the cells to be cultured are cultured in an atmosphere close to the in-vivo environment, so that the conditions for multiplying are established.

 BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram illustrating an embodiment of collecting skin pieces.

 FIG. 2 is a process diagram illustrating a processing procedure before culturing.

 FIG. 3 is a cross-sectional view illustrating a main part of the culture device of the present invention.

 FIG. 4 is a perspective view illustrating the structure of a culture case used in the present invention.

 FIG. 5 is a perspective view showing an embodiment in which fibroblasts are collected from a culture device.

 FIG. 6 is a view showing a step of mixing fibroblasts with a substrate.

 FIG. 7 is a view showing a step of mixing fibroblasts and a substrate, which is a step subsequent to FIG. 5;

 FIG. 8 is a view showing a state where a filling material is sucked into a syringe.

 FIG. 9 is a view showing a state where a filling material is injected subcutaneously.

 FIG. 10 is a view showing a filling material after subcutaneous injection.

 FIG. 11 is a diagram of a first stage showing an embodiment in which the proliferated fibroblasts are collected.

 FIG. 12 is a diagram of a second stage showing an embodiment of collecting the fibroblasts shown in FIG. 11.

 FIG. 13 is a diagram of a third stage showing an embodiment of collecting the fibroblasts shown in FIGS. 11 and 12. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

 [0036] As described above, when performing a treatment for filling a concave portion formed in the skin (treatment for cosmetic purposes: for example, removing a wrinkle line, repairing a dent portion due to an accident or the like), it is difficult to perform a treatment such as a rejection reaction. Since no inconvenience occurs, it is desirable to fill the recess with cells of the patient's own skin to perform such a treatment.

 [0037] From such a viewpoint, it is necessary to culture the cells of the skin of the patient performing the treatment.

When culturing the skin cells, as shown in FIG. From the back part (between the ear Y and the hairline H: indicated by reference numeral 12 in FIG. 1), a 3 mm square piece of skin (indicated by reference numeral 10 in FIG. 1) is collected or excised. Other parts that can be collected include, in addition to the ear, the oral cavity, axilla, and thigh, but are not limited to these.

 [0039] The skin piece 10 is collected (or excised) so as to include the dermis layer. As described later, this is for isolating fibroblasts that migrate by dermal layer culture.

 [0040] Here, the portion behind the ear Y as a place where the skin piece 10 is collected "(12) is usually a place which is not affected by ultraviolet rays, and therefore, skin cells including fibroblasts are included. This is because it is a place where the condition is well maintained. At the same time, the marks (scratches) from which skin pieces were collected are noticeable.

 [0041] In other words, a portion where the condition of skin cells including fibroblasts is not affected by ultraviolet rays and the condition of skin cells including fibroblasts is well maintained, and a trace (scratch) of a skin piece collected is inconspicuous. In this case, a 3 mm square piece of skin 10 may be collected from a portion other than the portion behind the ear Y.

 Here, if an excessively large amount of skin is collected, there is a possibility that the collected trace may remain in the patient. In addition, it is impossible to obtain an excessively large piece of skin from the portion behind the ear Y described above. Therefore, the area of the skin piece 10 to be collected cannot be made larger than necessary.

 [0043] On the other hand, if the skin pieces do not have a certain size, there is a possibility that a sufficient amount of fibroblasts cannot be secured when performing the pretreatment described below and culturing.

[0044] For the above reasons, the skin piece 10 to be collected was set to "3 mm square". However, based on the above reasons, the size of the skin piece to be collected is not limited to “3 mm square” unless it is too large and not too small.

Next, the collected (excised) skin piece 10 is subjected to pretreatment according to the procedure shown in FIG.

First, the collected skin pieces 10 are washed twice in 20 mL of a washing solution (RINSE solution) (FIG. 2: Step Sl).

 [0047] This is for cleaning and removing foreign substances attached to the skin piece 10, particularly foreign substances attached to the outer skin.

[0048] This RINSE solution is added to prevent the skin pieces from drying and killing the cells. Have been calo. However, if the amount of RINSE solution is too large, the skin pieces to be treated will sink in the RINSE solution, making the treatment difficult. From such a viewpoint, the amount of the RINSE solution of “3 m LJ” was set for the “60 mm dish”.

 [0049] If the processing operation is short, physiological saline can be used instead. However, if the operation is performed over a long period of time, general additives such as glucose and vitamins used for cell culture are used. It is preferable to use a RINSE solution containing nutrients such as foods.

 [0050] Here, the washing solution (RINSE solution) is a solution prepared for washing the skin pieces, and is adjusted so that the osmotic pressure and the mineral composition match the cells of the collected skin pieces.

 [0051] The RINSE solution can be prepared on a case-by-case basis, but a commercially available product can also be used. Such RINSE solutions include physiological saline, Kreps Ringer's solution, phosphate buffer solution (PBS), and the like, but are not limited thereto.

 [0052] In step S1, the amount of the RINSE solution "20 mL" is an amount exemplified as a necessary and sufficient amount for washing and removing foreign substances attached to the collected skin pieces 10. If the amount is too small, the skin piece 10 cannot be sufficiently washed. However, if the amount of use is increased too much, it is uneconomical because the skin piece 10 to be cleaned has a small size of 3 mm square.

 [0053] However, the amount of the RINSE solution is not limited to 20 mL as long as the skin pieces 10 can be sufficiently washed to remove the deposits, and the amount does not cause ecology. Absent

After the washing, the skin piece 10 from which the adhered substance has been removed by washing is put into a dish having an inner diameter of 60 mm containing 3 mL of the RINSE solution (Step S2).

