FR2876246A1 - Producing regenerated human bronchiolar epithelium uses cells implanted as xenograft in non-human mammal, e.g. rat - Google Patents

Abstract

Producing a regenerated human bronchiolar epithelium in one or more layers comprising implanting pathological or non-pathological epithelial cells as a xenograft in a non-human mammal such as a rat, is new. Producing a regenerated human bronchiolar epithelium in one or more layers comprises implanting pathological or non-pathological epithelial cells as a xenograft in a non-human mammal such as a rat. The graft is taken from the trachea of a first non-human mammal such as a rat and implanted in a second non-human mammal, such as a xeno-tolerant mouse, by isolating bronchioles from human lung tissue, separating the epithelial cells bordering the bronchioles and growing them in a culture medium to produce a confluent culture. At the same time, the trachea or other support is dissected from an animal such as a rat, is treated to eliminate living cells and is coated with the cultured cells to produce a graft that can be implanted under the skin of a second, xeno-tolerant mammal, such as a mouse, to produce partial or total bronchiolar epithelium regeneration. ACTIVITY : CNS-Gen.; Respiratory-Gen.; Antiinflammatory. No biological data given. MECHANISM OF ACTION : Cell Therapy.

Description

Method and model for obtaining regenerated human bronchiolar epithelium

  The present invention provides a non-human mammalian model comprising a xenograft comprising bronchiolar epithelial cells of human origin, capable of regenerating human bronchiolar epithelium, and an implant comprising or consisting of human bronchiolar epithelium seeded on a trachea or other carrier of a non-human mammal.

  The invention also relates to a method for obtaining a non-human mammalian model comprising a regenerated human bronchiolar epithelium, a method for obtaining regenerated human bronchiolar epithelium, a method for screening pharmacological compounds of interest in the field of respiratory pathologies, a method for studying the regeneration of the bronchiolar epithelium, the use of the non-human mammal model of the invention or the implant according to the invention for the study of pluripotent cells, and a method for evaluating the transfection of polynucleotides within the bronchiolar epithelium of the non-human mammalian model according to the invention.

  The normal bronchiolar human epithelium is monostratified, consisting of basal cells, hair cells, secretory cells like Clara cells and some secretory cells of the mucous type (Ebert and Terracio: The bronchiolar epithelium in cigarette smokers. electron microscope, Am.Rev. Respir.Fis., 1975, 111 (1): 4-11).

  In many human respiratory pathologies, of genetic origin such as cystic fibrosis or, acquired as chronic obstructive pulmonary disease, chronic bronchitis or bronchiolitis, which affect the most distal airways, called bronchioles, we are witnessing lesions and symptoms. reshaping the respiratory epithelium that borders these airways.

  These rearrangements and lesions may result in a change in the epithelial structure, loss of integrity of the epithelial barrier associated with partial or total desquamation of the epithelium, which is particularly true for bronchiolar distal airways. . In this type of pathological respiratory tissue, the bronchiolar human epithelium becomes pseudostratified and exhibits hyperplasia of the secretory cells of the mucosal type (Hubea et al: Quantitative analysis of inflammatory cells infiltrating the cystic fibrosis airway mucosa, Clin Imm Immolate, 2001). , 124 (1): 69-76).

  These rearrangements and lesions lead to a loss of functions (antioxidant, ion and water transport, anti-bacterial ...) of the respiratory epithelium which must regenerate to restore its functions.

  Dupuit et al have developed a model of humanized bronchial xenograft, the bronchial epithelium being very accessible and rich in cells (Dupuit et al: Differentiated and functional human airway epithelium regeneration in tracheal xenografts, Am J Physiol Lung Cell. Mol Physiol 278: LI 65-L176, 2000).

  Respiratory epithelium of the bronchiolar type, as opposed to the bronchial epithelium, is very inaccessible because of its localization, and very little rich in cells by the diameter of the bronchioles (1 mm in diameter).

  Bronchiolar cells have already been isolated from bronchioles and cultured to examine ionic fluxes therethrough (Blouquit et al., Characterization of ion and fluid transport in human bronchioles, Am J Respir Cell mol Biol., Vol. 27, pp. 503-510, 2002). The conditions of the cultivation carried out (maintenance of the cells in their initial state of differentiation) did not make it possible to envisage the study of the modifications of the state of these cells, in particular interesting modifications within the framework of the definition of the regeneration of the cells. a bronchiolar epithelium.

