Multicellular organotypic model of human intestinal mucosa

摘要

Disclosed are methods of preparing multi-cellular three-dimensional tissue constructs, that include fibroblasts, endothelial cells, lymphocytes and epithelial cells. The present methods may include embedding fibroblasts and endothelial cells in a matrix enriched with gut basement membrane proteins to form a cell containing matrix that is then added to a bioreactor and exposed to epithelial cells and activated lymphocytes as the cell cultures. Also provided are the tissue constructs formed from such methods, a matrix enriched with gut basement membrane proteins and kits that include the same. Further provided are methods of measuring toxicity of a pathogen or commensal organisms, chemosensitivity of tissues to a toxic material and inflammatory conditions, which use the present multi-cellular three-dimensional tissue constructs.

主权项

1. A method of preparing a multi-cellular three-dimensional construct, comprising
growing human intestinal epithelial cells, primary human umbilical vein endothelial cells (HUVEC), and primary human fibroblast cells separately as two-dimensional (2-D) confluent monolayers; embedding the fibroblast cells and the endothelial cells, in a collagen-I extracellular matrix mixture enriched with gut basement membrane proteins comprising laminin, collagen IV, fibronectin and heparin sulfate proteoglycan, to form a cell-containing gel; with a specific gravity similar to culture medium; adding the cell-containing gel to a rotating wall vessel bioreactor and suspending the human intestinal epithelial cells in medium and then adding the suspended cells to the rotating wall vessel to form a cell culture; and applying microgravity conditions that produce laminar flow and minimize shear force inside the culture, allowing the cells to culture, while adding primary human lymphocytes to the cell culture, to form a multi-cellular three-dimensional tissue construct; wherein the epithelial cells form a simple columnar epithelium and differentiate into multiple lineages.