JP2001211876A - Incubator to which adhesivity and proliferation property of cell have been imparted - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an incubator capable of producing an epidermal cell sheet and an epidermal cell suspension which are used for skin-lost wounds, such as burns, wounds, bedsores or skin ulcer, to reproduce or treat the lost tissues in their early storage. SOLUTION: This incubator to which the adhesivity and proliferativity of cell have been imparted by a method characterized by seeding, culturing, killing and at least partially peeling fibroblasts or by a method characterized by recovering the supernatant of the culture product of the fibroblasts, killing and/or removing the cells and then coating one of various incubators with the left solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an incubator provided with cell adhesion and proliferation. More specifically, the present invention relates to an incubator provided with adhesion and proliferation of epithelial cells and hepatocytes. More specifically, the present invention relates to an incubator which can be used for a skin defect wound such as a burn, a wound, a pressure sore or a skin ulcer to produce an epidermal cell sheet and an epidermal cell suspension for early reconstruction or treatment of a defective tissue.

[0002]

2. Description of the Related Art Conventionally, among epithelial cells, epithelial cells (also called epidermal keratinocytes) have been cultured using radiation such as gamma rays or a drug such as mitomycin C. A method of culturing epidermal cells using 3T3 mouse lung fibroblasts, etc., which have lost the ability to divide and proliferate, as feeder layers (James G. Reinwald and Howard Green, Cell, No. 6) Volume, 331-34
Page 4, (James G. Rheinwald and Howard Green. Cell
6: 331-344. Serial Cultivation of Strains of Human
Epidermal Keratinocytes: the Formation of Keratin
), and a method of culturing using a serum-free medium such as MCDB153 without using cells serving as feeder layers.

However, in the conventional feeder layer method using 3T3 mouse lung fibroblasts as a feeder layer, for example, it is necessary to prepare the 3T3 mouse lung fibroblasts immediately before seeding the epidermal cells, which is complicated, and the feeder layer is not effective. Certain periods are also limited. In the growth of epidermal cells other than mouse (eg, human epidermal cells) and preparation of epidermal cell sheets, heterogeneous cells such as 3T3 mouse lung fibroblasts may be mixed with these cells.
There is also concern about the retention of drugs such as mitomycin C, which is used in deficient division and proliferation of 3 mouse lung fibroblasts.

[0004] On the other hand, instead of 3T3 mouse lung fibroblasts, epidermal cells to be obtained and / or fibroblasts of the same kind as the epidermal cell sheet (for example, when a human epidermal cell sheet is prepared, human fibroblasts are used). When cells are used as a feeder layer, foreign cells are not contaminated, but it is still necessary to delete the fibroblast division / proliferation ability. Is concerned. In addition, when human fibroblasts and the like are used, 3T3 mouse lung fibroblasts are used,
Slow growth of epidermal cells.

A method using a serum-free medium is 3T3
Compared to methods using feeder layer cells such as mouse lung fibroblasts, epidermal cells grow more slowly and often take days to culture, and epidermal cells are superimposed because the medium has the effect of suppressing epidermal cell differentiation. In some cases, an epidermal cell sheet could not be produced without being transformed.

Japanese Patent Application Laid-Open No. Sho 62-285781 discloses a method using a feeder layer also in hepatocyte culture, but it cannot be ruled out that a drug may remain or a different type of cells may be mixed.

According to the present invention, it is possible to omit the steps of preparing cells to be a feeder layer such as fibroblasts and sterilizing the cells, which are required by the conventional feeder layer method.
Further, it is possible to obtain an epidermal cell sheet and an epidermal cell suspension free from contamination of foreign cells, and to provide an incubator with improved cell adhesion and proliferation as compared with the method. Aim.

Specifically, fibroblasts derived from mammals,
In particular, by culturing 3T3 mouse lung fibroblasts in an incubator and then killing the cells by treatment such as freezing and / or drying in the incubator and exfoliating at least a part thereof, substantially, By leaving components such as extracellular matrix secreted and deposited by the cultured cells on a culture surface in an incubator, that is, by leaving components required for cell adhesion and growth on the culture surface, It is an object of the present invention to provide an incubator to which the adhesiveness and the growth property are imparted.

[0009] More specifically, a culture supernatant obtained by culturing fibroblasts derived from mammals is collected to kill the cells or /
And, after removal, the components, such as extracellular matrix secreted by the cells, are substantially adhered to the incubator by coating in various incubators, thereby providing a culture in which hepatocyte adhesion and proliferation are imparted. The purpose is to provide a vessel.

[0010]

Means for Solving the Problems The present inventors have made intensive studies on the conditions for culturing epidermal cells and hepatocytes to form an epidermal cell sheet. As a result, (1) seeding mammalian fibroblasts; (2) a method in which, after the culture, the cells are killed, and at least a part of the cells is exfoliated, so that components such as extracellular matrix secreted and deposited by the cultured cells are substantially left on a culture surface in a culture vessel. The culture supernatant obtained by culturing the mammalian-derived fibroblasts is collected, and the fibroblasts mixed in the culture supernatant are killed and / or removed. Then, the cells are substantially coated with the cells by coating on various incubators. The method of attaching the secreted extracellular matrix and other components to the incubator allows the preparation of cells to be used as feeder layers, such as fibroblasts, required by the conventional feeder layer method, and the preparation of such cells. I was able to dispense with the sterility of the process. Further, the incubator provided with the adhesive ability and the proliferation ability by any of the above methods can be stored for a long period of time while maintaining the adhesiveness and the proliferation property of the cells. In addition, they have found that an incubator with improved cell adhesion and proliferation can be obtained as compared with the conventional method, and have completed the present invention.