 [0055] The RINSE solution is added to the skin in order to prevent the skin pieces from drying and killing the cells. However, if the amount of RINSE solution is too large, the skin pieces to be treated will sink in the RINSE solution, making the treatment difficult. From such a viewpoint, the amount of the RINSE solution of “3 m LJ” was set for the “60 mm dish”.

[0056] It is possible to substitute physiological saline if the processing operation is short, but if the operation is performed over a long period of time, a common addition such as glucose and vitamins used for cell culture is used. It is preferable to use a RINSE solution containing nutrients such as foods. Then, using a surgical knife or the like, the washed skin piece 10 is cut into small pieces having a diameter of about lmm (Step S3).

[0058] After the skin pieces have been minced, the minced skin pieces are transferred together with the RINSE solution to another container (for example, a 50-mL volume Serum tube, and pipetted with a 10-mL pipette (Step S4). This is performed to sufficiently disperse the minced skin pieces and thoroughly wash each of the minced skin pieces.

[0059] Then, centrifugation is performed for 5 minutes at 100,000 rpm using a centrifuge (not shown) (step S5). When the centrifugation is completed, discard the supernatant, add lOmL of new RINSE solution (Step S6), centrifuge again, and centrifuge at lOOOOrpm for 5 minutes (Step S7). .

 [0060] Steps S5 and S7 are performed to collect the skin pieces shredded by the surgical knife in step S3. These steps are performed to sufficiently collect only the desired shredded skin pieces.

 [0061] Since the shredded skin pieces are scattered in the RINSE solution, it is difficult to collect them as they are. However, if centrifugation is performed in the steps shown in steps S5 to S7, the shredded skin pieces are separated from the RINSE solution and the like, and are extremely easily collected. In addition, in order to efficiently collect only the intended skin pieces, the centrifugation is performed at 100 rpm for 5 minutes as described above. It is not limited.

 Here, after washing the 3 mm square test piece 10, it is not immediately cultured in the medium but cut into small pieces having a diameter of about lmm and collected. Compared to a case where the four sides of the skin pieces 10 were directly placed on the culture medium without performing the treatment shown in FIG. 2 and cultured, the skin pieces having a diameter of about lmm were This is because fibroblasts, which have a greater degree of freedom in cells in the dermis layer, are more likely to migrate when dispersed in the dermis.

[0063] When the centrifugation in Step S7 is completed, 3 mL of the RINSE solution is added to the precipitate containing the cells (Step S8), and the entire amount of the pellet containing the cells and the RINSE solution is transferred to a cell culture dish ( Step S 9). The petri dish for cell culture will be described later in detail. [0064] Thus, the pre-processing is completed.

 [0065] If the pretreatment is completed after step S9 is performed, the temperature is set to 37 ° C in an atmosphere of 5% CO.

 2. Leave to stand for at least 2 days, and then aseptically culture the cells in the petri dish for at least 2 weeks (step S10).

 Here, step S10 is a stage in which cells are already cultured, and it cannot be called pretreatment. In that sense, in FIG. 2, step S10 is only marked with parentheses, and is clearly different from steps S1 and S9 surrounded by a square frame.

 Here, a culture apparatus for culturing a Petri dish for cell culture (Step S9) to which the collected skin debris has been transferred will be described with reference to FIG.

 [0068] In Fig. 3 represented as a cross-sectional view, this culturing apparatus is indicated by reference numeral 10 as a whole, and a culturing surface 32 of a commercially available petri dish 20 made of glass, synthetic resin, or the like is provided with a high molecular material. (For example, fibrin sheet), a layer made of 22 is formed, and contains a mixture of a cell culture medium 24 and autologous blood 26, and a skin strip is formed on the layer of the polymer material 22. 30 is placed.

 [0069] More specifically, the culture surface 32 of the Petri dish 20, which serves as the casing of the culture device, is provided with a layer made of a polymer material (for example, fibrin, collagen, platelet-rich plasma, CMC, etc.) 22, and has a high density. A skin strip 30 is placed on a layer of molecular material 22.

 [0070] Then, a cell culture medium (basic culture medium for mammalian cells: hereinafter, simply referred to as "medium" in the present specification) 24 and a patient from whom skin pieces 10 were collected in the step shown in FIG. A mixture 25 in which the own blood (autologous blood) 26 is inseparably mixed is filled in an area above the layer of the polymer material 22 on which the skin strip 30 is placed. Autologous blood 26 does not clot because it is a serum component collected from heparin.

 Here, when the culture medium 24 and the autologous blood 26 are mixed, the ratio of the autologous blood 26 to the culture medium 24 is 4% 50%.

 [0072] In Fig. 3, reference numeral 27 denotes a migrating fibroblast.

 [0073] Then, an atmosphere with a temperature of 37 ° C and 5% CO (described in relation to step S10 in Fig. 2)

 Two

Leave the cells in the Petri dish under sterile culture for 2 weeks or more. As a result, fibroblasts migrate from the dermis layer of the skin strip 30. The culture vessel having the above-described configuration is accommodated in a culture case 100 as shown in FIG. 4, and the atmosphere in the culture case 100 is adjusted to various conditions as described with reference to step S10 in FIG. Is adjusted to an atmosphere that satisfies the conditions, ie, an atmosphere of a temperature of 37 ° C. and 5% CO.

