In vitro reconstructed mucosa‐integrating Langerhans' cells

Abstract

All three-dimensional in vitro mucosal models constructed, thus far, have only been reconstituted by epithelial cells. We have developed a reconstructed oral and vaginal epithelium that integrates Langerhans' cells (LC), the dendritic cells (DC) of malpighian epithelia. The epithelium was composed of gingival or vaginal keratinocytes seeded on a de-epidermized dermis (DED) and grown in submerged culture for 2 weeks. LC precursors, obtained after differentiation of cord blood-derived CD34+ hematopoietic progenitor cells (CD34+HPC) by granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and Flt3-ligand (Flt3-L), were introduced after 6-8 days of culture into the reconstituted epithelium. The in vitro reconstituted mucosal epithelium formed a multilayered, well-differentiated epithelial structure, confirmed by the immunohistochemical expression of cytokeratins 4, 6, 10, 13, 14, 16 and involucrin. LC were identified in the basal and suprabasal epithelial layers by CD1a antigen, S100 protein and Langerin/CD207 expression, and by transmission electron microscopy. Type IV collagen was expressed at the chorio-epithelial junction, and most ultrastructural features of this junction were visualized by electron microscopy. This in vitro reconstructed gingiva or vagina integrating LC represents interesting models very similar to native tissues. Because LC play an important role in the mucosal immune system, our models could be useful for conducting studies on interactions with pathogenic agents (viruses, bacteria etc.), as well as in pharmacological, toxicological and clinical research.

Characterization of CCL20 secretion by human epithelial vaginal cells: involvement in Langerhans cell precursor attraction

“…The SiHa cells are able to grow as a multilayer and express the CKs characteristic of the different degrees of cell differentiation [19]. Indeed, the SiHa multilayer grown on Matrigel™ support is representative of the in vivo vaginal situation, with proliferative epithelial cells and more superficial, differentiated ones [17]. The addition of LC (12-day-cultured cells obtained from hematopoietic progenitor CD34ϩ) to the SiHa multilayer demonstrated the ability of these immune cells to integrate and remain viable inside the multilayer for 96 h in the same proportion as that observed in vivo [20].…”

“…Normal human vaginal cells (NHVC) were obtained from women undergoing routine hysterectomies as already described [17]. Briefly, biopsies (2-5 mm 2 ) were washed several times with phosphate-buffered saline (PBS) containing an antibiotic-antimycotic solution (penicillin-streptomycin-amphotericin B, Sigma-Aldrich, St. Louis, MO) and were incubated with dispase II (Roche Diagnostics, Basel, Switzerland).…”

Selective sequestration of X4 isolates by human genital epithelial cells: Implication for virus tropism selection process during sexual transmission of HIV

“…Normal human vaginal cells were isolated and cultured as already described [Sivard et al, 2003]. Briefly, epithelial cells obtained from vaginal biopsy specimens after hysterectomy (from HIV-negative patients) were expanded by co-culture with NIH-3T3 cells treated previously with 5 mg/ml mitomycin C (Sigma-Aldrich, St. Louis, MO) for 2 hr, in DMEM medium and Ham's F12 (3:1, Cambrex BioScience) containing 10% FBS, 0,4 mg/ml hydrocortisone (Sigma-Aldrich), 5 mg/ml insulin (Sigma), and 10 ng/ ml EGF (BD Biosciences, Franklin Lakes, NJ).…”

MUTZ-3 Langerhans Cell maturation and CXCL12 independent migration in reconstructed human gingiva

“…However, none of these reconstructed models contain DC, and therefore their application as in vitro testing models is limited. Only one study has described integration of DC into a mucosa equivalent, and notably these were derived from primary CD34 + cells isolated from fresh cord blood, which creates a major logistical problem as well as donor variation (Sivard et al, 2003). Therefore, in this study, we developed a full thickness oral mucosa model with integrated human Langerhans-like cells derived from the human MUTZ-3 cell line.…”