WO2005071065A1 - Method for studying functional interactions between sensory neurons and keratinocytes or melanocytes - Google Patents

Method for studying functional interactions between sensory neurons and keratinocytes or melanocytes Download PDF

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WO2005071065A1
WO2005071065A1 PCT/EP2005/000364 EP2005000364W WO2005071065A1 WO 2005071065 A1 WO2005071065 A1 WO 2005071065A1 EP 2005000364 W EP2005000364 W EP 2005000364W WO 2005071065 A1 WO2005071065 A1 WO 2005071065A1
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neurons
keratinocytes
calcium
cell culture
melanocytes
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French (fr)
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Jean-Luc Rodeau
Laurianne Ulmann
Rémy SCHLICHTER
Jean-Luc Contet-Audonneau
Louis Danoux
Gilles Pauly
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Cognis France S.A.S
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Definitions

  • the present invention relates to a method for studying functional interaction s between sensory neurons and keratinocytes or melanocytes.
  • the human skin is the largest body organ which forms a metabolically active barrier protecting internal organs from the assaults of the external environment.
  • the skin can be classified in 2 histological compartments covering the sub-cutaneous adipose tissue: the dermis and the epidermis.
  • Epidermis is mainly formed by keratinocytes (95% of epidermal cells) which are arranged in 4 layers: firstly a basal layer whicn contains germinative cells, then a spinous layer where keratinocytes can sti II proliferate, a granulous layer where keratinocytes begin their terminal differentiation and the outermost, the horny layer formed by completely differentiated keratinocytes named corneocytes.
  • the sensory nervous system is mainly represented either by nerve fibers associated with receptors such as Merkel disks in epidermis, Meissner corpuscles and Ruffini end-organs in dermis and Pacinian corpuscles in subcutaneous tissue, or by a rich network of free nerve endings reaching the epidermis.
  • These free endings are small unmyelinated nervous fibers of type C and conduct signals for pain, pressure, vibration, touch, heat and pleasure.
  • CGRP Calcitonim- Gene-Related-Peptide
  • This sensory network has a specific topographical distribution: the sensory nervous fibers of the torso, extremities, posterior scalp and neck originate from a neuronal cell body located in the dorsal root ganglia (or DRG) of the spinal cord, while the face, most of the scalp, and upper anterior neck are innervated mainly by trigeminal nerve branches.
  • DRG dorsal root ganglia
  • These sensory neurons are pseudo-unipolar that means featured by an axon which is separated in 2 branches, a first peripheral branch which penetrates through the epidermis and a second central branch which reaches the superficial layer of the dorsal horn of the spinal cord.
  • the calcium ion represents the most versatile and universal messenger for relaying external and internal signals in human cells.
  • Calcium acts as an external mediator by modulating for instance wound healing and as internal messenger regulating functions such as vesicle secretions.
  • Calcium concentration inside the cells is about 0.0001 mM (mM means millimolar, 1 M means 1 mole per liter), whereas in extracellular medium it varies between 1 to 2 mM.
  • An enzymatic protein, the so called Ca-ATPase maintains this calcium gradient between each side of the cellular membrane by consuming one molecule of ATP to expulse 2 calcium ions out of the cytoplasm (BERRIDGE M.J,. BOOTMAN M.D., LIPP P.: Calcium - a life and death signal, Nature, 1998, volume 395, pages 645-648).
  • keratinocytes take an important part in the differentiation of keratinocytes: at calcium levels lower than 0.1 mM, keratinocytes are kept in an undifferentiated state with a proliferation permissive status. When the calcium level is raised to 1 mM, there is a rapid formation of desmosomal junctions that hold epithelial cells together (WATT F., MARREY D.L., GARROD D.R: Calcium induced reorganization of desmosomal components in cultured human keratinocytes, The J. of Cell Biology, 1984, volume 99, pages 2211-2215).
  • an elevated level of calcium induces a modification in the distribution of keratin filaments, the appearance of keratohyaline granules and cornified envelopes in keratinocytes culture.
  • the shift of calcium concentrations from 0.07 mM to 1.2 mM triggers the synthesis of differentiation products such as filaggrin, a histidin rich protein aggregating the keratin filament, which is characteristical for the terminal differentiation of human keratinocytes in vivo (HAUGEN SCOFIELD J.A., HENNINGS H., DALE B.A., YUSPA S.H., STANLEY J.R.: Identification of filaggrin in cultured mouse keratinocytes and its regulation by calcium, J. Invest. Dermatol., 1983, volume 81 , pages 90S-95S).
  • basal keratinocytes depends on the calcium ion concentration.
  • human epidermis presents a positive gradient of calcium concentration from the basal layer up to the superficial stratum corneum.
  • the extracellular concentration is low in the basal proliferating layer (20-5O ⁇ M) and reaches a maximal concentration of 600 ⁇ M in the superficial layer constituted of completely differentiated corneocytes (MENON G.K., ELIAS P.M.: Ultrastructural localization of calcium in psoriatic and normal human epidermis. Arc Dermatol, 1991 , volume 127, pages 57-63).
  • NGF Neve Growth Factor
  • BDNF Brain Derived Nerve Growth Factor
  • the activity of certain specific chemical messengers or physical stress can be studied only with a low level of calcium in the cell culture assay medium in order to demonstrate, for example, an induction of neurite and/or axonal outgrowth or a release of various mediators.
  • the calcium concentration in the assay medium By varying the calcium concentration in the assay medium, the various extracellular calcium concentrations that nerve fibers meet when they cross human tissue such as skin can be simulated.
  • the present invention is related to cell cultures of neurons or cell cultures of neurons and keratinocytes cultured separately or co-cultured either in the same cell culture medium (in the same compartment) or in 2 different compartments filled with different cell culture media.
  • WO 03/005023 discloses a process to analyze the interactions between keratinocytes and normal neurons from dorsal root ganglia (DRG) using a co-culture of these 2 cell types in 2 separated compartments, e. g. separated by a glass ring that allows the crossing of neuritis and/or soluble factors.
  • This model was extended to primary cultures of keratinocytes and neurons but also to cell lines of epithelial cells such as HeLa, NCTC2544 and A431 , or neuronal cells such as PC12, ND7-23 and ND8/32.
  • the present invention relates to the use of a cell culture medium, characterized by a low concentration of calcium, for culturing neurons.
  • the neurons can be cultured in this medium, alone or mixed with keratinocytes or melanocytes, for example.
  • the use of a cell culture medium for culturing neurons, whereby the cell culture medium has a calcium concentration of lower than 0.1 mM (preferably the calcium concentration is 0.001 to 0.03 mM) is a subject of the present invention.
  • a further subject of the present invention is a cell culture comprising a) neurons and b) a cell culture medium, whereby the cell culture medium has a calcium concentration of lower than 0.1 mM (preferably the calcium concentration is 0.001 to 0.03 mM).
  • the cell culture according to the present invention further comprising c) keratinocytes is one embodiment of the present invention.
  • the cell culture according to the present invention further comprising c) melanocytes is another embodiment of the present invention.
  • the cell culture according to the present invention comprising keratinocytes or melanocytes, wherein the keratinocytes or the melanocytes are obtainable from primary cultures or from cell lines and the neurons are obtainalble from dorsal root ganglia (DRG) or from cell lines is another embodiment of the present invention.
  • DRG dorsal root ganglia
  • Another subject of the present invention is the use of the cell culture according to the present invention comprising keratinocytes or melanocytes as an assay determining the biological cross-talk of a) the neurons with b) the keratinocytes or melanocytes
  • Another subject of the present invention is a process for identifying substances useful as active ingredients for cosmetic compositions comprising testing the substances with the assay according to the present invention and identifying those substances that have a positive effect on the metabolism of the neurons of the assay.
  • Another subject of the present invention is the use of a substance iden termeied with the assay according to the present invention for the cosmetic treatment of humans.
  • Another subject of the present invention is the use of a substance able to control calcium signalling in neuronal functions such as the release of factors like peptides or steroids for the cosmetic treatment of hu mans.
  • RPMI 1640 [0.1 g/l of Ca(N0 3 ) 2 x 4H 2 0]
  • nutrient mixture HAM F12
  • Hanks balanced salts [0.185 g/l of CaCI 2 2H 2 0]
  • PBS phosphate buffered saline
  • MCDB153 [0.004411 g/l of CaCI 2 x 2H 2 0].