That is, the present invention provides an epithelial cell and / or hepatic cell by a method for adhering and expanding epithelial cells and / or hepatocytes, which comprises the steps of inoculating, culturing, and killing mammalian fibroblasts. The incubator provided with cell adhesion and proliferation (Claim 1), the incubator according to Claim 1 wherein at least a part or all of the killed fibroblasts is peeled off, and the culture thereof. Freezer and / or
Alternatively, the incubator according to claim 1, which can be preserved by drying (claim 3), wherein the fibroblast derived from a mammal is 3T.
3. The incubator according to claim 1, which is 3 mouse lung fibroblasts (claim 4), and the epithelial cells are epidermal keratinocytes.
The incubator according to claim 5, wherein the incubator is an incubator made of glass, a synthetic polymer or a biopolymer. The incubator according to claim 1, wherein the incubator is a flask, a petri dish, a roller. The incubator according to claim 1, which is a bottle, a tray, a well plate, a bead, a film, a sheet, or a sponge, a glass or synthetic polymer incubator, a flask, a petri dish, a roller bottle,
The incubator according to claim 6, which is a tray, a well plate, a bead, a film, a sheet, or a sponge (claim 8), or the incubator made of a biopolymer is a sheet, a film, a sponge, or a bead. The incubator according to claim 9, wherein the incubator according to claim 1 is capable of producing an epidermal cell sheet.
0) The incubator according to claim 1, wherein the incubator according to claim 1 is capable of producing an epidermal cell suspension (invention 11), and a culture supernatant of fibroblasts derived from mammals. A culture vessel (claim 12) provided with hepatocyte adhesion and proliferation by the method of adhesion and proliferation of hepatocytes, characterized in that the fibroblasts derived from mammals are 3T.
The incubator according to claim 12, which is 3 mouse lung fibroblasts (claim 13).

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method for adhering and growing cells comprising the steps of inoculating and culturing mammalian fibroblasts and killing them, or culturing a culture supernatant of mammalian fibroblasts. The present invention relates to an incubator to which cell adhesion and proliferation are imparted by a cell adhesion and proliferation method characterized by coating a vessel. More particularly, the present invention relates to an epidermal cell sheet and an incubator for producing an epidermal cell suspension for reconstructing or treating defective tissue at an early stage, for use in a skin wound such as a burn, a wound, a pressure ulcer or a skin ulcer. Things.

The cells that adhere and proliferate in the incubator provided with the cell adhesion and proliferation of the present invention include cells that can be cultured by a conventional feeder layer method, such as epithelial cells and hepatocytes. The term "epithelial cells" refers to epithelial cells, such as mucosal epithelial cells and corneal epithelial cells, which are surface cells in the small intestine, oral cavity, and nasal cavity, and epidermal cells, which are cells that are present in the skin epithelium and undergo enucleation and keratinization after division Is included. Hepatocytes are cells constituting the hepatic lobule.

[0014] In the incubator provided with the cell adhesion and proliferation of the present invention, mammalian fibroblasts are killed after culturing, and at least a part thereof is peeled off and removed. Presence of extracellular matrix components necessary for cell adhesion and proliferation on the surface, or killing and / or removing cells in the culture supernatant of cultured mammalian fibroblasts, followed by cell adhesion and proliferation. It is important to have the necessary components such as extracellular matrix.

As fibroblasts, for example, mice,
Fibroblasts derived from mammals such as humans, rats, hamsters, rabbits and the like, preferably 3T3 mouse lung fibroblasts widely used in the conventional feeder layer method are used. The conditions for seeding and culturing the cells are not particularly limited, and a general method is used. For example, fibroblasts grown alone in an incubator were treated with sodium ethylenediaminetetraacetate (EDTA) at a concentration of 0.206 mg / m2.
1 in a phosphate buffer solution prepared in 1 l, with a trypsin solution in which trypsin is dissolved to a concentration of 0.25 w / v%, suspended in a medium containing 5 to 10% fetal bovine serum, and inoculated in an incubator. Then, it is left still in a carbon dioxide gas culturing apparatus.
As the incubator used at that time, a special cell incubator such as a cell incubator coated with an extracellular matrix such as collagen is not required, and any incubator to which 3T3 fibroblasts and the like can adhere and proliferate, The material and shape are not particularly limited. Commercially available adherent cell culture vessels (flasks, Petri dishes, roller bottles, well plates, trays) or carriers, such as membranes, films, plates, and biopolymer membranes, films, and microbeads made of ordinary synthetic polymers Can also be selected as an incubator, and the cost can be greatly reduced as compared with the related art.

The incubator of the present invention to which cell adhesion and proliferation are imparted by a cell adhesion and proliferation method including the steps of inoculating, culturing, and killing mammalian-derived fibroblasts will be described below. .

Killing fibroblasts cultured in an incubator,
Dead cells (hereinafter referred to as dead cells) are peeled off and removed, and components necessary for cell adhesion and proliferation are left on the culture surface of the incubator. As a result, adhesion and proliferation of cells are imparted to the culture surface. From the viewpoint of imparting the adhesive property and the proliferation property, preferably 1.0 × 10 ^{2 to} 1.0 ×
10 ⁵ cells / cm ² , more preferably 3.0 × 10 ³
１．０1.0 × 10 ⁵ cells / cm ² of fibroblasts,
Culture according to the above conditions.

In the subsequent step of killing fibroblasts,
It is necessary that the cells be killed under conditions that do not denature the components necessary for cell adhesion and growth, and that they are substantially completely killed. By performing the killing substantially completely, the cells can be easily and reliably removed. as a result,
It is possible to prevent heterogeneous cells from being mixed into the finally obtained epidermal cell sheet and epidermal cell suspension. In addition, mitomycin C was completely killed and was not sterilized in the past.
Since there is no need for chemical treatment such as the above, there is no risk of the chemicals remaining.

The method of killing is preferably freezing, freeze-drying, drying, drying at low temperature, ultraviolet ray, β-ray, X-ray,
The use of electromagnetic waves, such as radiation, gamma rays or electron beams, and combinations of their treatment. According to these methods, the cells can be substantially completely killed, and components necessary for cell adhesion and proliferation are not denatured, and adhesion and proliferation are maintained. When epidermal cells are seeded and cultured in such an incubator, the epidermal cells adhere and proliferate well, and a practically suitable epidermal cell sheet is formed. Still more preferably, use of freezing is mentioned. The freezing method is easy to operate, can kill a large amount of fibroblasts at a time, and is less expensive than the device used by other methods. In addition, by continuing freezing even after the cells have died, it is possible to store the cells for a long period of time while maintaining the adhesiveness and the proliferation of the cells. The freezing operation includes freezing and subsequent thawing, and repeated freezing and thawing operations.

