CN101347640A - Pigmented skin analogue model for screening depigmentation agents and construction method thereof - Google Patents

Abstract

The invention belongs to the field of biotechnology, and relates to a pigmented skin analogue that can be used for screening fading agents and a construction method thereof. The skin analogue layer is constructed from skin fibroblasts and collagen cultured in vitro, and an air-liquid plane is used to construct a skin analogue layer on it. It is constructed by inoculating keratinocytes and melanocytes with three-dimensional culture technology. The obtained pigmented skin analogs are similar in structure to skin, can simulate the transport process of melanosomes, and can be used to screen fading agents, providing a more scientific basis for the research of melanocyte biology in vitro, the screening of fading agents and whitening cosmetics model.

Description

Pigmented skin analogue model for screening depigmentation agents and construction method thereof

technical field

The invention belongs to the field of biotechnology, and relates to a pigmented skin analog that can be used for screening fading agents and a construction method thereof.

Background technique

Skin grafts are often used in plastic surgery to repair large areas of skin defects. The color of the transplanted area and the surrounding skin is often different after surgery, and the pigmentation is usually increased. In recent years, due to the destruction of the environment and the ozone layer caused by pollution, the metabolic disorder of melanin-forming enzymes has occurred, and the number of patients suffering from various dark spot symptoms such as freckles, chloasma, and age spots has increased. Therefore, the treatment of hyperpigmentation and other pigmented diseases has high social and economic benefits. How to solve the problem of pigmentation has become an important research topic for plastic surgeons and dermatologists.

Mature melanocytes are located in the basal layer of epidermal cells and are a type of dendritic cells. Each melanocyte and about 36 surrounding keratinocytes constitute an "epidermal melanin unit". Under physiological conditions, the proliferation of melanocytes is weak, and both basal layer keratinocytes and dermal fibroblasts can regulate the morphology of melanocytes, melanin synthesis and transport by secreting some growth factors or extracellular matrix components , keratinocytes can also affect the behavior of melanocytes through cell-cell contact. In wound repair after skin grafts and other transplants, not only the keratinocytes in the hair follicles migrate, but also the melanocytes in the outer root sheath of the hair follicles are activated, and there is an abnormality in a certain link in the activation and migration, and the change of local inflammatory mediators , may be the cause of hyperpigmentation. Melanin is a nitrogenous compound that is synthesized in melanosomes, the unique organs of melanocytes. The difference in skin color is mainly determined by factors such as the difference in the ratio of melanin to pheomelanin, the distribution in keratinocytes, the activity of melanocytes, the size, number, distribution, and environment of melanosomes.

Since the skin is an organ composed of a variety of cells, in addition to keratinocytes, Langerhans cells, fibroblasts, etc. can affect the function of melanin units and melanocytes, among which keratinocytes and fibroblasts regulate melanin. It plays an active role in the growth, morphology, pigmentation and differentiation of vegetative cells. Since the successful culture of melanocytes in vitro, people's research on fading agents has entered a stage of rapid development. Because this method has the advantages of convenient and rapid screening of decolorizing agents, it has become the main method for current research on decolorizing agents. However, due to the differences in the cultured cell lines and culture conditions, the experimental results vary widely. For example, the cell lines used in some experiments are human or mammalian melanoma cells, and the signal transduction pathways and responses to external stimuli of tumor cells are very different from normal somatic cells; prime cells, but the mitogen added in the medium is 12-0-tetradecanoylphorbol-13-ethyl ester (TPA), and TPA is a strong cancer-promoting agent, so it is safe, stable and close to the body The cultivation system of the environment will be more convincing.

In recent years, there are many models used to study melanocytes and evaluate various factors affecting melanocytes, but they are quite different from the skin structure. With the advancement of bioengineering technology and the deepening of people's research, the gas-liquid Planar technology is used to cultivate pigmented skin, so the construction of a model that is closer to skin will provide a more ideal model for the study of melanocyte biology in vitro and the screening of depigmentation agents, with a view to clinical application in the treatment of pigmentation such as pigmentation. Disease provides experimental methods and theoretical basis.

Contents of the invention

In order to overcome the above-mentioned deficiencies in the prior art, the present invention provides a pigmented skin analog model for screening fading agents and its construction method, which is closer to normal skin and can provide an ideal skin analog model for screening whitening and fading agents .

