CN1562392A - Method for fabricating activated artificial skin tissue in bilayer by using bioreactor - Google Patents

Abstract

The invention discloses a method for preparing double-layer active artificial skin tissue by adopting a bioreactor. It includes the following steps: using the composite collagen sponge as the scaffold material, placing it on the porous scaffold of the perfusion bioreactor, adding the fibroblast suspension into the reactor, perfusion inoculation and cultivation; , to complete the construction of the epidermis. The bioreactor system is used to prepare active artificial skin with a double-layer structure of dermis and epidermis, so that the artificial skin has a certain mechanical strength, delays the degradation speed, and the cells and extracellular matrix are evenly distributed in the material, the preparation cycle is short, and the production can be achieved. Repeated, small batch-to-batch differences, and the constructed tissue is closer to natural tissue in anatomical structure.

Description

Method for preparing double-layer active artificial skin tissue using bioreactor

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for preparing an active artificial skin tissue with a double-layer structure of dermis and epidermis, in particular to a method for preparing an active artificial skin tissue by using a bioreactor.

Background technique

The skin is the largest organ of the human body, which plays a role in protecting the internal environment of the body and maintaining its stability. Because it is located on the body surface of the human body, it is susceptible to various injuries and diseases. Skin transplantation has been used to treat various acute and chronic skin diseases for thousands of years. The traditional treatment method is autologous or allogeneic transplantation. However, due to the limited source of autologous skin, allogeneic skin grafts have problems such as rejection, disease transmission, and difficulty in preservation. , it is difficult to meet the transplantation needs of patients. In recent years, skin transplantation has begun to develop from autologous skin transplantation and allograft transplantation to tissue-engineered skin substitute transplantation. Seeking an ideal permanent skin substitute has become a dream goal.

Tissue-engineered skin is divided into: epidermal substitute, dermal substitute and double-layer active skin. In 1975, Rheinwald and Green established the in vitro culture and expansion technology of keratinocytes (Rheinwald JG, Green H. Serial cultivation of human epidermal keratinocytes: the formation of keratining colonies from single cell. Cell, 1975, 6: 331-336), and then O' Connor and Gallico respectively reported that epidermal substitutes cultured in vitro were used to treat burn patients and achieved success (O'Connor NE, Mulliken JB, Banks-Schlegel S, et al. Grafting of burns with cultured epithelium prepared form autologous epidermal cell. Lancet, 1981 , 1:75. Gallico GG, O'connor NE, Comton CC, et al. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med, 1984, 311: 448-451). At present, this type of substitute is still used in the treatment of large area burns, but it has the following disadvantages: long culture period, poor anti-infection ability, lack of dermal components, large shrinkage, not wear-resistant, easy to rupture, poor elasticity, transplantation The success rate is low, so it is limited in clinical application. In order to overcome these problems, researchers have developed dermal substitutes, which can increase the success rate of epidermal substitute transplantation and improve the quality of healing, and a variety of commercial dermal substitutes have been used clinically. Integra is a collagen-aminopolysaccharide dermal substitute (Phillips TJ, Arch Dermatol, 1998, 110 (6): 344-349.) produced by the U.S. Integra lifesciences company. Observe the early infection of the wound, especially the need for a second operation to seal the wound. Hansbrough et al. inoculated fibroblasts into polylactic acid mesh to construct a dermal substitute and named it Dermagraft, which has been successfully used clinically to treat diabetic ulcers. Pure dermal substitutes have a high infection rate due to the lack of protection of the epidermis.

The ideal skin substitute should be able to repair the missing dermis and epidermis at the same time. Apligraf produced by Organogenesis Company in the United States is the first double-layer active skin substitute. The dermis is composed of fibroblasts, bovine type I collagen , culture medium, etc., inoculated with keratinocytes on the surface, and cultured in vitro for about 20 days to form a double-layered skin tissue. It has been clinically proven to have a certain effect on various refractory and chronic skin ulcers. Defects hinder clinical application, such as severe shrinkage of collagen gel, poor ability to resist collagenase degradation, and because the artificial skin is constructed under static conditions in a petri dish, the cell culture environment cannot be controlled, and the delivery ability of nutrients and by-products Limited, it is not conducive to the growth of cells inside the collagen gel, coupled with manual operation, resulting in a complicated production process, tissue uniformity and quality stability cannot be guaranteed.