Specific conditions in the case of drying will be described. As the drying means, a general dryer may be used, and the drying temperature is preferably a temperature at which freezing does not occur (around 0 ° C.) to 6 ° C.
The temperature is preferably 0 ° C, more preferably 4 to 30 ° C. As mentioned above, it is important to suppress denaturation of the active ingredient, and thus kill fibroblasts under these mild conditions.

On the other hand, in the case of freezing, as a freezing means, a freeze dryer, a freezer, an ultra-low temperature freezer, a liquefied carbon dioxide gas, a liquefied nitrogen gas or the like is used. The freezing temperature may be any temperature as long as freezing is possible, and is usually 0 ° C. or less. In addition, since the freezing rate of the cells affects the death of the cells, it is preferable to perform freezing by slow cooling using a program freezer or the like.

Next, a method of killing by irradiating an electron beam, a gamma ray or an ultraviolet ray among electromagnetic waves will be described. The electron beam or gamma ray irradiation is preferably performed at 5 to 30 kGy. UV irradiation is preferably 50 to 5000 mW
・ Implement at sec / cm ² . From the viewpoint of imparting a property capable of improving the adhesiveness to cells and the proliferation of cells, preferably from 1.0 × 10 ^{2 to} 1.0 × 1.
0 ⁵ cells / cm ² , more preferably 1.0 × 10 ³ to
1.0 × 10 ⁵ cells / cm ² of fibroblasts are seeded and cultured according to the above-mentioned usual conditions. The culture supernatant of the fibroblasts thus collected was removed, and 5 to 30
Irradiate an electron beam or a gamma ray of kGy. Before irradiating with electron beam or gamma ray, that is, after removing the culture supernatant, it is preferable to keep the incubator at 4 ° C. or lower, and more preferably, to incubate the incubator after freezing it. This is to prevent denaturation of a component capable of improving the ability of the cell to secrete and adhere to the cell and proliferate the cell. For example, when the incubator is kept at about 4 ° C. and irradiated with an electron beam, the adhesion and proliferation of the cells are slightly inferior to those of the 3T3 feeder layer method, but as a result, they grow to the same level of the epidermal sheet. In addition, when the incubator is frozen, it exhibits the same cell adhesion and proliferation as the 3T3 feeder layer method. Further, electron beams and gamma rays are useful because they can completely kill fibroblasts and can sterilize them.

After the cells are killed by the above method, the dead cells are peeled off and removed. In addition, "peeling"
Is a concept that includes all methods that can remove dead cells, such as, for example, peeling off or washing away.

An epidermal cell sheet obtained by using an incubator provided with cell adhesion and proliferation by a cell adhesion and proliferation method comprising a step of seeding, culturing, and killing mammalian fibroblasts. In order to prevent 3T3 mouse lung fibroblasts and the like from being mixed into the epidermal cell suspension, it is necessary to completely remove the cells in this step.
However, it is not always necessary to completely remove cells from the viewpoint of the adhesion and proliferation of epidermal cells, and to the extent that they do not affect the adhesion and proliferation,
That is, it is sufficient that at least a part is peeled and removed.
The term “at least a part of the cells is detached”, which indicates the degree of removal of dead cells, is specifically, preferably 50% or more, more preferably 80%, based on the number of cells immediately before the cell death treatment. This means removing the above cells, and most preferably removing 100%.

The removal of dead cells is generally carried out by rinsing the culture surface with a so-called isotonic solution which does not denature the active ingredients necessary for cell adhesion and growth, such as a phosphate buffer, Hanks' solution, and physiological saline. Do.

The degree of removal of dead cells can be easily confirmed by a method such as observation with a phase contrast microscope. At that time, if 100% removal of dead cells is confirmed, there is no contamination of the resulting epidermal cell sheet and epidermal cell suspension with 3T3 mouse lung fibroblasts or the like.
However, 100% removal of dead cells is not a difficult operation, and cells can be almost completely easily removed by ordinary operations. In addition, cells after death are completely detached in a cell culture medium. The epidermal cell sheet and the epidermal cell suspension obtained from the incubator provided with the cell adhesion and proliferation of the present invention are substantially free from foreign cells. Conventionally,
An epidermal cell sheet that can be practically used only by the feeder layer method cannot be produced.In this case, when cells other than humans are used for the feeder layer, there was considerable contamination with heterologous cells, and thus the present invention An epidermal cell sheet and an epidermal cell suspension obtained from an incubator provided with cell adhesion and proliferation properties are excellent epidermal cell sheets and epidermal cell suspensions free from contamination with foreign cells.

Incubators provided with cell adhesion and growth properties, such as petri dishes obtained by removing the dead cells and leaving components necessary for cell adhesion and growth obtained by the above-described operations, are, for example, 2-8 ℃ refrigerator, or -30 ℃,-
In a deep freezer at 80 ° C. or the like, it can be stored while maintaining its performance for at least about six months to one year. Therefore, a feeder layer preparation work immediately before seeding of epidermal cells, which was necessary in the conventional feeder layer method, for example, 3T3 cells were placed in an incubator at 1 × 1 after mitotic ability was deleted by γ-rays, mitomycin C, or the like.
0 ⁴ such seeding to be about the cell number / cm ² can be omitted. Also, the conventional feeder layer could only maintain its performance for about two days,
The incubator provided with the cell adhesion and proliferation of the present invention can be stored for at least about six months to one year.
In addition, the incubator provided with the adhesion and proliferation of the cells of the present invention can be produced in a simple manner and can be produced in large quantities at a time with the same activity, thereby greatly reducing the cost. It is also possible. In addition, the need for routine passage of fibroblasts for feeder layer preparation is eliminated.

Hereinafter, a culture vessel provided with cell adhesion and proliferation by a method for adhesion and proliferation of hepatocytes, which is characterized in that a culture supernatant of fibroblasts derived from mammals is coated on the culture vessel. explain.

From the viewpoint of imparting adhesiveness and proliferation, preferably from 1.0 × 10 ^{2 to} 1.0 × 10 ⁵ cells / c
Fibroblasts of m ² , more preferably 1.0 × 10 ^{3 to} 1.0 × 10 ⁵ cells / cm ² are seeded and cultured according to the above-mentioned usual conditions.

