JP2002200161A - Method of manufacturing cultured skin substitute and cultured skin substitute obtained thereby - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cultured skin substitute having a safe, stable and high wound healing power. SOLUTION: A method of manufacturing a cultured skin substitute having stable wound healing power comprises allowing a matrix to contain a human fibroblast producing a material effective against wound healing in an amount effective against wound healing and the method of manufacturing further comprises specifying the production amount of a material effective against wound healing for the cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a pre-culture of human fibroblasts effective for wound healing, and a cultured skin substitute using cells having a high ability to produce a production substance effective for wound healing. It relates to a method of manufacturing.

[0002]

2. Description of the Related Art Conventionally, in the treatment of wounds, generally, a method of suppressing the growth of bacteria in an affected area by an antibiotic and waiting for natural healing, a method of administering a granulation and epidermal formation promoter, and a method of treating cells such as collagen, chitin and chitosan. A method of engrafting an outer matrix and a method of engrafting artificial skin have been used.

In recent years, cell growth factors and cytokines produced by specific cells have been reported to be used for wound healing because they are effective for wound healing.
However, since cytokines and cell growth factors form a complex network in cells, administration of a single cytokine or cell growth factor to a living body at a high concentration is extremely dangerous because it causes an increase in side effects.

[0004] On the other hand, there has been reported a method in which cells producing these substances are used instead of directly administering cytokines and cell growth factors to a living body. For example,
Japanese Patent Application Laid-Open No. 6-142185 discloses an osteogenesis-promoting agent in which human ovarian tumor-established cells are supported on a substrate as cells for producing a substance for growing intravascular cells. Also, a medical material incorporating cells producing cell growth factor, which is a kind of a substance effective for wound healing, is disclosed in Japanese Patent Application Laid-Open No. 8-19 / 1996.
8763.

[0005] JP-A-11-246420 discloses a wound healing promoter characterized in that platelets are embedded in a collagen matrix. This is because platelet-derived growth factor (PDG) contained in platelets
F) The effect of vascular endothelial cell growth factor (VEGF) and epidermal growth factor (EGF) on promoting wound healing. However, this method requires the inclusion of platelets, which adhere to the extracellular matrix and form thrombi. Although this is convenient for the coagulation reaction in the very early stage of wound healing, it is often inconvenient when the capillary is newly born and transports nutrients, or in wounds where cultured skin is used.
Also, if platelet degranulation occurs late in wound healing, the inclusion of excessive wound healing factors will not impair fibroblast proliferation, and the production of extracellular matrix will greatly exceed degradation, resulting in scar tissue Is formed.

[0006] In any of the methods using cells producing a substance effective for wound healing, since the substance is derived from a living body, the individual difference is remarkably large, and an artificial skin having a constant healing power can be stably obtained. There was a drawback that it was difficult to produce.

[0007]

Accordingly, it is an object of the present invention to provide a cultured skin substitute which is safe, has a stable and high wound healing power.

[0008]

Means for Solving the Problems The present inventors have made intensive efforts to solve the above-mentioned problems, and as a result, have used human fibroblasts producing substances effective for wound healing, and further effective for wound treatment. The present inventors have newly found that it is possible to provide a cultured skin substitute having stable wound healing power by specifying an effective production amount of a novel substance, and have completed the invention.