  There is currently no cellular model for preparing a human bronchiolar epithelium and, subsequently, to study the conditions (for example the kinetics) of its regeneration.

  The inventors have carried out numerous studies in order to obtain an in vivo chimeric model capable of regenerating a human bronchiolar epithelium, allowing, on the one hand, studies, including pre-clinical studies, of pharmacological molecules that can be used in respiratory pathologies involving the bronchiolar distal airways and secondly, the study of the regeneration of human bronchiolar epithelium from pathological cells or from normal cells (ie characteristics of a healthy tissue).

  The invention relates to a non-human mammal model comprising a xenograft comprising bronchiolar epithelial cells of human origin, capable of regenerating a human bronchiolar epithelium.

  The graft carried out to obtain the animal model according to the invention is a xenograft inasmuch as the grafted cells are of human origin while the model is a non-human animal (excluding humans) xenotolerant. According to a particular embodiment of the invention, the cells are grafted using a carrier, which may be of animal origin.

  This non-human mammal model makes it possible not only for the complete regeneration of the normal human bronchiolar epithelium from grafted bronchiolar cells, but also for the reproduction of the kinetics of the events leading to this regeneration, in particular a intermediate stage of the regeneration process with human bronchiolar epithelium similar to pathological human bronchiolar epithelium.

  In a specific embodiment of the invention, human bronchiolar epithelium is obtained from pathological epithelial bronchiolar cells. The model is then prepared from bronchioles derived from tissues of patients with diseases such as cystic fibrosis, chronic obstructive pulmonary disease, chronic bronchitis or bronchiolitis.

  In another specific embodiment of the invention, the human bronchiolar epithelium is obtained from non-pathological, so-called normal or healthy, bronchial epithelial cells.

  The cells are obtained by a dissociation step from bronchioles taken from tissues of human pulmonary pieces in areas remote from the pathological tissue, most often tumor.

  The term "pathological cells" is intended to denote cells which are differentiated and which come from pathological tissues, in particular from a respiratory epithelium of a patient affected by any respiratory disease. Non-pathological or normal cells are differentiated and come from healthy tissues.

  By the term epithelium is meant a vascularized or non-vascularized tissue consisting of one or more layers of cells adhered to each other.

  The invention makes it possible to carry out the various stages of the regeneration of the bronchiolar epithelium and consequently, a non-human mammalian model according to the invention is also characterized by the stage of regeneration of the epithelium, especially as a function of time. elapsed since the establishment of the graft and / or, if appropriate, the treatment of said graft with substances whose effect on the bronchiolar epithelium is tested.

  In a specific example of the invention, the regenerated human bronchiolar epithelium of the model is monostratified. The term monostratified epithelium denotes a normal or healthy epithelium having differentiated cells, notably some basal cells, numerous hair cells, some secretory cells of the mucous type and of Clara cell type.

  In a specific example of the invention, the regenerated human bronchiolar epithelium of the model is pseudostratified. The term pseudostratified epithelium refers to an epithelium that is histologically similar to the modified bronchiolar human epithelium observed during various respiratory pathologies affecting the bronchiolar airways. This epithelium has differentiated cells, including quite a number of basal and secretory mucosal cells, some Clara cells and many hair cells.

  In a specific example of the invention, the regenerated human bronchiolar epithelium of the model is pluristratified. The term "pluristratified epithelium" refers to an epithelium composed of undifferentiated cells, in particular composed of a few layers of cubic cells covered with a layer of flattened cells.

  The cell types present in the epithelium at the different stages of its regeneration are characterized by known cell markers (see examples below).

  The xenograft model according to the invention can be obtained by grafting the trachea or other support of a first non-human mammal in a second non-human mammal, said trachea or other support serving as support for the bronchiolar cells of origin. and providing these cells with appropriate matrix factors to contribute to the proliferation and differentiation of cells, such as, for example, basal lamina proteins such as collagens or laminins. Alternatively the trachea can be replaced by other supports, esophagus segments or deepithelialized intestine.

  Prior to their seeding on the trachea (or on another adapted support), the cells are cultured under conditions allowing their proliferation. Thus, during their seeding on the trachea (or other support), the bronchiolar cells are in essentially dedifferentiated forms.

