DESCRIPTION FIELD OF THE INVENTION

The present application concerns a method for preparing three-dimensional, scaffold- free and stratified microtissues comprising at least two types of cells, in which the first layer consists of at least one first type of cells and the second layer consists of at least one second type of cells. The present application further concerns the use of said microtissues for the preclinical screening of active substances.

STATE OF THE ART

In preclinical screening, the development of in vitro models for testing biologically active compounds from the point of view of their biocompatibility (in vitro toxicity), of the definition of their mechanism of action and of their biological effectiveness is extremely important.

To this end, the production of cellular aggregates (defined as microtissues or organlike spheroids) that synthesise their own extracellular matrix without using any scaffold {scaffold-free) represents an effective high-throughput system of in vitro testing, where it is possible to generate a high number of samples with the minimum expenditure in terms of used cells.

A further relevant fact is that, compared to the corresponding two-dimensional cell cultures or co-cultures (monolayer), three-dimensional systems better mimic the structure and morphology of the starting organ, significantly increasing the biological relevance and therefore the predictivity of the preclinical screening system, especially with regard to efficacy tests.

Moreover, various methods for forming three-dimensional scaffold-free microtissues are known.

The most famous is, for example, the hanging drop technique (with hanging/suspended drop), according to which drops of cell suspension are suspended near the lower side of the plate with pores through which the cell suspension is released. In each drop, the cells are concentrated at the bottom of this drop due to gravity and, thanks to the aggregative capacities of the cells, single spheroids are formed in the air-liquid interface.

The liquid overlay technique also allows obtaining microtissues having a spheroidal shape. It is essentially based on the fact that the plate is made non-adherent by covering it e.g. with agarose or by means of a chemical photodynamic pattern, before cell seeding.

Finally, a possible three-dimensional microtissue forming technique is the technique that employs micro-pattern plates or the magnetic aggregation technique.

The aforesaid techniques allow obtaining microtissues formed by a single type of cells, such as e.g. microtissues formed by hepatocytes, as for example described in WO2015/15877.

The aforementioned three-dimensional microtissue forming techniques have also been used for forming more complex tissues consisting of several different types of cells.

For example, EP2455111B1 discloses a method for producing a cellular mass working as a primitive organ structure in which cells are able to coexist in undifferentiated form in the presence of differentiated cells including combinations of outer root sheath (ORS) and hair papilla cells.

These microtissues are obtained with the three-dimensional technique that contemplates the use of hanging drops plates, by simultaneously seeding the two distinct cell cultures.

The same method is also described in EP2034011 where, however, in this case in the co-culture subjected to the three-dimensional seeding, keratinocytes are present instead of the ORS. In both the aforesaid anteriorities it is not clear how the micro- tissue is composed and whether it is as similar as possible to the one of the human organ.

In fact, the Applicant has verified in the comparative example IB below that this technique inevitably leads to keratinocyte clusters formed within the microtissue, namely cells that should be contained only in the outer layer of the microtissue.

The need is felt to have a more selective method that allows obtaining a microtissue with the desired characteristics, so that it is able to mimic as much as possible the organs or part of them formed by layered tissues of different types and arranged in the order in which natural tissues are arranged.

SUMMARY OF THE INVENTION

The Applicant has found that this is possible with the method according to the invention. Object of the present invention is a method for preparing a three- dimensional scaffold-free microtissue (I), preferably having a spheroid shape, comprising:

a) a core comprising at least a first type of cells,

b) an outer layer on said core a) comprising at least a second type of cells, wherein said method is characterized in that it comprises

• forming the core a) by seeding at least a first type of cells on plates for forming three-dimensional cell cultures, • forming the outer layer b) on the core a) by seeding cells of at least a second type on the plates for forming three-dimensional cell cultures and containing the core a).

A further object of the present invention is a screening method for active substances that comprises the use of the microtissue obtained by the method of the invention. DESCRIPTION OF THE FIGURES

Figure 1 is an optical microscope (40X magnification) image obtained by staining through haematoxylin and eosin a paraffin section of the hair proto-follicle μΙΤΕ at culture day 2 prepared as described in the method of Example 1.

Figure 2 are optical microscopy images (magnification 40X) obtained for the immunostaining of the epithelial compartment of keratinocytes by the specific marker Cytokeratin 6 (CK6) in brown, at different culture times (T) of paraffin sections of the proto-follicle (μΗΡ) prepared as described in Example 1.

Figure 3 shows the proto-follicle vitality graph (μΤΠ prepared as described in Example 1 during culture by measuring the ATP content.

Figure 4 shows the graph on the different culture days of the expression of the three genes involved in maintaining the active follicle growth phase (anagen) evaluated by RTqPCR. The value of relative gene expression (RQ) is calculated with respect to dermopapilla cells in a set monolayer equal to 1. The expression values and the error. Figure 5 shows the comparison between the hair proto-follicle produced by means of a simultaneous seeding method (A), reported in the comparative example 1-A 8 days after seeding, and the one obtained by means of the sequential method (B) according to the present invention, 8 days after seeding the cells constituting the outer layer. The first two images shown in Figure 5A and in Figure 5B were taken by inverted microscope in a light field with lOx magnification and the relative detail of the surface with the arrangement of the superficial cells is indicated by the arrow. The subsequent images shown in Figures 5A and 5B are photographic images under an electron microscope for immunostaining with CK6. In particular, those shown in Figure 5A have been taken from the literature (Havlikova et al) by immunostaining. Figure 6 shows the graph of metabolic activity in the follicles prepared as described in Example IB by measuring the ATP content in MTs during the culture days. For each experimental condition, n = 5 follicles were analyzed, reporting in the graph the average content and the standard deviation of ATP (nM).