U.S. Patent RE35,399 and Chinese Invention Patent (Application No.: 91109937.9) describe a method for preparing a double-layer skin substitute, which is composed of three parts: cultured keratinocytes form the epidermis, and the middle is non-porous collagen The dermis consists of fibroblasts cultured on a porous, cross-linked collagen sponge matrix. Firstly, keratinocytes and fibroblasts are isolated from skin samples, and after in vitro culture, the fibroblasts are seeded on a collagen sponge, and the other side of the sponge is made into non-porous collagen, and the keratinocytes are "dropped" onto the non-porous collagen , to form skin substitutes after culture. Because the skin substitute is produced in a culture dish, on the one hand, most of the inoculated fibroblasts are trapped on the surface of the collagen sponge, and the distribution in the collagen sponge is uneven, so that the constructed tissue is anatomically different from the natural tissue. There is a big difference. On the other hand, the static culture process is not conducive to cell growth and matrix secretion as mentioned above. Moreover, the differences between each culture dish and between batches are relatively large.

Halberstadt (Halberstadt CR, Hardin R, Bezverkov K, et al. The in vitro growth of a three-dimensional human dermal replacement using a single-passperfusion system, Biotechnol Bioeng, 1994, 43: 740-746.) et al. ⁵ cells/cm ² of human fibroblasts are inoculated on the porous mesh scaffold made of PGA/PLA material, through a closed one-way perfusion culture system, after 2-3 weeks of culture, a biological Active dermal substitute and efficient reproducible production of dermal tissue substitutes. The advantage is that compared with static culture, it can shorten the time of dermis construction; the degradation rate of PGA/PLA scaffold material is controllable; the constructed artificial dermis can be directly cryopreserved; but this system is limited to the construction of artificial dermis.

Prenosil and Villeneuve (Prenosil JE, Villeneuve PE. Automated production of cultured epidermal autografts and sub-confluent epiderlmal autografts in a computer controlled bioreactor. Biotechnol Bioeng, 1998, 59: 679-683.) directly inoculated human epidermal cells into FEP The FEP membrane is placed in a polycarbonate growth chamber, and multiple growth chambers can be stacked. By connecting to a multi-pipe peristaltic pump, flow control can be performed automatically. The advantage is that since the support plate made of polycarbonate material is transparent, the formation of the skin can be monitored. Also, this reactor system is limited to the construction of epidermal substitutes and cannot realize the in vitro construction of bilayer active skin.

Contents of the invention

The technical problem to be solved in the present invention is to disclose a method for preparing double-layered active artificial skin tissue using a bioreactor, so as to overcome the above-mentioned deficiencies in the prior art and meet the needs of related fields.

Method of the present invention comprises the steps:

(1) Use the composite collagen sponge as the scaffold material, place it on the porous scaffold of the perfusion bioreactor, add DMEM medium containing 5-15% newborn bovine serum, soak overnight, discard the medium, and press 1×10 ⁵ cells /cm ² ~5×10 ⁶ cells/cm ² cell density Add the fibroblast suspension into the reactor, perfuse and inoculate through the peristaltic pump, until the residual cell density in the culture medium is lower than 5-20% of the inoculation density, Perfusion culture is carried out, and the glucose concentration in the culture solution at the outlet of the reactor is detected at the same time. Adjust the medium perfusion rate to 0.5-4d-1, and control the glucose concentration at the outlet to not be lower than 1.0-2.0g/L. After 10-20 days of perfusion culture in this reactor, the construction of the dermis is completed;

(2) Replace the DMEM medium of newborn calf serum with keratinocyte serum-free medium, inoculate keratinocytes on the above-mentioned artificial dermis at a density of 1×10 ⁵ cells/cm ² to 5×10 ⁶ cells/cm ² , and culture them statically for 1 ~2 days later, the cells were perfused with keratinocyte culture medium for 2~5 days, and the perfusion rate was 0.5~2d ^-1 . Then remove the keratinocyte serum-free medium, use DMEM medium containing 2-10% newborn bovine serum for air-liquid interface culture, and simultaneously detect the glucose concentration in the culture solution at the outlet of the reactor, and control the perfusion rate to 0.5-4d ^-1 , Make the glucose concentration not lower than 1.0-2.0g/L. After culturing for 8-14 days, the epidermal construction was completed.