The culture supernatant of the fibroblasts thus cultured is collected using a pipette or the like. Since the culture supernatant thus collected often contains fibroblasts, the fibroblasts mixed in the culture supernatant are killed and / or removed. Specific examples include cell killing methods such as freezing and irradiation with electromagnetic waves such as electron beams and X-rays, cell removal methods using a membrane filter or a centrifugal separator, or a combination thereof. According to these methods, the cells can be substantially completely killed and / or removed, and components necessary for cell adhesion and proliferation are not denatured and cell adhesion and proliferation are maintained. . When the present invention is used, epidermal cells proliferate well, and an epidermal cell sheet suitable for practical use is formed. Even more preferably, use of freezing is mentioned. The freezing method is easy to operate,
Cells in a large volume of supernatant can be killed at one time, and the device is less expensive than what would otherwise be used. The freezing operation involves freezing and subsequent thawing,
This includes repeated freezing and thawing operations.

Specific conditions of a method for removing fibroblasts using a membrane filter will be described. As a material of the filter, it is preferable to use a material that does not easily adsorb a component effective for cell adhesion and proliferation, and specific examples thereof include PVDF (polyvinylidene difluoride) and hydrophilic polysulfone. The pore size of the filter should be such that cells do not pass through, and is preferably 10 μm or less because the contamination of cells can be completely denied.

When fibroblasts are killed by freezing,
Freezing means such as freeze dryer, freezer, ultra low temperature freezer,
Liquefied carbon dioxide gas, liquefied nitrogen gas and the like are used. The freezing temperature may be any temperature as long as freezing is possible, and is usually 0 ° C. or less. In addition, since the freezing rate of the cells affects the death of the cells, it is preferable to perform freezing by slow cooling using a program freezer or the like.

After the cells in the supernatant have been killed and / or removed by the above method, the supernatant is coated on various incubators. Examples of the coating method include drying and freeze-drying. Further, it may be left in a dryer or a carbon dioxide cultivator kept at 30 to 37 ° C. for one hour or more, or may be left in a refrigerator at 4 ° C. for one night or more. Furthermore, after mixing with an extracellular matrix such as collagen, chitosan, gelatin, hyaluronic acid, chondroitin, or chondroitin sulfate, the mixture may be gelled and then freeze-dried.

[0034] In the production of an incubator to which hepatocyte adhesion and proliferation are imparted by a cell adhesion and proliferation method, which comprises coating a culture supernatant of mammalian fibroblasts on a culture vessel, Since fibroblasts in the culture supernatant are killed and / or eliminated, hepatocytes grown using the incubator of the present invention are substantially free from contamination with 3T3 mouse lung fibroblasts or the like.

Conventionally, an epidermal cell sheet which can be practically used only by the feeder layer method has not been prepared. In this case, since cells used for the feeder layer have not been killed, when non-human cells are used for the feeder layer, Since there was considerable contamination with heterologous cells, the epidermal cell sheet, epidermal cell suspension and hepatocytes obtained from the incubator provided with the adhesion and proliferation of the cells of the present invention were not contaminated with heterologous cells. There are no excellent epidermal cell sheets, epidermal cell suspensions and hepatocytes.

The epidermis cell sheet produced by the incubator of the present invention can be easily and conveniently used when needed, by using an incubator provided with the cell adhesion and proliferation of the present invention. It is possible to make quantities. The specific method for producing the same is the same as the method for producing an ordinary epidermal cell sheet, except that the incubator provided with the cell adhesion and proliferation of the present invention is used. For example, it can be prepared by seeding epidermal cells at 1.0 × 10 ⁴ cells / cm ² and culturing for about 7 to 21 days while changing the medium at a rate of about twice a week. .

The epidermal cell suspension prepared by the incubator of the present invention is a normal epidermal cell suspension except that an incubator provided with the cell adhesion and proliferation of the present invention is used. Same as the method. For example, if epidermal cells are
0 ⁴ after seeding a cell number / cm ^2, with medium changes at a rate of about twice a week, and cultured about 3 to 21 days.
After that, it was made almost single cell using enzymes such as trypsin,
It can be produced by suspending in a neutral collagen solution or the like.

Hereinafter, the present invention will be described specifically with reference to Examples.
These examples do not limit the invention.

[0039]

EXAMPLES Example 1 3T established in a culture flask (culture area 25 cm ² )
Three mouse lung fibroblasts were seeded at 3 × 10 ³ cells / cm ² and cultured for 4 days in a carbon dioxide gas culturing apparatus (set at 37 ° C., 5% carbon dioxide gas concentration). The medium was Dulbecco's modified Eagle's basic medium containing 10% fetal bovine serum (DMEM + 10%
FBS) was used.

After the culturing, the culture supernatant in the culture flask was removed by suction, and left in a deep freezer at -85 ° C. for 12 hours to freeze. Then, the cells were thawed at room temperature, and the culture surface was rinsed with 5 ml of a phosphate buffer to remove dead cells. Thereafter, the culture flask was again placed in a deep freezer at −85 ° C. overnight and frozen.

The culture flask was thawed at room temperature, and epidermal cells collected from human skin were seeded at 1 × 10 ⁴ cells / cm ² (3T3 frozen).

On the other hand, as a control, 3T3 mouse lung fibroblasts, which had been treated with mitomycin C to give a cell culture flask (culture area 25 cm ² ) at 1 × 10 ⁴ cells / cm ² and lacking division ability, were used. Was prepared in advance by inoculating another culture flask.

These culture flasks were cultured for 8 days in a carbon dioxide gas culture apparatus (set at 37 ° C., 5% carbon dioxide gas concentration). The medium used was a green medium containing 3% fetal bovine serum (green + 3% FBS).

After culturing for 8 days, an epidermal cell sheet was formed on the culture surface in the incubator. This was treated with dispase, peeled off from the culture surface, and a trypsin solution in which trypsin was dissolved to a concentration of 0.25 w / v% in a phosphate buffer prepared by adjusting the concentration of sodium ethylenediaminetetraacetate (EDTA) to 0.206 mg / ml. The epidermal cells were converted into single cells by the method described above, and the number of cells was measured using a hemocytometer.

