Mixed Spheroids of Melanocytes and Keratinocytes

Abstract

The present invention concerns the development of a model to evaluate active substances targeting the epidermis. It more particularly relates to the preparation of mixed spheroids of melanocytes and keratinocytes reproducing cell interactions occurring in the epidermis, to the spheroids as such and to the uses thereof.

Claims

1. Method for preparing mixed spheroids of keratinocytes and melanocytes, characterized in that it comprises: (a) suspending keratinocytes with melanocytes in a culture medium supplemented with a preparation of extracellular matrix proteins at a concentration of between 1 and 95% by volume/volume and an amount of 10 mM or less of a salt selected from among calcium, manganese or magnesium; (b) forming spheroids from the mixture obtained at step (a); and (c) incubating the spheroids obtained at step (b).

2. The method according to claim 1, characterized in that said suspension at step (a) is prepared with 1 melanocyte per 1 keratinocyte to 1 melanocyte per 40 keratinocytes.

3. The method according to claim 1, characterized in that the keratinocytes and melanocytes are healthy primary cells and are seeded in a number of between 100 and 5000 cells per spheroid.

4. The method according to claim 1, characterized in that the preparation of extracellular matrix proteins is MATRIGEL.

5. The method according claim 1, characterized in that the salt is calcium.

6. The method according to claim 1, characterized in that the incubation at step (c) lasts between 1 and 10 days.

7. The method according to claim 1, characterized in that it further comprises a marking step.

8. The method according to claim 1, characterized in that it comprises a step (d) to wash the mixed spheroids.

9. A mixed spheroid of keratinocytes and melanocytes able to be obtained with the method according to claim 1.

10. The spheroid according to claim 9, characterized in that it has an angular diameter of between 100 and 1000 μm and is of regular shape.

11. The spheroid according to claim 9, characterized in that it has a central mass.

12. The spheroid according to claim 9, characterized in that at least one of the constituents thereof is marked.

13. Use of a spheroid according to claim 9 to evaluate the activity of compounds acting at the epidermis.

14. Use of a spheroid according to claim 9 to test the activity of compounds on melanin production and/or transfer of melanosomes and/or the expression of genes involved in melanogenesis.

15. Use of a spheroid according to claim 9 to test the activity of compounds on the differentiation of keratinocytes.

16. A method for screening molecules able to act on melanin production and/or on the transfer of melanosomes and/or on the expression of genes involved in melanogenesis, comprising: (i) preparing spheroids according to claim 9; (ii) placing the spheroids prepared at step (i) in contact with one or more molecules to be tested; (iii) selecting molecules which modulate melanin production and/or the transfer of melanosomes and/or the expression of genes involved in melanogenesis.

17. A method for screening molecules able to act on the differentiation of keratinocytes, comprising: (i) preparing spheroids according to claim 9; (ii) placing the spheroids prepared at step (i) in contact with one or more molecules to be tested; (iii) selecting molecules which modulate the differentiation of keratinocytes.

Description

FIGURES

[0048] FIG. 1: A. image illustrating a mixed keratinocyte-melanocyte spheroid under transmitted light, having a cell ratio of 1 NHEM per 4 NHEKs. The scale bar represents 100 μm. B. Graph showing changes in volume of the mixed spheroids over 10 days.

[0049] FIG. 2: Images showing a spheroid of the invention at D3.

[0050] A. Cytokeratin 5 marking to evidence keratinocytes.

[0051] B. NKIbeteb (Pme117) marking to evidence melanocytes.

[0052] C. DAPI marking of cell nuclei.

[0053] D. EdU marking of proliferating cells. Proliferating cells can be seen on the periphery of the mixed spheroid.

[0054] E. Overlaying of the different markings.

[0055] The scale bar represents 100 μm.

[0056] FIG. 3: Images showing a spheroid of the invention at D7.

[0057] A. Cytokeratin 5 marking to evidence the keratinocytes. A gradient of CK5 expression can be seen corresponding to a differentiation gradient. It can be noted that the morphology of the keratinocytes is modified as a function of the differentiation gradient with elongation of the cells (arrow).

