JP4838981B2 - A topical skin preparation containing mesenchymal cells derived from human placenta - Google Patents

Description

  The present invention relates to an external preparation for skin, particularly an external preparation for treating skin damage or burns, comprising mesenchymal cells derived from human placenta or the cell extract.

  In a general sense, mesenchymal cells constitute a connective tissue histologically, and unlike vascular cells such as vascular endothelial cells and vascular endothelial progenitor cells, they generally have multiple differentiation potentials. It is a capable cell. In particular, mesenchymal stem cells are known to be bone, cartilage, fat, heart, nerve, liver cells, and differentiated fibroblasts, hair papilla cells, adipocytes, dental pulp cells, etc. It belongs to the leaf cell.

  It has also been reported that some mesenchymal cells are involved in various diseases. For example, it has been reported that mesenchymal cells present in the synovium are involved in joint destruction such as rheumatoid arthritis and collagen disease. In this case, suppression of the activity of synovial mesenchymal cells has been attempted in the treatment of joint disorders and the like.

The present inventors pay attention to a group of stromal cells (cells other than vascular endothelial cells) existing in the human placenta, and transplant the mesenchymal cells contained therein to the living body, thereby providing an angiogenic action to the living body. It has been found that it can be urged, and a patent application has been filed (Patent Document 1). However, although the placenta is a place for nutrient exchange between the fetus and the mother and is an extremely important tissue during pregnancy, it is discarded along with the umbilical cord after childbirth. There are almost no examples other than the above-mentioned patent applications, and there is no prior art which shows the usefulness especially regarding skin diseases.
International Publication No. 03/082305 Pamphlet

  As a result of repeated investigations on the usefulness of human placenta-derived mesenchymal cells, the present inventors have found that the cells or the cell extract have a function of promoting skin regeneration, and completed.

  That is, the present invention is an external preparation for skin containing mesenchymal cells derived from human placenta or an extract of the cells. In particular, the present invention is an external preparation for skin that can promote early regeneration of skin that has been externally damaged such as skin peeling or skin that has been damaged by burns.

  Human placenta-derived mesenchymal cells can be isolated, for example, by collecting the human placenta on ice and then adding DMEM medium containing trypsin and EDTA to the placenta section obtained by collecting tissue from the mother side of the placenta with scissors Can do. In the present invention, mesenchymal cells isolated from human placenta as described above, or mesenchymal cells cultured in an appropriate medium thereafter, or an extract obtained by disrupting the cells, It is used as an external preparation in the form.

  The external preparation of the present invention has the ability to regenerate skin lost due to injury. For example, it has the effect of significantly promoting the regeneration of the skin at the exfoliated part in rats where the skin at the leg part has been exfoliated surgically. Since this skin regeneration is a therapeutic effect that is medically different from the treatment effect of ischemic disease reported in Patent Document 1, the mesenchymal cells produce some factor that acts on the skin regeneration. It is assumed that

A preferred form of the external preparation for skin according to the present invention is lotion or ointment. The content of mesenchymal cells derived from human placenta is 1 × 10 ⁴ to 1 × 10 ⁸ cells, preferably 1 × 10 ⁵ to 1 × 10 ⁷ cells, and more preferably 1 × 10 ⁵ to 1 × 10 ⁶ cells. It is. When a cell extract is used, an extract corresponding to the above number may be included in the external preparation.

  The external preparation for skin of the present invention has a regeneration and healing effect on damaged skin and can be used as a medicine. In addition, based on such skin regeneration ability, it can also be used as a cosmetic, for example, to prepare rough skin texture or to give skin tension or gloss. In particular, since the present invention uses extracted human tissue as a raw material, it can be safely used without causing an unfavorable immune reaction such as allergy when applied to humans.

  That is, the present invention is an external preparation for skin containing mesenchymal cells derived from human placenta or an extract of the cells. In particular, the present invention is an external preparation for skin that can promote early regeneration of skin that has been externally damaged such as skin peeling or skin that has been damaged by burns.

(Preparation, culture and storage of mesenchymal cells)
Mesenchymal cells derived from human placenta used in the present invention, for example, after collecting the human placenta on ice, add a DMEM medium containing trypsin and EDTA to a placenta section obtained by collecting tissue with scissors from the mother side of the placenta, Cells can be isolated at room temperature and no special manipulation is required. The human placenta itself is generally treated as a waste product during childbirth, and is a separation material that can be obtained on a daily basis in the medical field.

Mesenchymal cells isolated from human placenta can be preserved by a conventionally known method. For example, 10 ⁵ to 10 ⁸ cells / ml, preferably 10 ⁶ to 10 ⁷ cells / ml, more preferably 5 × 10 ⁶ cells in a nutrient medium containing 10% glycerin or 10% dimethyl sulfoxide and 10% serum. The cells can be stored frozen at −80 ° C. or in liquid nitrogen in a suspended state at a cell concentration of / ml. The stored cell line can be rapidly lysed (eg, immersed in a 37 ° C. water bath), 10 volumes of the same medium is added and stirred, and the cells recovered by centrifugation are added to the desired medium. Can be grown again.

Mesenchymal cells can also be grown in a suitable medium. The culture is generally performed at 33-39 ° C., preferably 37 ° C., and fetal bovine serum, preferably inactivated fetal bovine serum (fetal bovine serum in which complement has been inactivated by heat treatment), 3-10% A basal medium containing (preferably 10%) such as an α-MEM medium may be used. The aeration can be carried out using air containing 5% CO ₂ and maintaining the humidity at 80 to 120% (preferably 100%).

