FR2729076A1 - Nutrient medium adapted for growth of human epidermal keratinocytes - Google Patents

Abstract

Use of a complex nutrient medium (A) that, when used alone or in aq. medium, provides viable in vitro culture of an inoculum of human epidermal keratinocytes, for the prepn. of a topical compsn. is new. The medium ensures \-1 clonal proliferation of the cells during the first or last passage without a feeder layer of living cells. Also claimed are topical cosmetic and pharmaceutical compsns. biocompatible with the human body surface, contg. homogeneously distributed (A).

Description

 The present invention relates to a composition & topical use, generally comprising a phase which is bio-compatible with the surface parts of the human body, in which a nutritional agent of said surface parts is distributed or distributed in a homogeneous manner.

 More specifically, the subject of the invention is a composition for topical use, making it possible to create an extracellular environment perfectly suited to the epidermis.

 In general, in accordance with the invention, the nutritional agent consists of a complex nutritive medium, comprising compounds which are both biocompatible, biomimetic and bioavailable at the cutaneous level, to the exclusion of any biological extract of animal origin, such as fetal calf serum. And the complex nutritive medium selected according to the invention has a composition adapted to allow, apart from the phase in which said medium is distributed, a viable in vitro culture of a human epidermal keratinocyte inoculum with at least one clonal proliferation of these last on the first pass.

 By "biocompatible" is meant the property that the compound is harmless to the skin.

 By "biomimetic" is meant the fact that the compound is present naturally in the skin.

 By "bioavailable" is meant the property that the compound is assimilable by human epidermal keratinocytes, both in vitro and in vivo.

 By routine tests, the person skilled in the art is able to formulate a complex nutritive medium according to the invention, by proceeding in particular with said medium to in vitro cultures of keratinocytes, the growth of which can be observed, for example in microscope.

In this regard, the following documents have already described media suitable for in vitro cultures of keratinocytes, the viability and growth of which can be objectified by the tests currently in force, and be directly assessed by observation under a microscope.
- Boyce ST, Ham RG, Calcium-regulated differentiation of normal human epidermal keratinocytes in defined clonal culture and serum-free serial culture, J.

Invest. Dermatol. 1983; 81: 33S-40S
- Boyce ST, Ham RG, Cultivation, frozen storage, and clonal growth of normal human epiderial keratinocytesin serum-free media, J. Tissue Culture
Methods. 1985; 9: 83-93.

 As necessary, the content of these publications is incorporated into this description.

 The complex nutrient medium according to the invention comprises amino acids, one or more vitamins, one or more cell growth factors, and one or more mineral salts.

All of the compounds present in the nutrient medium according to the invention being water-soluble, two formulation routes can be used
1) Continuous aqueous phase, containing the nutritive medium according to the invention
- In the form of an aqueous gel, using a non-ionic water-soluble polymer of the polysaccharide or cellulose ether type (polymers compatible with the high ionic charge of the medium);
- in the form of an emulsified system (oil in water emulsion using surfactants resistant to high ionic charges);
- in the form of cosmetic serum.

2) Continuous oily phase, the discontinuous phase containing the nutritive medium according to the invention
- in emulsified form, it being understood that the ionic strength of the discontinuous phase implies the instability of the emulsion; it is however possible to formulate lamellar or cylindrical phases having better stability, or even a two-phase system put back into temporary use in emulsion by simple agitation;
- by encapsulation
* in a rigid capsule, of the polysaccharide type, dispersed in the lipid phase,
* in a soft capsule, of the gelatin type, dispersed in the discontinuous phase.

 The use of liposomes as an encapsulation vector can be envisaged in the form of a liposomal gel in the aqueous continuous phase.

 A composition according to the invention can serve as a cosmetic base. Its nutritional contribution is notably interesting for the improvement of the viability, the maintenance of the integrity and the balance of the superficial skin cells.

 The use of such a nutritive composition makes it possible to durably preserve the primary intrinsic qualities of the skin, to increase its resistance to attack and to promote, if necessary, its return to a state of equilibrium.