The dispase treatment was performed at 10,000
3 ml of a dispase solution in which PU dispase was dissolved in 10 ml of Dulbecco's modified Eagle's basic medium was added to a culture flask, and the mixture was allowed to stand in a carbon dioxide gas culturing apparatus for about 1 hour.

The number of epidermis cells of the control and 3T3 frozen was measured three times, and the average of the results of the three measurements is shown in FIG. Incubator of the present invention (3T3
In Frozen), as shown in FIG. 1, more epidermis was inoculated than in the case of seeding epidermal cells in a feeder layer method (control) using conventional 3T3 mouse lung fibroblasts as a feeder layer and cultured for 8 days. Cells were attached and proliferating.

Example 2 3T established in a culture flask (culture area 25 cm ² )
Three mouse lung fibroblasts were seeded at 3 × 10 ³ cells / cm ² and cultured for 4 days in a carbon dioxide culture device (set at 37 ° C., 5% carbon dioxide gas concentration). The medium was Dulbecco's modified Eagle's basic medium containing 10% fetal bovine serum (DMEM + 10
% FBS).

After the culture, the culture supernatant in the culture flask was removed by suction, and the culture flask was allowed to stand in a dryer kept at 30 ° C. for 48 hours.

Epidermal cells recovered from human skin were seeded in this culture flask at a density of 1 × 10 ⁴ cells / cm ² . The medium used was a green medium containing 3% fetal bovine serum. After 4 days, the colonies of epidermal cells were observed.
The appearance of epidermal cells proliferating without contamination of 3 mouse lung fibroblasts was observed.

Example 3 3T established in a culture flask (culture area 25 cm ² )
Three mouse lung fibroblasts were seeded at 3 × 10 ³ cells / cm ² and cultured for 4 days in a carbon dioxide culture device (set at 37 ° C., 5% carbon dioxide gas concentration). The medium was Dulbecco's modified Eagle's basic medium containing 10% fetal bovine serum (DMEM + 10
% FBS).

After the culture, the culture supernatant in the culture flask was removed by suction, and freeze-dried with a freeze dryer. The schedule is-
Hold at 30 ° C for 1 hour, dry by vacuum suction, 1.5 ° C
The temperature was raised at 20 ° C./min and maintained at 20 ° C. for 20 hours.

Epidermal cells collected from human skin were seeded at 1 × 10 ⁴ cells / cm ^{2 in} this culture flask. The medium used was a green medium containing 3% fetal bovine serum. After 4 days, the colonies of epidermal cells were observed.
The appearance of epidermal cells proliferating without contamination of 3 mouse lung fibroblasts was observed.

Example 4 3T established in a culture flask (culture area 25 cm ² )
Three mouse lung fibroblasts were seeded at 3 × 10 ³ cells / cm ² and cultured for 4 days in a carbon dioxide culture device (set at 37 ° C., 5% carbon dioxide gas concentration). The medium was Dulbecco's modified Eagle's basic medium containing 10% fetal bovine serum (DMEM + 10
% FBS).

After the culturing, the culture supernatant in the culture flask was removed by suction, left in a deep freezer at -85 ° C. for 12 hours, and frozen.

Thaw the culture flask at room temperature,
The culture surface was rinsed with a phosphate buffer solution to remove cell debris.

The culture flask was again placed in a deep freezer at -85 ° C. overnight and frozen.

This culture flask was thawed at room temperature, and epidermal cells recovered from human skin were seeded at 1 × 10 ⁴ cells / cm ² .

This culture flask was placed in a carbon dioxide gas culture device (3
(Set at 7 ° C., 5% carbon dioxide concentration) for 8 days.
The medium was a green medium containing 3% fetal bovine serum (green +
3% FBS).

Epidermal cells were detached from the culture surface by dispase treatment, and sodium ethylenediaminetetraacetate (ED
TA) was converted into single cells with a trypsin solution in which trypsin was dissolved to a concentration of 0.25 w / v% in a phosphate buffer having a concentration of 0.206 mg / ml, and the number of cells was measured using a hemocytometer. The cells were suspended in a neutral collagen solution to a concentration of 3 × 10 ⁵ cells / ml to prepare an epidermal cell suspension. The neutral collagen solution was prepared by adding adult swine-derived atelocollagen to Dulbecco's modified Eagle's basic medium in a volume of 0.1%.
The solution was dissolved to 2% and the pH was adjusted to 7.4.

Example 5 3T established in a culture flask (culture area: 80 cm ² )
3 mouse lung fibroblasts were seeded at 3 × 10 ³ cells / cm ² and cultured for 3 days in a carbon dioxide gas culture device (set at 37 ° C., 5% carbon dioxide gas concentration). The medium was Dulbecco's modified Eagle's basic medium containing 10% fetal bovine serum (DMEM + 10%
FBS) was used. After the culture, the culture supernatant in the culture flask was removed by suction, and 10 kGy and 25
Irradiation of kGy electron beam was performed.

The epidermal cells recovered from human skin were
The cells were seeded at a density of × 10 ⁴ cells / cm ² .

Thereafter, the epidermal cells adhered and proliferated to form an epidermal cell sheet. Culture flask without electron beam irradiation is 3T
Proliferation of three cells was observed, but no proliferation was observed in those irradiated with an electron beam.

[0063]

According to the present invention, fibroblasts are seeded, cultured, and then killed.
Prepared by a method characterized in that at least a part thereof is detached or a method characterized in that a culture supernatant obtained by culturing fibroblasts is collected and cells are killed and / or removed, and then coated on various incubators. With the incubator of the present invention, (1) the danger of living 3T3 mouse lung fibroblasts or the like used as a feeder layer for adhesion of epidermal cells to the culture surface can be avoided from being mixed into epidermal cells. (2) When 3T3 mouse lung fibroblasts or the like are used as a feeder layer, the danger of remaining a drug such as mitomycin C to be administered to lose division / proliferation ability can be avoided.