Figure 7 shows the viability of the endometrium microtissues ^endometrio) during treatment with estradiol or progesterone (10-100 nM) by measuring the ATP content. Figure 8 (A, B) shows the representative images of spheroids produced with two different ratios of keratinocytes (HHFK): fibroblasts (HHDPC) after 2 and 3 culture days respectively in Gravity TRAP. Figure 8A shows spheroids produced with 5000 HHDPC and 2500 or 500 HHFK, after 2 culture days in Gravity TRAP; Figure 8B shows spheroids produced with 5000 HHDPC and 2500 or 500 HHFK after 3 culture days in Gravity TRAP.

Figure 9 schematically shows the sequential seeding procedure followed in the case of the experimental study described in the embodiment Example No. 3; "+" corresponds to the end of the morphological/histochemical analysis; "*" corresponds to the time when the visual inspection of tissue formation was carried out, which was specifically carried out by inverted microscope.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of the present invention, the definition "comprising" does not exclude the presence of further components besides those listed after this definition, while the definitions "formed" and "made up" exclude the presence of further components besides those listed.

For the purposes of the present invention, active substance means one or more active ingredients, or a complete formulation comprising one or more active ingredients in combination with suitable excipients/eluents whose potential use is in the pharmaceutical field as a medicine or as a medical device, or in the cosmetic field. For the purposes of the present invention, outer layer b) arranged on core a) means a layer that partially or completely covers the surface of the core, depending on the physiology of the organ or part of the organ to be mimicked.

When the outer layer partially covers the core (a), this means that the uncoated core surface is between 10 and 70%, preferably between 10 and 30% of the total core surface. When the outer layer is completely covered, this means that the surface of the uncoated core is less than 1%.

The preparation method object of the present invention includes in particular the following steps:

A) seeding on two-dimensional plates a cell culture of said at least first type of cells in a specific medium for said cells and cultivating them in said medium up to reaching at least 80%, preferably 90%, confluency;

B) removing the culture of said at least first type of cells from the two-dimensional plates at the end of step A) and counting the viable cells;

C) seeding on two-dimensional plates a cell culture of said at least second type of cells in a specific medium for said cells and cultivating them in said medium up to reaching at least 80% preferably at least 90% confluency;

D) seeding the cell culture from step B) on plates for the formation of three- dimensional scaffold-free cell cultures;

E) forming the core a) of said at least first type of cells; F) removing from the two-dimensional plates the cell culture of said at least second type of cells at the end of step C) and counting the viable cells;

G) replacing part of the culture medium on the plates containing the core a), according to step E), with the cell culture of at least a second type of cells according to step F) and forming and cultivating the three-dimensional scaffold-free microtissue (I);

H) transferring the culture containing the three-dimensional scaffold-free microtissue (I) on three-dimensional collection plates suitable for the storage thereof and replacing the exhausted culture medium with maintenance culture medium. Preferably, the maintenance culture medium is the seeding culture medium used in step G).

Preferably, said maintenance culture medium contains hydrocortisone and insulin. According to an aspect of the present invention, insulin is present in concentrations ranging from 2.5 to 15 μg/ml, preferably from 5 to 10 μg/ml and according to a particularly preferred solution is 10 μg/ml.

According to a further aspect of the invention, the concentration of hydrocortisone is between 0.5 and 2.5, preferably between 1 and 2 μg/ml, more preferably 2 μg/ml. In particular, the Applicant found that when the concentration of hydrocortisone is 2 μg/ml and/or that of insulin is 10 μg/ml, the metabolic activity increases, thus extending the microtissue life.

According to a preferred embodiment, the method for preparing the three- dimensional scaffold-free microtissue (I) provides that the cells forming the core a) and the cells forming the outer layer b) are seeded with a seeding ratio (in terms of density of viable cells/well) comprised between 6: 1 and 17: 1.

More preferably, the seeding ratio between cells of the core a): cells of the outer layer b) is between 8: 1 and 13 : 1, and more preferably said seeding ratio is equal to 10: 1.

Preferably, in the method for preparing microtissues according to the present invention, the cells forming the core a) or the cells forming the outer layer b) are selected from primary cell cultures, immortalized cell lines, or cells from one or more donors obtained by biopsy.

According to another aspect of the present invention, the cells forming the core (a) and the cells forming the outer layer (b) are cells of mature tissue or undifferentiated cells, whose differentiation preferably occurs in steps A) and/or C) of the method according to the present invention.

The method object of the present invention can be carried out using the three- dimensional scaffold-free cell culture techniques, already known in the state of the art, namely the hanging drop technique, the liquid overlay technique, the micro- pattern plates technique and the magnetic aggregation technique.