 Likewise, the use of such a medium on weakened skin (irritated, dry skin, senescent skin, etc.) makes it possible to rediscover a satisfactory skin condition both in terms of trophicity and of hydration of the surface layers. of the epidermis.

 More generally, a composition according to the invention can be incorporated into any cosmetic preparation, as an active principle or with other active principles, which it is capable of potentiating.

A complex nutritive medium according to the invention recreates a suitable extracellular environment, by providing
- an optimized nutritional intake, both in vitamins, trace elements and in essential amino acids,
- cell growth factors, targeting & BR <substituting morphogenic cellular interactions,
- and pH and osmolarity characteristics close to physiological conditions.

 The characteristics, applications and advantages of the present invention are explained in more detail in Examples 1 and 2 and the following Figures 1 to 3.

 Example 1 gives an example of formulation of a composition of the invention.

Example 2 highlights the properties of a composition of the invention with respect to known media, in support of the appended drawing in which
FIG. 1 is a sectional view of human epidermis after 36 hours of culture in a standard commercial medium called MCDB 153,
FIG. 2 is a sectional view of human epidermis after 36 hours of culture in a buffered saline solution (PBS), a balanced saline solution commonly used in cell culture,
Fig 3 is a sectional view of human epidermis in culture in the nutrient medium of the invention described & BR <Example 1 at different culture times
A: after 12 hours
B: after 24 hours
C: after 36 hours
Example 1:
Formulation of a composition of the invention
TABLE 1
Concentration components
in mg / l.

Amino acids
L-Alanine 9.2
L-Arginine HCL 421.4
L-Asparagine (anhydrous) 14.2
L-aspartic acid 4.0
L-Cysteine HCL. H20 42.0
L-Glutamic acid 14.8
L-Glutamine 1754.4
Wisteria 7.6
L-Histidine HCL. H20 50.0
L-Isoleucine 6.0
L-Leucine 131.2
L-Lysine HC1 54.0
L-Methionine 13.5
L-Phenylalanine 10.0
L-Proline 34.6
L-Serine 126.1
L-Threonine 24.0
L-Tryptophan 9.3
L-Tyrosine 2 Na 2H20 11.7
L-Valine 70.3
Vitamins and cell growth factors d-Biotin 0.02
Folic acid 0.80
Nicotinamide 0.04
D-Ca Pantothenate 0.30
Pyridoxine HC1 0.06
Riboflavin 0.04
Thiamine HC1 0.30
Vitamin B12 0.41 i-Inositol 18.0
Putrescine 2 HC1 0.20
Sodium pyruvate 55.0
Thymidine 0.73
Adenine (HC1) 24.0
DL-lipoic acid 0.20
Inorganic components
Sodium chloride 6800.0
KC1 112.0
Na2 HPO4 284.0
CuS04.5H20 0.003
Sodium acetate 300.0 (anhydrous)
D-Glucose 1080.0
HEPES (piperazine) 6600.0
Phosphorylethanolamine 0.06768
Ethanolamine 0.04684
Sodium sulfate 3,4
Sodium bicarbonate 1160.0
FeS04.7H20 1.39
Mgcl2 6H20 120.0
CaC12.2H2O from 13.0 to 22.05
ZnSO4.7H2O 0.144
(NH4) 6 MO7O24.4H2O 0.00120
Na2SiO3. 5H20 0.142
MnC12.4H20 0.00002
SnC12. 2H20 0.00011
NH4 V03 0,00057
Example 2:
The cytocompatibility and the performances of the complex nutritive medium described in Example 1 were tested on cultures of human keratinocytes in monolayer, and on human epidermis reconstituted in vitro.

 The nutritive medium according to Example 1 allows the culture of keratinocytes in a monolayer under optimal conditions of viability, for at least 36 hours, without any slight cytotoxic effect being manifested.

 Conversely, a conventional survival solution such as PBS (Phosphate Buffered Saline, balanced saline solution commonly used in cell culture) proves to be cytotoxic after 12 hours of incubation.

