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WO2019092667A1 - Méthode de préparation de microtissus tridimensionnels exempts d'échafaudage destinés à être utilisés dans le criblage préclinique de substances actives - Google Patents

Méthode de préparation de microtissus tridimensionnels exempts d'échafaudage destinés à être utilisés dans le criblage préclinique de substances actives Download PDF

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WO2019092667A1
WO2019092667A1 PCT/IB2018/058870 IB2018058870W WO2019092667A1 WO 2019092667 A1 WO2019092667 A1 WO 2019092667A1 IB 2018058870 W IB2018058870 W IB 2018058870W WO 2019092667 A1 WO2019092667 A1 WO 2019092667A1
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cells
core
microtissue
medium
fibroblasts
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Elisa Caviola
Marisa MELONI
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Vitroscreen S.R.L.
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0627—Hair cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/092—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells hair cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/04—Screening or testing on artificial tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2513/00—3D culture

Definitions

  • TITLE "Method for preparing three-dimensional scaffold-free microtissues for use in the preclinical screening of active substances"
  • 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.
  • cellular aggregates defined as microtissues or organlike spheroids
  • 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.
  • 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.
  • 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.
  • a possible three-dimensional microtissue forming technique is the technique that employs micro-pattern plates or the magnetic aggregation technique.
  • 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.
  • 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.
  • ORS outer root sheath
  • 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 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.
  • Object of the present invention is a method for preparing a three- dimensional scaffold-free microtissue (I), preferably having a spheroid shape, comprising:
  • a core comprising at least a first type of cells
  • 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 ⁇ 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 ( ⁇ ) prepared as described in Example 1.
  • Figure 3 shows the proto-follicle vitality graph ( ⁇ 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.
  • 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.
  • those shown in Figure 5A have been taken from the literature (Havlikova et al) by immunostaining.
  • 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.
  • HHFK keratinocytes
  • HHDPC fibroblasts
  • 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.
  • 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.
  • 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.
  • 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.
  • the uncoated core surface is between 10 and 70%, preferably between 10 and 30% of the total core surface.
  • the surface of the uncoated core is less than 1%.
  • the preparation method object of the present invention includes in particular the following steps:
  • step 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;
  • step 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);
  • the maintenance culture medium is the seeding culture medium used in step G).
  • said maintenance culture medium contains hydrocortisone and insulin.
  • insulin is present in concentrations ranging from 2.5 to 15 ⁇ g/ml, preferably from 5 to 10 ⁇ g/ml and according to a particularly preferred solution is 10 ⁇ g/ml.
  • the concentration of hydrocortisone is between 0.5 and 2.5, preferably between 1 and 2 ⁇ g/ml, more preferably 2 ⁇ g/ml.
  • the Applicant found that when the concentration of hydrocortisone is 2 ⁇ g/ml and/or that of insulin is 10 ⁇ g/ml, the metabolic activity increases, thus extending the microtissue life.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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;
  • 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;
  • 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 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).
  • 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.
  • the hair proto-follicle (1-1) is characterized by a seeding ratio of human dermopapilla cells (HHDPC): keratinocytes (HHFK) equal to 10: 1.
  • HHDPC human dermopapilla cells
  • HHFK keratinocytes
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 ( ⁇ ) and its use as an in vitro growth/regression model.
  • MSCM Mesenchymal stem cell medium kit
  • P60115 basic culture medium + additives
  • FBS fetal bovine serum
  • mesenchymal stem cell growth additive Mesenchymal Stem Cell Growth Supplement MSCGS
  • Pen/Strep penicillin/streptomycin
  • Human hair keratinocytes Human Hair Follicle Keratinocytes - HHFK
  • Keratinocytes Growth Supplement KGS Keratinocytes Growth Supplement
  • Serum-free medium ready to use, suitable for defrosting and amplification of primary keratinocytes
  • Dermo papilla human cells human hair follicle dermal papilla, HHDPC
  • Innoprot cat. PI 0881 were grown in the presence of specific Innoprot medium, cat. P60157 at 37°C, 5% C0 2 in moisture saturation.
  • 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.
  • HHFK human hair follicle keratinocytes
  • the HHDPC cells 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 ⁇ /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.
  • Gravity PLUS InSPhero AG
  • 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 ⁇ 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).
  • the microtissues are transferred to the Gravity TRAP and cultured in 70 ⁇ of CnT Prime 3D Barrier Medium.
  • the medium is renewed every two or three days eliminating 70 ⁇ L 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.
  • 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.
  • microtissues can be collected and used for the following analyses:
  • the luminescence kit Celltiter GLO luminescent viability kit (Promega, P7571) is used. Briefly, 3-7 microtissues are taken individually in 20 ⁇ 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.
  • a pool of 12-15 microtissues per sample 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.
  • Microtissues can be included in OCT (cryomedium) and cold cut (-20°C) in sections of 5-7 ⁇ or fixed in formalin, pre-included in agarose and subsequently in paraffin for sectioning (5-7 ⁇ ).
  • Cold cut or paraffin sections can be used for morphological staining using classical histology (e.g. hematoxylin-eosin) or immunostaining (fluorescent or chromogenic).
  • 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 ⁇ g 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 ( ⁇ 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.
  • Fig. 3 shows the viability of ⁇ during 7 days culture. As shown by the graph, the transfer of the ⁇ 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 ⁇ 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.
  • BMP2 and FGF7 the genes involved in the active follicle growth phase
  • LAMC3 epithelial and dermal cells
  • 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.
  • ORSK Outer Root Sheath Keratinocytes
  • compositions of the culture media have been modified by increasing the concentrations of some hormones involved in the cell metabolism.
  • 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.
  • microtissues formed by fibroblasts of the dermopapilla were created.
  • 5000 micro- tissue fibroblasts were seeded in specific HHDPC medium.
  • the microtissues were transferred to the Gravity TRAP plate, the medium was renewed every 2-3 days with fresh medium containing:
  • 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.
  • 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
  • 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.
  • ATCC cat Provider: ATCC cat. ATCC-CLR-4003
  • HEC-l-A Epithelial cells (Adenocarcinoma of the uterus)
  • ATCC cat Provider: ATCC cat. ATCC-HTB-112
  • 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)
  • 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.
  • specific medium THESCs Medium
  • 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.
  • 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.
  • the THESCs cells 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 ⁇ /well of THESCs medium. The cells are placed in incubators under standard culture conditions (37°C,
  • 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
  • the microtissues are transferred into the Gravity TRAP and cultured in 70 ⁇ of HEC-1A medium.
  • the medium is renewed every two or three days, eliminating 70 ⁇ of exhausted medium and renewing it with an equal amount of fresh HEC-1 A medium.
  • the ⁇ -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 ⁇ -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
  • the micro endometrium tissues are produced as previously described. Once formed and transferred to the Gravity PLUS collection plate, the culture medium (70 ⁇ 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 ⁇ -endometrium that induces an increase in the protein expression Ki67 and ⁇ (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 ⁇ . ⁇ 1 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
  • 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.
  • the attached Figure 9 schematically shows the main steps of the seeding and cultivation protocol followed in the present study.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne une méthode de préparation d'un microtissu sans échafaudage tridimensionnel (I), de préférence ayant une forme sphéroïdale, comprenant : a) un cœur comprenant au moins un premier type de cellules, b) une couche externe sur ledit cœur a) comprenant au moins un second type de cellules, ladite méthode consistant à : • former le cœur a) par ensemencement d'au moins un premier type de cellules sur des plaques pour former des cultures cellulaires tridimensionnelles, • former la couche externe b) sur le cœur a) par ensemencement de cellules d'au moins un second type sur les plaques pour former des cultures cellulaires tridimensionnelles contenant le coeur a). Le microtissu obtenu par la méthode ci-dessus est utilisé pour effectuer un criblage préclinique de substances actives.
PCT/IB2018/058870 2017-11-10 2018-11-12 Méthode de préparation de microtissus tridimensionnels exempts d'échafaudage destinés à être utilisés dans le criblage préclinique de substances actives WO2019092667A1 (fr)