Technical scheme of the present invention is realized like this:

The present invention is a pigmented skin analog that can screen for depigmentation agents. The dermis analog layer is constructed from skin fibroblasts and collagen cultured in vitro, and keratinocytes and melanocytes are compounded thereon. The fibroblasts, ^{keratinocytes} and melanocytes are obtained from human foreskin tissue by expanding culture ^; After the rat tail type I collagen was mixed and solidified to form a dermis analog layer, keratinocytes (1.0×10 ⁷ /ml) and melanocytes (0.3×10 ⁷ /ml) were inoculated on the dermis analog layer.

The construction method of the present invention includes sterilization, digestion, separation, and cultivation, including the following steps:

(1) Source, isolation and culture of tissue-engineered skin seed cells:

a) Surgically excised human foreskin tissue soaked in 0.25% chlorhexidine solution;

b) 2.5g/L Dispase II enzyme digests the foreskin tissue, and separates the epidermis and dermis;

c) The dermal tissue is cut into a tissue block with a size of 1×1×1 mm ³ , and placed in a fibroblast culture medium to expand and culture fibroblasts;

d) Digest the epidermal tissue in 2.5g/L trypsin, centrifuge, place in melanocyte culture medium, and culture and expand melanocytes;

e) Culture fibroblasts in a culture flask covered with rat tail type I collagen to form a fibroblast trophoblast, separate the epidermal tissue and digest it in 2.5g/L trypsin, then centrifuge and place in a culture flask containing a trophoblast , add KGM-G3F culture medium to cultivate and expand keratinocytes;

(2) Construct a pigmented skin analog that can screen for fading agents:

a) Fibroblasts are mixed with rat tail type I collagen at a concentration of 20% at a density of 2×10 ⁵ /ml to 3×10 ⁵ /ml, and then solidified to form a dermis analog layer;

b) Add 1.0×10 ⁷ /ml keratinocytes and 0.3×10 ⁷ /ml melanocytes on the surface and culture them in a transwell culture chamber, add KGM-G3F culture medium;

c) After culturing for 1 week, conduct air-liquid plane culture, and continue culturing for 2 weeks to obtain a pigmented skin analog model that can screen for fading agents.

The composition of the fibroblast culture solution of the present invention is: DMEM commercial culture solution, 15% calf serum; the composition of the described melanocyte culture solution is: RMP1640 commercial culture, and contains: basic fibroblast growth factor (bFGF ) 2～3×10 ⁴ U/L, cholera toxin (CT) 0.20～0.25g/L, transferrin 5～10mg/L, calf serum 100～150ml/L, hydrocortisone 2～5mg/L , insulin 100～150U/L, gentamicin 100,000～150,000/L; the composition of the KGM-G3F culture medium is: DMEM/F12 (3:1) commercial culture medium, and contains 100ml～150ml/L fetal Bovine serum, adenine 0.18～0.20mM, epidermal growth factor EGF10～15ng/ml, hydrocortisone 0.4～0.50ug/ml, insulin 3～5ug/ml, isoproterenol 10～15uM, transferrin 5～ 10ug/ml, triiodothyronine 2~3nM, ampicillin 100~120ug/ml, streptomycin sulfate 80~100ug/ml.

The melanocyte culture method established by the present invention adopts the culture medium containing the natural mitogen of melanocytes bFGF and CT to successfully establish an in vitro culture system of normal human epidermal melanocytes, and the cell morphology is normal, which is a safe and stable culture system . The established method for culturing keratinocytes and fibroblasts can maintain high viability of the cells, and the purity can reach 100%. On the basis of the successful establishment of cell lines, the present invention successfully constructs a pigmented skin analog model by using the air-liquid three-dimensional culture technology, and the results show that the constructed pigmented skin analog has a complete structure, and the vitality of epidermal cells and fibroblasts is good. During the construction of pigmented skin analogues, the color of the analogues gradually deepened with the prolongation of culture time. Transmission electron microscopy shows that melanocytes are distributed in the basal layer of the epidermis and are in good condition. There are abundant melanin granules in the cytoplasm. The distribution of varying melanosomes was not regular and scattered throughout the cytoplasm, reflecting the transfer of melanosomes between melanocytes and keratinocytes. This phenomenon mimics the structure of the "epidermal melanin unit" in vivo. During the migration of epidermal cells from the basal layer to the cornified layer, the nuclei of keratinocytes gradually degenerated, consistent with the appearance of the epidermis in vivo. The pigmented skin analogs thus constructed mimic the function of the skin.