The method object of the present invention is particularly suitable for the preparation of the following microtissues:

(1-1) hair proto-follicle in which the cells of the core a) are cells of human dermopapilla and the cells of the outer layer b) are keratinocytes;

(1-2) microtissue that simulates the endometrium in which the cells of the core a) are stromal cells of the human endometrium hTERT and the cells of the layer b) are immortalized epithelial cells of the human endometrium,

(1-3) microtissue that simulates human dermis with underlying fat, in which the cells of the core a) are adipocytes obtained by differentiation of preadipocytes and the cells of the core b) are fibroblasts;

(1-4) microtissue that simulates whole skin and subcutaneous fat, in which the cells of the core a) are adipocytes and fibroblasts obtained by differentiation of preadipocytes and fibroblasts, while the cells of the layer b) are keratinocytes, (1-5) microtissue that simulates the pigmented skin, in which the cells of the core a) are fibroblasts, the cells of the layer b) are a mixture of keratinocytes and melanocytes;

(1-6) microtissue that simulates the innervated dermis, in which the cells of the core a) are fibroblasts, and the cells of the layer b) are nervous cells;

(1-7) microtissue that simulates the innervated skin, in which the cells of the core a) are a mixture of fibroblasts and neuronal cells and the cells of the layer b) are keratinocytes;

(1-8) microtissue that simulates cartilage, in which the cells of the core are chondroblasts and/or fibroblasts and the cells of the outer layer b) are chondrocytes. The micro-tissue (1-1) in which the outer layer b) comprises melanocytes either alone or mixed with keratinocytes, and the microtissues (1-2) - (1-8) have not yet been disclosed, therefore they constitute a further object of the present invention.

The aforementioned microtissues preferably have the core formed by at least one first type of cells and the outer layer b) formed by at least one second type of cells, therefore characterized by the fact that the core a) is free of clusters of cells of the second type, which form the outer layer b).

Also the micro-follicle (1-1), in which the core a) is completely free of keratinocyte clusters, was obtained for the first time by the method according to the present invention.

It should be noted that, according to a preferred embodiment, the hair proto-follicle (1-1) is characterized by a seeding ratio of human dermopapilla cells (HHDPC): keratinocytes (HHFK) equal to 10: 1. Likewise, the preferred embodiment of the endometrial simulating microtissue (1-2) is characterized by a seeding ratio of stromal cells of the human endometrium hTERT : immortalized epithelial cells of the human endometrium equal to 10: 1. This seeding ratio is also applicable to other types of microtissue (1-3) - (1-8).

Advantageously, the Applicant has noted that a high seeding ratio for the cells of the core a) with respect to those of the outer layer b) allows the formation of a more compact microtissue structure, in which the cells of the outer layer b) are more adherent to those of the core a).

The tissue microstructure is therefore more organized, from a morphological point of view, and therefore more similar to the structure of the reference tissue, in vivo.

The further advantage resulting from the use of such seeding ratios is the possibility of using a reduced amount of cells in the outer layer b). This aspect is particularly useful in the case of cell types, in particular epithelial, that are particularly rare or precious, such as primary cultures of tissues difficult to find and that can be scarcely amplified in the culture.

In particular, see Example No. 3 for the experimental study of morphological changes in the hair follicle model based on different seeding ratios.

The screening method that is the object of the invention can be used to evaluate the toxicity and/or activity of the active substance.

The preclinical screening method, in addition to the steps A) - H) of the preparation method that is the object of the present invention, comprises a further step J), in which the maintenance medium of the three-dimensional scaffold-free microtissue (I) is replaced with a medium containing the active substance to be screened.

It should be noted that, preferably, the substitution of the maintenance culture medium with the medium containing the active substance is carried out at times given by a protocol, after which the medium is replaced with a fresh maintenance culture medium.

The following examples of preparation of the method for preparing the hair proto- follicle and of the endometrial microtissue are given below, together with the screening methods which contemplate the use of such microtissues.

EXAMPLE 1. Lons-term co-culture of hair follicle cells and construction of a three-dimensional model of micro-follicle nHF.

The following example describes the procedure for sequential seeding of a hair micro-follicle model (μΗΤ) and its use as an in vitro growth/regression model.

Human Hair Dermal Papilla Cells - HHDPC

Supplier: Innoprot cat. PI 0881

Reagents for 100 ml of medium HHDPC:

Mesenchymal stem cell medium kit (MSCM) Innoprot, cat. P60115 (basal culture medium + additives)

- 93 ml basal medium MSCM

- 5 ml fetal bovine serum (FBS)

- 1 ml of mesenchymal stem cell growth additive (Mesenchymal Stem Cell Growth Supplement MSCGS)

- 1 ml solution Pen/Strep (penicillin/streptomycin)

- Insulin: final concentration in the medium 5 μg/ml

- Hydrocortisone: final concentration in the medium 1 μg/ml

Cell types and related media

Human hair keratinocytes (Human Hair Follicle Keratinocytes - HHFK)

Supplier: Innoprot cat. 10885

Reagents for 100 ml of medium HHFK: Keratinocyte medium kit (KM), Innoprot, cat. P60157 (basal medium + additives):

- 98 ml keratinocyte medium (KM)

- 1 ml of keratinocytes growth supplement (Keratinocytes Growth Supplement KGS)

- 1 ml solution Pen/Strep

Medium suitable for cell defrosting and amplification

CnT Prime Culture medium, Cellntech, cat. CnT-PR

Serum-free medium, ready to use, suitable for defrosting and amplification of primary keratinocytes

Hair Proto-follicle

Co-culture of HHDPC (5000 cells each) and HHFK (500 cells each)

HHDPC Medium

Medium used for the formation of the inner layer of HHDPC

CnT Prime 3D Barrier. Cellntech. cat. CnT-PR-3D

Medium used for the formation of the outer layer of HHFK and subsequent maintenance in culture and use as a screening model of the hair proto-follicle