Said composite collagen sponge is composed of amino polysaccharides and collagen solution, and the content of amino polysaccharides is 5-25% (g/g) of the dry weight of collagen; said amino polysaccharides include chitosan , 6-chondroitin sulfate, hyaluronic acid or sodium heparin or one or more.

Wherein: said collagen solution is the collagen-HAc solution obtained by soaking the tendon powder made of beef tendon without fascia and lipid in acetic acid solution, and its concentration is generally 5.0-10.0 mg/mL. It is prepared in the following way: remove fascia from tendon, make tendon powder from lipid, soak in acetic acid solution, and obtain collagen-HAc solution with a final concentration of 8.0 mg/mL. Alternatively, purchase commercial collagen directly and prepare a collagen-HAc solution with a final concentration of 5.0-10.0 mg/mL.

Said composite collagen sponge is prepared by adding one or more of chitosan, chondroitin 6-sulfate, hyaluronic acid and sodium heparin to the collagen-HAc solution, freezing overnight in a -20°C refrigerator, and vacuum freeze-drying 24hr, adopt 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma) to carry out chemical cross-linking to collagen sponge, wash, lyophilize, sterilize, promptly obtain said composite collagen sponge . After the composite collagen sponge is cross-linked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, it can overcome the insufficient mechanical strength, high shrinkage rate and excessive degradation speed of pure collagen gel or collagen sponge. Fast, glutaraldehyde cross-linking has disadvantages such as cytotoxicity, and can improve the biocompatibility of the material, and promote the attachment and proliferation of cells on the three-dimensional scaffold material;

Said keratinocytes are keratinocytes isolated from epidermal tissue, and the separation method is a conventional method, described in Johnson EW, Meunier SF, Roy CJ, Parenteau NL.Serial cultivation of normal human keratinocytes: a defined system for studying theregulation of growth and differentiation. In Vitro Cell Dev Biol, 1992, 28A: 429-435. There have been detailed reports in the literature;

Said fibroblasts are fibroblasts isolated from dermal tissue, and the separation method is a conventional method. In Halberstadt CR, Hardin R, Bezverkov K, et al.The in vitrogrowth of a three-dimensional human dermal replacement using a single-passperfusion system, Biotechnol Bioeng, 1994, 43: 740-746. There have been detailed reports in the literature;

Said perfusion bioreactor has a public description in Chinese Patent Application No. 200410016651.6.

Said keratinocyte serum-free medium is described in detail in Chinese Patent Application No. 200410016367.9, and it is characterized in that this serum-free medium is an aqueous solution, including basal medium and the following components:

Basal medium 12-18g/L

Histidine 0.1-1.0mmol/L

Isoleucine 0.1-0.6mmol/L

Tryptophan 0.05-0.3mmol/L

Phenylalanine 0.3-0.6mmol/L

Lysine 0.7-1.2mmol/L

Methionine 0.05-0.3mmol/L

Tyrosine 0.3-0.6mmol/L

Keratinocyte growth factor 20-200ng/L and/or epidermal growth factor 5-20

ng/L

Pantothenate 0.02-0.04mmol/L

Choline chloride 0.1-0.5mmol/L

Folic acid 0.01-0.03mmol/L

Inositol 0.1-0.4mmol/L

Nicotinamide 0.05-0.5mmol/L

Vitamin B6 0.02-0.06mmol/L

Riboflavin 0.001-0.01mmol/L

Thiamine 0.01-0.03mmol/L

Insulin 1-20μg/L

Transferrin 1-20μg/L

Ethanolamine 0.01-0.05mmol/L

Phosphoethanolamine 0.01-0.05mmol/L

Strontium chloride 1-5mmol/L

Bovine serum albumin 5-20μg/L

Hydrocortisone 0.1-1μg/L

Triiodothyronine 10-40pmol/L

Calcium chloride 0.03-0.5mmol/L

Magnesium chloride 5-10mmol/L

Antioxidant Appropriate amount

Penicillin 50-200U/L

Streptomycin 50-200U/L

The basal medium includes MCDB153, calcium-free DMEM or a mixture of calcium-free DMEM and Ham's F12 at a volume ratio of 1:0.3-3.

Said antioxidants include mercaptoethanol 0.01-0.05mmol/L and/or catalase 50-150U/L and/or superoxide dismutase 50-150U/L and/or sodium selenite 0.01-0.05mmol /L.