Description of drawings

Figure 1 Photos of fibroblast culture

A shows spiral growth, B vimentin staining is positive;

Figure 2 Culture photos of melanocytes

A primary culture for 3 days, B dopa staining positive;

Figure 3 Culture photo of keratinocytes

A primary culture for 9 days, B keratin staining positive;

Figure 4 Photo of dermis analogs

A without steel ring to limit shrinkage, B with steel ring to limit shrinkage, C leather analog HE staining;

The color of the pigmented skin analogue constructed in Figure 5 gradually deepens

A submerged culture for 3 days, B submerged culture for 7 days, C air-liquid plane culture for 4 days, D air-liquid plane culture for 9 days, E air-liquid plane culture for 14 days;

Figure 6 HE staining photos of pigmented skin analogs, the arrows indicate the keratinized beads of the epidermis stratum corneum;

Figure 7 Fontana Masson staining shows that melanocytes are located at the base of the skin

Figure 8 Transmission electron microscope pictures of pigmented skin analogs

A Keratinocyte nuclear migration, B melanocyte status;

Figure 9 electron microscope shows the transport process of melanosomes in pigmented skin analogs; the arrow shows melanosomes;

Figure 10 Electron microscope shows the photos after different fading agents

Detailed ways

1. Cell Isolation and Culture

(1) Separation of specimens

The specimens of healthy people after circumcision (age 5-10 years old) were soaked in 0.25% chlorhexidine solution for 10 minutes, rinsed in 0.01M PBS buffer for 3 times, and sterilized with instruments on the ultra-clean table. Remove the subcutaneous tissue and meat membrane as much as possible, then cut the specimen into thin strips of 1mm×2mm, wash 3 times in PBS solution for 5min each time, transfer to 2.5g/L Dispase II enzyme, and cool overnight in the refrigerator at 4°C Digestion 16-22h.

(2) Culture and identification of fibroblasts

Take out the cold-digested tissue, separate the epidermis and dermis with ophthalmic tweezers, add appropriate amount of calf serum to the separated dermis tissue, then cut it into 1×1×1mm ³ tissue pieces, put them in a 25cm ² culture medium Put the culture bottle upside down in an incubator at 37°C and 5% CO ₂ . After 30 minutes, add a small amount of DMEM culture solution containing 15% calf serum, then place it in the incubator, and take it out for culture after 4 hours. bottle, add 2ml of culture medium, turn the culture bottle over slowly, and culture statically. On the third day, 5ml of culture medium was added to continue culturing. The medium was changed every 3 days until the fibroblasts migrated out of the tissue mass and grew into a cell monolayer (Figure 1A). Cells of passage 5-15 were used in the experiment.

The subcultured fibroblasts were inoculated on the coverslips, cultured in DMEM medium containing 15% calf serum for 3 days, then the slides were taken out, fixed with pre-cooled acetone for 10 minutes, and peroxidase inhibitor was added dropwise on the slides. Block with 5% goat serum, incubate at room temperature for 10 minutes, discard the serum, do not wash, add primary antibody (mouse anti-human vimentin monoclonal antibody) dropwise on the glass slide, use PBS as negative control, vimentin staining is positive Fibroblasts were confirmed (Fig. 1B).

(3) Culture of melanocytes

Gently separate the epidermis from the dermis with tweezers for the above-mentioned cold-digested tissue, transfer the epidermal tissue to 2.5g/L trypsin at 37°C for 10min, and blow repeatedly with a pipette to form single cells. The liquid in the bottle is turbid That is, add melanocyte culture medium equal to that of trypsin to stop the action of trypsin. After filtering through a 280-mesh sieve, draw the cell suspension into a centrifuge tube, centrifuge at 1000r/min for 10min, discard the supernatant, add culture solution and pipette To form a single-cell suspension, repeat centrifugation once, add culture medium and pipette to form a single-cell suspension, and count the cells. Inoculate in a 25ml culture bottle at a density of 2-4×10 ⁴ /ml, place in 50ml/L CO ₂ , and culture in a 37°C incubator. After 24 hours, the medium was changed for the first time to remove unattached cells, and the medium was changed once in the next 2-3 days (Figure 2A). In this experiment, 3-4 generations were selected. Subcultured 3-4 generation melanocytes were inoculated on coverslips, fixed with 10% formaldehyde for 1 hour, rinsed with double distilled water for 3 times, added 0.1% levodopa solution, incubated at 37°C for 45 minutes, and then rinsed with double distilled water After rinsing, add fresh levodopa staining solution and continue to incubate at 37°C for 4h. Observation under an inverted microscope, the cytoplasm of melanocytes in positive cases can be seen to be gray-brown or brown (Figure 2B).