[0009] That is, the present invention provides a method for producing a cultured skin substitute, characterized in that human fibroblasts producing a substance effective for wound treatment in an amount effective for wound healing are contained in a matrix. (1) The production method according to (1), wherein the amount of a production substance effective for wound healing is specified before the human fibroblasts are contained in a matrix. The above-mentioned human fibroblasts are pre-cultured at a seeding density of 2 × 10 ^{3 to} 5 × 10 ³ cells / cm ² for a period of 4 days or more and 9 days or less, and before being contained in a matrix, a production substance effective for wound treatment. 2. The production method according to claim 1, wherein the production amount is specified. (Claim 3), wherein the production substances effective for wound healing are vascular endothelial cell growth factor (VEGF), interleukin-6 (IL)
-6) and type I collagen (claim 4), wherein the effective substance for wound healing is vascular endothelial cell growth factor (VEGF), and the cell density during preculture is 6 ×. 3. The production method according to claim 2, wherein the production amount is 0.1 to 1 ng / ml / day in a cell culture solution of 10 ⁴ to 4 × 10 ⁵ cells / ml, which is effective for the wound healing. The substance produced is interleukin-6 (IL-
6) and the cell density in the pre-culture period of 6 × 10 ⁴ to
3. The method according to claim 2, wherein the production amount is 0.1 to 10 ng / ml / day in a cell culture solution of 4 × 10 ⁵ cells / ml. It is type I collagen and has a cell density of 6 × 1 during the pre-culture period.
0 In ^{4 ~4} × 10 ⁵ cells / ml of cell culture method according to claim 2, wherein the production amount is 100~1000ng / ml / day (claim 7), wherein the matrix atelocollagen, collagen, gelatin The method according to claim 1, wherein the wound is chondroitin sulfate or hyaluronic acid (claim 8), (1) a wound prepared by preculturing human fibroblasts in a serum-containing medium, and (2) culturing in (1). A human fibroblast producing a therapeutically effective product in an amount effective for wound healing is seeded on a matrix, and the human fibroblast on the matrix according to (3) or (2) is subjected to serum-free or serum-containing medium. The production method according to claim 1, which comprises a step of culturing the dermis, and the production method according to claim 1, wherein the cultured skin substitute is a cultured dermis substitute.
The present invention also relates to a cultured skin substitute obtained by the production method according to claim 11 (claim 11), and a cultured skin substitute according to claim 11 wherein the skin is dermis (claim 12).

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The method for producing a cultured skin substitute of the present invention comprises, in a matrix, human fibroblasts producing a substance effective for wound treatment and producing an effective amount for wound healing. Features.

[0011] Specific examples of the substance effective for wound healing include, for example, vascular endothelial cell growth factor (hereinafter, VEG).
F), interleukin-6 (hereinafter referred to as IL-6) or type I collagen, fibronectin, platelet-derived growth factor (PDGF), epidermal growth factor (EG)
F), fibroblast growth factor (FGF), interleukin-1 (IL-1), and the like, and any type may be used as long as the object of the present invention can be achieved.

The wound healing effective amount of the wound healing substance can be appropriately determined according to the substance. For example, a cell density of 6 × 10 ⁴ to
In a 4 × 10 ⁵ cells / ml cell culture, VEGF is preferably 0.1 to 1 ng / ml / day, more preferably 0.1 to 0.5 ng / ml / day, and 0.1 ng / ml. If it is less than ml / day, there is a tendency that a sufficient wound healing effect cannot be obtained,
Above 1 ng / ml / day, the ability of cells at the wound site to produce VEGF tends to decrease due to the negative feedback mechanism and fail to promote wound healing. In the case of IL-6, preferably 0.1 to 10 ng / ml /
Days, more preferably 0.1 to 1 ng / ml / day;
At 1 ng / ml / day or less, a sufficient wound healing effect tends not to be obtained. At 10 ng / ml / day or more, the ability of cells at the wound site to produce IL-6 decreases due to a negative feedback mechanism. Consequently, there is a tendency that wound healing cannot be promoted. In the case of type I collagen, preferably 1
00-1000 ng / ml / day, more preferably 100-5
At 100 ng / ml / day or less, a sufficient wound healing effect tends not to be obtained.
Above ml / day, the ability of cells at the wound site to produce type I collagen tends to decrease due to the negative feedback mechanism and fail to promote wound healing.

