Thesis presented October 17, 2014

Abstract :
In all secretory epithelia from glandular tissues, there is a common structural and functional unit, the acinus. It is a well polarized and organized pluricellular structure that is spontaneously reconstructed in 3D culture, therefore closely mimics the real structure we find in vivo. For my purpose, acini are used as models for tumor initiation and cancer development. One of the objectives of Biomics laboratory is to identify the genetic and micro environmental determinants of prostate acini morphogenesis and polarity. The strategy is based on High-Throughput (HT) RNA interference (RNAi)-based screening. To meet this objective, my project was to develop appropriate 3D cell models which closely mimic the cyst-like and duct-like structure of prostate. By optimizing conventional 3D culture in Matrigel, I could recapitulate prostate acini morphogenesis and showed that lumen formation is independent to the polarity, which appears later. However, the conventional 3D cell culture formats and analytical tools are not suited for HT Screening (HTS). They lack control over acini size, are label-dependent​ and therefore time-consuming and labor intensive. Also, classical microscopy offers a very limited field of view and hence does not allow observing a large amount of 3D structures for statistical analysis. To overcome these limitations, I have first developed a new approach based on cell encapsulation using microfluidics to handle acini in flux. Single prostate cells are encapsulated into mono-dispersed Matrigel droplets. After 6 days, these droplets containing growing acini are collected in culture media and are now easy to handle, to store for long-term and to sort for analysis thus allowing RNAi HTS on acini with flow-based detection. By properly adjusting microfluidics parameters, this encapsulation process provides a mean to control mechanical environment through regulation of the capsule size and cell distribution through our ability to encapsulate one singe acinus. Then, to rapidly and directly (label-free) image 3D structures, we performed image acquisition with lens-free microscopy. We demonstrated that fully well differentiated acini with lumen could be distinguished from tumor-like spheroids (without lumen) only on the basis of their specific optical (holographic) signatures. This method allows fast tracking of the effects of siRNA with an extensive statistical analysis of the percentage of acini and spheroids. By carrying out 3D lens-free video microscopy​ for 72 hours, we went further in 3D observations and could observe at the same time many dynamic phenotypes in 3D and branching-like processes. In conclusion, the technologies developed during my PhD provide a way to control and standardize 3D cell models which thus become useful models for drug discovery in oncology, since acini well mimic tumor initiation and development. Microfluidics and lens-free imaging enable new opportunities in HTS in 3D cultures.


Keywords:
Polymeric capsules, microenvironment, cell culture, differentiation, proliferation, microfluidics

On-line thesis.