  The first mammal used to make this model may be a rodent, for example a Wistar rat or a Fisher rat. The second mammal may be a xenotolerant rodent, for example a xenotolerant mouse, such as a NUDE mouse or SCID mouse.

  By xenotolerant mammal or xenotolerant rodent is meant a mammal or a rodent capable of receiving foreign or heterologous cells or tissues, without rejection of this graft.

  The invention also relates to an implant comprising or consisting of a human bronchiolar epithelium seeded on a trachea or other support of a non-human mammal, obtainable from a non-human animal model according to the invention. invention.

  The trachea (or other support) can be mounted on a device and maintained by ligatures. The rat tracheal device assembly may be subject to freeze thawing cycles.

  The device is for example a catheter.

  The bronchiolar human cells are inoculated into the assembly constituted by the trachea (or other appropriate support) by injecting the cells in the device located in the immediate vicinity of the trachea (or other support).

  The invention also relates to a method for obtaining a non-human mammal model capable of regenerating a human bronchiolar epithelium comprising the following steps: a) isolation of bronchioles from human lung parts, b) dissociation of epithelial cells bronchiolar bordering said bronchioles, c) seeding of said bronchiolar cells and culture in an appropriate culture medium allowing their proliferation to obtain a confluent culture, d) in parallel with step c), dissection of a tracheae or other support of an animal, for example a rodent, treatment for eliminating living cells and mounting on a device, e) after detachment, seeding said bronchiolar cells in the trachea or in another support of the animal, f) grafting the assembly formed of the device and the trachea or other support under the skin of a xenotolerant animal, for example a rodent, the animal for a period of time allowing total or partial regeneration of the bronchiolar epithelium.

  In a specific example of the invention, the culture medium used in step c) makes it possible to obtain a proliferation of bronchiolar epithelial cells.

  The culture medium used in step c) may comprise proliferation factors, in particular Epidermal Growth Factor (EGF), bovine pituitary extract, epinephrine and 3, 3 ', 5- triido-Lthyronine.

  In particular, the culture medium for the proliferation of epithelial cells results in a dedifferentiation of these cells.

  In a specific example of the invention, the cells of step c) are seeded at very low density. For example, the cells are seeded at a density of between 13,000 and 70,000 cells per cm 2, preferably between 20,000 and 40,000 cells per cm 2.

  The device has the following characteristics: hollow tubing, sterile, partially flexible, that is to say except at the junction area of the trachea (or other support) which must be more rigid.

  The device is for example on a catheter.

  The following examples describing the use of a trachea and a catheter can be provided respectively for any type of support replacing the trachea, and for any type of device replacing the catheter adapted to the operation of steps d) to g) of the process according to the invention.

  The animal used in steps f) and g) may be a rodent, in particular a mouse, for example a NUDE or SCID mouse. The animal used in steps d), e) and f) can be a rodent, in particular a rat, for example a Wistar rat or a Fisher rat.

  According to a particular embodiment of the invention, the step of isolating bronchioles is carried out by microdissection. The step consists of micro-dissecting the human bronchioles, for example from surgical lung resections under binocular loupes.

  The dissociation step of the epithelial cells can be carried out by enzymatic process.

  According to a particular embodiment of the invention, step d) comprises steps of freezing, thawing, after dissection of the rat trachea or other support, which is then mounted on the device and then refrozen.

  According to a particular embodiment of the invention, step e) comprises a step of thawing the rat trachea or other support before inoculation of bronchiolar cells therein.

  In a specific embodiment of the invention, human bronchiolar epithelium is obtained from pathological epithelial bronchiolar cells. The model is then prepared from bronchioles derived from tissues of patients with diseases such as cystic fibrosis, chronic obstructive pulmonary disease, chronic bronchitis or bronchiolitis.

  In another specific embodiment of the invention, the human bronchiolar epithelium is obtained from non-pathological, so-called normal or healthy, bronchial epithelial cells.

  The invention also relates to a method for obtaining regenerated human bronchiolar epithelium comprising the following steps: a) obtaining a non-human mammal model capable of regenerating a human bronchiolar epithelium according to the invention, b) recovery of the assembly formed by the device and the trachea or other support carrying the regenerated epithelium under the skin of the animal; c) optionally recovery of the regenerated human bronchiolar epithelium.