In the early stage of pre-culture in which the cell density is lower than 6 × 10 ⁴ cells / ml, the stimulation of cell growth due to the cell-cell interaction is not sufficiently transmitted and the cells are not sufficiently activated. However, even if a product in the above concentration range is produced, when it is used as a cell for a cultured skin substitute, subsequent cell growth and the physiological activity of the produced substance tend to decrease. Therefore, it is preferable to use cells having a cell density of 6 × 10 ⁴ to 4 × 10 ⁵ cells / ml during preculture, and more preferably 1 × 10 ⁵ to 2 × 10 ⁵ cells / ml.
It is preferred that

The "matrix" must be a material excellent in biocompatibility so that when the artificial skin is applied (applied) to the patient, it is not rejected by the patient's immune function. Also, if it is a biodegradable material, there is no need to remove it after application. The matrix is composed of collagen, gelatin, chitin, chitosan, fibrin, mucopolysaccharides such as chondroitin or chondroitin sulfate or hyaluronic acid, biodegradable polymers such as polyglycolic acid, polylactic acid, and mixtures thereof as biological materials. Polyurethane among non-biodegradable polymers,
Those having good cell adhesion, such as polystyrene and polyacrylate, or those comprising a mixture thereof are preferred. More preferred are atelocollagen, collagen, gelatin, chondroitin sulfate, and hyaluronic acid which are more biocompatible and have a porous and spongy form so that cells can easily stay in the matrix. The holes are preferably perpendicular to the cell seeding surface so that the cells can easily penetrate into the sponge when seeding the cells. The pore size is usually preferably in the range of 20 μm to 1000 μm in order to allow cells to easily penetrate into the seeding surface and prevent air bubbles from entering. However, it is not necessarily limited to these numerical ranges as long as the object of the present invention can be achieved.

As the form of the matrix, any form can be used as long as the object of the present invention can be achieved. Preferably, the matrix is used in the form of a porous sheet, sponge or gel.

The pre-culture period of the human fibroblasts is a period in which the cells retain a high substance-producing ability, preferably 4 to 9 days, more preferably 5 to 9 days, and still more preferably 6 to 9 days. 8 days. When the preculture period is shorter than 4 days and longer than 9 days, the ability to produce a substance effective for wound healing tends to be inferior. In the early stage of preculture, a sufficient number of mature cells is not obtained, and as described above, even when producing a sufficient amount of a production substance, when used as cells for a cultured skin substitute, bioactivity is low. Substances tend to be produced.

In the early stage of the preculture, a high result may be obtained when a substance effective for wound healing of cells is measured. This is because in the measurement of the produced substance, the precursor substance of the produced substance stored in the cell membrane and the inside thereof is measured together, but when measured at this time, the precursor stored until immediately before the preculture is measured. This is because the ratio of the substance is high. Therefore, there is a tendency that the production amount of a production substance effective for wound healing cannot be specified accurately.

The production ability of a production substance effective for wound healing is measured by an enzyme immunoassay using a specific antibody (hereinafter referred to as EL).
ISA method) by colorimetry.

The medium used in the present invention may be any medium used by those skilled in the art when culturing fibroblasts. A specific example is Dulbecco's modified Eagle's medium (hereinafter referred to as DE medium). Examples of the medium containing serum include, for example, a DE medium supplemented with 10% by volume of bovine serum (hereinafter, referred to as FBS) (hereinafter, referred to as DE + 10% FBS).

The cultivation in the present invention can be all carried out in a medium containing serum, but the preculture is carried out in a medium containing serum, and then the medium is replaced to remove the serum.
It is preferable that the cells are seeded on a matrix and cultured in a serum-free medium.

Hereinafter, the present invention will be described in detail.

[0022] Human fibroblasts are pre-cultured using a medium containing serum. The time when the most effective product for producing wound healing is produced is selected and collected. The collected cells were applied to 1 × 10 ^{4 to} 5 × 10 ⁵ cells / cm ² per 1 cm ² of matrix.
At a density of 2 to 20 hours, preferably 4 to 16 hours, at room temperature (about 20 ° C.) to 37 ° C., preferably 3
Leave at 7 ° C. After that, the culture medium is 0.1 to 1.0 ml, preferably 0.1 to 0.25 ml per 1 cm ^{2 of} the matrix.
At room temperature to 37 ° C, preferably at 37 ° C,
Culture for 0 days, preferably 1 to 3 days, to obtain the intended cultured skin substitute.

The obtained cultured skin substitute can be used clinically as it is. If there is time before clinical use, store at -20 to -196 ° C, preferably -20 ° C by replacing the medium with 4 ° C or a cryopreservation medium.
At -20 ° C or higher, the survival rate tends to decrease when stored for a long period (several months or more). When stored at such a low temperature, return to room temperature before use for transplantation.