  • MEM minimum essential medium of eagle
  • DMEIvl Dynamic Modulator's modified eagle's media
  • RPMI, HAM F12 etc. are culture media well known to the skilled artisan they are available commercially. They can be purchased from companies like SIGMA or In Vitrogen (SIGMA ALDRICH CHIMIE SARL, L'lsle D'Abeau Chesnes - B.P. 701 , 38297 Saint-Quentin Fallavier Cedex and Invitrogen S.A.R.L., BP 030096, 95613 Cergy Pontoise Cedex).
  • low calcium media are media that have a low concentration of calcium
  • co- cultivate i. e. co-culture
  • proliferating keratinocytes of the human epidermis are in an intercellular medium characterized by a low concentration of calcium ion (around 20 ⁇ M).
  • This co-culture with a low calcium concentration allows for evaluating the effects of proliferating keratinocytes on axonal development and neuronal functions.
  • BDNF neurotrophins
  • cell culture media with a low concentration of calcium allow for the investigation of the positive effect of keratinocytes on the length of axonal extensions in co-cultures of neurons and keratinocytes. These investigations can be used to discover new active ingredients for cosmetic applications, wherein these active ingredients have the same activity on neurons as said neurotrophins or others molecules secreted by keratinocytes.
  • Another improvement provided by the use of low calcium media is the possibility to co-cultivate neurons with melanocytes because it is well known that melanocytes are located in the basal layer of the epidermis where the concentration of calcium is low.
  • cell culture media with a low calcium concentration the interactions between neurons and melanocytes in a status similar to the normal in vivo conditions can be studied in vitro.
  • the present invention is related to the development of a coculture model for the functional study of interactions between sensory neurons and keratinocytes.
  • mammal skin is made of three layers: the hypodermis deep down, the dermis and the epidermis at the surface.
  • Keratinocytes represent 95% of the cells of the epidermis, which is divided up into 4 layers, successively: the basal (proliferative) layer, the spiny layer, the granular layer and the horny layer at the surface.
  • nerve endings that are specialized in the detection of physical and chemical stimuli originating from the surrounding environment. These endings belong in particular to unmyelinated sensory neurons called nociceptors, the cell body of which is located in the spinal ganglia. These pseudounipolar neurons have a peripheral branch which penetrates into the skin so as to reach the various layers of the epidermis, and a central branch which reaches the superficial layers of the dorsal horn of the spinal cord.
  • the extracellular calcium concentration is also a determining factor in the control of the growth and of the development of the first axonal projections (neurites) (Fields RD, Guthrie PB, Russell JT, Kater SB, Malhotra BS, Nelson PG: Accommodation of mouse DRG growth cones to electrically induced collapse: kinetic analysis of calcium transients and set-point theory. J Neurobiol 1993 24(8): 1080-98).
  • the medium used in the cocultures is MCDB 153 medium (Sigma, M7403).
  • the medium is entirely controlled (serum-free) and has already been described and used for keratinocyte cultures in the literature (Oku H, Kumamoto C, Miyagi T, Hiyane T, Nagata J, Chinen I: Serum-free culture of rat keratinocytes. In Vitro Cell Dev Biol 1994 30A ⁇ 96-503).
  • the calcium contained in the culture medium is chelated using Chelex-100 resin (BIO-RAD ref. 142-2822) at a concentration of 5 g/100 ml for 1 hour with stirring. The medium is then recovered and the calcium (CaCI 2 ) concentration is adjusted to the chosen value.
  • the epidermis detaches from the dermis and is recovered in DMEM supplemented with foetal calf serum using fine forceps, and stirred for 20 min.
  • the dissociated cells which are recovered from the supernatant are centrifuged for 10 min at 1000 rpm. The pellet is taken up in MCDB 153 medium.
  • Three newborn rats are sacrified by decapitation.
  • the skin is removed from the back by means of iridectomy scissors.
  • the ganglia visible in the vertebral cavities are removed under a binocular magnifying lens using fine forceps, and placed in a culture dish 35 mm in diameter containing sterile PBS.
  • the central and peripheral projections are removed using a scalpel.
  • the ganglia are then placed in a 15 ml tube and the PBS is replaced with medium without Ca 2+ or Mg 2+ , in which the ganglia are washed twice.
  • the ganglia are subjected to enzymatic dissociation with 2 ml of trypsin (0.5 mg/ml trypsin, 0.2 mg/ml EDTA, IBCO).
  • the tube is placed in a waterbath for 25 minutes at 37°C and regularly agitated.
  • the reaction is stopped by adding MeM ⁇ (GIBCO, ref. 2561-021 ) supplemented with 10% vol/vol horse serum.
  • MeM ⁇ GIBCO, ref. 2561-021
  • the ganglia are then dissociated mechanically in MEM by means of three Pasteur pipettes of decreasing diameter, the tips of which have been smoothed off in a flame. When the medium becomes cloudy, this indicates the presence of dissociated cells.
  • the cells are recovered and centrifuged at 400 rpm for 5 minutes. The pellet is taken up in the medium chosen for the seeding.
  • the cultures are seeded into glass-bottom Petri dishes (imaging) or onto glass cover slips (immunocytochemistry) pretreated with rat tail collagen.
  • the kerati nocytes are seeded first and the neurons are ad ded after 3-4 hours.
  • the keratinocyte (A431 , ECACC 85090402) or neuron (ND7-23, ECACC 92090903) lines proliferate in a flask containing DMEM medium supplemented with 1 0% (vol/vol) foetal calf serum; 2 ml of trypsin are added in order to detach the cells. The cells are centrifuged at 1000 rpm for 5 min. , and the pellet is then taken up in the chosen seeding medium.
  • the same seeding protocol as for the primary cells is used: the cells are centrifuged and the pellet is taken up in the seeding medium.
  • the ND7-23 cells are seeded after the A431 cells have adhered to the collagen substrate.
  • the MCDB 153 medium makes it possible to maintain the two cell types in culture for more than a week, but reveals differences in the ability of the neurons to put out axonal projections.
  • the criterion used to evaluate the interactions between keratinocytes and neurons is the length of the axonal projections.
  • the measurements were carried out after 1 , 3 and 7 days of seeding for the sensory neurons from the line, and after 1 , 3 and 6 days for the primary neurons.
  • the neuronal projections are visualized after immunocytochemical labelling with an anti-Smi antibody (Sternberger Monoclonals Incorporated) specific for axonal proteins, or an anti-PGP 9.5 antibody (Chemicon) specific for unmyelinated sensory fibres.
  • the keratinocytes are labelled with an anti- cytokeratin 14 antibody (Chemicon) specifically expressed by keratinocytes during the proliferative phase, or with an anti-cytokeratin 10 antibody (Chemicon) specific for differentiated keratinocytes of the granular and spiny layers.
  • an anti- cytokeratin 14 antibody specifically expressed by keratinocytes during the proliferative phase
  • an anti-cytokeratin 10 antibody Chemicon
  • Control 1 neurons alone cultured in the usual medium; effect of calcium
  • the axonal projections were measured in cultures of neurons carried out in the medium commonly used: MEM ⁇ + 10% vol/vol horse serum, for the primary neurons, and DMEM for the neurons from the line.
  • MEM ⁇ + 10% vol/vol horse serum for the primary neurons
  • DMEM for the neurons from the line.
  • Control 2 neurons cultured alone in MCDB 153 medium; effect of calcium The neurons were cultured in the MCDB 153 medium used for the cocultures (medium, 20 ⁇ M Ca 2+ ). The lengths of the axonal projections measured in this medium with calcium concentrations of 20 ⁇ M and 2 mM, respectively, were compared.
  • the axons show a considerable growth retardation in the case of the neurons from the line ND7-23, and are absent in the case of the primary sensory neurons throughout the duration of the cultures.
  • Cocultures neurons cultured in MCDB medium in the presence of keratinocytes
  • the neurons are added to a culture of keratinocytes cultured in MCDB 153 medium containing a low calcium concentration (20 ⁇ M). Unlike control 2, in the presence of keratinocytes, the length of the axons tends to be similar to that measured in the optimum neuron culture medium (MEM ⁇ or DMEM), or to that measured in an MCDB 153 medium containing 2 mM Ca 2+ . The results obtained are similar whatever the combination of neurons (primary/line) and keratinocytes (primary/line).