  This method of obtaining regenerated human bronchiolar epithelium comprises the same steps a) to g) as the method for obtaining a non-human mammalian model comprising a regenerated human bronchiolar epithelium cited above. All remarks made about the method of obtaining the model are applicable to the process for obtaining the epithelium concerning steps a) to g).

  The invention also relates to a method for screening pharmacological compounds of interest in the field of respiratory pathologies comprising the following steps: contacting the non-human mammal model according to the invention or the implant according to the invention comprising or consisting of a human bronchiolar epithelium regenerated with a determined pharmacological compound, - detecting the change in the state of said epithelium.

  The pharmacological molecules of interest are, for example, anti-inflammatory, cytoprotective and pro-regenerative molecules of the epithelium.

  The invention also relates to a method for screening pharmacological compounds of interest in the field of respiratory pathologies comprising the following steps: contacting the pathological bronchiolar epithelium from the non-human mammalian model with a specific pharmacological compound, - Detecting the change in the state of said epithelium.

  The subject of the invention also relates to a method of studying the regeneration of the bronchiolar epithelium comprising the following steps: contacting the non-human mammal model according to the invention or the implant according to the invention invention comprising or consisting of a human bronchiolar epithelium regenerated with a specific pharmacological compound, - detecting the change in the state of said epithelium.

  The subject of the invention also relates to a method for studying the regeneration of the bronchiolar epithelium comprising the following steps: the determined pharmacological compound is brought into contact with the bronchiolar epithelium from the non-human mammalian model. The second step concerning the detection of the modification of the state of said epithelium is identical.

  In a specific example of the invention, the bronchiolar epithelium is monostratified.

  In another specific example of the invention, the bronchiolar epithelium is pseudostratified.

  In another specific example of the invention, the bronchiolar epithelium is pluristratified.

  The subject of the invention also relates to the use of the non-human mammal model of the invention or of the implant according to the invention for the study of pluripotent cells.

  The invention also relates to a method for evaluating the transfection of polynucleotides within the bronchiolar epithelium of the non-human mammalian model according to the invention, comprising: the transfection of the cells of the bronchiolar epithelium with a determined polynucleotide the observation of the effects of the transfection carried out on the cells of the epithelium.

  Other features and advantages of the invention appear in the examples and figures which follow.

LEGEND OF FIGURES

  Figure 1: catheter consisting of an elastic tubing (inner diameter 0.66 mm, outer diameter 1.65 mm) and a polyethylene tubing (internal diameter, 14 mm, outer diameter 1.57 mm), nested one in the other.

  Figure 2: thawed rat trachea mounted on the catheter and maintained by ligatures and tissue glue.

  Figure 3: bronchiolar human cells inoculated into the assembly by injecting the cells into the catheter located in the immediate vicinity of the trachea.

  Figure 4: graft montages on a NUDE mouse. an incision is made behind each of the ears of the mouse. Using a cannula inserted into the incision, we take off the skin from the back of the mouse. An assembly is inserted in each incision. The incisions are then sutured using non-surgical suture. Each mouse carries two grafts.

  Figure 5a: Bronchiolar epithelium in xenograft after 4 days.

  Figure 5b: Bronchiolar epithelium in xenograft after 13 days (pluristratified epithelium).

  Figure 5c: Bronchiolar epithelium in the xenograft after 25 to 35 days (pseudostratified epithelium).

  Figure 5d: Bronchiolar epithelium in the xenograft after 35 to 45 days (monostratified epithelium).

  Figure 6a: CK13 basal cell marker.

  Figure 6b: CK18 marker of cylindrical cells (hair cells, mucous membranes and Clara.

  Figure 6c: CCI O Clara cell marker.

  Figure 6d: MUC 5AC mucosal cell marker. EXAMPLES Example 1: microdissection of human bronchioles.

  The method consisted in microdisscribing the human bronchioles resulting from surgical pulmonary resections under binocular loupes.

  Example 2: Dissociation of bronchiolar epithelial cells.

  The bronchioles were then maintained in DMEM / Ham F12 culture medium (Gibco BRL) supplemented with penicillin (100 U / mL), streptomycin (100 μg / mL), gentamycin (50 μg / mL) and amphotericin B (2.5 μg / ml) (Gibco BRL) until epithelial cell dissociation.