[0024] The medium for cryopreservation used in the present invention includes, specifically, a DE medium containing 20% by volume of FBS and 10% by volume of glycerol. Anything you can get.

[0025]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Preculture of Examples 1 to 22 and Comparative Examples 1 to 15 In Examples 1 to 13 and Comparative Examples 1 to 8, human fibroblasts (HF21, PN6) were used, and Examples 14 to 24 and Comparative Examples 9 to 17 is human fibroblasts (HF8, PN21)
In Examples 25 to 27, human fibroblasts (HF8
(2), PN6) was treated with 0.25% trypsin, collected, seeded in a T80 flask, and pre-cultured. Seeding density is 5 × 10 ³ cells / cm ² (4 × 10 ⁵ cells
/ Sheet), and 15 ml / sheet of DE + 10% FBS was used as a medium.

Examples 1 to 3 and Comparative Examples 1 to 5 <Measurement of VEGF Production Amount> Each time, 15 ml of the culture supernatant cultured for the period shown in Table 1 under the same conditions as the above-mentioned preculture was collected, and The cell number was measured after detachment after treatment with 0.25% trypsin. In addition, the previously collected culture supernatant (15 ml)
Was centrifuged at 3000 rpm at 4 ° C. for 5 minutes to remove unnecessary substances, and then the concentration of VEGF was measured by ELISA. In addition, as a culture method, after the start of the culture period, 1,
New DE + on 2,3,4,5,6,7,8
This was performed by replacing the medium with a 10% FBS medium.

Table 1 and FIG. 1 show the culture period, the number of cells in 15 ml of medium in a T80 flask, and the amount of VEGF produced per ml of medium secreted by the cells 24 hours before the end of the culture period. In FIG. 1, the cell number is shown by a logarithmic line graph, and the VEGF production is shown by a bar graph.

[0029]

[Table 1]

The amount of VEGF produced for 24 hours immediately after seeding was 2
It is very high at 48 pg / ml, and the yield 8 hours after seeding is 215 pg / ml, which is about 87% of the yield during 24 hours.
Was secreted. After 2 days, 3 days, and 4 days of culture, which are considered to be in the active logarithmic growth phase, the concentration dropped to about 30 to 90 pg / ml, and after about 7 days in the stagnation phase, it became about 400 pg / ml again.
More than that. That is, it was produced in a very large amount immediately after seeding, hardly produced during the logarithmic growth phase, and when the cells became confluent and the growth rate of the cells was reduced, the production gradually started again. After culturing 7 to 9 days after seeding, the number of cells is about 5 × 10 ⁶ cells / 15 ml, and the amount of VEGF produced is about 400
６００600 pg / ml.

Examples 4 to 9 and Comparative Examples 6 and 7 <Measurement of IL-6 Production Amount> Except that the pre-culture period was the number of days shown in Table 2 under the above culture conditions, Examples 1 to 3 and Comparative Examples The cells were cultured in the same manner as in Nos. 1 to 5, and the cell number and I
The amount of L-6 production was measured.

Table 2 and FIG. 2 show the culture period, the number of cells in 15 ml of medium in a T80 flask, and the amount of IL-6 produced per ml of medium secreted by the cells 24 hours before the end of the culture period. In FIG. 2, the number of cells is indicated by a logarithmic line graph, and the amount of produced IL-6 is indicated by a bar graph.

[0033]

[Table 2]

The amount of IL-6 produced for 24 hours immediately after seeding was 3
It is very high at 467 pg / ml, and the production 8 hours after seeding is 2371 pg / ml, which is about 7
0% was produced. After 2 or 3 days of culture, which is considered to be in the active logarithmic growth phase, it is reduced to about 400 pg / ml, which is about a tenth, and after about 7 days in the stagnant phase, about 2 pg / ml again.
000 pg / ml. That is, it was produced in a very large amount immediately after the seeding, hardly produced during the logarithmic growth phase, and resumed production when the cells became confluent and the growth rate of the cells decreased.