 Two

 [0075] In such an atmosphere (temperature of 37 ° C, 5% CO 2), leave still for 2 days or more, and then

 Two

 The cells in the jar are cultured aseptically. As a result, the dermal layer fibroblasts of the skin strip 30 migrate.

 [0076] The reason for allowing the fibroblasts to stand still for 2 days or more is to release the fibroblasts, solidify the scaffold, and stabilize the original culture state. It is necessary at least for culturing. During the culture period, the mixture 25 is replaced with a fresh one at least twice a week, preferably daily.

 [0077] In FIG. 4, a plurality of Petri dishes 20 for culturing fibroblasts by filling the above-described medium with autologous blood therein are accommodated in a culture case 100. This petri dish 20 is placed on the bottom 104 of the culture case 100 via a door 102.

 [0078] The culture case 100 has a temperature sensor 106, and the inside of the culture case 100 communicates with an analysis means 108 for analyzing the air composition in the case 100.

 [0079] Then, the internal temperature and composition of the culture case 100 are set to predetermined values (temperature 37 ° C, 5% CO 2).

 A setting means 110 is provided for maintaining at 2.

[0080] The adjusting means 110 is in communication with the carbon dioxide supply source 112 via a pipe 114, and the pipe 1

An adjustment valve 116 is interposed in 14.

[0081] The analysis means 108 oscillates a control signal based on the analysis result of the air composition in the culture case 100, and the preparation valve 116 receives the control signal and determines the opening. As a result, the carbon dioxide concentration in the culture case 100 was 5. / ₀ so that it is maintained at

From two, carbon dioxide is supplied.

[0082] Further, the measurement result of the temperature sensor 106 is used as a control unit of the adjusting means 110 (for example,

It has a HEPA filter 120 and a temperature control means (for example, an air conditioner) 122, and the heating amount of the heater 122 is controlled by the control unit 118.

[0083] Outside air is supplied to the adjusting and setting means 110 by a blower (not shown). Conditioning and installation means 11

The air (outside air) supplied inside is sterilized and dust-removed by the filter 120 and is suitable for cell culture. The air is cleaned. On the other hand, the carbon dioxide supplied from the carbon dioxide supply source 112 via the pipe line 114 mixes with the cleaned air.

[0084] Then, the temperature of the cleaned air and carbon dioxide is adjusted to 37 ° C by air conditioner 122. Then, the solution is supplied into the culture case 100 through the conduit 124.

[0085] As a result, the inside of the culture case 100 is maintained in an atmosphere of 37 ° C and 5% CO.

 Two

 is there.

 [0086] The culture case 100 is not limited to the size shown in FIG.

 For example, a case where the entire room (sterile room) is used as the culture case 100 is also included.

[0087] In this case, only fibroblasts proliferate. Even if epidermal cells or dermis components are contaminated, they do not have the activity to survive and proliferate under these conditions. Therefore, only fibroblasts are cultured and proliferated.

[0088] Fibroblasts constitute interstitial tissue interposed between parenchymal cells of each organ having a high proliferative capacity, have a spindle type, and are derived from the mesenchymal system (in the future, interstitial cells). Cells), which mainly produce collagen proteins.

[0089] Fibroblasts are differentiated into fibroblasts. Usually, fibroblasts and fibroblasts are mixed, and both synthesize and secrete collagen fibers inside the cells.

[0090] The function of fibroblasts in vivo is to repair and maintain stromal cells, and in the process of repairing wounds and inflammation, they proliferate vigorously and produce elastic fibers such as collagen and proteodalican. And responds to repair and defense reactions of the body.

[0091] If the fibroblasts survive, they will stay in place and become a source of collagen.

 [0092] Such properties are favorable for the above-mentioned treatment, ie, treatment for filling a concave portion formed in the skin (treatment for cosmetic purposes: for example, removing wrinkle lines, repairing a dent part due to an accident or the like).

[0093] Here, the medium 24 contains some nutrients necessary for culturing skin cells. Specifically, it contains liquid nutrients such as amino acids and carbohydrates.

[0094] However, the conventional culture medium 24 does not include proteins contained in blood or components (albumin) necessary for maintaining the osmotic pressure of blood or culture solution. [0095] Therefore, the autologous blood 26 is filled.

 [0096] In other words, the medium 24 and / or the autologous blood 26 alone do not satisfy the conditions necessary for culturing skin cells (fibroblasts).

[0097] As a medium, DMEM (Dulbecco's Modified Eagle Medium: commercially available from Invitrogen Corporation) was used.

[0098] Here, the table shows the DMEM compositions of usable culture media.

[0099] [Table 1]

However, if the composition of the medium, such as the amount of glucose, the amount of amino acids (particularly glutamic acid), vitamins, mineralella, etc., matches the conditions of fibroblasts, a medium other than DMEM, such as ME

It is also possible to use M, RPMI1640, HANKS, F12, etc.

[0100] In addition to the illustrated embodiment, the inventor also carried out experiments in a case where the culture was performed only with a medium (DMEM) and in a case where the culture was performed with only autologous blood.

[0101] Table 2 below shows a comparison between the results of such an experiment and the case where fibroblasts were cultured by adding autologous blood to the medium 24 as shown in Fig. 3.

[0102] [Table 2] Petri dish contents Result of culture

 Medium only does not grow

 Only autologous blood died

 Medium + autologous blood growth

Subject: 3 women in their 20s

 Women in their 30s 2

 40s male 1 person

 Medium: DMEM

 14 days sterile culture at 37 ° C, 5% C02

 As is evident from Table 2, the culture results of fibroblasts when the autologous blood 26 was added to the culture medium 24 (see Fig. 3) were very good, whereas the culture medium 24 alone or the autologous blood 26 was used. Alone, the fibroblast culture failed.