  For the dissociation of the epithelial cells, the bronchioles were incubated for 16 hours at 4 C in RPMI / HEPES medium (Gibco, BRL) supplemented with 0.1% of pronase E (Sigma Aldrich). At the end, the bronchioles were manually shaken in the dissociation medium. The action of pronase E was stopped by the addition of 10% fetal calf serum (Gibco BRL). The remains of bronchioles were removed with sterile forceps and the supernatant was centrifuged for 5 minutes at 250 g. The cell pellet was then resuspended and incubated for 5 minutes at 37 ° C. in a solution of PBS (Phosphate Buffered Saline, pH 7.4, devoid of Cal + and Mg 2+, Gibco BRL) containing 25% of 2.5% trypsin. (Sigma Aldrich) and 25% versene (solution consisting of PBS containing 8.5 g / L of NaCl (sodium chloride, Sigma Aldrich) and 2 g / L of EDTA (ethylene-diamino-tetraacetic, Sigma). of the solution was stopped by addition of 10% fetal calf serum The supernatant was centrifuged for 5 minutes at 250 g.

  Example 3: Inoculation of bronchiolar cells in an appropriate culture medium allowing their proliferation until a confluent culture is obtained.

  The cell pellet was then resuspended and inoculated into culture medium consisting of 75% DMEM medium (Gibco BRL) and 25% F12 medium (Gibco BRL) supplemented with 0.87 μM bovine insulin (Sigma Aldrich), 65 nM human transferrin (Sigma Aldrich), 1.6 nM EGF (Epidermal growth factor) recombinant human (Sigma Aldrich), 1.38 pM hydrocortisone (Sigma Aldrich), 30 nM retinyl acetate (Sigma Aldrich), 9.7 nM of 3, 3 ', 5-triiodo-L-thyronine (Sigma Aldrich), 2.7 μM epinephrine (Sigma Aldrich), 35 μg / ml bovine pituitary extract (Sigma Aldrich), 5 μM ethanolamine (Sigma Aldrich), pM o-phosphorylethanolamine (Sigma Aldrich), 30 nM sodium selenite (Sigma Aldrich), 1 nM manganese chloride tetrahydrate (Sigma Aldrich), 0.5 μM sodium metasilicate nonahydrate (Sigma Aldrich), 1 nM ammonium molybdate tetrahydrate (Sigma Aldrich), 5 nM ammonium vanadate (Sigma Aldrich), 1 nM nickel sulfate hexahydrate (Sigma Aldrich), 0.5 nM stannous c hydrochloride dihydrate (Sigma Aldrich), 100 U / ml penicillin and 100 μg / ml streptomycin (Gibco BRL) as well as 10% fetal calf serum. The cells are then cultured in a 37 C incubator in the presence of 5% CO 2. After 24 hours, the medium was changed and replaced by the same medium but devoid of fetal calf serum. The culture was continued in this environment, with a renewal of the medium every three days, until a confluent culture was obtained.

  Example 4: Dissection of a rat trachea and mounting on a catheter.

  In parallel, the trachea of male Wistar rats (220-250 g, Charles River) was collected after sacrifice of the animals, and frozen at -80 c. A catheter was manufactured: it consists of elastic tubing (internal diameter 0.76 mm, outer diameter 1.65 mm, Silastic) and polyethylene tubing (internal diameter 1.14 mm, external diameter 1.57 mm, Aubry establishment), nested one inside the other according to FIG.

  The rat trachea was then thawed and mounted on the catheter where it was held by ligations (non-resorbable braided silk thread, Look Suture) and tissue glue (Histoacryl, B Braun Medical) according to Figure 2.

  The whole was then refrozen at -80c until use.

  Example 5: Detachment of the cells and seeding in the rat trachea.

  When the culture of human bronchiolar cells was confluent, the cells were rinsed twice with PBS devoid of Cal + and Mg 2 +, and then detached with a trypsin / EDTA solution (0.5 g of trypsin and 0.2 g of EDTA per liter of PBS, Gibco BRL). The detached cells were taken up in DMEM / Ham F12 medium containing 10% FCS, then counted, centrifuged at 250 g for 5 minutes and then resuspended at a rate of 12.5 million cells / mL of DMEM / Ham F12 medium containing 10% of fetal calf serum.