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WO2022155708A1 (fr) * 2021-01-22 2022-07-28 Skin2Neuron Pty Ltd Procédés de fabrication de précurseurs neuronaux

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EP2455111A2 (fr) * 2006-06-27 2012-05-23 Shiseido Company, Limited Groupe de cellules renfermant des types diversifiés de cellules dérivées du soma, capables de former une structure primitive de type organique
WO2013014435A1 (fr) * 2011-07-27 2013-01-31 University Of Durham Microorgane comprenant des cellules mésenchymateuses et épithéliales
FR3041656A1 (fr) * 2015-09-29 2017-03-31 Oreal Utilisation des cellules de la matrice pour la preparation d'un microfollicule pileux

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EP2455111A2 (fr) * 2006-06-27 2012-05-23 Shiseido Company, Limited Groupe de cellules renfermant des types diversifiés de cellules dérivées du soma, capables de former une structure primitive de type organique
WO2013014435A1 (fr) * 2011-07-27 2013-01-31 University Of Durham Microorgane comprenant des cellules mésenchymateuses et épithéliales
FR3041656A1 (fr) * 2015-09-29 2017-03-31 Oreal Utilisation des cellules de la matrice pour la preparation d'un microfollicule pileux

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WANG H ET AL: "A novel model of human implantation: 3D endometrium-like culture system to study attachment of human trophoblast (Jar) cell spheroids", MOLECULAR HUMAN REPRODUCTION., vol. 18, no. 1, 11 October 2011 (2011-10-11), pages 33 - 43, XP055496408, ISSN: 1360-9947, DOI: 10.1093/molehr/gar064 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022155708A1 (fr) * 2021-01-22 2022-07-28 Skin2Neuron Pty Ltd Procédés de fabrication de précurseurs neuronaux

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