With the new bioreactor system, a double-layer active artificial skin with dermis and epidermis can be constructed in the same reactor, which avoids the limitation that the previous bioreactor system can only construct artificial dermis or artificial epidermis. The superposition of the device enables multiple pieces of artificial skin to be constructed at the same time in one preparation process. Compared with manual operation, the preparation process is more effective and repeatable.

The bioreactor system is used to prepare active artificial skin with a double-layer structure of dermis and epidermis, so that the artificial skin has a certain mechanical strength, delays the degradation speed, and the cells and extracellular matrix are evenly distributed in the material, the preparation cycle is short, and the production can be achieved. Repetition, small batch-to-batch differences, etc.

Compared with previous preparation methods, perfusion inoculation and culture methods are adopted. On the one hand, the material transfer mode is changed from passive diffusion to active transfer, which makes the distribution of cells in the collagen sponge more uniform, which is beneficial to tissue formation, cell growth and cell proliferation. The secretion of the extracellular matrix, in this dynamic culture environment, can alleviate the diffusion limitation of nutrients and by-products to a certain extent, and reduce the impact of nutrient depletion and by-product toxicity on the normal growth and metabolism of cells in the three-dimensional material, thereby further Improve the structure, composition and function of the engineered tissue; on the other hand, adopt the perfusion culture method, adjust the perfusion strategy of the medium according to the change of the glucose concentration at the inlet and outlet of the reactor, maintain the culture environment at a relatively stable level, and make it closer to The stable internal environment of cells and tissues in vivo promotes cell proliferation and functional expression, shortens the culture time, and makes the constructed tissues closer to natural tissues in anatomical structure.

Description of drawings

Figure 1 is a structural diagram of a perfusion bioreactor system.

Figure 2 is a schematic diagram of the reactor results.

Detailed ways

Referring to Fig. 1 and Fig. 2, the perfusion bioreactor system used in the in vitro construction of double-layer active skin tissue according to the present invention includes: a liquid addition device, a perfusion bioreactor, a liquid collection device, a liquid return device and an air supply device;

Said perfusion bioreactor comprises: a closed container 2 provided with a loam cake 1; a porous support plate 5 arranged in the closed container 2 to separate the closed container 2 into an upper chamber 3 and a lower chamber 4, arranged on the porous support plate 5 above the fastening seal 6 close to the inner wall of the closed container 2; the scaffold material or cell material composite 7 for skin construction is placed flat on the porous support plate 5 and fixed by the fastening seal 6, and the epidermis is porous when it is constructed The support plate 5 can provide a gas-liquid interface; the upper chamber 3 is provided with a liquid inlet 8 and a liquid outlet 9, the lower chamber is provided with a liquid inlet 8 and a liquid outlet 9, and the upper cover 1 is provided with a liquid inlet 8, a gas inlet 10 and a gas outlet 11 , A liquid distributor 101 is provided under the upper cover 1 for distributing the culture medium entering from the liquid inlet 8 of the upper cover 1 .

The liquid feeding device is connected with the liquid inlet 8 of the perfusion bioreactor, the liquid receiving device is connected with the liquid outlet 9 of the perfusion bioreactor, and the liquid return device is respectively connected with the liquid feeding device through pipelines. The device is connected with the liquid receiving device, and said gas supply device is connected with the gas inlet 10 of the perfusion bioreactor through a pipeline;

Said liquid addition device comprises liquid addition peristaltic pump 12 and the liquid addition liquid storage bottle 13 that is connected with it, and liquid addition liquid storage bottle 13 is communicated with the liquid inlet 8 of perfusion bioreactor through pipeline, because said biological The reactor has three liquid inlets 8. The liquid inlet 8 of the upper cover 1 is used for inoculation, and the liquid inlet 8 on the side may be selected for perfusion culture. A control valve 26 is set between the connected pipelines to control which inlet 8 the liquid enters from;

Said liquid collection device comprises: liquid collection peristaltic pump 14, liquid collection storage bottle 15, liquid collection control valve 16 and liquid collection bottle 17, the inlet of liquid collection peristaltic pump 14 is connected with the liquid outlet 9 of perfusion bioreactor The outlet of the receiving liquid peristaltic pump 14 is connected with the inlet of the receiving liquid storage bottle 15, and the outlet of the receiving liquid storage bottle 15 is connected with the receiving liquid bottle 17 through the liquid storage peristaltic pump 25, and the liquid receiving control valve 16 is arranged on The pipeline between the liquid collection storage bottle 15 and the liquid storage peristaltic pump 25 is used to control the liquid collection volume, and a control valve 26 is provided between each outlet of the bioreactor and the liquid collection peristaltic pump 14 .