  The rat tracheal catheter + set assembly was thawed in DMEM / Ham F12 medium. DMEM / Ham F12 medium was passed three times through the assembly using a 1 ml syringe with a 21 Gx11'2 (0.8x40 mm, Terumo) needle to remove debris from the body. the epithelium of the rat trachea that peeled after both freeze / thaw cycles. The assembly was then emptied of its liquid contents by air injection.

  The bronchiolar human cells were then inoculated into the assembly at a rate of 80 μL of cell suspension by assembly, injecting the cells into the catheter located in the immediate vicinity of the trachea, according to FIG.

  The ends of the catheters were then closed. The whole was kept in DMEM / Ham F12 medium for 2 hours in a 37 c incubator in the presence of 5% CO 2, returning the assembly every 15 minutes.

  Example 6: Grafting of the assembly formed of the catheter and the trachea under the skin of a mouse.

  Female NUDE mice (nu / nu, 8 weeks, Charles River), were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg / kg, Centravet). The montages were then grafted: an incision was made behind each of the ears of the mouse. Using a cannula inserted into the incision, the skin of the back of the mouse was peeled off. A montage was inserted into each of the incisions. The incisions were then sutured using nonabsorbable surgical suture (black monofilament polyamide mounted on triangular vectral 3/8 circle and 16 mm triangular needle, Ethicon). Each mouse had two grafts (Figure 4).

  Example 7 Sacrifice of the mice and analysis of the bronchiolar human epithelium.

  The mice were raised in a sterile atmosphere until sacrifice, excision of the graft and analysis of the bronchiolar human epithelium which borders the rat trachea.

  After 4, 13, 25 and 35 days of transplantation, mice are sacrificed by pentobarbital sodium surdose and excised grafts.

Example 8: Histology of grafts.

  After 4 days of transplantation, a monolayer of undifferentiated flattened cells was observed. After 13 days of grafting, a pluristratified epithelium consisting of a few layers of cubic cells covered with a layer of flattened cells was observed. After 25 to 35 days of transplantation, a pseudostratified epithelium with differentiated cells was observed: quite a few basal and secretory mucosal cells, some Clara cells and many hair cells were present. After 35 to 45 days of transplantation, an epithelium with a phenotype comparable to that of a normal native epithelium was observed. This differentiated epithelium had some basal cells, many hair cells, some mucosal secretory cells and Clara cells.

  It is important to note that between 25 and 35 days following the graft, the epithelium present in the grafts is histologically similar to the reworked bronchiolar human epithelium observed during various bronchiole-type respiratory pathologies.

  It is important to note that between 35 and 45 days following the graft, the epithelium present in the grafts is histologically similar to the normal bronchiolar human epithelium (Figures 5a, 5b, 5c and 5d).

  Example 9 Immunohistochemical Characterization of Grafts.

  The presence of the different cell types constituting the bronchiolar human epithelium was evaluated by immunofluorescent staining with mouse monoclonal antibodies directed against cytokeratin 13 (Sigma Aldrich) showing the intermediate filaments of the cytoskeleton of the basal cells, the mouse monoclonal antibodies. directed against cytokeratin 18 (Sigma Aldrich) showing the intermediate filaments of the cytoskeleton of the cylindrical cells (secretory cells of the mucous or Clara type, and hair cells), polyclonal rabbit antibodies directed against the MUC-5AC protein (Santa Cruz) highlighting mucosal secretory cells and mouse monoclonal antibodies to the CC10 protein (Singh et al A Quantitative assay for a Clara cell-specific protein and its application in the study of pulmonary airways in rats. Pediatr Res., 1986, 20: 802-805) the secretory cells of the Clara cell type are shown (FIGS. 6a, 6b, 6c and 6d).

  For this, a part of the grafts was included in a resin (OCT, Optimal Cutting Temperature, Tissue Tek) and then frozen in the liquid nitrogen vapor. Sections of 5pm thickness were made from these blocks using a cryostat (Cryocut, Reichert Jung) then collected on slides previously covered with a solution of gelatin and chromium alum (gelatin 0.5% (Prolabo) and chromium alum (KCr (SO4) 2 Fluka) in deionized water and then air dried).