Production procedure of the hair proto-follicle

Cell culture

Dermo papilla human cells (human hair follicle dermal papilla, HHDPC) commercially available Innoprot cat. PI 0881 were grown in the presence of specific Innoprot medium, cat. P60157 at 37°C, 5% C0 ₂ in moisture saturation. During amplification, the cells were detached from the culture plates by means of 0.025% trypsin and re-seeded in a medium analogously to what described. The cells were cultured for a passage higher than the third P3 (up to 10-12 steps) and used for the production of microtissues or cryopreserved in specific medium containing 5% of DMSO. Human hair follicle epithelial cells (human hair follicle keratinocytes, HHFK) commercially available Innoprot cat. 10885 were grown in the presence of specific Innoprot medium, cat. P60157 at 37°C, 5% C0 ₂ in moisture saturation. During amplification, the cells were detached from the culture plates by means of 0.05% trypsin and re-seeded in a medium analogously to what described. The cells were cultured up to step 4 (P4) and used for the production of microtissues or cryopreserved in specific medium containing 5% of DMSO.

Production procedure

Example of timing for the production of microtissues of hair follicle

P = days in GRAVITY PLUS T = days in GRAVITY TRAP

For the production of the microtissues, the HHDPC cells, once reached 90% confluency, are separated by 0.025% trypsin. A vital cell count is performed by staining with trypan blue and Burker's Chamber, and the cells are seeded in a Gravity PLUS plate (InSPhero AG) at a density of 5,000 viable cells per well in 40 μΐ/well of HHDPC medium. The cells are placed in incubators under standard culture conditions (37°C, 5% C0 ₂ in moisture saturation). At day 3, after having verified the formation of a compact spheroid of HHDPC cells, it is carried out the sequential seeding of HHFK cells in Gravity PLUS. For this purpose HHFK cells at 80-90% confluency are separated by 0.05% trypsin, and vital cells are counted with Tripan blue and Burker's chamber. 20 μΐ of medium are eliminated from the Gravity PLUS plate and replaced with an equal volume of HHFK cells in CnT Prime 3D Barrier Medium at a density of 500 cells/well. The cells are put back into the incubator and grown under standard conditions (37°C, 5% CO2 in moisture saturation).

On day 6, after having verified the correct formation of a compact spheroid, the microtissues are transferred to the Gravity TRAP and cultured in 70 μΐ of CnT Prime 3D Barrier Medium.

For the culture of microtissues, the medium is renewed every two or three days eliminating 70μL of exhausted medium and renewing it with an equal amount of fresh CnT Prime 3D Barrier Medium. The described procedure allows the formation of microtissues containing two different types of cells with a sequential technique (designed to optimize a morphological-functional compartmentalization) through the hanging drop technology. This sequential procedure can be adapted in volumes and cell seeding densities to other scaffold-free spheroid forming technologies, such as e.g. lin liquid overlays or micro-pattern plates.

Application methods as a screening model

Once that the microtissues formed from two cell types by means of a sequential seeding procedure have been transferred to the Gravity TRAP collection plate, the culture medium is replaced with medium containing the active substance at the concentrations and for times set according to the experimental protocol. The treatments are performed in CnT Prime 3D Barrier Medium and the medium change is performed every 2-3 days according to the experimental protocol.

At the given time points, the microtissues can be collected and used for the following analyses:

1. viability (measurement of ATP content)

2. evaluation of gene expression by RTqPCR

3. localization of protein markers by immunohistochemistry

4. evaluation of structure and morphology using classical histochemical techniques

5. semi-quantitative analysis of proteins by Western Blot

In particular:

1. Viability (measurement of ATP content)

For the measurement of the ATP content, the luminescence kit Celltiter GLO luminescent viability kit (Promega, P7571) is used. Briefly, 3-7 microtissues are taken individually in 20 μΐ of culture medium and placed in as many wells of a 96- well luminescence plate. An equal volume of reagent is added to each microtissue and is incubated for 20 minutes at 37°C. The luminescence is read by a plate reader and the ATP content in each microtissue is calculated based on a calibration curve formed by known concentrations of ATP and expressed in nM.

2. Evaluation of gene expression by RTqPCR

For the analysis of gene expression, a pool of 12-15 microtissues per sample (experimental treatment or control) is formed by collecting the microtissues. Total RNA is extracted from the microtissue pool, retrotranscribed in cDNA and subjected to a Real Time PCR reaction for the relative evaluation of the expression of genes of interest.

3. Localization of protein markers by immunohistochemistry 4. Evaluation of structure and morphology using classical histochemical techniques A pool of 20-25 microtissues is formed for each sample (experimental or treated control). Microtissues can be included in OCT (cryomedium) and cold cut (-20°C) in sections of 5-7 μιη or fixed in formalin, pre-included in agarose and subsequently in paraffin for sectioning (5-7 μπι). Cold cut or paraffin sections can be used for morphological staining using classical histology (e.g. hematoxylin-eosin) or immunostaining (fluorescent or chromogenic).