[0058] B. DAPI marking of cell nuclei. Modification of the morphology of the nuclei can be seen with elongation (arrow) and finally loss of nuclei with the differentiation gradient.

[0059] C. EdU marking of the nuclei of proliferating cells. Proliferating cells can be seen on the periphery of the mixed spheroid.

[0060] D. Overlaying of the different markings.

[0061] The scale bar represents 100 μm.

[0062] FIG. 4: Keratinocyte differentiation.

[0063] A. Diagram of epidermis organisation.

[0064] B. Skin section with Fontana Masson staining.

[0065] C. Detail of a cross-section of mixed spheroids at D7 with cytokeratin 5 marking in grey and EdU marking (nuclei of proliferating cells) in white.

[0066] FIG. 5: images of spheroids of the invention prepared with various melanocyte/keratinocyte ratios (1:20, 1:5 and 1:2). The scale bar represents 100 μm.

[0067] FIGS. 6A to 6E: images showing objects obtained with various attempts to prepare spheroids such as described in Example 2.

[0068] FIG. 7: Visualisation of melanin transfer in the mixed spheroids.

[0069] A. Analysis of a section of mixed spheroids with cytokeratin 5 marking (dark grey) and NKIbeteb (Pme117) marking (light grey). In detail, on the right, doubly marked cells can be seen (arrow). The scale bar represents 100 μm.

[0070] B. Analysis of a whole spheroid by SPIM imaging and 3D reconstitution using IMARIS software, with CellMask marking of the keratinocytes (dark grey) and CFDA marking of the melanocytes (light grey). In detail, on the right, doubly marked cells can be seen (arrow).

[0071] C. Analysis of spheroids disrupted by flow cytometry with cytokeratin 5 (FL4) and CFDA marking of the melanocytes (FL1). A double marked population can be seen (population surrounded by a dotted line).

[0072] FIG. 8: histogram representing the melanin concentration on mixed spheroids after IBMX treatment (compared with the solvent control used and PTU).

[0073] FIG. 9: histogram representing gene expression of the cells of a mixed spheroid of the invention quantified as explained in Example 6. The expression of the MITF gene directly involved in IBMX treatment is increased. Expression of the genes TYR, TYRP1 and PMEL under the control of MITF is also increased after IBMX treatment.

EXAMPLE 1—PREPARATION OF SPHEROIDS OF THE INVENTION

Method

[0074] The cells are detached by trypsinization and counted to prepare a mixed suspension of melanocytes et keratinocytes with 60 000 cells/mL in medium supplemented with 10% Matrigel® and 1 mM calcium. This suspension is then seeded in a 96-well plate previously coated with polyHEMA and centrifuged 6 min at 190 g at 4° C. The plate is incubated 1 h at 37° C., 5% CO.sub.2, to obtain Matrigel® congealing, and culture medium is added to each well to hydrate the Matrigel®.

EXAMPLE 2—CHARACTERIZATION OF THE SPHEROIDS OF THE INVENTION

[0075] The spheroids thus produced have a diameter of about 250 μm after 3 days' culture and their volume doubles in 10 days (FIG. 1).

[0076] At D3, the marking method with NucView (caspase 3 marking) shows that no apoptosis occurs in the mixed spheroids. EdU markings allowed determination that the cells proliferate on the periphery of the spheroid. These cells are also marked with cytokeratin 5 and therefore correspond to keratinocytes (FIG. 2).

[0077] It is to be noted that, after several culture days, a central mass is seen in the spheroid and the morphology of the cells is modified (FIG. 3); this central mass is the result of differentiation of the keratinocytes (FIG. 4).

[0078] Spheroids of the invention were also prepared with various melanocyte/keratinocyte cell ratios (1:20, 1:5 and 1:2); they all exhibited satisfactory characteristics as illustrated in FIGS. 5A, 5B and 5C.

[0079] Other spheroids of the invention were prepared as in Example 1 using 50% and 75% Matrigel®; here again, the mixed spheroids obtained exhibited satisfactory characteristics; FIGS. 5D, 5E and 5F are photographs of these spheroids respectively prepared with 10%, 50% and 75% Matrigel®.