  After fixing in cold methanol for 10 minutes at -20 ° C., the slides were rinsed with PBS and then incubated for 10 minutes in a solution of PBS containing 3% of serum bovine albumin (Sigma Aldrich) before being incubated for one hour. with the various previously mentioned antibodies diluted in the same solution of PBS and serum bovine albumin. After a further PBS wash, the slides were incubated for one hour with a relevant secondary antibody (directed against mouse or rabbit antibodies) coupled to biotin molecules (Amesham) diluted in the same solution of PBS and Serum bovine serum albumin. After washing with PBS, the slides were then incubated for 30 minutes in the dark with a conjugate consisting of streptavidin, having a strong affinity for biotin, coupled to a fluorochrome (Alexa Fluor 488, Molecular Probe) diluted in PBS. After washing with PBS, the slides were incubated with a nuclear dye, Harris hematoxylin (Shandon), rinsed with PBS and then with deionized water and then mounted under coverslips using a mounting solution (Aqueous Mounting Medium, Biomeda ). The slides were observed under an Axiophot microscope (Zeiss).

  Example 10 Functional Characterization of Regenerated Epithelia

  The presence of the CC10 protein secreted specifically by Clara cells was detected by the ELISA method. For this, at the different regeneration times (4, 13, 25 and 35 days of grafting), the grafts were washed by passing DMEM / Ham F12 medium in the montages on the back of the live mice. The fixtures were then emptied of their liquid contents by passing air, and then filled again with DMEM / Ham F12 medium. After 4 hours of contact with the bronchiolar human epithelium lining grafts during regeneration, the mouse was sacrificed and the liquid was collected. The concentration of CCIO protein was then determined using a specific ELISA kit (Biovendor). The results showed the presence, with increasing concentration, of the CC10 protein in the collected fluids, attesting to the functional secretion of this protein by Clara cells present in the regenerated epithelia.

  The bioelectrical properties of regenerated epithelia were studied. For this, grafts of 35 days were taken, opened in the direction of the length then assembled in the so-called Ussing chambers making it possible to study the current generated by the movements of ions through the epithelium (Isc), thus than the transepithelial potential difference (ddp). The results obtained showed a ddp consistent with the ddp generated by a native human bronchiolar epithelium and variations of Isc, following stimulation by drugs, similar to those obtained with a native bronchiolar epithelium, that is to say decreased after incubation with amiloride (10-5 M, Sigma Aldrich), and increased following incubation with forskolin (10-5 M, Sigma Aldrich) and ATP (10 -4 M Sigma Aldrich).

Claims (28)