[0103] By using the autologous blood 26, it is possible to prevent the inconvenience that cells constituting the cultured skin strips cause a rejection reaction or the like. It is also possible to prevent the proliferated fibroblasts from being infected with various diseases such as various blood-borne infectious diseases.

[0104] Furthermore, as a result of using autologous blood 26, there is no possibility of so-called "mad cow disease" (BSE) infection, unlike the conventional method of culturing using bovine serum.

[0105] The amount of autologous blood 26 is sufficient for 10 mL, which is known from the experimental results of the inventors in the past. The amount of autologous blood 26 may be less than 10 mL. During the cultivation of fibroblasts, a situation in which proteins and components (albumin) necessary to maintain the osmotic pressure of blood and culture medium are insufficient. Use 10 mL of autologous blood 26 to prevent this.

As described above with reference to step S10 in FIG. 2, the Petri dish 20 as shown in FIG. 3 is operated in an atmosphere at a temperature of 37 ° C. and 5% C〇2 (similar to the environment in a human body). Incubate for at least 2 days and then aseptically culture for about 2 weeks. As described above, the Petri dish 20 as shown in FIG. 3 is placed in a culture case 100 (FIG. 4) at a temperature of 37 ° C. under an atmosphere of 5% CO (an atmosphere similar to the environment in a human body). And conventional

 Two

 The culture conditions are the same, as shown in the results of Table 1 above), and the cells are allowed to stand for 2 days or more, and then are subjected to aseptic culture for about 2 weeks.

[0108] During the aseptic cultivation, fibroblasts of the skin strip 30 divide and proliferate, and migrate from the dermis layer of the collected skin strip.

 Here, when fibroblasts divide and proliferate, they extend downward (so-called “stretched” state) and reach the bottom of petri dish 20. Then, when the fibroblasts are further divided and proliferated, when the cultured fibroblasts are collected from the petri dish 20, the cultured fibroblasts adhere to the bottom of the petri dish 20 and cannot be removed therefrom. If such a situation occurs and the cultured fibroblasts cannot be collected, or if the fibroblasts attached to the bottom of the Petri dish 20 are forcibly removed and the fibroblasts are destroyed. In addition, the fibroblasts cultured for a long time cannot be used effectively.

 [0110] Therefore, conventionally, when such a situation occurs, the cultured fibroblasts were peeled off from the bottom of the petri dish using enzymes (trypsin, ligase). However, a great deal of labor had to be spent to perform the treatment using the enzyme.

 On the other hand, in the illustrated embodiment, as shown in FIG. 3, a layer made of a high molecular material (eg, fibrin sheet) 22 is formed on the bottom of the petri dish 20. Proliferated fibroblasts grow downward and attach on a layer of polymeric material (eg, fibrin sheet) 22. Few fibroblasts that have grown downward penetrate the layer of polymeric material 22 and reach the bottom of the Petri dish 20.

 Therefore, in the container of FIG. 3, the inconvenience caused by the fibroblasts that have proliferated and grown downward adhere to the bottom of the petri dish 20 is prevented.

 [0113] As a result, the proliferated fibroblasts can be easily peeled off from the Petri dish 20 without destroying the fibroblasts and recovered.

[0114] Table 3 shows the results of recovering (proliferating) fibroblasts when the fibrin sheet 22 was spread on the bottom of the Petri dish 20, and the Petri dish 20 was directly filled with the medium 24, and the fibrin sheet 22 was not spread. The recovery results in the case are shown in comparison. [0115] In Table 3, the "recovery rate" was determined by the naked eye by preparing 20 Petri dishes in which fibroblasts were cultured, and those with fibrin sheets spread and those without fibrin sheets. The number of Petri dishes in which fibroblasts cultured within the possible range were collected was expressed as a percentage of the total number of Petri dishes (20).

 [0116] Similarly, the "fraction of cells destroyed" refers to the number of petri dishes that fibroblasts adhered to the bottom wall or side wall and could not be detached and collected as a percentage of the total number of petri dishes (20). It is expressed by

 [0117] [Table 3]

It is clear from Table 3 that the disadvantage of cells with fibrin sheet being crushed (crushed) is suppressed.

Next, an embodiment in which a layer made of the polymer material 22 is formed of a so-called “fibrin sheet” will be described.

 In FIG. 3, in order to form a layer of the fibrin sheet 22 (polymer material) on the bottom of the petri dish 20, self-blood-strength-collected fibrin is spread in the form of a film on the bottom of the petri dish 20 and formed as a fibrin sheet. You do it.

[0120] Specifically, 10 mL of autologous blood is coagulated, and the coagulated autologous blood is collected by centrifugation. Here, the numerical value “10 mL” is exemplified as a numerical value considered necessary and sufficient, and has no limiting meaning.

[0121] The collected (coagulated) autologous blood is placed in a syringe and physically crushed by pushing it out of a syringe needle. In this state, if the petri dish is spread on the bottom, a layer of the fibrin sheet 22 is formed on the bottom of the petri dish.

 [0123] When the fibrin sheet 22 is formed by such a method, it has been experimentally proved that 10 mL of the amount of autologous blood to be coagulated for preparing the fibrin sheet is sufficient. That is, a total of 20 mL of the autologous blood of the patient from whom the skin piece was collected is collected in addition to the 10 mL required for the culture described above.