  • This model indicates that the presence of keratinocytes allows axon development, even in a "low calcium" MCDB 153 medium which reduces (ND7-23 neurons) o r blocks (primary sensory neurons) axonal growth in the absence of keratinocytes.
  • the keratinocytes appear to secrete one or more factors capable of acting on the sensory neurons and of controlling axonal development.
  • the calcium imaging technique with the Fura2 probe which is sensitive to variations in intracellular calcium, has shown that neurons cultured in the presence of keratinocytes express membrane receptors identical to those characterized in the neurons cultured in the control medium (ME IVI ⁇ or DMEM). In fact, we have demonstrated voltage-dependent Ca 2+ channels, and receptors for ATP (P2X and P2Y), for bradykinin (B2), and for capsaicin (VR1
  • Anti-cytokeratin 14 labelling and anti-PGP 9.5 labelling were observed by confocal microscopy and made it possible to demonstrate apparent physical contacts between the neurons and keratinocytes.
  • this coculture model demonstrates: 1 ) the important role of extracellular calcium in the axonal development of sensory neurons; 2) the effect of keratinocytes on axonal growth (compensation for the inhibitory effect of a low extracellular calcium concentration). It makes it possible to study neurons under extreme growth conditions (neurons without keratinocytes/with keratinocytes, low/high calcium concentration).
  • the invention has been carried out by comparing the activity of neurotrophins (BDNF, NGF for example) in media with a low calcium concentration versus the activity of these neurotrophins in mediawith a high calcium concentration.
  • BDNF neurotrophins
  • these neurotrophins do not present any distinct effects
  • media with a low concentration of calcium the neurotrophins enhance markedly the length of axonal extensions. Therefore, the use of media with a low concentration of calcium constitutes a model to study the activity of various molecules on the length of axonal extensions.
  • PBS Phosphate Buffered Salt solution: it is a standardized well known solution for cell cultures
  • DMEM Dulbecco's Modified Eagle's Media: DMEM is a well known and standardized cell culture medium provided by the company SIGMA or In Vitrogen
  • Liquid culture medium used for cultures of neurons and keratinocytes or melanocytes
  • the medium with low calcium concentration used has been MCDB 153 (purchasable from the company Sigma). This medium is completely defined (without serum) and has been used for the cultures of keratinocytes before (OKU H., KZUMAMOTO C. MIYAGI T., HIYANE T., NAGATA J., CHINEN I.: Serum-free culture of rat keratinocytes, In Vitro Cell Dev. Biol,. 1994 volume 30A, pages 496-503).
  • the calcium present in the medium can be removed by chelating it with chelex 100 resin (purchasable from the company BIO-RAD) used at the dose of 1 to 10 g/100 ml for 1 hour with shaking. Then the calcium (CaCI 2 ) concentration of the medium can be adjusted to a chosen value.
  • Cells (neurons and keratinocytes or melanocytes) have been seeded in Petri dishes with a glass bottom for microscope imaging or on glass slides for immuno-cyto- chemistry coated with collagen type I from rat tails. Keratinocytes or melanocytes have been seeded some hours before neurons (1 to 72 hours).
  • the cells for the cell-culture have been separated from the tissues of new-born rats according to the modification of dissociation technique (SCOTT B.S.: Adult mouse dorsal root ganglia neurons in cell culture, J. Neurobiol., 1977, volume 8, pages 417- 427 ; DICHTER MA, FISHBACH G.D.: The action potential of chick dorsal root ganglion neurones maintained in cell culture, J. Physiol, 1977, volume 267, pages 281-298). DRG have been obtained from new-born rats (not older than one week), placed into a Petri dish with PBS on ice and cleaned with a scalpel in order to suppress nervous ramifications.
  • dissociation technique SCOTT B.S.: Adult mouse dorsal root ganglia neurons in cell culture, J. Neurobiol., 1977, volume 8, pages 417- 427 ; DICHTER MA, FISHBACH G.D.: The action potential of chick dorsal root ganglion neurones maintained in cell culture,
  • Keratinocytes have been prepared according to the technique of RHEINWALD J.G., GREEN H.: Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinising colonies from single cells, Cell, 1975, volume 6, pages 331- 434. Rats which are not older than 24 hours have been anesthetized and killed. Then skin pieces of about 1 cm 2 have been cut out and have been submitted to enzymatic digestion (trypsin at a concentration of 0.2 % by weight).
  • Cell lines of keratinocytes such as A431 , NCTC2544 and PAM-212, of neurons such as ND7-23, ND8-32 and PC12, of melanocytes such as B16 and Cloudmann S91 have been cultivated in DMEM with FCS (fetal calf serum) at 10 % by volume. Cells have been detached by trypsin ization and recovered by centrifugation at 1000 rpm (rounds per minute) for 5 min, then the pellet of cells formed during centrifugation has been diluted in cell culture medium with a low calcium concentration.
  • FCS fetal calf serum
  • A431 , NCTC2544, PAM-212 are well known cell lines available from various companies, e. g. ATCC or ECACC.
  • the 2 types of cells can either be introduced in the same compartment or in 2 different compartments, e. g. two compartments separated by a glass ring (as disclosed in WO 03/005023).
  • a glass ring as disclosed in WO 03/005023.
  • neurons of the cell line used have been seeded on a culture of keratinocytes or melanocytes either on plastic or collagen coating.
  • This model makes it possible to study the effect of a chemical or physical stress on neurons cultivated in a low calcium medium, too.
  • the patch-clamp method is a technique that allows to record the electric activity of neurons. With the aid of a microelectrode in glass directed with a micromanipulator, variations of the electric potential between the inside and the outside o-f a neuron are visualized. The signals are amplified with an amplifier of patch and visualized with an oscilloscope. The data can be recorded on a video tape and analysed with specific software.
  • Example 1 effect of the concentration of calcium in the culture medium on the axonal development of neurons cultivated alone
  • anti-Smi (Stemberger Monoclonals Incorporated) is specific for certain proteins, which are a characteristic feature of neuronal axons.
  • anti-Pgp9.5 (protein gene product 9.5) is an antibody specific for another protein characteristic of neuronal axone.
  • Neurons have been seeded and cultivated for 7 days. At days 1 , 3 and 7 (for cell lines of neurons), and at days 1 , 3 and 6 (for primary cultures of sensory neurons), the axonal extensions have been immunocytochemistry-stained with a specific antibody of axonal proteins: anti-Smi (purchasable from Stemberger Monoclonals Incorporated) or anti-PGP 9.5 (purchasable from Chemicon) specific of the non myelinisated sensory fibers. Then the length of stained axonal extensions has been measured by image analysis.
  • anti-Smi purchasable from Stemberger Monoclonals Incorporated
  • anti-PGP 9.5 purchasable from Chemicon
  • axonal extensions have been measured on neurons cultivated in media with a concentration of calcium of 20 ⁇ M and 2 mM and then the results of these measurements have been compared.
  • axonal extensions show an important delay of growth in the case of neurons of the cell line, and are absent in the one of the primary sensory neurons during all the period of cultures.
  • Example 2 effect of primary keratinocytes and cell line keratinocytes on axonal growth of the primary neurons and cell line neurons
  • the criterion used to evaluate interactions between keratinocytes and neurons has been the length of axonal extensions. Measures have been done after 1 , 3 and 7 days after neuronal cells seeding for the sensory neurons of cell line and after 1 , 3 and 6 days for the primary sensory neurons. Axonal extensions of neurons have been visualized after immunocytochemistry-staining with a specific antibody of axonal proteins: anti-Smi (Stemberger Monoclonals Incorporated) or anti-PGP 9.5 (Chemicon) specific of the non myelinisated sensory fibers.
  • Keratinocytes have been marked by an anti-cytokeratine 14 antibody (purchasable from Chemicon) expressed specifically by keratinocytes during the proliferative phase or by an anti-cytokeratine 10 antibody (purchasable from Chemicon), specific for keratinocytes differentiated of the granular and spinous layers.
  • Neurons have been added to keratinocytes cultivated in the medium MCDB 153 containing a low concentration of calcium (20 ⁇ M).
