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ID: 78 (Conflict of Interest: K)

Entwicklung eines humanen 3D Schilddrüsenmodells in vitro

Ö.Vural1, S.Kadler1, M.Rosowski1, M.Mogl2, C.Bures2, R.Lauster1
1Technische Universität Berlin, Berlin
2Charité - Universitätsmedizin Berlin, Berlin

Einleitung

The thyroid gland plays an important role during embryonic development as well as in the adulthood by regulating metabolism of various organs. The thyroid is composed of numerous follicles that are smallest in vivo bioreactors to produce substantially important thyroid hormones T3 and T4 which are supplied to the body. Embedded in extracellular matrix substitutes, such as Matrigel, thyrocytes can arrange follicles in vitro. Such in vitro systems are utilized for functional studies. Due to the lack of representative human thyroid models, a vast number of animal studies are conducted.

Organogenesis can be mimicked in vitro by 3D culture. This 3D microenvironment provides cells with important cell-cell-contacts that drive cellular differentiation and maturation.

Our hypothesis comprises that primary human thyrocytes are able to aggregate to thyroid-like organoids without an artificial matrix when co-cultured with cells of mesenchymal origin. Furthermore, 3D printing technology is involved to allow the development of a reproducible and representative organ model.

Material und Methoden

Initially, cells from normal thyroid tissue were isolated (n > 34) and expanded as monolayer culture in H6 media. After 3 passages, thyrocytes were co-cultured for up to 4 weeks with fibroblasts of dermal origin or endothelial cells isolated from human umbilical cord. Thyroid-organoids were subsequently analyzed on transcriptional (qPCR) and protein (immunohistology) level.

 

Ergebnisse

Initially, cells from normal thyroid tissue were isolated (n > 34) and expanded as monolayer culture in H6 media. After 3 passages, thyrocytes were co-cultured for up to 4 weeks with fibroblasts of dermal origin or endothelial cells isolated from human umbilical cord. Thyroid-organoids were subsequently analyzed on transcriptional (qPCR) and protein (immunohistology) level.

In primary culture, cells of 14 out of 34 donors were used for experiments. Thyrocytes change their morphology quickly and initiate senescence early in culture (P4/P5). Our results confirm this statement: In 2D, thyrocytes de-differentiate and lose their phenotype. Compared to native thyroids, thyroid-specific markers, such as NKX2-1, PAX8, TSHR, SLC5A5, are significantly down-regulated up to 1000-fold. However, when cultured in 3D conditions, thyrocytes induce a re-differentiation process and upregulate their markers to levels similar to native thyroid.

Schlussfolgerung

Using 3D printing, this effect can be emphasized. Bioprinting of single cells offers the possibility to print organ models with high resolution, thus facilitating the generation of a reproducible and representative thyroid model.

The aim of our project is to mimic the impact of thyroid hormones on other organ models, such as the liver or the heart, on a multi-organ-chip platform. Microfluidics on the multi-organ-chips allow organ interaction that enable substance testing.