(4) Culture of keratinocytes

Take the above-mentioned cultured fibroblasts, when the growth is close to 80% confluent, add mitomycin to HDF, the final concentration is 10ug/ml, incubate in 50mL/L CO ₂ , 37°C incubator for 4h, use without Wash twice with calcium and magnesium PBS, add 1ml of 0.25% trypsin, digest in 50ml/L CO ₂ , 37°C incubator for 5min, under an inverted microscope, the cells shrink and the intercellular space increases, then add the culture medium to stop the digestion, the cells Transfer the suspension into a centrifuge tube, centrifuge at 1000r/min for 5min, discard the supernatant, resuspend the cells in the culture medium and count them, and inoculate them at 1×10 ⁴ /ml in a culture bottle pre-coated with collagen. The culture medium is DMEM+15 % calf serum, used every other day.

Take the above-mentioned cold digested tissue, gently separate the epidermis from the dermis with tweezers, transfer the epidermal tissue to 2.5g/L trypsin at 37°C for 10 minutes, and blow it repeatedly with a straw to form single cells. Add the same amount of KGM-G3F culture medium as that of trypsin to stop the action of trypsin. After filtering through a 100-mesh sieve, draw the cell suspension into a centrifuge tube, centrifuge at 1000r/min for 10min, discard the supernatant, and add KGM-G3F culture medium Pipette into a single cell suspension, inoculate the cells in a 25cm ² culture flask containing a HDF feeder layer at a density of 2-4×10 ⁴ /ml, put in 50ml/L CO ² , and culture in a 37°C incubator for 48 hours Change the medium afterward, and change the medium every 3 days thereafter. Cell morphology was observed under an inverted microscope every day (Fig. 3A). The 2nd to 3rd passage cells were used for experiments.

Adjust the cell suspension concentration of the primary cultured keratinocytes to 1×10 ⁵ /ml, draw 1ml of the cell suspension and inoculate it on the glass slide pre-coated with collagen and HDF feeder layer in the culture dish, put it in 37°C, 5% Cultivate in _CO2 incubator, take out the slide after 4 days, fix with pre-cooled acetone for 10min, add peroxidase blocking solution dropwise on the slide, block with 5% positive goat serum, incubate at room temperature for 10min, pour off the serum, do not After washing, the primary antibody (broad-spectrum mouse anti-human keratin monoclonal antibody AE ₁ /AE ₃ ) was added dropwise on the glass slide, and PBS was used as a negative control, and the positive was confirmed to be keratinocytes ( FIG. 3B ).

2. Construction of pigmented skin analogue model:

(1) Culture of dermal analogs

Take 7 parts of rat tail type I collagen solution and 2 parts of 5×DMEM solution, mix well and place in ice bath, adjust the pH of the solution to neutral with 0.5mol/L NaOH, then add 1 part of calf serum, and mix well in ice bath , add an appropriate amount of the suspension into a 12-well transwell culture chamber, place it in a 50ml/L CO ₂ , 37°C incubator, and wait for the collagen solution to solidify. At the same time, take 7 parts of rat tail collagen solution and 2 parts of 5×DMEM solution, mix them evenly and place them in an ice bath, adjust the pH of the solution to neutral with 0.5mol/L NaOH, and then add 1 part of fibroblast solution (cell number 2× 10 ⁵ /ml-3×10 ⁵ /ml, resuspended with calf serum), mixed in an ice bath, and added the suspension to the 12-well transwell culture chamber in which the above-mentioned collagen gel had solidified, 500ul per well, and placed In a 50ml/L CO ₂ , 37°C incubator, after the collagen solution is solidified, add DMEM culture solution containing 15% calf serum for immersion culture, change the medium every two days, and cultivate for one week. The dermis analogs cultured for two weeks were fixed with 10% formaldehyde fixative at room temperature for 3 hours, 25% sucrose overnight, frozen sections, thickness 10um, conventional HE staining (Figure 4), the results showed that fibroblasts were evenly distributed in the collagen, Compared with the normal dermal tissue, the fibroblasts in the dermis were spindle-shaped and irregular, and the cells were arranged in disorder. Small, with oval nuclei and little cytoplasm. The scaffolds in the gel are light red, wavy, often branched and intertwined.