  • the length of axons has the tendency to come closer to the one measured for neurons cultivated in a medium with a high calcium concentration such as MEM or DMEM, or MCDB 153 containing 2 mM of Ca 2+ .
  • the obtained results have been similar whatever the combination of neurons (primaries / cell line) and of keratinocytes (primaries / cell line) was.
  • Keratinocytes seem to secrete one or several factors susceptible to act on the sensory neurons or to be metabolised by neurons and finally to control axonal development.
  • Table 2 Effect of primary keratinocytes and cell line keratinocytes on axonal growth of the primary neurons and cell line neurons ND7-23 (mean ⁇ SEM (standard error mean) on 5 assays)
  • Example 3 effect of neurotrophins and steroid on the axonal cell growth
  • keratinocytes have been replaced by specific substances like neurotrophins (BDNF, NGF (purchasable from Sigma)) or steroid such as DHEA (Dehydroepiandrosterone) (purchasable from Sigma).
  • BDNF neurotrophins
  • NGF purchasable from Sigma
  • DHEA Dehydroepiandrosterone
  • neurotrophins such as BDNF and NGF have a similar effect on axonal cell growth as primary keratinocytes or as cell line keratinocytes after 6 days of culture.
  • DHEA In low calcium media, DHEA has a similar effect on axonal cell growth as primary keratinocytes or as cell line keratinocytes after 7 days of culture.
  • Example 4 evaluation by calcium imaging and by immunocytochemistry of membrane receptors of neurons in the presence of keratinocytes and in high calcium media

Abstract

The present invention relates to a cell culture comprising neurons and a cell culture medium, whereby the cell culture medium has a calcium concentration of lower than 0.1 mM (preferably the calcium concentration is 0.001 to 0.03 mM).

Description

Method for studying functional interactions between sensory neurons and keratinocytes or melanocytes
The present invention relates to a method for studying functional interaction s between sensory neurons and keratinocytes or melanocytes.
The human skin is the largest body organ which forms a metabolically active barrier protecting internal organs from the assaults of the external environment. The skin can be classified in 2 histological compartments covering the sub-cutaneous adipose tissue: the dermis and the epidermis. Epidermis is mainly formed by keratinocytes (95% of epidermal cells) which are arranged in 4 layers: firstly a basal layer whicn contains germinative cells, then a spinous layer where keratinocytes can sti II proliferate, a granulous layer where keratinocytes begin their terminal differentiation and the outermost, the horny layer formed by completely differentiated keratinocytes named corneocytes.
Therefore it is critical to maintain the skins structural integrity and this requires an efficient skin sensory equipment to recognize and integrate appropriate signals. This sensory function is realised in skin by the activity of the local neuronal system whicfi is able to signal physical stress (pressure, heat, cold) as well as chemical stress (acid, irritant).
In human skin, the sensory nervous system is mainly represented either by nerve fibers associated with receptors such as Merkel disks in epidermis, Meissner corpuscles and Ruffini end-organs in dermis and Pacinian corpuscles in subcutaneous tissue, or by a rich network of free nerve endings reaching the epidermis. These free endings are small unmyelinated nervous fibers of type C and conduct signals for pain, pressure, vibration, touch, heat and pleasure.
Evidence suggests that cutaneous inflammation induced by a trauma such as scratching, originates from cell necrosis and from neuropeptides like Calcitonim- Gene-Related-Peptide (CGRP) released from nervous endings in skin during the so called "axon reflex flare".
This sensory network has a specific topographical distribution: the sensory nervous fibers of the torso, extremities, posterior scalp and neck originate from a neuronal cell body located in the dorsal root ganglia (or DRG) of the spinal cord, while the face, most of the scalp, and upper anterior neck are innervated mainly by trigeminal nerve branches.
These sensory neurons are pseudo-unipolar that means featured by an axon which is separated in 2 branches, a first peripheral branch which penetrates through the epidermis and a second central branch which reaches the superficial layer of the dorsal horn of the spinal cord.
In order to study the very close interactions between keratinocytes and sensory neurons, previous studies were carried out on human skin slices but with fragmented nervous fibers and with a persistent inflammatory status (HILLIGES M., WANG L. JOHANSSON O.: Ultrastructural evidence for nerve fibers within all vital layers of the human epidermis, J. Invest. Dermatol., 1995, volume 104, number 1 , pages 134- 137). Moreover isolated nerve cells appear fastidious to cultivate because they will not proliferate in vitro and they survive only on specific substrates such as collagen or poly-D-lysine. However, it was observed in these cell cultures, that neurite outgrowth was stimulated by a polypeptide known as Nerve Growth Factor (NGF). The concentration of calcium in the cell culture medium is not mentioned in said publication.
The calcium ion represents the most versatile and universal messenger for relaying external and internal signals in human cells. Calcium acts as an external mediator by modulating for instance wound healing and as internal messenger regulating functions such as vesicle secretions. Calcium concentration inside the cells is about 0.0001 mM (mM means millimolar, 1 M means 1 mole per liter), whereas in extracellular medium it varies between 1 to 2 mM. An enzymatic protein, the so called Ca-ATPase, maintains this calcium gradient between each side of the cellular membrane by consuming one molecule of ATP to expulse 2 calcium ions out of the cytoplasm (BERRIDGE M.J,. BOOTMAN M.D., LIPP P.: Calcium - a life and death signal, Nature, 1998, volume 395, pages 645-648).
Calcium takes an important part in the differentiation of keratinocytes: at calcium levels lower than 0.1 mM, keratinocytes are kept in an undifferentiated state with a proliferation permissive status. When the calcium level is raised to 1 mM, there is a rapid formation of desmosomal junctions that hold epithelial cells together (WATT F., MARREY D.L., GARROD D.R: Calcium induced reorganization of desmosomal components in cultured human keratinocytes, The J. of Cell Biology, 1984, volume 99, pages 2211-2215). Moreover, an elevated level of calcium induces a modification in the distribution of keratin filaments, the appearance of keratohyaline granules and cornified envelopes in keratinocytes culture. The shift of calcium concentrations from 0.07 mM to 1.2 mM, triggers the synthesis of differentiation products such as filaggrin, a histidin rich protein aggregating the keratin filament, which is characteristical for the terminal differentiation of human keratinocytes in vivo (HAUGEN SCOFIELD J.A., HENNINGS H., DALE B.A., YUSPA S.H., STANLEY J.R.: Identification of filaggrin in cultured mouse keratinocytes and its regulation by calcium, J. Invest. Dermatol., 1983, volume 81 , pages 90S-95S).
Moreover, numerous works have shown that the proliferation of basal keratinocytes depends on the calcium ion concentration. In fact, human epidermis presents a positive gradient of calcium concentration from the basal layer up to the superficial stratum corneum. The extracellular concentration is low in the basal proliferating layer (20-5O μM) and reaches a maximal concentration of 600 μM in the superficial layer constituted of completely differentiated corneocytes (MENON G.K., ELIAS P.M.: Ultrastructural localization of calcium in psoriatic and normal human epidermis. Arc Dermatol, 1991 , volume 127, pages 57-63).
Unexpectedly, the same type of calcium regulation on cell differentiation was observed for neurons cultured in media with different calcium levels. Several classical functions were described in neurons, e. g. vesicle secretions. Furthermore the calcium concentration is also an important factor determining the development and the growth of primary axonal extension (FIELDS R.D, GUTHRIE P.B, RUSSELL J.T., KATER S.B., MALHOTRA B.S., NELSON P.G.: Accomodation of mouse DRG growth cones to electrically induced collapse: kinetic analysis of calcium transients and set- point theory, J. Neurobiol., 1 993 volume 24 number 8, pages 1080-1098). Numerous studies have shown the crucial role of calcium in the building of cone proteins and axonal cyto-skeletons and for the expression of genes involved in development mechanisms. The calcium concentration required in cell culture media for axonal development was generally assumed to be between 1 and 2 mM (SPIRA M.E., OREN R., DORMANN. A, IILOUZ N., LEV S.: Calcium, protease activation and cytoskeleton remodeling underlie growth cone formation and neuronal regeneration, Cell. Mol. Neurobio, 2001 volume 21 number 6, pages 591-604 ; WEST A.E., CHEN W.G., DALVA M.B., DOLMETSCH R.E., KORNHAUSER J.M., TAKASU M.A., TAO X., GREENBERG M.E : Calcium regulation of neuronal gene expression, Proc. Natl. Acad. Sci. USA, 2001 volume 98, number 20, pages 11024-11031).
