Three dimensional vaginal tissue model containing immune cells

Abstract

Disclosed is a cervico-vaginal tissue equivalent comprised of vaginal epithelial cells and immune cells, cultured at the air-liquid interface. The tissue equivalent is capable of being infected with a sexually transmitted pathogen such as a virus (e.g., HIV), a bacteria, a helminthic parasite, or a fungus. The tissue equivalent is also capable of undergoing an allergic-type reaction or an irritant-type reaction. The tissue equivalent is characterized as having nucleated basal layer cells and nucleated suprabasal layer cells, and further as having cell layers external to the suprabasal layer progressively increasing in glycogen content and progressively decreasing in nuclei content. Immune cells of the tissue equivalent are primarily located in the basal and suprabasal layers. Also disclosed are methods for producing the tissue equivalent. The methods involve providing vaginal epithelial cells and immune cells, seeding the cells onto a porous support, and co culturing the seeded cells at the air-liquid interface under conditions appropriate for differentiation. One such method disclosed is for generation of the tissue equivalent in serum free medium. Specific cells from which the tissue equivalent is generated, and also specific preferred components of the medium in which the tissue equivalent is generated are provided. Also disclosed is a cervico-vaginal tissue equivalent produced by the methods disclosed herein.

1 . A cervico-vaginal tissue equivalent comprised of vaginal epithelial cells, and immune cells, cultured at the air-liquid interface.  
     
     
         2 . The cervico-vaginal tissue equivalent of  claim 1  which further comprises a support on which it is cultured.  
     
     
         3 . The cervico-vaginal tissue equivalent of  claim 1  which is capable of being infected with a sexually transmitted pathogen selected from the group consisting of a virus, a bacteria, a helminthic parasite, and a fungus.  
     
     
         4 . The cervico-vaginal tissue equivalent of  claim 3  wherein the sexually transmitted pathogen is HIV.  
     
     
         5 . The cervico-vaginal tissue equivalent of  claim 1  which is capable of undergoing an allergic-type reaction or an irritant-type reaction.  
     
     
         6 . The cervico-vaginal tissue equivalent of  claim 1  which is generated in serum free medium.  
     
     
         7 . The cervico-vaginal tissue equivalent of  claim 1  wherein the vaginal epithelial cells, the immune cells, or both, are of human origin.  
     
     
         8 . The cervico-vaginal tissue equivalent of  claim 1  wherein the vaginal epithelial cells, the immune cells, or both, are selected from the group consisting of primary cells, passaged primary cells, transformed cells, and immortalized cells.  
     
     
         9 . The cervico-vaginal tissue equivalent of  claim 8  wherein the primary or passaged primary vaginal epithelial cells are derived from tissue selected from the group consisting of normal human ectocervical tissue, normal human endocervical tissue, pathological human ectocervical tissue, and pathological human endocervical tissue.  
     
     
         10 . The cervico-vaginal tissue equivalent of  claim 1  wherein the immune cells comprise Langerhans cells, Langerhans precursor cells (CD34+), monocytes (CD14+), immature dendritic cells (CD1a+, CD4+), mature dendritic cells (CD86+, HLA-DR++), T cells (CD3+), macrophages, or any combination thereof.  
     
     
         11 . The cervico-vaginal tissue equivalent of  claim 1  wherein the immune cells are generated in vitro from Langerhans precursor cells or monocytes.  
     
     
         12 . The cervico-vaginal tissue equivalent of  claim 2  wherein the support is selected from the group consisting of an artificial membrane, an extracellular matrix component, a collagen mixture, in vivo derived connective tissue, a mixed collagen-fibroblast lattice, mixed extracellular matrix-fibroblast lattice, and plastic.  
     
     
         13 . The cervico-vaginal tissue equivalent of  claim 12  wherein the mixed collagen-fibroblast lattice is comprised of vaginal fibroblasts.  
     
     
         14 . The cervico-vaginal tissue equivalent of  claim 13  wherein the mixed collagen-fibroblast lattice is further comprised of T cells (CD3+).  
     
     
         15 . The cervico-vaginal tissue equivalent of  claim 1  wherein the immune cells express HLA-DR.  
     
     
         16 . The cervico-vaginal tissue equivalent of  claim 1  which is characterized as having nucleated basal layer cells and nucleated suprabasal layer cells.  
     
     
         17 . The cervico-vaginal tissue equivalent of  claim 16  which is characterized as having cell layers external to the suprabasal layer progressively increasing in glycogen content and progressively decreasing in nuclei content.  
     
     
         18 . The cervico-vaginal tissue equivalent of  claim 16  which is characterized as having immune cells primarily located in the basal and suprabasal layers.  
     
     
         19 . A cervico-vaginal tissue equivalent produced by a method, comprising the steps: 
 providing vaginal epithelial cells and immune cells;    seeding the cells;    co-cultivating the seeded cells submerged in growth medium under conditions appropriate for cell propagation, and    co-culturing the seeded cells at the air-liquid interface under conditions appropriate for differentiation.    
     
     
         20 . The cervico-vaginal tissue equivalent of 19 wherein the method further comprises the step of co-cultivating the seeded cells submerged in growth medium under conditions appropriate for cell propagation, prior to the co-culturing step.