Said liquid backflow device includes a backflow control valve 18 arranged between the liquid collection storage bottle 15 and the liquid addition storage bottle 13 through the return line 19;

Said air supply device comprises an air pump 20, an air flow meter 21 and an air filter 22 interconnected in sequence, and the air filter 22 is connected with the perfusion bioreactor;

Said detection device comprises the dissolved oxygen electrode 23 inserted in the liquid storage bottle 13, the liquid storage bottle 15 and the pH electrode 24 in the liquid storage bottle 15, and the dissolved oxygen electrode 23 and the pH electrode 24 are respectively connected with The dissolved oxygen meter is connected with the pH meter to detect and control the dissolved oxygen concentration and pH of the culture medium in the liquid storage bottle 13 and the liquid storage bottle 15 .

It can be seen from Fig. 1 that a plurality of said perfusion bioreactors can be connected in parallel to meet the needs of expanding production capacity.

Example 1

Take the foreskin of the child, cut it into a skin block of about 1 cm ² , wash it three times with PBS containing 200 U/mL gentamycin, wash it twice with 75% alcohol, and wash it twice with PBS. The skin tissue was added to 200 U/mL neutral protease (Sigma) and digested at 4°C for 18 hours to separate the dermis and epidermis. Keratinocytes are obtained from epidermal tissue and fibroblasts are obtained from dermal tissue.

Cut the epidermis into small pieces, act with 6 mL of 0.25% trypsin (Sigma)/0.06% EDTA (the volume of trypsin/EDTA digestion solution is about 3 times the volume of the digested tissue) for 8 minutes, and add an equal amount of calf serum and trypsin activity.

Filter through a 150-mesh sieve to remove epidermal fragments and obtain keratinocyte suspension. Centrifuge at 2000r/min for 5min, pour off the supernatant, wash twice with PBS, add keratinocyte serum-free medium (Gibco) containing 200U/mL gentamicin, the cell inoculation volume is 2×10 ⁴ cells/cm ² , Add 5 mL of culture medium to a 25 cm ² culture flask (Nunc), culture in an incubator (Revco) at 37° C. and 5% CO ₂ , and change the medium every three days to obtain primary cultured cells. When the cells covered 75% of the bottom area of the square flask, subculture was performed, the supernatant was discarded, and 1.5 mL of trypsin was added for digestion. When the cells became round and fell off, an equal amount of calf serum was added to terminate the trypsin activity. Centrifuge the cell suspension at a speed of 2000r/min for 5min, pour off the supernatant, wash twice with PBS, add medium, inoculate into a square bottle or a bioreactor system for subculture and expansion, and obtain the cutin required for the construction of the epidermis cell.

Cut the dermis into pieces, add 0.05% collagenase (Gibco) solution, and enzymatically hydrolyze at 37°C for 40 minutes; filter to remove residual debris, wash the cells with PBS and collect by centrifugation, and the fibroblast suspension is 5×10 ⁴ according to the inoculum size /cm ² were inoculated in square flasks for primary culture, using DMEM (Gibco) containing 10% calf serum as the medium, and changing the medium every 3 days. After the fibroblasts cover about 80% of the bottom of the square bottle, they are digested with 0.25% trypsin solution, subcultured and amplified in the square bottle or bioreactor according to the inoculation amount of 2×10 ⁴ /cm ² to obtain the constructed skin Fibroblasts required to organize the dermis.

Example 2

The fascia and lipids were removed from the tendon to make tendon powder, which was stored at -20°C for later use. Soak the tendon end in 0.1mol/L HAc solution to prepare a collagen-HAc solution with a final concentration of 8.0mg/mL. Chitosan solution, heparin sodium solution, and chondroitin sulfate solution were added to the collagen solution so that the final concentrations were 10% (g/g), 7% (g/g) and 3% (g/g) of the dry weight of collagen, respectively. g), frozen at -20°C overnight, and vacuum freeze-dried for 24 hours to obtain a collagen sponge. Collagen sponges were chemically cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Sigma), rinsed thoroughly with distilled water, freeze-dried, and sterilized with ethylene oxide to make usable Composite collagen sea surface matrix material for preparing double-layer active skin tissue.