Surprisingly it has been found now by in vitro experiments that the activity of certain mediators belonging to the neuropeptide family such as NGF (Nerve Growth Factor), BDNF (Brain Derived Nerve Growth Factor)..., depends on the concentration of calcium. As a consequence the activity of certain specific chemical messengers or physical stress can be studied only with a low level of calcium in the cell culture assay medium in order to demonstrate, for example, an induction of neurite and/or axonal outgrowth or a release of various mediators. By varying the calcium concentration in the assay medium, the various extracellular calcium concentrations that nerve fibers meet when they cross human tissue such as skin can be simulated.
The present invention is related to cell cultures of neurons or cell cultures of neurons and keratinocytes cultured separately or co-cultured either in the same cell culture medium (in the same compartment) or in 2 different compartments filled with different cell culture media.
WO 03/005023 discloses a process to analyze the interactions between keratinocytes and normal neurons from dorsal root ganglia (DRG) using a co-culture of these 2 cell types in 2 separated compartments, e. g. separated by a glass ring that allows the crossing of neuritis and/or soluble factors. This model was extended to primary cultures of keratinocytes and neurons but also to cell lines of epithelial cells such as HeLa, NCTC2544 and A431 , or neuronal cells such as PC12, ND7-23 and ND8/32. The present invention relates to the use of a cell culture medium, characterized by a low concentration of calcium, for culturing neurons. The neurons can be cultured in this medium, alone or mixed with keratinocytes or melanocytes, for example.
The use of a cell culture medium for culturing neurons, whereby the cell culture medium has a calcium concentration of lower than 0.1 mM (preferably the calcium concentration is 0.001 to 0.03 mM) is a subject of the present invention.
A further subject of the present invention isa cell culture comprising a) neurons and b) a cell culture medium, whereby the cell culture medium has a calcium concentration of lower than 0.1 mM (preferably the calcium concentration is 0.001 to 0.03 mM).
The cell culture according to the present invention further comprising c) keratinocytes is one embodiment of the present invention.
The cell culture according to the present invention further comprising c) melanocytes is another embodiment of the present invention.
The cell culture according to the present invention comprising keratinocytes or melanocytes, wherein the keratinocytes or the melanocytes are obtainable from primary cultures or from cell lines and the neurons are obtainalble from dorsal root ganglia (DRG) or from cell lines is another embodiment of the present invention.
Another subject of the present invention is the use of the cell culture according to the present invention comprising keratinocytes or melanocytes as an assay determining the biological cross-talk of a) the neurons with b) the keratinocytes or melanocytes
(preferably through soluble compounds such as neurotrophins or steroids) comprising the evaluation of the metabolism of the neurons, preferably by methods such as patch-clamp and/or calcium imaging and/or immunocytochemistry and/or molecular biology.
Another subject of the present invention is a process for identifying substances useful as active ingredients for cosmetic compositions comprising testing the substances with the assay according to the present invention and identifying those substances that have a positive effect on the metabolism of the neurons of the assay.
Another subject of the present invention is the use of a substance identiefied with the assay according to the present invention for the cosmetic treatment of humans.
Another subject of the present invention is the use of a substance able to control calcium signalling in neuronal functions such as the release of factors like peptides or steroids for the cosmetic treatment of hu mans.
Several standard cell culture media containing different calcium concentrations can be used:
- examples of media with a low concentration of calcium are RPMI 1640 [0.1 g/l of Ca(N03)2 x 4H20], nutrient mixture (HAM F12) [0.0441 g/l of CaCI2 x 2H20], Hanks balanced salts [0.185 g/l of CaCI2 2H20], PBS (phosphate buffered saline) [0.133 g/l of CaCI2 x 2H20] and MCDB153 [0.004411 g/l of CaCI2 x 2H20].
- examples of media with a high concentration of calcium are MEM (minimum essential medium of eagle) and DMEIvl (Dulbecco's modified eagle's media) which contain 0.265 g/l of CaCI2 x 2H2O.
RPMI, HAM F12 etc. are culture media well known to the skilled artisan they are available commercially. They can be purchased from companies like SIGMA or In Vitrogen (SIGMA ALDRICH CHIMIE SARL, L'lsle D'Abeau Chesnes - B.P. 701 , 38297 Saint-Quentin Fallavier Cedex and Invitrogen S.A.R.L., BP 030096, 95613 Cergy Pontoise Cedex).
Another improvement provided by the use of "low calcium media" ("low calcium media" are media that have a low concentration of calcium), is the possibility to co- cultivate (i. e. co-culture) neurons with proliferating keratinocytes, because it has been established that proliferating keratinocytes of the human epidermis are in an intercellular medium characterized by a low concentration of calcium ion (around 20 μM). This co-culture with a low calcium concentration, allows for evaluating the effects of proliferating keratinocytes on axonal development and neuronal functions.
It is possible to investigate the influence of important biological molecules such as peptides or steroids on neuronal functions using culture media with a low calcium concentration. These investigations are not possible using culture media with high calcium concentrations.
In media with high concentrations of calcium, molecules such as neurotrophins (BDNF, NGF for example), do not shown any effects on axonal extensions of neurons, whereas in media with a low concentration of calcium, the same neurotrophins provoke a distinct effect on axonal extensions of neurons. Therefore the use of cell culture media with a low concentration of calcium enable the skilled artisan to demonstrate the activity of these neurotrophins on neurons.
Another advantage of cell culture media with a low concentration of calcium is that these media allow for the investigation of the positive effect of keratinocytes on the length of axonal extensions in co-cultures of neurons and keratinocytes. These investigations can be used to discover new active ingredients for cosmetic applications, wherein these active ingredients have the same activity on neurons as said neurotrophins or others molecules secreted by keratinocytes.
Another improvement provided by the use of low calcium media, is the possibility to co-cultivate neurons with melanocytes because it is well known that melanocytes are located in the basal layer of the epidermis where the concentration of calcium is low. Through the use of cell culture media with a low calcium concentration the interactions between neurons and melanocytes in a status similar to the normal in vivo conditions can be studied in vitro.
The following paragraphs (until the listing of the "advantages of the model") give more information in relation to the present invention. These paragraphs are essentially the text of a summary describing a preferred embodiment of the present invention including background information (on the state of the art etc.) and on requirements and their results.
The present invention is related to the development of a coculture model for the functional study of interactions between sensory neurons and keratinocytes.
More information on state of the art:
Histologically, mammal skin is made of three layers: the hypodermis deep down, the dermis and the epidermis at the surface. Keratinocytes represent 95% of the cells of the epidermis, which is divided up into 4 layers, successively: the basal (proliferative) layer, the spiny layer, the granular layer and the horny layer at the surface.
In the skin there are nerve endings that are specialized in the detection of physical and chemical stimuli originating from the surrounding environment. These endings belong in particular to unmyelinated sensory neurons called nociceptors, the cell body of which is located in the spinal ganglia. These pseudounipolar neurons have a peripheral branch which penetrates into the skin so as to reach the various layers of the epidermis, and a central branch which reaches the superficial layers of the dorsal horn of the spinal cord.
The relationships between keratinocytes and sensory neurons are therefore very close, but the existing morphological or functional studies, characterizing the contacts between nociceptors and keratinocytes, have been carried out mainly on human skin slices with nerve sections or induction of chronic inflammation (Hilliges M, Wang L, Johansson O: Ultrastructural evidence for nerve fibers within all vital layers of the human epidermis. J Invest Dermatol 1995 104(1):134-7; Kennedy WR, Wendelschafer-Crabb G: The innervation of human epidermis. J Neurol Sci 1993 115(2): 184-190).
We have developed a coculture model in which keratinocytes and sensory neurons are cultured in the same medium. This model has been optimized not only for rat primary keratinocytes and primary sensory neurons, but also for cells of a keratinocyte line (A431) and of a sensory neuron line (ND7-23).
