Perfusion System for Modification of Luminal Contents of Human Intestinal Organoids and Realtime Imaging Analysis of Microbial Populations.

Nicholas J Ginga,Sha Huang,Veda K Yadagiri,Shuichi Takayama,Ge-Ah Kim,Vincent B Young,Raleigh Slyman,Eric Parigoris,Jason R Spence

doi:10.3390/mi13010131

Abstract

Intestinal organoids are 3D cell structures that replicate some aspects of organ function and are organized with a polarized epithelium facing a central lumen. To enable more applications, new technologies are needed to access the luminal cavity and apical cell surface of organoids. We developed a perfusion system utilizing a double-barrel glass capillary with a pressure-based pump to access and modify the luminal contents of a human intestinal organoid for extended periods of time while applying cyclic cellular strain. Cyclic injection and withdrawal of fluorescent FITC-Dextran coupled with real-time measurement of fluorescence intensity showed discrete changes of intensity correlating with perfusion cycles. The perfusion system was also used to modify the lumen of organoids injected with GFP-expressing E. coli. Due to the low concentration and fluorescence of the E. coli, a novel imaging analysis method utilizing bacteria enumeration and image flattening was developed to monitor E. coli within the organoid. Collectively, this work shows that a double-barrel perfusion system provides constant luminal access and allows regulation of luminal contents and luminal mixing.

Highlights

Organoids are 3D cell cultures that arise from the self-organization of adult or pluripotent stem cells into organotypic structures [1,2,3]
The results show that the perfusion device was able to modulate luminal contents and bacterial proliferation
The main function of the human intestinal organoids (HIOs) holder is to immobilize the HIO while providing access to the HIO for a horizontally positioned double-barrel glass capillary controlled by a micromanipulator to pierce and inject/withdraw material from the HIO (Figure 1)

Summary

Introduction

Organoids are 3D cell cultures that arise from the self-organization of adult or pluripotent stem cells into organotypic structures [1,2,3]. Accessing the apical surface is required for some studies, such as host–microbe interactions which take place on the luminal surface of the epithelium. For such studies, growth of apical-out enteroids has been developed, but other methods are still needed [4,5]. Growth of apical-out enteroids has been developed, but other methods are still needed [4,5] One such method to access the apical cell surface is to create 2D cell cultures from 3D. This enables the use of established 2D cell culture test methods, it adds additional steps and processes to create 2D cell cultures from 3D organoids [6,7,8]

Methods

Results

Conclusion