(2) Culture of pigmented skin analogs

Take melanocytes and keratinocytes in good growth state, routinely digest and centrifuge, resuspend in KGM-G3F culture medium after counting the cells, mix the two cell suspensions, adjust the concentration of keratinocytes to 1.0×10 ⁷ / ml, and the concentration of melanocytes was 0.3×10 ⁷ /ml. Aspirate and discard the culture medium in the transwell culture chamber, take 150ul of the two cell suspensions and add them to the groove formed on the surface of the dermis analog after culture in the transwell chamber, in 50ml/L CO ₂ , in a 37°C incubator After standing for 2 hours, add an appropriate amount of KGM-G3F culture medium to the transwell culture chamber for submerged culture, change the medium every two days, and submerge for one week.

One week later, the culture solution in the transwell culture chamber was aspirated and discarded, and an appropriate amount of KGM-G3F culture solution was added to each well, and the liquid level of the culture solution in the well was adjusted to the membrane bottom level of the transwell culture chamber to carry out the aeration of the construct. - Liquid surface culture, continue to culture for two weeks, in order to promote the mature keratinization of keratinocytes. The state of the culture was recorded every day during the construction of the pigmented skin analog (Figure 5). The results showed that after inoculation of melanocytes and keratinocytes on the dermal analog, slight pigmentation could be seen on the third day of immersion culture. Appeared in light brown-black or yellow-brown color, and the color of the analog gradually deepened with the prolongation of culture time.

The cultured pigmented skin analogs were fixed overnight, decolorized with 70% alcohol, stained with HE, and observed under a light microscope. The results showed that the pigmented skin analogs cultured in vitro were similar in structure to normal skin, with epithelial layers and Dermis, in which the epidermis is thinner and the dermis is thicker, but lacks skin appendages such as hair follicles and sweat glands; the granular layer is discontinuous, partly absent, and consists of about 1-3 layers of relatively flattened spindle cells Composition, located above the spinous layer, most of the nucleus has degenerated, the shape of the cells is irregular, the size is different, and the cytoplasm contains more particles; the cuticle is thin, some parts are missing, the cells are homogeneous, and the outline is unclear . Most of the stratum corneum is orthokeratosis, and a small part is parakeratosis (Fig. 6). The results of Fontana Masson staining showed that the basal layer contained black-stained melanocytes, and the melanocytes were in good shape (Figure 7).

The pigmented skin analogs were observed under a transmission electron microscope, and the results showed that the nuclei of keratinocytes gradually degenerated during the process of migrating from the basal layer to the cornified layer ( FIG. 8A ). Melanocytes were distributed in the basal layer of the epidermis, and there were abundant melanin granules in the cytoplasm, and the protrusions penetrated deep into the keratinocytes (Fig. 8B). Pigmented skin analogue models can allow transfer of melanosomes. In the model, we can see that melanosomes gradually approach the membrane of melanocyte dendrites from the dendrites of melanocytes (Figure 9A, B), and finally enter keratinocytes (Figure 9C, D)

The above results show that the present invention successfully constructs a pigmented skin analog that can simulate the transport process of melanosomes and has a structure similar to skin.

3. Effect of different depigmenting agents on pigmented skin analogues

The experimental drugs were divided into three groups: aloesin, androsin, and tea polyphenols. The supernatant was discarded, and 450 μl of K6M-G3F and 50 μl of the drug solution were added to each well. The final concentrations of the drugs in each group were 1, 0.1, and 0.01, respectively. g/L, 7 wells for each concentration. In the blank group, 450 μl of KGM-G3F and 50 μl of 100ml/L PEH were added to each well of 7 wells. Continue culturing for one week in a 50ml/L CO ₂ incubator at 37°C.

After one week of drug action, one hole was taken from the wells with the highest concentrations of the above three drugs and one from the blank group, fixed, dehydrated, embedded, routinely made ultra-thin sections, and observed with a transmission electron microscope. The results showed that the arbutin group and the aloesin group (both 1g/L): the melanosomes in the keratinocytes were less than those in the blank group, the diameter of the melanosomes became smaller, the keratinocytes were in good condition, and the cytoplasm There are a small amount of vacuoles inside. Tea polyphenol group (1g/L): the melanosomes in the keratinocytes were less than those in the arbutin group and the aloesin group, the state of the keratinocytes was not good, and there were a large number of vacuoles in the cytoplasm (Fig. 10).