Many studies have shown the importance of the extracellular Ca2+ ion concentration in keratinocyte proliferation (basal layer). In fact, under base conditions, human epidermis exhibits an increase in calcium gradient in its various layers: the extracellular concentration is low (20-50 μM) in the deep (proliferative) layers and a maximum extracellular concentration (600 μM) is reached in the superficial layers (Menon GK, Elias PM: Ultrastructural localization of calcium in psoriatic and normal human epidermis. Arc Dermatol 1991 127:57-63). As a result, cultures of keratinocytes in the basal layer of the epidermis require a culture medium containing a low calcium concentration (20 μM).
The extracellular calcium concentration is also a determining factor in the control of the growth and of the development of the first axonal projections (neurites) (Fields RD, Guthrie PB, Russell JT, Kater SB, Malhotra BS, Nelson PG: Accommodation of mouse DRG growth cones to electrically induced collapse: kinetic analysis of calcium transients and set-point theory. J Neurobiol 1993 24(8): 1080-98). Many studies have in particular shown the essential nature of the extracellular calcium in the organizing of the proteins of the growth cone and of the axonal cytoskeleton, and also for the expression of genes involved in developmental mechanisms (Spira ME, Oren R, Dormann A, llouz N, Lev S: Calcium, protease activation and cytoskeleton remodeling underlie growth cone formation and neuronal regeneration. Cell Mol Neurobiol 2001 21(6):591-604; WestAE, Chen WG, Dalva MB, Dolmetsch RE, Ko nhauser JNl, Takasu MA, Tao X, Greenberg ME: Calcium regulation of neuronal gene expression. Proc Natl Acad Sci USA 2001 98{20):11024-31). The extracellular calcium concentration required for axonal development is generally between 1 and 2 mM.
Originality of the model
The originality of our model lies in the fact that it is the first model consisting of the coculturing of keratinocytes and sensory neurons in "low calcium" medium. It endeavours to conserve the conditions observed in vivo in the epidermis, while maintaining an extracellular calcium concentration of e.g. 20 μM. As a result, it makes it possible to study the morphological and functional consequences of a low extracellular calcium concentration on axonal development, and to study and characterize the effects of keratinocytes on this development.
Culture model
The medium used in the cocultures is MCDB 153 medium (Sigma, M7403). The medium is entirely controlled (serum-free) and has already been described and used for keratinocyte cultures in the literature (Oku H, Kumamoto C, Miyagi T, Hiyane T, Nagata J, Chinen I: Serum-free culture of rat keratinocytes. In Vitro Cell Dev Biol 1994 30AΑ96-503). The calcium contained in the culture medium is chelated using Chelex-100 resin (BIO-RAD ref. 142-2822) at a concentration of 5 g/100 ml for 1 hour with stirring. The medium is then recovered and the calcium (CaCI2) concentration is adjusted to the chosen value.
Example: Culturing of primary keratinocytes
Six newborn rats are sacrificed by euthanasia. The dorsal skin is removed, cleaned in 70% ethanol and rinsed twice in PBS (Phosphate Buffer Saline). The skin is cut into 1 cm2 pieces, which are placed in a Petri dish containing trypsin (0.5 mg/ml trypsin, 0.2 mg/ml EDTA, GIBCO) for 1 h at 37°C. The action of the trypsin is stopped in a DMEM medium (GIBCO ref. 41965-039) supplemented with foetal calf serum (10% vol/vol). The epidermis detaches from the dermis and is recovered in DMEM supplemented with foetal calf serum using fine forceps, and stirred for 20 min. The dissociated cells which are recovered from the supernatant are centrifuged for 10 min at 1000 rpm. The pellet is taken up in MCDB 153 medium.
Example: Culturing of sensory neurons
Three newborn rats are sacrified by decapitation. The skin is removed from the back by means of iridectomy scissors. After having removed the spinal cord, the ganglia visible in the vertebral cavities are removed under a binocular magnifying lens using fine forceps, and placed in a culture dish 35 mm in diameter containing sterile PBS. The central and peripheral projections are removed using a scalpel. The ganglia are then placed in a 15 ml tube and the PBS is replaced with medium without Ca2+ or Mg2+, in which the ganglia are washed twice. The ganglia are subjected to enzymatic dissociation with 2 ml of trypsin (0.5 mg/ml trypsin, 0.2 mg/ml EDTA, IBCO). The tube is placed in a waterbath for 25 minutes at 37°C and regularly agitated. The reaction is stopped by adding MeMα (GIBCO, ref. 2561-021 ) supplemented with 10% vol/vol horse serum. The ganglia are then dissociated mechanically in MEM by means of three Pasteur pipettes of decreasing diameter, the tips of which have been smoothed off in a flame. When the medium becomes cloudy, this indicates the presence of dissociated cells. The cells are recovered and centrifuged at 400 rpm for 5 minutes. The pellet is taken up in the medium chosen for the seeding.
Example: Seeding
The cultures are seeded into glass-bottom Petri dishes (imaging) or onto glass cover slips (immunocytochemistry) pretreated with rat tail collagen. The kerati nocytes are seeded first and the neurons are ad ded after 3-4 hours.
Example: Cells from a line
The keratinocyte (A431 , ECACC 85090402) or neuron (ND7-23, ECACC 92090903) lines proliferate in a flask containing DMEM medium supplemented with 1 0% (vol/vol) foetal calf serum; 2 ml of trypsin are added in order to detach the cells. The cells are centrifuged at 1000 rpm for 5 min. , and the pellet is then taken up in the chosen seeding medium.
The same seeding protocol as for the primary cells is used: the cells are centrifuged and the pellet is taken up in the seeding medium. The ND7-23 cells are seeded after the A431 cells have adhered to the collagen substrate. Results
Cell survival
The MCDB 153 medium makes it possible to maintain the two cell types in culture for more than a week, but reveals differences in the ability of the neurons to put out axonal projections.
Morphometric study
The criterion used to evaluate the interactions between keratinocytes and neurons is the length of the axonal projections. The measurements were carried out after 1 , 3 and 7 days of seeding for the sensory neurons from the line, and after 1 , 3 and 6 days for the primary neurons. The neuronal projections are visualized after immunocytochemical labelling with an anti-Smi antibody (Sternberger Monoclonals Incorporated) specific for axonal proteins, or an anti-PGP 9.5 antibody (Chemicon) specific for unmyelinated sensory fibres. The keratinocytes are labelled with an anti- cytokeratin 14 antibody (Chemicon) specifically expressed by keratinocytes during the proliferative phase, or with an anti-cytokeratin 10 antibody (Chemicon) specific for differentiated keratinocytes of the granular and spiny layers.
1 ) Control 1 : neurons alone cultured in the usual medium; effect of calcium
The axonal projections were measured in cultures of neurons carried out in the medium commonly used: MEMα + 10% vol/vol horse serum, for the primary neurons, and DMEM for the neurons from the line. We compared the effect on axonal development of a calcium concentration of 2 mM, which is normal for neurons, and of a concentration of 20 μM required for the cocultures with keratinocytes.
There is less axonal development and it is slower with 20 μM of calcium than with a concentration of 2 mM, both for the primary neurons and for the neurons from the line.
2) Control 2: neurons cultured alone in MCDB 153 medium; effect of calcium The neurons were cultured in the MCDB 153 medium used for the cocultures (medium, 20 μM Ca2+). The lengths of the axonal projections measured in this medium with calcium concentrations of 20 μM and 2 mM, respectively, were compared.
In the medium containing a calcium concentration of 20 μM, the axons show a considerable growth retardation in the case of the neurons from the line ND7-23, and are absent in the case of the primary sensory neurons throughout the duration of the cultures.
Conclusion: extracellular calcium is a factor that is essential to the axonal development of primary neurons or neurons from a line, whatever the medium used.
3) Cocultures: neurons cultured in MCDB medium in the presence of keratinocytes
The neurons are added to a culture of keratinocytes cultured in MCDB 153 medium containing a low calcium concentration (20 μM). Unlike control 2, in the presence of keratinocytes, the length of the axons tends to be similar to that measured in the optimum neuron culture medium (MEMα or DMEM), or to that measured in an MCDB 153 medium containing 2 mM Ca2+. The results obtained are similar whatever the combination of neurons (primary/line) and keratinocytes (primary/line).