 [0124] Autologous blood 26 (Fig. 3) used for culture together with medium 24 is subjected to "heparin blood sampling" so as not to coagulate.

 Next, another embodiment of the production of a fibrin sheet will be described.

 [0126] Blood is collected from the patient from whom the skin fragments have been collected, and centrifuged at the stage where coagulation has started 2-3 hours after the blood collection. If the coagulated clot (coagulated clot) of the collected blood has settled down, the upper uncoagulated component is spread on a medium (eg, a nutrient medium such as a common amino acid).

 [0127] The "non-coagulated component" is mostly serum, plasma, and other small amounts of leukocytes.

, Platelets and erythrocytes, and a necessary and sufficient amount of uncoagulated components (including fibrin) remain in serum.

Therefore, when the “non-coagulated component” is spread on a medium, the “non-coagulated component” is dispersed.

The remaining amount of fibrin contained in the "" is gradually solidified on the ground, forming a thin film, that is, a fibrin sheet.

[0129] Then, the fibrin sheet thus formed may be spread on the bottom of the petri dish as shown in FIG.

 [0130] Culture of fibroblasts (of skin strips 30) is performed until the number of cells grows from about 10 million to about 100 million. However, the number of cells grown is not a strict condition for determining the culture period. This is because even if the order of cell number is different by one digit, there is almost no change in capacity.

[0131] Initially, the number of fibroblasts is about several hundred at the beginning of the culture, and after several hundred fibroblasts are released and proliferated sufficiently to reach the above-mentioned number of cells, The patient from whom the skin strip was collected). That is, the cultured or proliferated fibroblasts are injected from a petri dish 20 as shown in FIG. The injected fibroblasts grow at the injected site and produce collagen to repair wounds, wrinkles, and other dents in the patient.

 [0133] Here, in order for fibroblasts to grow, a three-dimensional structure (matrix) that serves as a "scaffold" is required even after being injected into a patient (living body). In other words, even if only fibroblasts are injected into a patient (living body) without providing such a stereostructure, the injected fibroblasts do not grow sufficiently and the amount of collagen production is low. It will be connected.

 Therefore, before injecting only cultured fibroblasts into a patient (living body), the fibroblasts are mixed with a substrate for providing a three-dimensional structure (matrix) for fibroblast growth.

 [0135] The base materials used are as follows.

 [0136] (l) Hespander (Product name: Kyorin Constraint Co., Ltd.) / Dextran (Dextran: Product name: Otsuka Pharmaceutical Co., Ltd.)

 (2) Collagen or Hyaluronic acid

(3) Fibrin

 (4) PRP (Platelet Rich Plasma)

 (5) CMC (Carboxy Methyl Cellulose)

(6) PLA (Poly lactic acid)

 “Hespander / Dextran” in (1) above has an osmotic pressure equal to or higher than that of blood and is commercially available as a plasma substitute. When commercialized, commercially available amino acid preparations as nutrients for fibroblast growth, such as moripron (trade name: product of Ajinomoto Pharma Co., Ltd.), Hybleamine (hy-pleamin: trade name: Fuso Pharmaceutical Co., Ltd.) It is preferable to use a mixture of products of the formula company).

[0137] Collagen or hyaluronic acid of the above (2) is a common injection used for injection into wrinkles. It has the advantage that a temporary anti-wrinkle effect can be obtained even if fibroblasts do not proliferate.

[0138] Since those used in the prior art are derived from animals, sufficient treatment to prevent infection and other pathogens was essential. On the other hand, in the present invention, Fibroblasts produce collagen, but the fibroblasts of the patient's own are cultured in the patient's own blood (autologous blood), and the fibroblasts proliferated in this way produce collagen. Disease and other pathogens. "

 [0139] The fibrin of the above (3), particularly the fibrin generated from the autologous blood of the patient, is most suitable as the base material.

 [0140] The PRP in the above (4) is plasma containing a large amount of platelets and platelet-derived growth factors contained in autologous blood. (How to collect PRP from patients. Claim!)

 CMC in item (5) above is a material used in breast augmentation, for example. Having biocompatibility has the following advantages.

 [0141] (6) PLA (Poly lactic acid) is a substance produced by fermenting starch and sugars such as corn potatoes with lactic acid bacteria and combining them by chemical treatment, and is derived from plants. As a biocatalyst with low allergenicity, it has the effect of promoting the self-production of collagen.

 [0142] Next, the case of reimplantation into a living body using the fibroblasts cultured as described above and the above-described base material will be described with reference to Figs.

 [0143] When the above-described base material is mixed with the cultured fibroblasts, as shown in FIG. 5, the fibroblasts 31 cultured in the dish 20 are aspirated by an instrument 40 such as a pipette or a syringe. Do At this time, as shown in FIG. 3, a fibrin sheet is spread on the bottom of the petri dish 20, so that the fibroblasts 30 are easily detached from the petri dish 20 without adhering to the petri dish, and the inside of the instrument 40 is removed. Is sucked.

 [0144] The fibroblasts aspirated by the device 40 are transferred into a shaking container 42 such as a test tube as shown in FIG. The above-described base material 34 is further put into the container 42 by an instrument 44 such as a pipette or a syringe.

 [0145] The ratio of fibroblasts to the substrate can be up to about 1: 4 to 4: 1, and can be mixed, for example, at 1: 1. However, this varies depending on the type of the base material and other conditions. For example, the mixing amount of a viscous base material such as hyaluronic acid may be small.