Example 3

The same method as in Example 2 is adopted, but in the composite collagen sponge, the amino polysaccharide substance is hyaluronic acid, and the final concentration is 20% (g/g) of the dry weight of the collagen.

Example 4

Place the collagen sponge on the porous stainless steel spacer support of the perfusion bioreactor constructed in vitro with double-layer active skin, add DMEM medium containing 10% newborn bovine serum, and soak overnight. Abandon the medium, add the fibroblast suspension into the reactor at a cell density of 5×10 ⁵ cells/cm ² , and perform perfusion inoculation through a peristaltic pump until the residual cell density in the culture medium is lower than 10% of the inoculation density, then carry out Perfusion culture.

Control the perfusion rate to 1d ^-1 , and detect the glucose concentration in the culture solution at the outlet of the reactor. When the glucose concentration is lower than 2g/L, increase the perfusion rate to 2.5d ^-1 . After 16 days of perfusion culture in this reactor, the construction of the dermis was completed.

Use keratinocyte serum-free medium instead of DMEM medium with 10% newborn bovine serum, inoculate keratinocytes on the above-mentioned artificial dermis at a density of 5×10 ⁵ cells/cm ² , after static culture for 1 day, perfuse culture with keratinocyte medium for 2 days , the perfusion rate is 1d ^-1 . Then remove the keratinocyte serum-free medium, adopt DMEM medium containing 10% newborn bovine serum for air-liquid interface culture, the position of the liquid level is controlled by the upper outlet, and the medium is perfused from the lower inlet and outlet, and the perfusion rate is controlled to be 1d ^-1 , while detecting the glucose concentration in the culture solution at the outlet of the reactor, when the glucose concentration is lower than 2g/L, increase the perfusion rate to 3d ^-1 . After 14 days of culture, the epidermal construction was completed.

Claims (6)

1. a composite collagen sponge that is used for external structure double-layer active artificial skin tissue is characterized in that being made up of aminopolysaccharide class material and collagen solution, and aminopolysaccharide class content of material is 5～25% (g/g) of collagen dry weight; Said aminopolysaccharide class material comprises in receiving one or more of chitosan, 6-chondroitin sulfate, hyaluronic acid or heparin, said collagen solution is that the tendon powder that tendon Bovis seu Bubali goes fascia, lipid to make is soaked in the collagen-HAc solution that obtains in the acetum, or by the collagen-HAc solution of business-like collagen product preparation.

2. a method that adopts bioreactor to prepare double-layer active artificial skin tissue is characterized in that, comprises the steps:

(1) be timbering material with the composite collagen sponge, place on the porous support of filling type bioreactor, adding the DMEM culture medium that contains 5～15% new-born calf serum soaks, abandon culture medium, perfusion inoculation fibroblast, to the culture fluid residual cell density be lower than inoculum density 5～20% after, carry out perfusion cultures, in reactor,, finish corium and make up through the perfusion cultures of 10～20d;

(2) inoculate horn cell on above-mentioned artificial dermis, replace containing the DMEM culture medium of new-born calf serum with the horn cell serum-free medium, behind static cultivation 1～2d, with horn cell culture medium perfusion cultures 2～5d, irrigation rate is 0.5～2d ^-1, then remove the horn cell serum-free medium, adopt the DMEM culture medium that contains 2～10% new-born calf serum to carry out gas-liquid interface and cultivate, and adjust irrigation rate (0.5～4d as required ^-1), finish epidermis behind 8～14d and make up.

Said horn cell is an isolating horn cell from epidermal tissue, and said fibroblast is an isolating fibroblast from dermal tissue.

3. method according to claim 2 is characterized in that, with 1 * 10 ⁵Cells/cm ²～5 * 10 ⁶Cells/cm ²Cell density adds the fibroblast suspension in the reactor.

4. method according to claim 2 is characterized in that, the irrigation rate of step (1) is 0.5～4d ^-1, make that concentration of glucose is not less than 1.0～2.0g/L in the reactor exit culture fluid.

5. method according to claim 2 is characterized in that, presses density 1 * 10 on artificial dermis ⁵Cells/cm ²～5 * 10 ⁶Cells/cm ²The inoculation horn cell.

6. method according to claim 2 is characterized in that, step (2) is carried out perfusion cultures with the DMEM that contains serum, and the determining of irrigation rate makes the concentration of glucose in the reactor exit culture fluid be not less than 1.0～2.0g/L.

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