This model indicates that the presence of keratinocytes allows axon development, even in a "low calcium" MCDB 153 medium which reduces (ND7-23 neurons) o r blocks (primary sensory neurons) axonal growth in the absence of keratinocytes. The keratinocytes appear to secrete one or more factors capable of acting on the sensory neurons and of controlling axonal development.
Functional study
The calcium imaging technique with the Fura2 probe, which is sensitive to variations in intracellular calcium, has shown that neurons cultured in the presence of keratinocytes express membrane receptors identical to those characterized in the neurons cultured in the control medium (ME IVIα or DMEM). In fact, we have demonstrated voltage-dependent Ca2+ channels, and receptors for ATP (P2X and P2Y), for bradykinin (B2), and for capsaicin (VR1
Immunocytochemical study
Anti-cytokeratin 14 labelling and anti-PGP 9.5 labelling were observed by confocal microscopy and made it possible to demonstrate apparent physical contacts between the neurons and keratinocytes.
In conclusion, this coculture model demonstrates: 1 ) the important role of extracellular calcium in the axonal development of sensory neurons; 2) the effect of keratinocytes on axonal growth (compensation for the inhibitory effect of a low extracellular calcium concentration). It makes it possible to study neurons under extreme growth conditions (neurons without keratinocytes/with keratinocytes, low/high calcium concentration).
Advantage of the model
This model makes it possible to study the interactions and the reciprocal signalling between keratinocytes and sensory neurons by means of various experimental approaches: patch-clamp, calcium imaging, imm nocytochemistry, molecular biology, in particular:
1 ) The setting up of neuron-keratinocyte relationships (development stage).
2) The effect of molecules secreted by the keratinocytes on the development of neurons (cytokins, interleukins, growth factors, hormones, etc.).
3) The morphological characterization of the physical contacts which exist (synapses, adjoining of membranes, tight j unctions, gap junctions, etc.)
4) The effect of the presence of neurons on the mitotic activity of keratinocytes.
5) The effect of the presence of neurons on the differentiation of keratinocytes.
6) The effect of substances known to be secreted by neurons, on the metabolism of keratinocytes. 7) The effect of a physical stress (UV, pressure, temperature, etc.) or a chemical stress (capsaicin) or of an inflammation on keratinocytes and the functional consequences thereof on neurons.
8) To screen substances having an activity on neurons and on keratinocytes in a dermatology and cosmetics context.
9) To characterize and measure the substances secreted by the two cell types and the receptors involved (neuropeptides, cytokins, neurotransmitters, etc.).
10) To study the skin diseases related to the interactions between keratinocytes and neurons.
11) To study functional keratinocyte-neuron interactions at the adult stage (culturing of adult rat sensory neurons).
Examples
General Information concerning the experiments that have been carried out
The invention has been carried out by comparing the activity of neurotrophins (BDNF, NGF for example) in media with a low calcium concentration versus the activity of these neurotrophins in mediawith a high calcium concentration. In fact in media with a high concentration of calcium, these neurotrophins do not present any distinct effects, whereas in media with a low concentration of calcium, the neurotrophins enhance markedly the length of axonal extensions. Therefore, the use of media with a low concentration of calcium constitutes a model to study the activity of various molecules on the length of axonal extensions.
The following abbreviations are used in the text:
PBS = Phosphate Buffered Salt solution: it is a standardized well known solution for cell cultures
DMEM = Dulbecco's Modified Eagle's Media: DMEM is a well known and standardized cell culture medium provided by the company SIGMA or In Vitrogen
Liquid culture medium used for cultures of neurons and keratinocytes or melanocytes
The medium with low calcium concentration used has been MCDB 153 (purchasable from the company Sigma). This medium is completely defined (without serum) and has been used for the cultures of keratinocytes before (OKU H., KZUMAMOTO C. MIYAGI T., HIYANE T., NAGATA J., CHINEN I.: Serum-free culture of rat keratinocytes, In Vitro Cell Dev. Biol,. 1994 volume 30A, pages 496-503). The calcium present in the medium can be removed by chelating it with chelex 100 resin (purchasable from the company BIO-RAD) used at the dose of 1 to 10 g/100 ml for 1 hour with shaking. Then the calcium (CaCI2) concentration of the medium can be adjusted to a chosen value. Substrate of the culture
Cells (neurons and keratinocytes or melanocytes) have been seeded in Petri dishes with a glass bottom for microscope imaging or on glass slides for immuno-cyto- chemistry coated with collagen type I from rat tails. Keratinocytes or melanocytes have been seeded some hours before neurons (1 to 72 hours).
Method of culturing of primary cells
Cell-culture of neurons from DRG (dorsal root ganglia) (as disclosed in WO 03/005023)
The cells for the cell-culture have been separated from the tissues of new-born rats according to the modification of dissociation technique (SCOTT B.S.: Adult mouse dorsal root ganglia neurons in cell culture, J. Neurobiol., 1977, volume 8, pages 417- 427 ; DICHTER MA, FISHBACH G.D.: The action potential of chick dorsal root ganglion neurones maintained in cell culture, J. Physiol, 1977, volume 267, pages 281-298). DRG have been obtained from new-born rats (not older than one week), placed into a Petri dish with PBS on ice and cleaned with a scalpel in order to suppress nervous ramifications. Then, they have been washed twice with PBS without Ca2+ and Mg2+ and submitted to an enzymatic dissociation with trypsin at a concentration of 0.05 % by weight. When the cell suspension has been obtained, the supernatant has been centrifuged for 8 minutes at 150 g. The residue has been washed and has been put into DMEM-serum. The cellular suspension containing neurons and glial cells has been counted on a Mallassey slide and cultured at a density of 150 000 cells/cm2.
Cell-culture of keratinocytes (as disclosed in WO 03/005023)
Keratinocytes have been prepared according to the technique of RHEINWALD J.G., GREEN H.: Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinising colonies from single cells, Cell, 1975, volume 6, pages 331- 434. Rats which are not older than 24 hours have been anesthetized and killed. Then skin pieces of about 1 cm2 have been cut out and have been submitted to enzymatic digestion (trypsin at a concentration of 0.2 % by weight).
Culture of cell lines
Cell lines of keratinocytes such as A431 , NCTC2544 and PAM-212, of neurons such as ND7-23, ND8-32 and PC12, of melanocytes such as B16 and Cloudmann S91 have been cultivated in DMEM with FCS (fetal calf serum) at 10 % by volume. Cells have been detached by trypsin ization and recovered by centrifugation at 1000 rpm (rounds per minute) for 5 min, then the pellet of cells formed during centrifugation has been diluted in cell culture medium with a low calcium concentration.
A431 , NCTC2544, PAM-212 are well known cell lines available from various companies, e. g. ATCC or ECACC.
Co-culture of neurons and keratinocytes or melanocytes
The 2 types of cells can either be introduced in the same compartment or in 2 different compartments, e. g. two compartments separated by a glass ring (as disclosed in WO 03/005023). For co-cultures of neurons with keratinocytes, the same protocol as above has been used and neurons of the cell line used have been seeded on a culture of keratinocytes or melanocytes either on plastic or collagen coating.
This model makes it possible to study the effect of a chemical or physical stress on neurons cultivated in a low calcium medium, too.
This model allows to study interactions and reciprocal signalling between sensory neurons and keratinocytes or melanocytes. This study can use different technical methods such as the patch-clamp method or methods well-known to the skilled artisan like calcium imaging, immunocytochemical methods or methods of molecular biology. The patch-clamp method is a technique that allows to record the electric activity of neurons. With the aid of a microelectrode in glass directed with a micromanipulator, variations of the electric potential between the inside and the outside o-f a neuron are visualized. The signals are amplified with an amplifier of patch and visualized with an oscilloscope. The data can be recorded on a video tape and analysed with specific software. This technique is highly resolutive and allows a pushed analysis of synaptic currents, direct reflect of communication between neurons but also to determine the characteristics of cell receptors. For pharmacologic studies, substan ces/drugs are applied topically (microperfusion) or by bath. The patch-clamp method is described in: HAMILL O.P., MARTY A., NEHER E., SACKMANN B., SIGΛ/VORTH F.J.: Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfϋggers Arch., 1981 , volume 391 : pages 85-100.
This study notably encompasses:
1 ) the establishing of interactions between neurons and keratinocytes and/or melanocytes (stadium of development/differentiation).