Here, at the stage shown in FIG. 5, together with the cultured fibroblasts 30, the fibrin sheet (reference numeral 22 in FIG. 3; not shown in FIG. 5) spread on the bottom of the Petri dish 30 is also used with the instrument 40. When sucked, it is not necessary to newly add the substrate 34 to the container 44.

[0147] The fibrin itself constituting the fibrin sheet acts extremely effectively as a base material (as described above), so it is sufficient to put the fibrin sheet sucked by the device 40 into the container 44 together with the fibroblasts 30. Because.

[0148] After the fibroblasts 30 and the base material 34 (including the case of the fibrin sheet) are charged, the container

 42 is shaken by hand HH as shown in FIG.

[0149] Here, a machine such as a shaker is not used because there is a risk that fibroblasts may be crushed by mechanical shaking. Of course, if the bottom side can be shaken to the extent that cells are not crushed, hygiene considerations are sufficient, and if the labor can be reduced compared to manual HH, mechanical shaking should be performed. Is also good.

[0150] The substrate (or matrix) and the fibroblasts, that is, the mixture of the fibroblasts and the substrate, which have been shaken by the process shown in Fig. 7, are inhaled into the syringe as the filling material 50 (Fig. 8).

 [0151] As shown in Fig. 9, the filling material 50, which is a mixture of fibroblasts and the base material, is provided with a concave portion 55 (for example, a wound, a wrinkle, a defect caused by an accident, etc.) formed on the skin S of a living body. Is injected into the area below (the subcutaneous part).

 [0152] As a result of injecting the filling material 50 subcutaneously, the concave portion on the skin S (the concave portion 55B shown by a dotted line in Fig. 9) is the same as the other portions on the skin S as shown by reference numeral 55A in Fig. 9. It rises to the level and is restored.

 [0153] The fibroblasts injected subcutaneously as a composition of the filling material 50, because of the presence of the base material, grow steadily even at the injected site and produce collagen, which is an elastic fiber. Therefore, even if the injected fibroblasts have reached their end of life, they will divide and produce another fibroblast, and collagen production will continue. Then, the repaired portion 55A maintains the same level as other portions on the skin S.

[0154] Here, in the reimplantation into the living body shown in Fig. 8 and Fig. 10, the cultured fibroblasts are removed from the Petri dish 20 (or collected and recovered: the process of Fig. 5) within a predetermined time (6 (Within hours), preferably immediately after mixing, is injected subcutaneously into a predetermined part of the living body (wounds, wrinkles, depressions, etc.) and filled with a syringe (FIG. 10). [0155] The fibroblasts recovered from the culture medium 24 of the Petri dish 20 are not supplied with necessary nutrients (unlike on the culture medium 24 containing autologous blood), and thus are recovered from the culture medium 24. Then, the cell activity decreases. Therefore, in order for the fibroblasts returned to the living body to grow and function sufficiently, it is more advantageous that the shorter the time from the recovery from the medium 24 to the return to the living body, the shorter the time.

 According to experiments performed by the inventors, fibroblasts cultured in the medium 24 containing autologous blood 26 were recovered from the medium, and after 6 hours or more, they were returned to the living body. Cell activities necessary for growth have been lost.

 [0157] However, when fibrin or PRP is used as the base material, such a base material can provide favorable conditions for cell growth, and thus attenuates the deterioration of cell activity and reduces the medium power. After collecting fibroblasts, it is possible to specify the critical time until it is returned to the living body.

 Table 4 shows the experimental results regarding the critical time.

 [0159] Table 4 shows a comparison of the time required for fibroblasts to lose the required activity after recovery from the medium.

[0160] [Table 4]

As is evident from the experimental results shown in Table 4, collagen, hyaluronic acid, The use of Blin or PRP can extend the time it takes for fibroblasts to recover the required activity from the culture medium.

 [0161] Therefore, the fibroblasts recovered from the medium can be easily stored.

 [0162] The mixture of fibroblasts and substrate recovered from the medium can be stored frozen. In other words, if cultured fibroblasts, serum for cell preservation obtained from autologous blood, DMSO (Dimethyl Sulfoxide), and fibrin sheet (substrate) are mixed and rapidly cooled with liquid nitrogen, In that state, frozen storage becomes possible.

 [0163] Serum for cell preservation obtained from autologous blood can be obtained by adding "inactivation" to the serum. The operation of “inactivation” is an operation in which serum is heated at, for example, 55 ° C for 30 minutes. By such an operation, a factor (for example, TGF-) that suppresses cell growth is inactivated and components in the serum are removed. Use efficiency as nutrients can be increased.

 [0164] For frozen storage, put the proliferating fibroblasts in a stock solution containing 10% serum, medium, and 10% DMSO for storage, cool at 1 ° C per minute, and finally add liquid nitrogen. It is stored inside.

 [0165] Instead of DMSO, it is also possible to use a solvent used for storing cultured cells in liquid nitrogen. For example, glycerin (grycerin, glycerol) or HES (Hydroxy Ethyl Starch) may be used. It is also possible to use.

 [0166] After being mixed with the base material serving as a scaffold and injected into a predetermined location in a living body, fibroblasts grow smoothly and proliferate, and at the same time, produce collagen to produce wounds, wrinkles, and injuries. It fills the internal space, such as the resulting recess, and restores it to the state (in a living body) before such scratches, wrinkles, and recesses were formed.