Examples 10 to 13 and Comparative Example 8 <Measurement of Type I Collagen Production Amount> After culturing for the number of days shown in Table 3 under the above pre-culture conditions, the human type I collagen measurement system contains FBS in the medium. Therefore, the medium was exchanged with a fresh FBS-free DE medium and cultured for 24 hours. Next, after collecting the culture supernatant, the number of cells and the amount of type I collagen produced were measured in the same manner as in Examples 1 to 3 and Comparative Examples 1 to 5.

Table 3 and FIG. 3 show the culture period, the number of cells in 15 ml of medium in a T80 flask, and the amount of type I collagen produced per ml of medium secreted by the cells 24 hours before the end of the culture period. In FIG. 3, the number of cells is indicated by a logarithmic line graph, and the amount of type I collagen produced is indicated by a bar graph.

[0037]

[Table 3]

As for the amount of type I collagen produced, the amount produced per cell number increased about twice from day 3 to day 8 of culture.

Examples 14 to 16 and Comparative Examples 9 to 12 <Measurement of VEGF> Cells were cultured in the same manner as in Examples 1 to 3 and Comparative Examples 1 to 5 except that the culture period was changed to the number of days shown in Table 4. After culturing, the cell number and VEGF production were measured.

Table 4 shows the culture period, the number of cells in 15 ml of medium in a T80 flask, and the amount of VEGF produced per ml of medium secreted by the cells 24 hours before the end of the culture period.

[0041]

[Table 4]

From the results shown in Table 4, the amount of VEGF produced was PN
21 also showed the same tendency as the VEGF production of the cells in PN6.

Examples 17 to 20 and Comparative Examples 13 to 15 <Measurement of IL-6> Except that the culture period was changed to the number of days shown in Table 5, it was carried out in the same manner as in Examples 4 to 9 and Comparative Examples 6 to 7. The cells were cultured, and the number of cells and the amount of IL-6 produced were measured.

Table 5 shows the culture period, the number of cells in 15 ml of medium in a T80 flask, and the amount of IL-6 produced per ml of medium secreted by the cells 24 hours before the end of the culture period.

[0045]

[Table 5]

From the results shown in Table 5, the amount of IL-6 produced was PN
21 also showed the same tendency as the cell IL-6 production in PN6.

Examples 21 to 24 and Comparative Examples 16 and 17 <Measurement of Type I Collagen> After culturing for the number of days shown in Table 6, the number of cells and the amount of type I collagen produced were obtained in the same manner as in Examples 10 to 13 and Comparative Example 8. Was measured.

Table 6 shows the culture period, medium 1 in T80 flask.
The number of cells in 5 ml and the amount of type I collagen produced per ml of medium secreted by the cells 24 hours before the end of the culture period are shown.

For the measurement of the amount of type I collagen produced in the present example and the comparative example group, the concentration was determined from the data of the 2-fold diluted solution. Therefore, the measurement range is 2
~ 200ng / ml, the detection limit is 400ng / ml
It became.

[0050]

[Table 6]

The amount of type I collagen production rapidly increased from day 4 of culture, and reached the detection limit (400 ng / ml) on day 8 of culture.
The above production amount was shown. Also in this example and the comparative example group, the same tendency as the type I collagen production amount of cells in PN6 described above was shown.

In addition to the above Examples and Comparative Examples, PN1
VEG was performed on 2, 15, and 18 cells.
The production amounts of F, IL-6 and type I collagen showed the same results as those in the above examples in relation to the culture period (not shown).

As described above, the present invention has a stable effect even when cells having a large number of passages are used.

Example 25 <VEGF production of cultured skin substitute> 2.5 cm × 2.5
One piece of collagen sponge cut into cm (area: about 6.3 cm ² ) was placed in each hole of a 6-well plate for floating cells and sterilized by electron beam.

Next, human fibroblasts (HF8 (2), PN6) cultured for 8 days under the above preculture conditions were treated with 0.25% trypsin, and suspended with 10 ml of DE + 10% FBS to inactivate trypsin. did. This cell suspension was centrifuged at 300 to 500 rpm at 4 ° C. for 5 minutes, and the obtained cells were suspended in DE + 10% FBS. The cell suspension was dispensed into three 50 ml centrifuge tubes, and after further centrifugation, the obtained cells were subjected to 1 × 10 5
^The cells were suspended at ⁷ cells / ml. This cell suspension was seeded on 15 sponges in an amount of 600 μl each. Using a continuous pipetting pipette, 50 μl per sponge (50 μl / well) was dropped 12 times so that the cells adhered evenly. Add 300 more medium to allow cells to soak into the entire sponge.
μl / well was added.