The present invention utilizes a small amount of skin tissue by means of bioengineering to amplify and cultivate melanocytes and other two important cells that affect skin pigmentation, fibroblasts and keratinocytes, and successfully construct pigments similar to skin structure by using these three types of cells. The skin analogue model can simulate the transport process of melanosomes, which is similar to the skin structure, and can observe the mechanism of action of different fading agents, providing an ideal experimental model for further research on the fading mechanism of fading agents and cosmetics. Although the model training process is complicated, its good physiological function has become the best in vitro model for studying pigment problems at home and abroad, and it will have important clinical value and socioeconomic value.

Claims (3)

1. A pigmented skin analog model that can screen fading agents, is characterized in that the dermis analog layer is constructed from skin fibroblasts cultured in vitro and rat tail type I collagen, on which keratinocytes and melanocytes are compounded The fibroblasts, keratinocytes and melanocytes are obtained after being separated from human foreskin tissue by expanding culture; the fibroblasts are 2×10 ⁵ /ml～3×10 ⁵ /ml After mixing with 20% rat tail type I collagen and coagulating to form a dermis analog layer, 1.0×10 ⁷ /ml of keratinocytes and 0.3×10 ⁷ /ml of melanocytes were inoculated on the dermis analog layer. 2. A method for building a pigmented skin analog model capable of screening depigmentation agents as claimed in claim 1, comprising the following steps: (1) Source, isolation and culture of tissue-engineered skin seed cells: a) Surgically excised human foreskin tissue soaked in 0.25% chlorhexidine solution; b) 2.5g/L Dispase II enzyme digests the foreskin tissue, and separates the epidermis and dermis; c) Cut the dermal tissue into 1×1×1mm ³ tissue pieces, put them into culture flasks, wait for the fibroblasts to migrate out of the tissue pieces after 30 minutes, and expand and culture the fibroblasts; d) Digest the epidermal tissue in 2.5g/L trypsin, centrifuge, place in melanocyte culture medium, and culture and expand melanocytes; e) Fibroblasts were cultured in a collagen-containing culture flask to form a fibroblast trophoblast, and the epidermal tissue was digested in 2.5g/L trypsin, then centrifuged and placed in a culture flask containing a trophoblast, and KGM-G3F was added Cultivate and expand keratinocytes in culture medium; (2) Construct a pigmented skin analog that can screen for fading agents: a) Fibroblasts were mixed with 20% rat tail type I collagen at a density of 2×10 ⁵ /ml to 3×10 ⁵ /ml and coagulated for 30 minutes, then added to culture medium and cultured for one week to construct a dermal analog layer ; b) Add 1.0×10 ⁷ /ml keratinocytes and 0.3×10 ⁷ /ml melanocytes on the surface and culture them in a transwell culture chamber, add KGM-G3F culture medium; c) After culturing for 1 week, conduct air-liquid plane culture, and continue culturing for 2 weeks to obtain a pigmented skin analog model that can screen for fading agents. 3. The construction method according to claim 2, characterized in that: the transwell culture chamber is a commercial culture plate; the components of the fibroblast culture medium are: DMEM commercial culture medium, 15% calf serum; The composition of the melanocyte culture solution described above is: RMP1640 commercial culture, and contains: basic fibroblast growth factor (bFGF) 2～3×10 ⁴ U/L, cholera toxin (CT) 0.20～0.25g/L, transferrin Protein 5-10mg/L, calf serum 100-150ml/L, hydrocortisone 2-5mg/L, insulin 100-150U/L, gentamicin 100-150,000/L; the KGM-G3F The composition of the culture medium is: DMEM/F12 (3:1) commercial culture medium, containing 100ml-150ml/L fetal bovine serum, adenine 0.18-0.20mM, epidermal growth factor EGF10-15ng/ml, hydrocortisone 0.4- 0.50ug/ml, insulin 3~5ug/ml, isoproterenol 10~15uM, transferrin 5~10ug/ml, triiodothyronine 2~3nM, ampicillin 100~120ug/ml, streptomycin sulfate Su 80 ~ 100ug/ml.

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