[0080] The following spheroid preparation conditions were also tested but did not allow spheroids to be obtained: [0081] centrifugation method of a mixed NHEM-NHEK suspension in NHEK medium without Matrigel® and without calcium; with this method, the cells group together but do not form three-dimensional structures (FIG. 6A); [0082] centrifugation method of mixed NHEM-NHEK suspension in NHEK medium supplemented with 1 mM CaCl.sub.2 or in F12+SVF medium without Matrigel®: with this method, the cells form three-dimensional structures but remain segregated per cell type. The structure is not spherical and insufficiently reproducible for screening (FIG. 6B); [0083] confrontation method of a NHEK-only spheroid with a suspension of NHEM in NHEK medium supplemented with 1 mM CaCl.sub.2 or in F12+SVF medium without Matrigel®; the previously formed NHEK-only spheroids were prepared using the centrifugation method: with the confrontation method of a pre-existing spheroid with a cell suspension in a medium with calcium or in F12+SVF medium without Matrigel®, the cells form three-dimensional structures but remain segregated per cell type. The structure is not spherical and insufficiently reproducible for screening (FIG. 6C); [0084] confrontation method of a NHEM-only spheroid with NHEK suspension in NHEK medium supplemented with 1 mM CaCl.sub.2 or in F12+SVF medium without Matrigel®: the previously formed NHEM-only spheroids were prepared using the centrifugation method; with the confrontation method of a pre-existing spheroid with a cell suspension in a medium with calcium or in F12+SVF medium without Matrigel®, the cells form three-dimensional structures but remain segregated per cell type. The structure is not spherical and insufficiently reproducible for screening (FIG. 6D); [0085] confrontation method of single spheroids (spheroids of NHEM and spheroids of MHEK respectively) in NHEK medium supplemented with 1 mM CaCl.sub.2 without Matrigel®; the two previously formed single spheroids were prepared using the centrifugation method; with the confrontation method of two spheroids of each cell type in a medium with calcium but without Matrigel®, the cells form three-dimensional structures but remain segregated per cell type. The structure is not spherical and insufficiently reproducible for screening (FIG. 6E); [0086] droplet method from a mixed NHEM-NHEK suspension in NHEK medium without calcium, supplemented or not with Matrigel® (10 to 50%). The conditions with Matrigel® did not form 3D structures; with this method the cells form three-dimensional structures but the structure is not spherical and insufficiently reproducible for screening; [0087] centrifugation method of a mixed NHEM-NHEK suspension in NHEK medium without calcium and with a Matrigel® percentage varying from 10 to 50%: with this method, the cells form three-dimensional structures but remain segregated per cell type. The structure is not spherical and insufficiently reproducible for screening.

EXAMPLE 3—ASSAY PROTOCOL OF MELANIN BY PHOTOMETRY

[0088] The cells were lysed in a solution of 1M NaOH+10% DMSO and incubated 30 min at 90° C. A standard range was prepared with synthetic melanin and read-out performed at a wavelength of 405 nm.

[0089] Assay of total proteins was performed on the lysate of melanin assay using the microBCA kit in accordance with the supplier's instructions. A standard range was prepared with BSA and read-out was performed at a wavelength of 605 nm.

[0090] The results are expressed in the form of a ratio between the assay of melanin and assay of total proteins.

EXAMPLE 4—EXAMPLE OF A MARKING PROTOCOL FOR MIXED SPHEROIDS OF THE INVENTION

[0091] The mixed spheroids were prepared as explained in Example 1 with melanocytes previously marked with CFDA (carboxy-fluorescein diacetate, succinimidyl ester). The mixed spheroids thus formed were marked with CellMask. They were then washed, fixed in formalin, dehydrated and made transparent in a mixture of Benzyl-alcohol and benzyl-benzoate (BaBb). Analysis was performed using the light-sheet imaging technique.