5. Semi-quantitative analysis of proteins by Western Blot (future application)

A pool of 50 microtissues is formed for each sample (experimental or treated control). The total proteins are extracted from the microtissue pool by using specific Biorad Kits for CHEMIDOC XRS System and quantified. 20-50 μg of total proteins are submitted to a denaturing-reducing SDS page for protein separation based on molecular weight. The proteins are then transferred onto a nitrocellulose membrane and the proteins of interest are identified by staining with specific antibody and visualized by chemiluminescence. A relative quantification of the signal is carried out by semi-quantitative image analysis of the labelled protein bands.

Figure 1 shows the morphology of the hair proto-follicle (μΤΠ ⁷ ) in section. The cells of the dermopapilla (HHDPC) form a compact spheroid in the centre of the micro- tissue and are surrounded by a multilayer of keratinocytes (HHFK).

Figure 2 shows by specific staining of HHFK keratinocytes by means of CK6 marker as these cells are arranged around the compact central core of dermopapilla fibroblasts so as to mimic the structure of the hair bulb. The evaluation of CK6 expression carried out on samples at different culture times shows that there is a temporal evolution of the model in which the epithelial part of keratinocytes tends to decrease in size and colour by indicating a cycle comparable to a phase of transition growth = regression (less signal at 7 days time).

Fig. 3 shows the viability of μΗΡ during 7 days culture. As shown by the graph, the transfer of the μΗΡ in the maintenance plates increases the cell viability (T2 compared to Tl), which remains high and constant up to the day T6. At day T7, a more variable vitality could indicate the beginning of a regressive phase of the μΗΡ model.

Fig. 4 shows how the expression of the genes involved in the active follicle growth phase (BMP2 and FGF7) as well as the gene involved in the interaction between epithelial and dermal cells (LAMC3) is much more expressed in three-dimensional cultures than in cells of HHDPC dermopapilla grown in monolayer. During the culture of there is an evolution in gene expression that shows an increase between day 1 and 3 and a subsequent decrease until day 7, thus confirming the possibility of using this model as a dynamic model of transition from a phase of active proliferation to a phase of regression.

Example 1A SIMULTANEOUS METHOD vs SEQUENTIAL METHOD

Rational:

In the hair bulb the spatial organization of the cells of the various compartments is well defined and involves a precise morphology where the stromal component (dermopapilla cells) are grouped to form a compact core surrounded by the epithelial cells that form the follicle matrix and differentiate in the follicle layers (inner and outer root sheath) and in the hair stem.

For this reason, in the production of a model of hair follicle it is of particular importance to adopt a cell seeding procedure that determines as much as possible a spatial separation between the two cell types (fibroblasts and keratinocytes) and in particular determines a central core of stromal cells (fibroblasts) surrounded by a multi-stratified compartment of epithelial cells.

Two seeding procedures have been adopted for the two different cell types called:

1. Simultaneous: in which 500 keratinocytes and 5000 fibroblasts (ratio 1 : 10) were simultaneously seeded in CNT 3D Barrier medium (in order to favour the epithelial component, with greater nutritional and metabolic requirements). After 4 days of culture in Gravity PLUS, the microtissues were transferred in Gravity TRAP and cultured for a further 4 days and visualized under a light field microscope.

2. Sequential: in which 5000 dermopapilla fibroblasts were seeded in Gravity PLUS with specific medium (HHDPC) and kept in culture for 3 days in order to allow the formation of the central spheroid in the optimal culture conditions for such cells. Subsequently 500 keratinocytes were added to the Gravity PLUS culture with CNT 3D Barrier medium. The culture lasted for a further 3 days after which the microtissues were transferred to Gravity TRAP and cultured for a further 2 days (with CNT 3D Barrier Medium). The microtissues were then photographed under a light field microscope and used to evaluate their internal structure by means of histological analysis of the CK6 epithelial marker (cytokeratin 6).

As shown by the first two images of Figure 5 A and the corresponding ones of Figure 5B, which report the results of the analysis of the whole microtissue in the light field with the sequential method, it is obtained a better separation between the central compact core of fibroblasts and the superficial layer of keratinocytes, which are evenly distributed to form a light-transparent lamellar layer unlike what happens with the microtissue obtained by contemporary simultaneous seeding, where a translucent area on the surface of the spheroid is not equally well distinguished.

The morphological analysis carried out by immunostaining with cytokeratin 6, whose results are evident in the remaining images of Figures 5 A and 5B respectively, shows that this protein is exclusively present in the outer layer of the sequentially produced spheroid, while the central part is formed by a compact agglomerate of CK6-negative fibroblasts (stained in contrast with hematoxylin). There are no keratinocyte clusters within the central core as seen in the images shown in Figure 5A taken from Havlickova's 2009 work in the case of simultaneous seeding. These clusters, identified in the figure with the acronyms ORSK (Outer Root Sheath Keratinocytes) are groups of keratinocytes trapped in the fibroblast core and are visible both by ematoxylin eosin and by immunofluorescence for CK6.

These evidences clearly show that the sequential seeding of the two cell types guarantees the formation of multilayered microtissues in which there is a clear spatial division between the different cellular compartments as in the hair bulb in vivo, and in which the core does not contain cellular aggregations of keratinocytes of the outer layer b), but on the contrary the core is formed only by the cells of the first type.

EXAMPLE IB

In order to obtain a model of cultivable microtissues for an extended period of time (18 days) to better mimic or miniaturize the physiological/natural cycle of the manufactured models, the compositions of the culture media have been modified by increasing the concentrations of some hormones involved in the cell metabolism. In particular, insulin and hydrocortisone, either singly or in mixture, were used at double concentrations compared to what is already present in the medium in order to promote the absorption of the metabolites (in particular glucose), support cell metabolism and guarantee cell homeostasis.