 [0167] When the polymer sheet (eg, fibrin sheet) indicated by the reference numeral 22 in Fig. 3 is not used, the migrated fibroblasts are attached to the bottom of the Petri dish 20 and grow while solidifying the scaffold.

[0168] After a predetermined culture period has elapsed, the grown fibroblasts are peeled off from the bottom of the Petri dish 20, and in this case, the enzyme Trypsin (trade name: "Roche Inc. as a trypsin recombinant") was used. (Manufactured and sold by the company)), add about 25 mg if the diameter is 10 cm in a Petri dish, and incubate at 10 ° C-37 ° C for 10-20 minutes. Peel from the bottom of layer 20.

[0169] Trypsin causes cytotoxicity when it is allowed to act for a long period of time. Therefore, trypsin should be used with the minimum necessary processing time. For example, the timing of removing the fibroblasts from the bottom of the Petri dish is not determined by the temperature or time, but by observing them under a microscope. When the blasts have become round, detach them from the bottom of the Petri dish.

 [0170] The detached fibroblasts are collected by suction with a pipette, and centrifuged at 800 rpm for 5 minutes using a centrifuge. Since the fibroblasts sediment, collect them and make them turbid with fresh medium (approximately 510 mL). The fibroblasts, which are newly turbid in the medium, can be grown 10-fold by adjusting the relative concentration by, for example, dispersing them on 10 separate dishes and collecting the grown cells. .

 [0171] Hereinafter, a predetermined number of fibroblasts are obtained by repeating the above procedure of collecting, centrifuging, turbidity, and culturing in a plurality of different dishes.

 [0172] In the embodiment described above with reference to Fig. 5 to Fig. 10, there are cases where cells cultured on the polymer sheet 22 do not pass through the needle of the syringe, making it difficult to collect. As mentioned above, there is a demand that trypsin should not be used as much as possible if trypsin can be used to recover it.

 [0173] In order to cope with a strong case, for example, the polymer sheet 22 may be a low-temperature sensitive polymer. Hereinafter, a case where the low-temperature sensitive polymer is a polymer sheet will be described with reference to FIGS.

 If the fibroblasts 27 proliferated over the entire surface of the polymer sheet 22 as shown in FIG. 11 as a result of culturing in the state shown in FIG. 3 and FIG. 5, the control unit in FIG. A control signal is sent from 118 to the heating heater 122 to lower the internal temperature of the culture case 100 in which the Petri dish 20 is placed (the ambient temperature in which the Petri dish 20 is placed) to 37 ° C and 32 ° C. I do.

When the ambient temperature was lowered to 32 ° C., the state of attachment of the fibroblasts 27 to the sheet 22 was released based on the characteristics of the low-temperature sensitive polymer, as shown in FIG. Float in a mixture of medium and autologous blood 25 (liquid phase). [0176] In Fig. 12, fibroblasts detached from the sheet 22 and floated are indicated by reference numeral 27A.

[0177] Then, as shown in Fig. 12, if the total amount of the proliferating fibroblasts detached from the low-temperature-sensitive polymer sheet 22 and floated (fibroblasts indicated by reference numeral "27A"), a pipette-like shape was obtained. What is necessary is just to collect the floating fibroblasts 27A by using a large-diameter instrument 64.

 In the above description of FIG. 3, the ratio of autologous blood 26 to culture medium 24 in the mixture of cell culture medium 24 and autologous blood 26 contained in culture apparatus 10 according to the present invention is 4% — It was stated that it was 50%, but the ratio was determined from the experimental results shown in Table 5.

 [0179] In the experiments shown in Table 5, the ratio of the autologous blood 26 to the culture medium 24 was set to 1% for every 2% to 52%. The results of 7% and 48% are omitted in Table 5 because they are all good.

 [0180] It should be noted that the force S was determined to be "good" when culturing was performed in an amount necessary for transplantation, and "poor" when the required amount was not obtained.

 [0181] [Table 5]

Autologous blood to medium ratio Culture results

 2 Bad

 3 Bad

 4 Good

 5 Good

 6 Good

 49 Good

 50 Good

 51 Bad

52 Bad As is evident from Table 5, fibroblast cultivation failed when the ratio of autologous blood 26 to medium 24 was less than 4% or greater than 50%. The reason is presumed as follows.

 [0182] That is, if the ratio of autologous blood 26 to culture medium 24 is less than 4%, the amount of blood (autologous blood 26) collected from a living body with respect to culture medium 24 is too small, and proteins contained in blood and blood Or the components (albumin) required to maintain the osmotic pressure of the culture medium. On the other hand, if the ratio of autologous blood to the culture medium 24 is greater than 50%, the amount of blood (autologous blood 26) collected from a living body with respect to the culture medium is too large, and the amount of the culture medium 24 becomes relatively small. Of nutrients required for culturing skin cells, such as liquid nutrients such as amino acids and carbohydrates.

[0183] Therefore, the ratio of autologous blood 26 to medium 24 was 4. / ₀ — 50. / o is preferred.

 [0184] Before injecting only cultured fibroblasts into a patient (living body), the fibroblasts are mixed with a base material for providing a three-dimensional structure (matrix) for fibroblast growth. As described above, the ratio of fibroblasts to the above-described base material can be up to about 1: 4 to 4: 1, but such a ratio was obtained from the results shown in Table 6.

 [0185] In Table 6, the ratio between the fibroblasts and the above-described base material is shown as a percentage of the fibroblasts relative to the mixture of the fibroblasts and the base material. The experiments were carried out in the range of fibroblasts to the above-mentioned substrate in the ratio of 1: 9) to 90% (the ratio of fibroblasts to the above-mentioned substrate was 9: 1). The results between 35% and 65% are “good” and are omitted in Table 6.