By inoculating and culturing fibroblasts, they are killed, detached and removed, and the components such as extracellular matrix secreted and deposited by the cultured cells are substantially left on the culture surface of the incubator. Alternatively, by coating the culture supernatant with the culture supernatant of fibroblasts, use a culture vessel that allows 3T3 mouse lung fibroblasts and the like to adhere and grow without using a special cell culture container. And cost can be reduced.

After killing and exfoliating cells such as 3T3 mouse lung fibroblasts, or coating the culture supernatant of fibroblasts with the culture supernatant, the culture vessel is preserved while maintaining epidermal cell adhesion. Therefore, the conventional operation of preparing a feeder layer immediately before seeding epidermal cells can be omitted. In addition, since incubators with the same epidermal cell adhesion can be produced in large quantities at a time,
Cost can be reduced.

Furthermore, the need for routine passage of fibroblasts used as a feeder layer (subsequent passage in another container so that the cells do not become too dense in the incubator) is eliminated.

[Brief description of the drawings]

FIG. 1 is a bar graph showing the average value of the results of three measurements of the number of epidermal cells recovered from each culture flask (culture area 25 cm ² ) of the control and 3T3 frozen of Example 1. It is.

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[Procedure amendment]

[Submission date] February 23, 2001 (2001.2.2)
3)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 4

[Correction method] Change

[Correction contents]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] Claim 13

[Correction method] Change

[Correction contents]

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0002

[Correction method] Change

[Correction contents]

[0002]

2. Description of the Related Art Conventionally, among epithelial cells, epithelial cells (also called epidermal keratinocytes) have been cultured using radiation such as gamma rays or a drug such as mitomycin C. A method of culturing epidermal cells using 3T3 mouse embryonic fibroblasts or the like that have lost the ability to divide and proliferate as feeder layers (James G. Reinwald and Howard Green, Cell, No. 6) Volume, 331-34
Page 4, (James G. Rheinwald and Howard Green. Cell
6: 331-344. Serial Cultivation of Strains of Human
Epidermal Keratinocytes: the Formation of Keratin
), and a method of culturing using a serum-free medium such as MCDB153 without using cells serving as feeder layers.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0003

[Correction method] Change

[Correction contents]

However, in the conventional feeder layer method using 3T3 mouse embryo fibroblasts as a feeder layer, for example, it is necessary to prepare the 3T3 mouse embryo fibroblasts immediately before seeding the epidermal cells, which is complicated, and the feeder layer is not effective. Certain periods are also limited. In the growth of epidermal cells other than mouse (for example, human epidermal cells) and preparation of epidermal cell sheets, heterogeneous cells such as 3T3 mouse embryonic fibroblasts may be mixed with these cells.
There is also concern about the retention of drugs such as mitomycin C, which is used in deficient division and proliferation of mouse embryo fibroblasts.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0004

[Correction method] Change

[Correction contents]

On the other hand, instead of 3T3 mouse embryonic fibroblasts, epidermal cells to be obtained and / or fibroblasts of the same kind as the epidermal cell sheet (for example, when a human epidermal cell sheet is prepared, human fibroblasts are used). When cells are used as a feeder layer, foreign cells are not contaminated, but it is still necessary to delete the fibroblast division / proliferation ability. Is concerned. In addition, when human fibroblasts and the like are used, 3T3 mouse embryo fibroblasts are used,
Slow growth of epidermal cells.

[Procedure amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0005

[Correction method] Change

[Correction contents]

A method using a serum-free medium is 3T3
Compared to methods using feeder layer cells such as mouse embryo fibroblasts, the growth of epidermal cells is slow and often takes days to culture, and the effect of suppressing the differentiation of epidermal cells is in the medium, so that epidermal cells are overlaid. In some cases, an epidermal cell sheet could not be produced without being transformed.

[Procedure amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction contents]

Specifically, fibroblasts derived from mammals,
In particular, by culturing 3T3 mouse embryonic fibroblasts in an incubator and then killing the cells by treatment such as freezing and / or drying in the incubator and exfoliating at least a part thereof, substantially, By leaving components such as extracellular matrix secreted and deposited by the cultured cells on a culture surface in an incubator, that is, by leaving components required for cell adhesion and growth on the culture surface, It is an object of the present invention to provide an incubator to which the adhesiveness and the growth property are imparted.

[Procedure amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction contents]

That is, the present invention provides an epithelial cell and / or hepatic cell by a method for adhering and expanding epithelial cells and / or hepatocytes, which comprises the steps of inoculating, culturing, and killing mammalian fibroblasts. The incubator provided with cell adhesion and proliferation (Claim 1), the incubator according to Claim 1 wherein at least a part or all of the killed fibroblasts is peeled off, and the culture thereof. Freezer and / or
Alternatively, the incubator according to claim 1, which can be preserved by drying (claim 3), wherein the fibroblast derived from a mammal is 3T.
The incubator according to claim 1, which is 3 mouse embryo fibroblasts (Claim 4), and the epithelial cells are epidermal keratinocytes.
The incubator according to claim 5, wherein the incubator is an incubator made of glass, a synthetic polymer or a biopolymer. The incubator according to claim 1, wherein the incubator is a flask, a petri dish, a roller. The incubator according to claim 1, which is a bottle, a tray, a well plate, a bead, a film, a sheet, or a sponge, a glass or synthetic polymer incubator, a flask, a petri dish, a roller bottle,
The incubator according to claim 6, which is a tray, a well plate, a bead, a film, a sheet, or a sponge (claim 8), or the incubator made of a biopolymer is a sheet, a film, a sponge, or a bead. The incubator according to claim 9, wherein the incubator according to claim 1 is capable of producing an epidermal cell sheet.
0) The incubator according to claim 1, wherein the incubator according to claim 1 is capable of producing an epidermal cell suspension (invention 11), and a culture supernatant of fibroblasts derived from mammals. A culture vessel (claim 12) provided with hepatocyte adhesion and proliferation by the method of adhesion and proliferation of hepatocytes, characterized in that the fibroblasts derived from mammals are 3T.
The incubator according to claim 12, which is 3 mouse embryo fibroblasts (claim 13).