 In accordance with FIG. 3, the nutritive medium according to the example allows a culture of normal human epidermis reconstituted under optimal conditions of viability, without cytotoxic manifestations even after 36 hours (FIG. 3C) of contacting. The cultures presented intact basal, spinous, granular and corneal layers, orthokeratotic, of regular and normal stratification.

 By comparing FIG. 3C with FIG. 1, the latter illustrating the use of a standard MCDB 153 medium, marketed in particular by IRVINE SCIENTIFIC, it can be seen that the performance of the medium of the invention is also good.

 On the other hand, the use of PBS induces, in accordance with FIG. 2, the appearance of terminal keratinocytes of differentiation at the level of the basal and spinous foundations, with more or less pronounced signs of necrosis, detachment of the epidermis from its support and a complete destruction of the various cellular foundations.

Claims (7)

RIVINDICATIONS  1 / Composition for topical use, comprising a phase biocompatible with the surface parts of the human body, in which a nutritional agent of said surface parts is distributed in a homogeneous manner, characterized in that the nutritional agent consists of a complex nutritive medium, comprising compounds that are both biocompatible, biomimetic and bioavailable in the skin, & BR <excluding any biological extract of animal origin, said complex nutritive medium having a composition suitable for allowing, outside the phase in which said medium is distributed , a viable in vitro culture of an inoculum of human epidermal keratinocytes, with at least one clonal proliferation of the latter on the first pass.  2 / Composition according to claim 1, characterized in that the complex nutrient medium comprises amino acids, at least one vitamin, at least one cell growth factor, and at least one mineral salt.  3 / Composition according to Claim 1, characterized in that the complex nutritive medium has the following composition, the concentration of the components being expressed in mg / l  Amino acids  L-Alanine 9.2  L-Arginine HCL 421.4  L-Asparagine (anhydrous) 14.2  L-aspartic acid 4.0  L-Cysteine HCL. H20 42.0  L-Glutamic acid 14.8  L-Glutamine 1754.4  Wisteria 7.6  L-Histidine HCL.H20 50.0 L-Isoleucine 6.0 L-Leucine 131.2 L-Lysine HC1 54.0 L-Methionine 13.5 L-Phenylalanine 10.0 L-Proline 34.6 L-Serine 126.1 L-Threonine 24.0 L-Tryptophan 9.3 L-Tyrosine 2 Na 2H20 11.7 L-Valine 70.3 Vitamins and cell growth factors d-Biotin 0.02 Folic acid 0.80 Nicotinamide 0.04 D-Ca Pantothenate 0.30 Pyridoxine HC1 0.06 Riboflavin 0.04 Thiamine HC1 0.30 Vitamin B12 0.41 i-Inositol 18.0 Putrescine 2 HC1 0.20 Sodium pyruvate 55.0 Thymidine 0.73 Adenine (HC1) 24.0 DL-lipoic acid 0.20 Inorganic components Sodium chloride 6800.0 KC1 112.0 Na2 HPO4 284.0 CuS04.5H20 0.003 Sodium acetate 300.0 (anhydrous) D-Glucose 1080.0 HEPES (piperazine) 6600.0  Phosphorylethanolamine 0.06768  Ethanolamine 0.04684  Sodium sulfate 3,4  Sodium bicarbonate 1160.0  FeS04.7H20 1.39  MgC12.6H20 120.0  CaC12.2H20 from 13.0 to 22.05  ZnSO4.7H20 0.144  (NH4) 6 M07024.4H20 0.00120  Na2SiO3. 5H20 0.142  MnC12.4H20 0.00002  SnC12.2H20 0.00011  NH4 V03 0,00057  4 / Composition according to claim 1, characterized in that it is in biphasic form, with an aqueous continuous phase containing the complex nutrient medium, and in particular in the form of an aqueous gel, or of an oil in water emulsion .  5 / A composition according to claim 1, characterized in that it is in biphasic form, with a continuous oily phase, in particular in the form of an emulsion, the discontinuous phase containing the complex nutritive medium.  6 / cosmetic base comprising a composition according to any one of claims 1 to 3.  7 / Cosmetic preparation comprising, in particular as active principle, a composition according to any one of claims 1 to 5.