To adhere the cells to the sponge, 37
° C., and cultured overnight in 5% CO ₂ incubator. Thereafter, a medium was added in an amount of 2.1 ml / well so that the sponge was sufficiently immersed, and the culture was continued in an incubator to prepare a cultured skin substitute. After culturing for the number of days shown in FIG. 4 from the start of culturing, the medium was replaced by 2 ml each. When replacing the medium,
Each of the prepared cultured skin substitutes was taken out as a sample for counting the number of cells three by three. Cell supernatant was also collected for VEGF measurement.

<Measurement of Cell Number> One cultured skin substitute was placed in 6 ml of a 1000 units / ml collagenase solution (for a 2.5 cm × 2.5 cm sponge) and slowly shaken in a 37 ° C. water bath. did. Shaking time is 30
Minutes to 2 hours.

The collagen was dissolved and visually observed, and centrifuged (1500 rpm, 4 ° C., 5 minutes).

The cells were suspended in DE + 10% FBS, and the number of cells and viability were measured by trypan blue exclusion using a hemocytometer.

<Measurement of VEGF Production Amount> ELISA was performed on the culture supernatant of the cultured skin substitute collected at the time of cell measurement.
The VEGF concentration was measured using the method.

FIG. 4 shows the change in the number of cells with respect to the number of days of culture after seeding on the matrix, and the amount of VE per 1 ml of the medium secreted during the period from the exchange of the medium to the number of days of culture.
The GF production was shown. In FIG. 4, the number of cells is indicated by a logarithmic line graph, and the amount of VEGF produced is indicated by a bar graph.

It was shown that human fibroblasts pre-cultured for 8 days continue to produce VEGF at a high level even after seeding on a matrix.

Example 26 <Amount of IL-6 Produced by Cultured Skin Substitute> The cytokine to be measured was IL-6, and the number of days from the start of culture to medium exchange and sample collection for measurement was changed to the number of days shown in FIG. Was performed in the same manner as in Example 25.

<Measurement of Cell Number> The cell number was measured in the same manner as in Example 25.

<Measurement of IL-6 Production Amount> The culture supernatant of the cultured skin substitute collected at the time of cell measurement was analyzed by ELISA.
The amount of IL-6 produced was measured using the method.

FIG. 5 shows the change in the number of cells with respect to the number of culture days after seeding on the matrix and the change in the number of culture mediums.
-6 production amount was shown. In FIG. 5, the number of cells is indicated by a logarithmic line graph, and the amount of produced IL-6 is indicated by a bar graph.

It was shown that human fibroblasts pre-cultured for 8 days continue to produce IL-6 at a high level even after seeding on a matrix.

Example 27 <Amount of Type I Collagen Produced by Cultured Skin Substitute> In the human type I collagen measurement system, a cross reaction occurs due to the presence of FBS in the medium. The cell supernatant was collected for measurement of type I collagen in the same manner as in Example 25, except that the number of days from the start of culture to the exchange of the medium and the collection of the sample for measurement was changed to the number of days shown in FIG.

<Measurement of Cell Number> The cell number and viability were measured in the same manner as in Example 25 except that DE medium was used as the medium.

<Measurement of Type I Collagen Production Amount> The concentration of type I collagen was measured for the culture supernatant of the cultured skin substitute collected at the time of cell measurement.

FIG. 6 shows the change in the number of cells with respect to the number of culture days after seeding on the matrix, and the amount of type I collagen produced per ml of the secreted medium until the number of culture days after the medium was replaced. In FIG. 6, the number of cells is shown by a logarithmic line graph, and the amount of produced IL-6 is shown by a bar graph.