[Procedure amendment 9]

[Document name to be amended] Statement

[Correction target item name] 0015

[Correction method] Change

[Correction contents]

As fibroblasts, for example, mice,
Fibroblasts derived from mammals such as humans, rats, hamsters, rabbits and the like, preferably 3T3 mouse embryo fibroblasts widely used in the conventional feeder layer method are used. The conditions for seeding and culturing the cells are not particularly limited, and a general method is used. For example, fibroblasts grown alone in an incubator were treated with sodium ethylenediaminetetraacetate (EDTA) at a concentration of 0.206 mg / m2.
1 in a phosphate buffer solution prepared in 1 l, with a trypsin solution in which trypsin is dissolved to a concentration of 0.25 w / v%, suspended in a medium containing 5 to 10% fetal bovine serum, and inoculated in an incubator. Then, it is left still in a carbon dioxide gas culturing apparatus.
As the incubator used at that time, a special cell incubator such as a cell incubator coated with an extracellular matrix such as collagen is not required, and any incubator to which 3T3 fibroblasts and the like can adhere and proliferate, The material and shape are not particularly limited. Commercially available adherent cell culture vessels (flasks, Petri dishes, roller bottles, well plates, trays) or carriers, such as membranes, films, plates, and biopolymer membranes, films, and microbeads made of ordinary synthetic polymers Can also be selected as an incubator, and the cost can be greatly reduced as compared with the related art.

[Procedure amendment 10]

[Document name to be amended] Statement

[Correction target item name] 0024

[Correction method] Change

[Correction contents]

An epidermal cell sheet obtained by using an incubator provided with cell adhesion and proliferation by a cell adhesion and proliferation method comprising a step of seeding, culturing, and killing mammalian fibroblasts. In order to prevent 3T3 mouse embryonic fibroblasts and the like from being mixed in the epidermal cell suspension, it is necessary to completely remove the cells in this step.
However, it is not always necessary to completely remove cells from the viewpoint of the adhesion and proliferation of epidermal cells, and to the extent that they do not affect the adhesion and proliferation,
That is, it is sufficient that at least a part is peeled and removed.
The term “at least a part of the cells is detached”, which indicates the degree of removal of dead cells, is specifically, preferably 50% or more, more preferably 80%, based on the number of cells immediately before the cell death treatment. This means removing the above cells, and most preferably removing 100%.

[Procedure amendment 11]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction contents]

The degree of removal of dead cells can be easily confirmed by a method such as observation with a phase contrast microscope. At that time, if 100% removal of dead cells is confirmed, 3T3 mouse is added to the obtained epidermal cell sheet and epidermal cell suspension.
This means that there is no contamination of embryo fibroblasts.
However, 100% removal of dead cells is not a difficult operation, and cells can be almost completely easily removed by ordinary operations. In addition, cells after death are completely detached in a cell culture medium. The epidermal cell sheet and the epidermal cell suspension obtained from the incubator provided with the cell adhesion and proliferation of the present invention are substantially free from foreign cells. Conventionally,
An epidermal cell sheet that can be practically used only by the feeder layer method cannot be produced.In this case, when cells other than humans are used for the feeder layer, there was considerable contamination with heterologous cells, and thus the present invention An epidermal cell sheet and an epidermal cell suspension obtained from an incubator provided with cell adhesion and proliferation properties are excellent epidermal cell sheets and epidermal cell suspensions free from contamination with foreign cells.

[Procedure amendment 12]

[Document name to be amended] Statement

[Correction target item name] 0034

[Correction method] Change

[Correction contents]

[0034] In the production of an incubator to which hepatocyte adhesion and proliferation are imparted by a cell adhesion and proliferation method, which comprises coating a culture supernatant of mammalian fibroblasts on a culture vessel, Since the fibroblasts in the culture supernatant are killed and / or removed, hepatocytes grown using the incubator of the present invention are substantially free from contamination with 3T3 mouse embryo fibroblasts and the like. Not at all.

[Procedure amendment 13]

[Document name to be amended] Statement

[Correction target item name] 0039

[Correction method] Change

[Correction contents]

[0039]

EXAMPLES Example 1 3T established in a culture flask (culture area 25 cm ² )
Three mouse embryo fibroblasts were seeded at 3 × 10 ³ cells / cm ² and cultured in a carbon dioxide culture device (37 ° C., 5% carbon dioxide concentration) for 4 days. The medium was Dulbecco's modified Eagle's basic medium containing 10% fetal bovine serum (DMEM + 10%
FBS) was used.

[Procedure amendment 14]

[Document name to be amended] Statement

[Correction target item name] 0042

[Correction method] Change

[Correction contents]

On the other hand, as a control, 3T3 mouse embryonic fibroblasts, which had been treated with mitomycin C to be 1 × 10 ⁴ cells / cm ^{2 in a} culture flask (culture area: 25 cm ² ) and lacking division ability, were used. Was prepared in advance by inoculating another culture flask.

[Procedure amendment 15]

[Document name to be amended] Statement

[Correction target item name] 0046

[Correction method] Change

[Correction contents]

The number of epidermis cells of the control and 3T3 frozen was measured three times, and the average of the results of the three measurements is shown in FIG. Incubator of the present invention (3T3
In Frozen), as shown in FIG. 1, more epidermis was inoculated than in the case where seed cells were seeded by a feeder layer method (control) using conventional 3T3 mouse embryo fibroblasts as a feeder layer and cultured for 8 days. Cells were attached and proliferating.

[Procedure amendment 16]

[Document name to be amended] Statement

[Correction target item name] 0047

[Correction method] Change

[Correction contents]

Example 2 3T established in a culture flask (culture area 25 cm ² )
Three mouse embryo fibroblasts were seeded at 3 × 10 ³ cells / cm ² and cultured in a carbon dioxide culture device (37 ° C., 5% carbon dioxide concentration) for 4 days. The medium was Dulbecco's modified Eagle's basic medium containing 10% fetal bovine serum (DMEM + 10
% FBS).

[Procedure amendment 17]

[Document name to be amended] Statement

[Correction target item name] 0049

[Correction method] Change

[Correction contents]

Epidermal cells recovered from human skin were seeded in this culture flask at a density of 1 × 10 ⁴ cells / cm ² . The medium used was a green medium containing 3% fetal bovine serum. After 4 days, the colonies of epidermal cells were observed.
The appearance of epidermal cells proliferating without contamination of 3 mouse embryo fibroblasts was observed.