  However, considering that the cultured cells are finally administered to humans, it is preferable to cultivate mesenchymal cells under conditions that do not include foreign substances that cause an immune response to humans. For example, human serum albumin and cell growth factor are added to a basal medium in which vitamins are added to amino acids, such as MEM medium, RPMI 1640, RITC80-7, MCDB series, HamF-12 / DME 1: 1 mixed medium, and the like. It is preferable to culture in a medium that does not contain added protein components derived from non-human animals.

  Growth factors include platelet-derived growth factor (PDGF, Platelet-Derived Growth Factor), fibroblast growth factor (bFGF, basic Fibroblast Growth Factor), epithelial growth factor (EGF, epidermal growth factor group) and insulin. One or more selected. These growth factors may be, for example, commercially available as research reagents from R & D or Sigma, or may be separated and purified from living organisms or produced by genetic recombination techniques. The addition amount is 0.01 to 1000 ng / ml, preferably 0.1 to 100 ng / ml, and most preferably 1 to 10 ng / ml.

  Further, human serum can be prepared by centrifugation from donated blood or the like. The amount of human serum added is about 1-20%, preferably 2-15%, most preferably 5-10% of the culture medium. When using formulated human albumin, it is 0.01 to 10%, preferably 0.05 to 1%, and most preferably 0.1 to 0.5%.

  Furthermore, cell growth can be further enhanced by adding an antioxidant, particularly an antioxidant selected from the group consisting of 2-mercaptoethanol, ascorbic acid and vitamin E, to the medium. In the case of 2-mercaptoethanol, the addition amount of the antioxidant is 0.01 to 100 μM, preferably 0.1 to 50 μM, and most preferably 1 to 10 μM. For ascorbic acid or vitamin E, an amount equivalent to the 2-mercaptoethanol can be used.

(Preparation of external preparation for skin)
What is necessary is just to prepare the skin external preparation of this invention by the general method, using the raw material normally used for an external preparation. For example, surfactants (nonionic surfactants such as lipophilic glycerin monotesaleate and self-emulsifying glycerin monostearate, anionic surfactants such as sodium stearate, triethanolamine palmitate, sodium N-acyl glutamate, Natural and synthetic higher alcohols such as cationic surfactants such as stearyldimethylbenzylammonium chloride, amphoteric surfactants, etc., vegetable oils such as castor oil, waxes such as lanolin, hydrocarbons such as petrolatum, and cetanol Humectants such as glycerin, thickeners such as sodium alginate, preservatives such as benzoate, antioxidants such as dibutylhydroxytoluene, chelating agents such as ethylenediaminetetraacetate, pH adjusters such as citric acid, Other perfumes, pigments, UV absorbers / scatterers, vitamins It can be prepared by blending the amino acids, water and the like.

  As specific forms, hydrophilic ointments, lotions, sprays, creams, emulsions, lotions, cosmetics, packs, under-makeups, foundations, jellies and the like can be fried. Preferred are hydrophilic ointments and lotions. Each dosage form can be prepared by a general method shown in, for example, the pharmacopoeia.

  Moreover, you may add the cell growth factor which promotes skin reproduction | regeneration to the skin external preparation of this invention. These cell growth factors include acidic and basic fibroblast growth factor (FGF), epidermal growth factor (EGF), and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited only to these Examples.

Preparation of hydrophilic ointment 1 × 10 ⁵ mesenchymal cells isolated from human placenta were inoculated into DME medium supplemented with 10% FCS, and the condition was 3 under conditions of 37 ° C., 5% CO ₂ and 100% humidity. The cells were cultured for a day, and finally 3 × 10 ⁵ cells were collected in PBS (phosphate buffer).

  The cells were repeatedly frozen and thawed for destruction, and the supernatant was collected by centrifugation at 5000 rpm for 20 minutes to obtain a cell extract. 10 g of a hydrophilic ointment having the following composition containing this cell extract was prepared.

Composition (per 100g of hydrophilic ointment)
25g white petrolatum
Stearyl alcohol 20g
12g propylene glycol
Polyoxyethylene hydrogenated castor oil 60 4g
1g glyceryl monostearate
Methyl paraoxybenzoate 0.1g
Propyl paraoxybenzoate 0.1g
Cell extract 50mL
Purified water

Preparation of Lotion 1 × 10 ⁶ cultured cells prepared in the same manner as in Example 1 were suspended in 1 mL of PBS, and then frozen and thawed repeatedly to prepare 1 mL of lotion containing a cell extract.

[Test example]
After the skin of the side of the mouse (20 bodies) was excised with a 12 mm drawing, 50 mg of the ointment prepared in Example 1 was applied only once, and the area of the wound after 6 days was measured using calipers. As a control, models (20 bodies) coated with hydrophilic ointment containing only PBS under the same conditions were prepared, and the areas of the wounds were measured and compared using calipers.

  The skin external preparation of the present invention significantly repaired skin damage compared to the control (p <0.05, Man-Whitney u test, FIG. 1). The vertical axis represents the wound area, and the bar represents the standard error.

FIG. 1 shows the effect of skin regeneration by the external preparation for skin of the present invention.

Claims (1)

An external preparation for skin containing an extract of mesenchymal cells derived from human placenta for the treatment of skin damage or burns .

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