EXAMPLE 5—ASSAY OF MELANIN ON A MIXED SPHEROID AFTER TREATMENT WITH IBMX and PTU

[0092] 24 h after seeding, the mixed spheroids of the invention such as prepared in Example 1, and after removal of Matrigel® by washing with PBS, were treated with 300 μM IBMX or 500 μM PTU for 5 days with renewal of treatment at 4 days. The melanin was assayed as explained in Example 3.

[0093] As illustrated in FIG. 8, the results obtained show that treatment with PTU leads to a decrease in the global amount of melanin in the mixed spheroid, in comparison with the solvent control (EtOH), and treatment with IBMX causes an increase in the total amount of melanin in the mixed spheroid as compared with the solvent control (DMSO).

EXAMPLE 6—QUANTIFICATION OF GENE EXPRESSION BY QPCR

[0094] 48 h after their seeding the mixed spheroids of the invention such as prepared in Example 1, and after removal of Matrigel® by washing with PBS, were treated with 300 μM IBMX for 24 h. The spheroids were collected and washed with PBS after 24 h treatment with IBMX. The RNAs were extracted and reverse-transcribed to cDNA. Quantitative PCR was then performed to quantify the expression of the genes MITF, TYR, TYRP1 and PMEL. The expression of these genes was normalised by expression of the GAPDH housekeeping gene and compared with the non-treated control.

[0095] The results obtained show that the MITF gene, directly involved in IBMX treatment, has its expression increased. The expression of the genes TYR, TYRP1 and PMEL, under the control of MITF, is also increased after treatment with IBMX (FIG. 9).

EXAMPLE 7—EVALUATION OF THE GENE EXPRESSION PROFILE WITH GENOME CHIP

[0096] The gene expression profile of the spheroids was evaluated after 3 and 7 culture days on a genome chip following the protocol detailed below.

[0097] The mixed spheroids of the invention prepared as described in Example 1 were collected, washed in PBC at 3 days and 7 days respectively after seeding. The mRNAs were extracted followed by reverse transcription. Finally, the specific preparation steps for the chip (96 genes) applying the Fluidigm protocol were carried out. A pre-amplification step in the presence of all the primers used on the chip was performed. Each pre-amplified cDNA sample was then deposited with the mix in a 96-well plate following the plate layout allowing real-time PCR. In parallel, on another 96-well plate, each pair of primers was placed in a well following the plate layout. Each mix was then deposited either side of the chip. Mixing of the two mixes was performed by the IFC Controller and the chip then placed in the BioMark to conduct real-time PCR. The genome chip was used on 64 genes specific to the metabolism of keratinocytes of which 15 are also expressed by the melanocytes. Fluidigm software was used for data analysis. The results obtained are given in Table 1 below which shows that several genes involved in epidermal cohesion and differentiation are over-expressed after spheroid development between D3 and D7 as illustrated in FIG. 3; the inflammation genes are also over-expressed:

TABLE-US-00001 TABLE I Table showing genes over-expressed between culture Days 3 and 7 % over- Gene expression Action CTNNA1  +50% Alpha-catenin binds to Cadherin to promote cell interactions CDLN1 +280% Claudin is a membrane protein acting at tight junctions OCLN +270% Occludin is a membrane protein acting at tight junctions EVPL  +60% Envoplakin is a protein involved in the formation of desmosomes EPPK1 +540% Epiplakin is a protein involved in the formation of desmosomes. It also seems to be involved in cell differentiation PXN  +40% Paxillin acts in protein complexes belonging to the cytoskeleton. It is located on the cytoplasmic surface of regions specialised in the attachment of cells to the extracellular matrix AQP3  +90% In man, the circulation of water within the skin occurs via a specific Aquaporin called AQP3. It plays an essential role in the formation of the hydrolipid barrier TGM1 +110% Transglutaminase is involved in the formation of the cornified envelope creating cross-links between structural proteins including involucrin and thereby rigidifying the stratum corneum. STPL Cl  +40% Serine palmitoyltransferase is an enzyme acting in the biosynthesis of sphingolipids which concentrate in the stratum corneum. It is therefore a marker of epidermal differentiation Il8 +390% Pro-inflammatory cytokine IL6 +710% Pro-inflammatory cytokine TNFA +710% Pro-inflammatory cytokine