  A non-human mammal model comprising a xenograft comprising bronchiolar epithelial cells of human origin grafted under the skin of said non-human mammal under conditions for regenerating a human bronchiolar epithelium.   2- Non-human mammal model comprising a xenograft according to claim 1, characterized in that the human bronchiolar epithelium is obtained from pathological bronchiolar epithelial cells.   3- Non-human mammal model comprising a xenograft according to claim 1, characterized in that the human bronchiolar epithelium is obtained from non-pathological bronchiolar epithelial cells.   4- Non-human mammal model comprising a xenograft according to claims 1 or 2, characterized in that the regenerated human bronchiolar epithelium is monostratified.   5- Non-human mammal model comprising a xenograft according to claims 1 or 2 characterized in that the regenerated human bronchiolar epithelium is pseudostratified.   6. Non-human mammal model comprising a xenograft according to claims 1 or 2 characterized in that the regenerated human bronchiolar epithelium is pluristratified.   7- A non-human mammal model comprising a xenograft according to any one of claims 1 to 6, characterized in that it is obtained by grafting the trachea or other support of a first non-human mammal in a second non-human mammal.   8- Non-human mammal model comprising a xenograft according to claim 7, characterized in that the first mammal is a rodent, for example a rat.   9- A non-human mammal model comprising a xenograft according to claims 7 or 8, characterized in that the second mammal is a xenotolerant rodent, for example a xenotolerant mouse.   Implant comprising or consisting of a human bronchiolar epithelium inoculated on a trachea or other support of a nonhuman mammal, obtainable from a non-human animal model   according to any one of claims 1 to 9.   11- A method for obtaining a nonhuman mammalian model intended to be sacrificed, capable of regenerating a human bronchiolar epithelium, comprising the following steps: a) isolation of bronchioles from human lung parts, b) dissociation of cells bronchiolar epithelial lining bordering said bronchioles, c) seeding said bronchiolar cells and culturing in an appropriate culture medium allowing their proliferation until a confluent culture is obtained.   d) in parallel with step c), dissection of a trachea or other support of an animal, for example a rodent, treatment to eliminate living cells and mounting on a device, e) after detachment, seeding said bronchiolar cells in the trachea or other support of the animal, f) grafting the assembly formed of the device and the trachea or other support under the skin of a xenotolerant animal, for example a rodent, g) rearing the animal during a delay allowing the total or partial regeneration of the bronchiolar epithelium.   12- Method according to claim 11, characterized in that the culture medium used in step c) comprises proliferation factors to obtain proliferation of bronchiolar epithelial cells.   13. Method according to claim 12, characterized in that the proliferation factors are in particular Epidermal Growth Factor (EGF), bovine pituitary extract, epinephrine and 3, 3 ', 5- triiodo-LThyronine.   14- Method according to any one of claims 11 to 13, characterized in that said cells of step c) are seeded at very low density, for example at a density between 13,000 and 70,000 cells per cm2, preferably between 20,000 and 40,000 cells per cm2.   15- Method according to any one of claims 11 to 14, characterized in that the rodent used in steps f) and g) is a NUDE mouse.   16- Method according to any one of claims 11 to 15, characterized in that the rodent used in steps d), e) and f) is a Wistar rat.   17- Method according to any one of claims 11 to 16 characterized in that the step of isolating bronchioles is carried out by microdissection.   18- Method according to any one of claims 11 to 17, characterized in that step d) comprises steps of freezing, thawing, after dissection of the rat trachea or other support, which is then mounted on the device and The method according to any one of claims 11 to 18, characterized in that step e) comprises a step of thawing the rat trachea or other support before seeding the bronchiolar cells therein.   20. A process for obtaining regenerated human bronchiolar epithelium comprising the steps of: a) obtaining a nonhuman mammalian model capable of regenerating a human bronchiolar epithelium, according to any one of claims 11 to 19; b) recovering the assembly formed from the device and the trachea or other support carrying the regenerated epithelium under the skin of the animal, after the sacrifice of said animal, c) optionally recovering the regenerated human bronchiolar epithelium.   21- A method of screening pharmacological compounds of interest in the field of respiratory diseases comprising the following steps: contacting the non-human mammal model according to any one of claims 1 to 9 or the implant according to claim 10, comprising or consisting of a regenerated human bronchiolar epithelium, with a determined pharmacological compound, - detecting the change in the state of said epithelium.   22- A method of screening pharmacological compounds of interest in the field of respiratory diseases comprising the following steps: contacting the pathological bronchiolar epithelium from the non-human mammal model according to any one of claims 1 to 9, with a determined pharmacological compound, - detecting the change in the state of said epithelium.   23- A method of studying the regeneration of the bronchiolar epithelium comprising the following steps: - contacting the model of. non-human mammal according to any one of claims 1 to 9 or the implant according to claim 10, comprising or consisting of a regenerated human bronchiolar epithelium, with a determined pharmacological compound, - detecting the modification of the state of said epithelium.   24. A method of studying the regeneration of bronchiolar epithelium comprising the steps of: contacting the bronchiolar epithelium from the non-human mammalian model according to any one of claims 1 to 9, with a determined pharmacological compound, - detection of the change in the state of said epithelium.   25- Method according to any one of claims 21 to 24 characterized in that the bronchiolar epithelium is monostratified.   26- Method according to any one of claims 21 to 24 characterized in that the bronchiolar epithelium is pseudostratified.   27- A method according to any one of claims 21 or 24 characterized in that the bronchiolar epithelium is pluristratified.   28. Use of the non-human mammal model according to any one of claims 1 to 9 or the implant according to claim 10 for the study of pluripotent cells.   29. A method for evaluating the transfection of polynucleotides within the bronchiolar epithelium of the non-human mammalian model according to any one of claims 1 to 9, comprising: transfection of the cells of the bronchiolar epithelium with a determined polynucleotide, -observation of the effects of transfection carried out on the cells of the epithelium.