As compared with VEGF and IL-6, the amount of type I collagen produced gradually decreased after seeding on a matrix because a serum-free medium was used. However, it was shown that human fibroblasts produced about 150 ng / ml of type I collagen even for 4 days between 4 and 8 days of matrix seeding.

Example 28 <Preparation of Cultured Dermal Sheet and Collection of Culture Supernatant> Human fibroblasts (HF5, HF5,
PN6) was treated with 0.25% trypsin. The recovered cell suspension was suspended with 10 ml of DE + 10% FBS to inactivate trypsin, and centrifuged to obtain only cells. Furthermore, in order to remove FBS, the suspension was suspended in 30 ml of DE medium and centrifuged. 6. Suspend again in DE medium.
It was adjusted to 34 × 10 ⁶ cells / ml.

Collagen sponge without mesh (9 cm)
X 9cm) 2 sheets (hereinafter referred to as CD1 and CD2 respectively)
Was seeded with 15 ml of the cell suspension (1 × 10 ⁵ cel).
ls / cm ² ). Four hours after sowing, the DE medium was further added to each 15 m.
l was added.

After culturing for 3 days, each culture supernatant was collected, sponge CD1 and CD2 were digested with collagenase, and the number of each cell was measured. Unnecessary substances were removed from the collected culture supernatant by centrifugation (3000 rpm, 4 ° C., 10 minutes) and stored frozen at −30 ° C. CD1 or CD
The culture supernatants obtained from 2 were used as CDS1 and CS, respectively.
It is called DS2. The number of cells in CD1 is 3.92 × 10 ⁶ cel.
By ls, the number of cells in CD2 was 4.61 × 10 ⁶ cells.

<Vascular endothelial cell proliferation test> Vascular endothelial cells
[Human skin microvascular cells (HMVEC)]
1 × 10 ^Fourcells / cm^TwoSeeded on a 24-well plate
Seeded. EC medium was used as the medium, and the medium volume was 500 μl /
It was a hole.

After confirming that the vascular endothelial cells had adhered, CDS1 or CDS2, a DE medium and an EC medium + 10% FBS were added at respective ratios, and the mixture was finally adjusted to have a mixing ratio shown in Table 7. .

[0078]

[Table 7]

The number of vascular endothelial cells was determined by measuring M
TT (3- (4,5-dimethylthiazol-2-yl)
-2,5-diphenyltetrazolium bromide) assay. After adding 100 μl / well of a 5 mg / ml MTT solution and culturing for 4 hours, the supernatant was removed and DMSO 2
Formazan was extracted and performed at 50 μl / well. The absorbance of the extract at 540 nm was measured using a plate reader (manufactured by Nippon Intermed Co., Ltd.).

FIG. 7 and Table 8 show the addition ratio of the culture supernatant CDS1 or CDS2 to the medium during the culture of vascular endothelial cells.
The number of vascular endothelial cells after 5 days of culture in each medium is shown. CDS1
In both CDS2 and CDS2, the higher the addition rate, the more the number of vascular endothelial cells was observed. This indicates that the amount of cytokine contributes to the proliferation of vascular endothelial cells.

[0081]

[Table 8]

[0082]

As described above, a cultured skin substitute having a high wound healing effect can be produced by using human fibroblasts secreting a large amount of a substance effective for wound healing. In addition, by specifying the production amount of a substance produced during preculture and effective for wound healing, it can be incorporated into the matrix at the best timing. Before using it further for wound treatment,
Since it is possible to culture in a serum-free medium, it is possible to produce a safe cultured skin substitute with low risk of contamination with various harmful substances.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a bar graph showing the amount of VEGF produced in 1 ml of culture supernatant secreted in 24 hours from the change of the medium to the number of culture days, and the number of cells in a T80 flask containing 15 ml of medium is expressed as a logarithm. It is the figure shown by the line graph.

FIG. 2 is a bar graph showing the amount of IL-6 produced in 1 ml of culture supernatant secreted in 24 hours from the change of the medium to the number of culture days, and the number of cells in a T80 flask containing 15 ml of medium is shown. It is the figure shown by the logarithmic line graph.