[Procedure amendment 18]

[Document name to be amended] Statement

[Correction target item name] 0050

[Correction method] Change

[Correction contents]

Example 3 3T established in a culture flask (culture area 25 cm ² )
Three mouse embryo fibroblasts were seeded at 3 × 10 ³ cells / cm ² and cultured in a carbon dioxide culture device (37 ° C., 5% carbon dioxide concentration) for 4 days. The medium was Dulbecco's modified Eagle's basic medium containing 10% fetal bovine serum (DMEM + 10
% FBS).

[Procedure amendment 19]

[Document name to be amended] Statement

[Correction target item name] 0052

[Correction method] Change

[Correction contents]

Epidermal cells collected from human skin were seeded at 1 × 10 ⁴ cells / cm ^{2 in} this culture flask. The medium used was a green medium containing 3% fetal bovine serum. After 4 days, the colonies of epidermal cells were observed.
The appearance of epidermal cells proliferating without contamination of 3 mouse embryo fibroblasts was observed.

[Procedure amendment 20]

[Document name to be amended] Statement

[Correction target item name] 0053

[Correction method] Change

[Correction contents]

Example 4 3T established in a culture flask (culture area 25 cm ² )
Three mouse embryo fibroblasts were seeded at 3 × 10 ³ cells / cm ² and cultured in a carbon dioxide culture device (37 ° C., 5% carbon dioxide concentration) for 4 days. The medium was Dulbecco's modified Eagle's basic medium containing 10% fetal bovine serum (DMEM + 10
% FBS).

[Procedure amendment 21]

[Document name to be amended] Statement

[Correction target item name] 0060

[Correction method] Change

[Correction contents]

Example 5 3T established in a culture flask (culture area: 80 cm ² )
Three mouse embryo fibroblasts were seeded at 3 × 10 ³ cells / cm ² , and cultured in a carbon dioxide gas culturing apparatus (set at 37 ° C., 5% carbon dioxide gas concentration) for 3 days. The medium was Dulbecco's modified Eagle's basic medium containing 10% fetal bovine serum (DMEM + 10%
FBS) was used. After the culture, the culture supernatant in the culture flask was removed by suction, and 10 kGy and 25
Irradiation of kGy electron beam was performed.

[Procedure amendment 22]

[Document name to be amended] Statement

[Correction target item name] 0063

[Correction method] Change

[Correction contents]

[0063]

According to the present invention, fibroblasts are seeded, cultured, and then killed.
Prepared by a method characterized in that at least a part thereof is detached, or a method characterized in that a culture supernatant obtained by culturing fibroblasts is collected, cells are killed and / or removed, and then coated on various incubators. With the incubator of the present invention, it is possible to avoid the danger that (1) live 3T3 mouse embryo fibroblasts used as a feeder layer for adhesion of epidermal cells to the culture surface are mixed into epidermal cells. (2) When 3T3 mouse embryonic fibroblasts or the like are used as a feeder layer, it is possible to avoid the danger of remaining a drug such as mitomycin C to be administered to lose the division / proliferation ability.

[Procedure amendment 23]

[Document name to be amended] Statement

[Correction target item name] 0064

[Correction method] Change

[Correction contents]

By inoculating and culturing fibroblasts, they are killed, detached and removed, and components such as extracellular matrix secreted and deposited by the cultured cells are substantially left on the culture surface of the incubator. Alternatively, by coating the culture supernatant with the culture supernatant of fibroblasts, use a culture vessel that allows 3T3 mouse embryonic fibroblasts to adhere and grow without using a special cell culture vessel. And cost can be reduced.

[Procedure amendment 24]

[Document name to be amended] Statement

[Correction target item name] 0065

[Correction method] Change

[Correction contents]

After killing and exfoliating cells such as 3T3 mouse embryo fibroblasts, or coating the culture supernatant of fibroblasts on the incubator, preserve the incubator while maintaining epidermal cell adhesion. Therefore, the conventional operation of preparing a feeder layer immediately before seeding epidermal cells can be omitted. In addition, since incubators with the same epidermal cell adhesion can be produced in large quantities at a time,
Cost can be reduced.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Tetsu Kanan 5-1-1 Takamoridai, Kasugai-shi, Aichi F-term in Menicon Research Institute, Inc. 4B029 AA08 AA21 BB11 CC02 CC11 CC12 GA01 GA02

Claims (13)

[Claims] 1. An adhesive method for epithelial cells and / or hepatocytes by a method for adhering and proliferating epithelial cells and / or hepatocytes, comprising the steps of inoculating, culturing, and killing mammalian-derived fibroblasts. And an incubator provided with proliferation. 2. The incubator according to claim 1, wherein at least a part or all of the killed fibroblasts are detached. 3. The incubator according to claim 1, wherein the incubator can be preserved by freezing and / or drying. 4. The incubator according to claim 1, wherein the mammal-derived fibroblasts are 3T3 mouse lung fibroblasts. 5. The incubator according to claim 1, wherein the epithelial cells are epidermal cells. 6. The incubator according to claim 1, wherein the incubator is made of glass, synthetic polymer or biopolymer. 7. The incubator according to claim 1, wherein the incubator is a flask, a petri dish, a roller bottle, a tray, a well plate, a bead, a film, a sheet, or a sponge. 8. The incubator according to claim 6, wherein the incubator made of glass or synthetic polymer is a flask, a Petri dish, a roller bottle, a tray, a well plate, a bead, a film, a sheet, or a sponge. 9. An incubator comprising a biopolymer, a sheet,
The incubator according to claim 6, which is a film, a sponge, or a bead. 10. The incubator according to claim 1, wherein an epidermal cell sheet can be produced. 11. The incubator according to claim 1, wherein an epidermal cell suspension can be prepared. 12. An incubator provided with hepatocyte adhesion and proliferation by a method for adhesion and proliferation of hepatocytes, which comprises coating a culture supernatant of fibroblasts derived from mammals onto the incubator. 13. The incubator according to claim 12, wherein the mammal-derived fibroblast is a 3T3 mouse lung fibroblast.