For this purpose, a sequential seeding method previously described for microtissues formed by fibroblasts of the dermopapilla was created. In particular, 5000 micro- tissue fibroblasts were seeded in specific HHDPC medium. After 3 days in Gravity PLUS, the microtissues were transferred to the Gravity TRAP plate, the medium was renewed every 2-3 days with fresh medium containing:

• the same maintenance cell culture medium used in Example 1 (1 μg/ml of hydrocortisone (HC) and 5 μg/ml of insulin (Control -CN),

· 1 μ^πιΐ of HC and 10 μg/ml of INS (ins),

• 2 μ^πιΐ of HC and 5 g/ml of (HC),

• 2 μ^πιΐ of HC 10 μg/ml of insulin (HC + INS).

At given time intervals TO, T4, T8, Ti l, T15, T18 (where T is the culture days in Gravity TRAP), the analysis of the ATP content was established as a parameter of metabolic activity. The following table shows the preparation and maintenance

(screening) times of the microtissues stored in the four different cell culture media.

P = Days IN GRAVITY PLUS T = Days IN GRAVITY TRAP

Fig. 6 shows the content of ATP (nM) as a parameter to evaluate the metabolic activity and, consequently, the viability of the MTs.

The graph in Figure 6 shows how the first maintenance culture period (T0-T8 days) corresponds to the phase of increased metabolic activity of the dermopapilla microtissues with an increase in ATP at day 4 and an apparent stimulatory activity of the treatments compared to the control CN). From T8 to T15 there is a stabilization of the ATP content for all the experimental samples (control and those in which at least one of insulin and hydrocortisone had a doubled concentration with respect to the concentration of control hydrocortisone and/or insulin) at values lower than those seen in the previous period. At day 18 of culture, there is a further decrease in the ATP content in the control, while in the other three cases the values remain at levels comparable or slightly higher than those seen in the previous days.

It can be hypothesized that the addition of insulin and/or hydrocortisone, in concentrations higher than those of the control, to preferably double values with respect to what already present in the culture medium, can in the long term favour the absorption of metabolites and the maintenance of homeostasis, slowing the involution of the microtissue and prolonging its culture life.

EXAMPLE 2. Long-term co-culture of line cells from epithelial tissue and endometrial stromal tissue for the production of a micro endometry endometrial model

In the field of reproductive biology and its clinical applications, the study of diseases affecting the reproductive system, fertility and pregnancy, has become increasingly important. For this reason, the interest in developing predictive and versatile models is high. However, obtaining representative material for the study of the embryo implant and the interaction with the embryo-endometrium remains a challenge and arises many ethical issues since an endometrial tissue biopsy cannot be explored during the implant window without interrupting the planting process. Furthermore, the possibility of evaluating the secretion profile and the paracrine cross-talk between the uterine stroma and the epithelium is also of fundamental importance in the development of in vitro models. A spheroidal micro endometrial model is currently being developed in our laboratory using drop-feed technology. Cells are two given lines of endometrial and stromal epithelial cells (to produce multilayer spheroid in which a stromal cell core is surrounded by a layer of epithelial cells to mimic the endometrial structure.

In this model, the epithelial-mesenchymal interaction is guaranteed by the optimized co-culture that allows the "miniaturization" of the system with a low use of cells and a consequent formation of a high-speed analysis system.

T-HESCs Human Endometrium hTERT - Stromal cells

Provider: ATCC cat. ATCC-CLR-4003

Reagents for 100 ml medium for THESCs

Mixture 1 : 1 Eagle medium modified with Dulbecco medium and Ham F12

medium with 3, 1 g/L glucose and ImM of pyruvate sodium and without phenol

red (Sigma Cat# D 2906) added with 1.5 g / L of sodium bicarbonate

1% ITS+ Premix (BD Cat# 354352) 1 ml

500 ng/mL puromycin (stock 100X) 1 ml

Sodium bicarbonate l,5g/l 150 mg

10 % coal/dextran treated with fetal bovine serum (HyClone Cat# 10 ml SH30068.03),

Cell types and related medium

HEC-l-A Epithelial cells (Adenocarcinoma of the uterus)

Provider: ATCC cat. ATCC-HTB-112

Reagents for 100 ml medium for HEC1A

The basic medium for this cell line is the medium formulated as modified 90 ml ATCC medium of McCoy 5a, Catalogue No. 302007 Fetal bovine serum (FBS)

PREPARATION PROCEDURE

Cell culture

Commercially available THESCs stromal cells ATCC cat. ATCC-CLR-4003 were grown in the presence of specific medium (THESCs Medium) at 37°C, 5% C0 ₂ in moisture saturation. During amplification, the cells were detached from 0.025% trypsin mediated culture plates and seeded again in a similar medium to the one described.