2) the effect of secreted molecules by keratinocytes or melano cytes on the development/differentiation of neurons (cytokines, interleukines, growth factors, hormones, steroids...), as well as the functional interactions between steroids and neurotrophins or other factors at the level of their respective receptors and/or their signalling pathways.
3) the morphological characterization of physical contact (synapses, joined membrane, tight junctions, gap junction....) between neurons from one part, and keratinocytes or melanocytes in the other part.
4) the effect of the neurons on mitotic activity of keratinocytes or melanocytes.
5) the effect of the neurons on the differentiation of keratinocytes or melanocytes.
6) the effect of substances known to be secreted by neurons, on the metabolism of keratinocytes or melanocytes
7) the effect of a physical stress (ultraviolet radiations (UVR), pressure, temperature...) or a chemical stress (capsaicine ...) on keratinocytes r melanocytes and the consequences of these effects on neuronal functions.
8) to screen products such as peptides, steroids, lipids, sugars, alkaloids, flavonoids, polyphenols, sterols, isoflavones having an activity on neurons and on keratinocytes or melanocytes in a context of dermatology and cosmetic applications. 9) to characterize and to measure substances secreted by the two cellular types and receptors implied (neuropeptides, cytokines, neuromediators....).
10) to study the cutaneous diseases associated to interactions between keratinocytes or melanocytes and neurons.
11 ) to study functional interactions of keratinocytes or melanocytes with neurons.
Example 1 : effect of the concentration of calcium in the culture medium on the axonal development of neurons cultivated alone
The following abbreviations are used in the text: anti-Smi: (Stemberger Monoclonals Incorporated) is specific for certain proteins, which are a characteristic feature of neuronal axons. anti-Pgp9.5: (protein gene product 9.5) is an antibody specific for another protein characteristic of neuronal axone.
Neurons have been seeded and cultivated for 7 days. At days 1 , 3 and 7 (for cell lines of neurons), and at days 1 , 3 and 6 (for primary cultures of sensory neurons), the axonal extensions have been immunocytochemistry-stained with a specific antibody of axonal proteins: anti-Smi (purchasable from Stemberger Monoclonals Incorporated) or anti-PGP 9.5 (purchasable from Chemicon) specific of the non myelinisated sensory fibers. Then the length of stained axonal extensions has been measured by image analysis.
The lengths of axonal extensions have been measured on neurons cultivated in media with a concentration of calcium of 20 μM and 2 mM and then the results of these measurements have been compared. In the medium containing a concentration of 20 μM calcium, axonal extensions show an important delay of growth in the case of neurons of the cell line, and are absent in the one of the primary sensory neurons during all the period of cultures.
Conclusion: extracellular calcium is a crucial factor to axonal development of the primary or cell line neurons, whatever the medium used for cultivation is. Table 1 : Effect of the calcium concentration in culture medium on the length of axonal extensions of primary sensory neurons and cell line neurons (mean ± SEM (standard error mean) on 5 assays)
Figure imgf000022_0001
Example 2: effect of primary keratinocytes and cell line keratinocytes on axonal growth of the primary neurons and cell line neurons
The criterion used to evaluate interactions between keratinocytes and neurons has been the length of axonal extensions. Measures have been done after 1 , 3 and 7 days after neuronal cells seeding for the sensory neurons of cell line and after 1 , 3 and 6 days for the primary sensory neurons. Axonal extensions of neurons have been visualized after immunocytochemistry-staining with a specific antibody of axonal proteins: anti-Smi (Stemberger Monoclonals Incorporated) or anti-PGP 9.5 (Chemicon) specific of the non myelinisated sensory fibers. Keratinocytes have been marked by an anti-cytokeratine 14 antibody (purchasable from Chemicon) expressed specifically by keratinocytes during the proliferative phase or by an anti-cytokeratine 10 antibody (purchasable from Chemicon), specific for keratinocytes differentiated of the granular and spinous layers. Neurons have been added to keratinocytes cultivated in the medium MCDB 153 containing a low concentration of calcium (20 μM). In the presence of keratinocytes, the length of axons has the tendency to come closer to the one measured for neurons cultivated in a medium with a high calcium concentration such as MEM or DMEM, or MCDB 153 containing 2 mM of Ca2+. The obtained results have been similar whatever the combination of neurons (primaries / cell line) and of keratinocytes (primaries / cell line) was.
This model indicates that the presence of keratinocytes permits a development of axons, even in a MCDB 153 medium with a low concentration of calcium that otherwise reduced (cell line neurons) or blocked (primary sensory neurons) axonal growth in the absence of keratinocytes. Keratinocytes seem to secrete one or several factors susceptible to act on the sensory neurons or to be metabolised by neurons and finally to control axonal development.
Table 2: Effect of primary keratinocytes and cell line keratinocytes on axonal growth of the primary neurons and cell line neurons ND7-23 (mean ± SEM (standard error mean) on 5 assays)
Figure imgf000024_0001
Example 3: effect of neurotrophins and steroid on the axonal cell growth
With the same technique as the one described in examples 1 and 2, keratinocytes have been replaced by specific substances like neurotrophins (BDNF, NGF (purchasable from Sigma)) or steroid such as DHEA (Dehydroepiandrosterone) (purchasable from Sigma). Table 3: Effect of neurotrophins on axonal growth of the primary neurons (mean ± SEM (standard error mean) on 4 assays)
Figure imgf000025_0001
In low calcium media, neurotrophins such as BDNF and NGF have a similar effect on axonal cell growth as primary keratinocytes or as cell line keratinocytes after 6 days of culture.
Table 4: Effect of steroid on axonal growth of the primary neurons (mean ± SEM (standard error mean) on 4 assays)
Figure imgf000026_0001
In low calcium media, DHEA has a similar effect on axonal cell growth as primary keratinocytes or as cell line keratinocytes after 7 days of culture.
Example 4: evaluation by calcium imaging and by immunocytochemistry of membrane receptors of neurons in the presence of keratinocytes and in high calcium media
The technique of calcium imaging with the probe Fura2 (sensitive to intracellular calcium variation) showed that the cultivated neurons in low calcium media, in the presence of keratinocytes express some identical membrane receptors to those characterized in the cultivated neurons in the control media with a high calcium concentration. We have put in evidence the voltage-dependent channel Ca2+, the receptors of the ATP (P2X and P2Y), of the bradykinine (B2), and of the capsaicine (VR1 ).
In conclusion, these results show: 1 ) the important role of extracellular calcium for the axonal development of sensory neurons; 2) the effect of keratinocytes on axonal growth (compensation of the inhibitory effect of a low extracellular concentration of calcium). This permits to study the neuron functions in cell cultures of neurons alone or in co-culture with melanocytes or keratinocytes, in the specific conditions of growth.

Claims

Claims
1. The use of a cell culture medium for culturing neurons, whereby the cell culture medium has a calcium concentration of lower than 0.1 mM (preferably the calcium concentration is 0.001 to 0.03 mM).
2. A cell culture comprising c) neurons and d) a cell culture medium, whereby the cell culture medium has a calcium concentration of lower than 0.1 mM (preferably the calcium concentration is 0.001 to 0.03 mM).
3. The cell culture according to claim 2 further comprising c) keratinocytes.
4. The cell culture according to claim 2 further comprising c) melanocytes.
5. The cell culture according to claim 3 or 4, wherein the keratinocytes or the melanocytes are obtainable from primary cultures or from cell lines and the neurons are obtainable from dorsal root ganglia (DRG) or from cell lines.
6. The use of the cell culture according to any of claims 3 or 5 as an assay determining the biological cross-talk of c) the neurons with d) the keratinocytes or melanocytes (preferably through soluble compounds such as neurotrophins or steroids) comprising the evaluation of the metabolism of the neurons, preferably by methods such as patch-clamp and/or calcium imaging and/or immunocytochemistry and/or molecular biology.
7. A process for identifying substances useful as active ingredients for cosmetic compositions comprising testing the substances with the assay according to claim 6 and identifying those substances that have a positive effect on the metabolism of the neurons of the assay.
8. The use of a substance identified with the assay according to claim 6 for the cosmetic treatment of humans.
9. The use of a substance able to control calcium signalling in neuronal functions such as the release of factors like peptides or steroids for the cosmetic treatment of humans.
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