FIG. 3 is a bar graph showing the amount of type I collagen produced in 1 ml of culture supernatant secreted for 24 hours from the change of the medium to the culture days, and the number of cells in a T80 flask containing 15 ml of medium is shown. It is the figure shown by the logarithmic line graph.

FIG. 4 is a logarithmic line graph showing the change in cell number with respect to the number of culture days after seeding human fibroblasts that had been precultured for 8 days into a matrix.
It is the figure which showed the change of the amount of production with the bar graph.

FIG. 5 is a logarithmic line graph showing the change in cell number with respect to the number of culture days after seeding human fibroblasts that had been precultured for 8 days into a matrix.
It is the figure which showed the change of the amount of production with the bar graph.

FIG. 6 is a logarithmic line graph showing a change in the number of cells with respect to the number of culture days after seeding human fibroblasts that had been precultured for 8 days on a matrix, and a change in the amount of type I collagen produced in 1 ml of culture supernatant. Is a diagram showing a bar graph.

FIG. 7 shows a culture supernatant (CDS) of a cultured skin substitute obtained by inoculating a matrix with human fibroblasts producing a substance effective for wound treatment in an amount effective for wound healing.
FIG. 3 is a graph showing the proliferation of endothelial cells with respect to the amount of 1 (-●-) or CDS2 (-O-)).

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Go Moriyama 5-1-1 Takamoridai, Kasugai City, Aichi Prefecture Inside Menicon Research Institute, Inc. (72) Inventor Yasuko Inai 5-1-1 Takamoridai Kasugai City, Aichi Prefecture F-term in Menicon Research Institute, Inc. (reference) 4C081 AB19 BA12 CD071 CD081 CD121 CD131 CD151 CD34 CE02 DA02 EA02

Claims (12)

[Claims] 1. A method for producing a cultured skin substitute, wherein a matrix contains human fibroblasts, which produce a substance effective for wound treatment in an amount effective for wound healing. 2. The production method according to claim 1, wherein before the human fibroblasts are contained in the matrix, the production amount of a production substance effective for wound healing is specified. 3. The method according to claim 1, wherein the human fibroblasts are seeded at a seeding density of 2 × 1.
Claims characterized in that after the preculture for 4 to 9 days at 0 ^{3 to} 5 × 10 ³ cells / cm ² , the amount of a production substance effective for wound healing is specified before being contained in the matrix. Item 10. The production method according to Item 1. 4. The product effective for wound healing is vascular endothelial cell growth factor (VEGF), interleukin-6
The production method according to claim 1, wherein the production method is (IL-6) and type I collagen. 5. The production substance effective for wound healing is vascular endothelial cell growth factor (VEGF), and in a cell culture solution having a cell density of 6 × 10 ⁴ to 4 × 10 ⁵ cells / ml during a pre-culture period, 3. The production method according to claim 2, wherein the production amount is 0.1 to 1 ng / ml / day. 6. The production substance effective for wound healing is interleukin-6 (IL-6), and in a cell culture solution having a cell density of 6 × 10 ⁴ to 4 × 10 ⁵ cells / ml in a pre-culture period, The production method according to claim 2, wherein the production amount is 0.1 to 10 ng / ml / day. 7. The product effective for wound healing is type I collagen, and has a cell density of 6 × 10
⁴ in cell cultures of ~4 × 10 ⁵ cells / ml, the production method according to claim 2, wherein the production amount is 100~1000ng / ml / day. 8. The method according to claim 1, wherein the matrix is atelocollagen,
2. The production method according to claim 1, wherein the production method is collagen, gelatin, chondroitin sulfate or hyaluronic acid. 9. A method comprising: (1) pre-culturing human fibroblasts in a serum-containing medium; and (2) producing a substance effective for wound healing, which is cultured in (1), in an amount effective for wound healing. 2. The method according to claim 1, further comprising the steps of: (3) seeding the human fibroblasts on a matrix, and (3) and (2) culturing the human fibroblasts on the matrix in a serum-free or serum-containing medium. 10. The method according to claim 1, wherein the cultured skin substitute is a cultured dermis substitute. 11. A cultured skin substitute obtained by the production method according to claim 1. 12. The cultured skin substitute according to claim 11, wherein the skin is dermis.