Being immortalized line cells, the cells can be kept in culture for an indefinite number of passages. Working batches of cells were produced and frozen in a number of two millions per vial in complete medium with 5% of DMSO to be stored in liquid nitrogen until use. Cell culture was restarted by defrosting the cells and re-seeding them in fresh medium, maintaining the standard culture conditions. Commercially available HEC-1A (adenocarcinoma of the uterus) line epithelial cells (ATCC cat. ATCC-HTB-112) were grown in the presence of specific medium (HEC-1A Medium) at 37°C, 5% CO2 in moisture saturation. During the amplification, the cells were detached with 0.05% trypsin from the culture plates and re-seeded in a similar medium as described. Being immortalized line cells, the cells can be kept in culture for an indefinite number of passages. Working batches of cells were produced and frozen in the number of two million for vial in complete medium with 5% of DMSO to be stored in liquid nitrogen until use. Cell culture was restarted by defrosting the cells and re-seeding them in fresh medium, maintaining the standard culture conditions.

Production procedure

Example of timing for the production of MTs of micro endometrium Days 1 2 3 4 5 6 7 8 9 10

P0 PI P2 P3 P4 P5

MTs Culture

TO Tl T2 T3 T4

THESCS seeding X

HEC1A seeding X

Transfer into GravityTrap X

Medium change/ X X X

Treatment application (screening)

Control collection:

- IHC/H&E X X X - PCR

- ATP

Treated collection:

- IHC/H&E X X X - PCR

-ATP

P = days in GRAVITY PLUS T = days in GRAVITY TRAP

For the production of microtissues, the THESCs cells, once reached 90% confluency, are separated by 0.025% trypsin. A vital cell count is performed by staining with trypan blue and Burker's Chamber, and the cells are seeded in a Gravity PLUS plate (InSPhero AG) at a density of 5000 viable cells per well in 40 μΐ/well of THESCs medium. The cells are placed in incubators under standard culture conditions (37°C,

5%> C0 ₂ in moisture saturation). At day 3-4, after having verified the formation of a compact spheroid of HHDPC cells, it is carried out the sequential seeding of HEC-

1 A cells in Gravity PLUS. For this purpose, the HEC-1 A cells at 80-90%) confluency are separated by 0.05% trypsin, and the vital cells are counted with trypan blue and

Burker's chamber. 20 μΐ of medium are removed from the Gravity PLUS plate and replaced with an equal volume of HEC-1 A solution in HEC-1 A Medium at a density of 500 cells/well. The cells are put back into the incubator and grown under standard conditions (37°C, 5% C0 ₂ in moisture saturation).

At day 6-7, after verifying the correct formation of a compact spheroid, the microtissues are transferred into the Gravity TRAP and cultured in 70 μΐ of HEC-1A medium. To keep the microtissues in culture, the medium is renewed every two or three days, eliminating 70 μΐ of exhausted medium and renewing it with an equal amount of fresh HEC-1 A medium. The μ-endometers can be kept in culture for up to 10 days in the Gravity TRAP and used as a screening model for the test of active compounds. The described procedure allows the formation of microtissues containing two different types of cells with a sequential technique (designed to optimize a morphological-functional compartmentalization) through the hanging drop technology. This sequential procedure can be adapted in volumes and cell seeding densities to other scaffold-free spheroid forming technologies such as lin liquid overlays or micro-pattern plates.

The μ-endometrium model can be grown in Gravity TRAP for 10 days.

The micro endometrial product was characterized during culture by verifying the behaviour of individual cell types in the hanging fall system and their ability to aggregate in co-culture in a well-defined spatial structure, expressing specific histochemical markers for the cell (E-cadherin, F-actin and vimentin, cytokeratin-7, integrin beta-1). The stability and profitability of the model was evaluated in a time experiment up to 10 days in culture (Fig. 7) Production and treatment procedure

Example of timing for the production of micro endometrium microtissues and treatment in particular with oestrogens

P0 P3 P7

Microtissues culture

TO T3 T6

T-HESCs seeding X HEC-l-A seeding X

Transfer into Gravity Trap X

Medium change/

X X

Treatment

MTS collection:

- Histology X - PCR

Viability X

Condition analysis X X X

The micro endometrium tissues are produced as previously described. Once formed and transferred to the Gravity PLUS collection plate, the culture medium (70 μΐ is replaced with fresh medium for untreated controls or with medium containing estradiol or progesterone at a concentration of 10 and 100 nM, respectively. The medium is renewed on the third day. After 6 total days of culture in Gravity PLUS, the microtissues are collected to perform histological and gene expression analyses. Fig.7 shows the viability of the micro endometrium during oestrogen treatment for 6 days. No decrease in viability of cell metabolism (measured with ATP content) was detected for the tested concentrations of each oestrogen.

Estradiol has shown a pro-proliferative activity on the μ-endometrium that induces an increase in the protein expression Ki67 and ΠΌβΙ (immunofluorescence data), especially at physiological dose of 10 nM. At the gene expression level, its effect led to a significant reduction in the regulation of pro-inflammatory cytokine ILpi . All these data confirm that HEC-1A respond to estradiol stimulation and have a pro- proliferative profile.

Progesterone has shown an inhibitory action on proliferation, particularly on the Ki67 protein, whose expression has been reduced. The pro-secretive action of progesterone has been demonstrated at the level of gene expression by the over- expression of the pro-inflammatory cytokine Π.β1 and, presumably, also of the angiogenic factor VEGF and LIF. HEC-1 A cells are also sensitive to the stimulation of progesterone.

EXAMPLE 3. Analysis of the morphology of a model of hair follicle based on two different seeding ratios

During the development of the spheroid model consisting of a core of a first cell type and of an outer layer of a second cell type (in particular a hair follicle model with keratinocytes and fibroblasts), sequential seeding tests were performed with different keratinocytes: fibroblasts ratios in order to optimize the structure of the organoid, thus reducing the use of cells.