 [0186] In addition, when the reproduced portion can maintain the same level as the other region after transplantation, the force S is "good", and when a dent that can be discerned by the naked eye occurs, it is determined as "poor". Is

[0187] [Table 6] Fibroblast binding to the mixture ^!

 Percentage

 1 0 Bad

 1 5 Bad

 20 Good

 25 Good

 30 Good

 70 Good

 75 Good

 80 Good

 85 Bad

 90 Bad

Thus, the ratio of fibroblasts to substrate is 1: 4 (the ratio of fibroblasts in the mixture of fibroblasts and substrate is 20%) and -4: 1 (fibroblasts to substrate). The ratio of fibroblasts in the mixture with the above is about 80%) is presumed to be due to the following reasons.

 [0188] That is, if the proportion of fibroblasts is more than 80%, the amount of the base material is relatively too small, and a three-dimensional structure that becomes a "scaffold" necessary for fibroblasts to grow is obtained. The structure (matrix) becomes insufficient, and the fibroblasts injected into the living body do not grow sufficiently, and the production of collagen is reduced. On the other hand, if the proportion of fibroblasts is less than 20%, the absolute amount of fibroblasts is small in the first place, so that the amount of collagen production is also reduced.

 [0189] Therefore, the above-mentioned ratio of fibroblasts to the substrate was 1: 4 (the ratio of fibroblasts in the mixture of fibroblasts and substrate was 20%) and -4: 1 (the ratio of The ratio of fibroblasts in the mixture with the substrate is preferably 80%).

[0190] Although not shown in Table 6, the ratio of fibroblast to base material was 1: 1 (fibroblast Particularly good results were obtained when the ratio of fibroblasts in the mixture of the substrate and the substrate was 50%).

[Example]

In accordance with the present invention, skin pieces (3 x 3 mm) were collected from the subject's ears. In addition, 50 mL of blood was collected from the above subjects. This blood was coagulated, and the obtained coagulated mass was pulverized by the above-mentioned method and applied to a cell culture dish to prepare a cell culture member. On the other hand, the collected blood was mixed with an equal amount of the DMEM medium to prepare a cell culture solution, which was cultured together with the skin pieces. Subculture was repeated as appropriate, and 1.5 × 10 ⁷ obtained fibroblasts were suspended in 2 mL of physiological saline. 0.5 mL of PLA was added to this suspension to prepare a filling material. The entire amount was injected into the eye of the subject according to the method described above. Before injection, wrinkles due to aging were observed in the eyes. After about 8 weeks, the wrinkles disappeared. No significant inflammatory effect was observed at the injection site.

 [0192] Similarly to this method, injection was also carried out into the nasolabial sulcus where the prominence was not remarkable, and the forehead where wrinkles due to aging were seen, but similar to the results obtained at the eyes, the remarkable after 8 weeks in the former Ridges were seen, and wrinkles due to aging in the forehead had disappeared significantly. No significant inflammatory effect was observed at the injection site.

 [0193] The illustrated embodiment is merely an example, and does not purport to reduce the technical scope of the present invention.

 [0194] The effects of the present invention described above are listed below.

 (1) Since the blood (autologous blood) of the living body containing the fibroblasts to be cultured is added to a commercially available medium, it is necessary to maintain the necessary nutrients, amino acids, and osmotic pressure. Fibroblasts can be cultured in an environment where components (albumin) are present.

 [0196] (2) Since autologous blood is used, it is possible to surely prevent, for example, various infectious diseases and pathogens mediated by blood of another living body from contaminating fibroblasts grown in the culture container. can do.

(3) By laying a sheet of polymer (fibrin) on the bottom of the culture vessel in which the fibroblasts are to be cultured, the cultured fibroblasts adhere to the bottom of the culture vessel and peel off. It is prevented that it cannot be released. (4) If a three-dimensional structure (matrix) that becomes a “scaffold” is mixed with cultured fibroblasts, the fibroblasts detached from the culture solution are returned to the body when the fibroblasts are removed. Grows smoothly.

Claims

The scope of the claims  [1] A method for culturing fibroblasts collected from a collection subject, comprising:  Filling a culture medium having fibrin deposited on a culture surface with a liquid medium containing at least a part of blood collected from the same collection target as the collection target;  A step of introducing the fibroblasts into the incubator filled with the liquid medium; and a step of culturing the introduced fibroblasts;  A culture method, comprising:  [2] The culture method according to claim 1, wherein the fibrin is a fibrin from which the same power to be collected is also collected. [3] A method for culturing fibroblasts collected from a collection subject, comprising:  Filling a culture vessel having a biocompatible polymer mounted on a culture surface with a liquid medium containing at least a part of the collected blood having the same collection target power as the collection target;  A step of introducing the fibroblasts into the incubator filled with the liquid medium; and a step of culturing the introduced fibroblasts;  A culture method, comprising: [4] The culture method according to claim 3, wherein the polymer is a low-temperature sensitive polymer.  [5] The method according to any one of claims 1 to 4, wherein at least a part of the blood is serum collected from the same collection target.  [6] A fibroblast derived from the same collection target as the collection target, which is cultured using a liquid medium containing at least a part of blood collected from the collection target.  [7] The fibroblast according to claim 6, wherein at least a part of the blood is serum collected from the same collection target.