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Subject of the invention is a method for long-term culture of columnar epithelial cells and the application thereof. The cells in culture survive more than 6 months and they can be expanded due to their regenerative capacity. The epithelial cells are seeded on a thin matrix-coated solid semi-permeable filter and cultivated in an air liquid interface to form a coherent monolayer. The cultivation method allows for the regeneration and expansion of the culture by enzymatic removal of the cells from the filters and seeding on a new filter. The proliferation, longevity and regenerative capacity of the cells rely on the maintenance of the stem cell niche in vitro. The stem cells are capable of multi-lineage differentiation. The cultures are sensitive to morphogens and grow factors that are inducing proliferation and/or differentiation into the specific cell lineages typical of the healthy tissue of origin. Specialized cell lineages maintain their function of origin also in the culture of the invention. By mimicking a pathological condition in the cultures of the invention, the distribution of the cells in the specific cell lineages can be altered and other lineages that are not typical of the tissue of origin may emerge (e.g. Metaplasia, neoplasia). Cultured cells of the invention maintain features of the tissue of origin including height and polarization. Polarization segregates the apical side from the basal side wherein the basal side is adjacent to the semi-permeable surface of the filter and in contact with the culture medium. The cells of the culture of invention are producing and accumulating mucus on the apical side. The apical and the basal side of the cells represent two different locations where the epithelial cells can conduct their secretory and absorptive function. The combination of these two functions may result in an active transport of substances form the apical to the basal side or vice-versa. The cells in the culture are able to respond to a variety of stimuli. The stimulus can be applied in the culture at the apical, basal or both sides. Result of the stimulation may be the activation of a specific signaling pathway that would promote the transcription of genes involved in a specific cellular function (e.g. regulation in: inflammation, differentiation, proliferation, migration, metabolism, transport, secretion, absorption and others). The stimulus can be of various sources including physical stimulation (e.g., heat, electromagnetic radiation, contact and others) chemical stimulation (e.g. natural or synthetic molecules) or biological stimulation (e.g., whole or part of organisms such as gamete, zygotes, embryos, bacteria, viruses or parasites). Other types of non-epithelial cells can be co-cultured. For example, stromal cells isolated from the same tissue can be co-cultivated below the epithelium or below the porous filters support in the same vessel reproducing the complexity of the whole mucosa. Similarly, cells of the hematopoietic lineage can be co-cultivated within or below the epithelium or below the porous filters support. We name the epithelial culture of the invention“mucosoid culture”, in reference to the fact that they recapitulate key characteristics of the polarized, mucus-secreting epithelial layers and they can optionally include other cells from the mucosa. The cultures originate from explanted epithelial cells and columnar epithelial cells. The epithelial cells can be derived from primary cells isolated from any embryonic or adult vertebrate, preferentially mammalian or human tissue; alternatively, the cell can be derived from other types of epithelial cultures that allow maintenance of stem cells in vitro, preferentially organoids or planar feeder supported culture. Cells prior cultivation into mucosoid culture can be genetically modified (e.g., using recombinant nucleic acids). The mucosoid culture of this invention can also be derived from other types of non-epithelial cells, providing they have been transformed or re-programmed to become epithelial cells, reminiscent to the authentic cells described in this invention. For example, fibroblasts can be converted into induced pluripotent cells (iPS) and the re-differentiated to form polarized epithelial cells. While the induction of pluripotency and the process of redifferentiation are not the topics of this invention, however, the outcome of the redifferentiation process, i.e. the epithelial cells, is part of the invention providing they are used as mucosoid cultures, as described herein. The mucosoid cultures are a source of mucus, cells and substances secreted in the culture medium. Analysis or direct use of these tree components, as a result of the mucosoid cultivation or of the stimulation of the mucosoid cultures, is part of this invention. The established mucosoid cultures serve a variety of purposes, including (i) the long-term maintenance or amplification of epithelial cells used in this invention, (ii) the exploration of the behavior of the cells in response to extrinsic stimuli, (iii) the transport of substances or factors from apical to basolateral directions, or vice versa, or the translation of biological, chemical and physical signals arriving from either direction, (iv) their usage as a production tool or factory for biological factors useful for technical and medical purposes, (v) their usage as an apical platform to facilitate and reproduce various extracorporeal biological processes, (vi) their usage in more complex biological settings with additional cell populations beyond the semi-permeable filter to reproduce various intracorporeal processes, and (vii) human diagnostic or therapeutic applications.



Publication Date



Francesco Boccellato, University of Oxford, Nuffield department of medicine, off Roosevelt Drive, Oxford, Oxfrordshire, OX3 7DQ, United Kingdom


Mucosoid culture, epithelium, stem cells, regeneration, in-vitro