Experimental drawing

Tested seeding conditions: HHDPC:HHFK RATIO 10: 1 (5000:500)

2: 1 (5000:2500)

Sequential seeding procedure

The attached Figure 9 schematically shows the main steps of the seeding and cultivation protocol followed in the present study.

For sequential seeding, at day 0 HHDPC cells are seeded at the density of 5000 cells/well (well) in Gravity PLUS Plate with complete MSCM medium (Innoprot) and incubated at 37°C, 5% CO2. The spheroid formation was monitored under a microscope.

On day 3 or 4 HFD F keratinocytes were seeded at densities of 500 or 2500 cells/well (well) in CnT Prime Medium (Cell'nTech) incubated at 37°C, 5% C0 ₂ . The placement of HHFK cells on the surface of the spheroid was monitored under the microscope until day 6-7 when the microtissues were transferred into Gravity TRAP plates for culture and manipulation.

From the experiments conducted, it results that an HHDPC:FIHFK 2: 1 ratio involves an excess of epithelial cells that are not able to be evenly distributed on the surface of the inner core of fibroblasts, forming aggregates of variable form. On the other hand, a lower number of keratinocytes (1/10 compared to fibroblasts), allows the formation of an outer layer of homogeneous thickness, in which the epithelial cells are arranged as a foil and undergo morphological changes similar to a differentiation (squamous and translucent cells ) that is more accentuated at day 8 if compared to day 7.

These results have selected as the best seeding ratio between the two HHDPC:HHFK 10: 1, which optimizes the morphology of the spheroid and has the advantage of saving more epithelial cells, often available in limited amounts for experimental use. BIBLIOGRAPHIC REFERENCES

1. Higgins (2013). Microenvironmental reprogramming by 3D culture enables DP cells to induce de novo human HF growth, doi: 10.1073/pnas.1309970110

2. Havlickova (2009). A Human Folliculoid Microsphere Assay for Exploring epit-mes interaction in the HF. Journal of Investigative Dermatology 129, 972-983

3. Biernaskie (2014). HF Dermal Stem Cells regenerate the dermal sheath repopulate the dermal papilla and modulate hair type Developmental. Cell 31, 543-

558

4. Jahoda (1992). Changes in fibronectin, laminin and type IV collagen distribution relate to basement membrane restructuring during the rat vibrissa follicle hair growth cycle. J Anat; 181(Pt 1): 47-60.

5. Wong (2003). Loss of keratin 6 (K6) proteins reveals a function for intermediate filaments during wound repair J Cell Biol. 2003 Oct 27; 163(2): 327- 337.

6. Woijc (2001). Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b J Cell Biol. 2001 Aug 6; 154(3): 619-630. 7. Kishimoto (1999). Selective activation of the versican promoter by epithelial - mesenchymal interactions during hair follicle development, Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7336-41.

8. J Invest Dermatol. 2003 Dec; 121(6): 1267-75.

9. McElwee (2003). Cultured peribulbar dermal sheath cells can induce hair follicle development and contribute to the dermal sheath and dermal papilla. J Invest Dermatol. 2003 Dec; 121(6): 1267-75.

10. Qiao J (2008). Hair morphogenesis in vitro: formation of hair structures suitable for implantation. Regen Med. Sep;3(5):683-92.

11. Ohyama M (2010). The mesenchymal component of hair follicle neogenesis: background, methods and molecular characterization. Exp Dermatol. Feb; 19(2):89- 99.

12. Chao-Chun Y (2010). Review of hair follicle dermal cells. J Dermatol Sci. Jan; 57(1): 2.

13. Rahmani W (2014). Hair follicle dermal stem cells regenerate the dermal sheath, repopulate the dermal papilla, and modulate hair type. Dev Cell. Dec 8;31(5):543-58.

14. Krikun et al: A novel immortalized human endometrial stromal cell line with normal progestational response. Endocrinology, 2004 May; 145(5):2291-6. Epub 2004 Jan 15

15. Krikun et al: Endometrial angiopoietin expression and modulation by thrombin and steroid hormones: a mechanism for abnormal angiogenesis following long-term progestin-only contraception. Am J Pathol, 2004 Jun; 164(6):2101-7 16. Wang et al: A novel model of human implantation: 3D endometrium-like culture system to study attachment of human trophoblast (Jar) cell spheroids Mol Hum Reprod, 2012 Jan; 18(l):33-43. doi: 10.1093/molehr/gar064. Epub 2011 Oct 11

17. Way et al: Characterization of a new human endometrial carcinoma (RL95-2) established in tissue culture. In Vitro, 1983 Mar; 19(3 Pt 1): 147-58

18. Chen et al. Cocultivating human endometrial epithelial cells and stromal fibroblasts alters cell-specific gene expression and cytokine production. Fertil Steril 2013 Oct; 100(4): 1132-43

19. Patel et al: A role for two-pore Potassium (K2P) channels in endometrial epithelial function. J Cell Mol Med 2013 Jan; 17(1): 134-46.

20. Heneweer et al: Adhesiveness of human uterine epithelial RL95-2 cells to trophoblast: Rho protein regulation . Mol Hum Reprod 2002; 8(11): 1014-1022

21. Arnold et al: Endometrial stromal cells regulate epithelial cell growth in vitro: a new co-culture model. Hum. Reprod. (2001) 16 (5): 836-845.