DEVELOPMENT OF A TWO PHASE

Jeni

Description

Niveau: Supérieur, Doctorat, Bac+8
DEVELOPMENT OF A TWO-PHASE MICROFLUIDIC PLATFORM FOR DRUG SCREENING THÈSE Présentée à l'Université de Strasbourg Ecole Doctorale des Sciences Chimiques Pour obtenir le grade de docteur de l'Université de Strasbourg Par JENIFER CLAUSELL-TORMOS Strasbourg 2010

• aqueous plugs


• high-throughput cell-based


• phase microfluidic


• platform sequentially


• ecole doctorale des sciences chimiques


• assays require


• require direct compound

Sujets

• De Mello
• Griffiths
• Institut Curie
• Université de Strasbourg
• Marquet
• Romero

Informations

• Publié par: profil-zyak-2012
• Nombre de lectures: 29
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

DEVELOPMENT OF A TWO-PHASE
MICROFLUIDIC PLATFORM FOR DRUG
SCREENING




THÈSE

Présentée à l’Université de Strasbourg
Ecole Doctorale des Sciences Chimiques

Pour obtenir le grade de docteur de l’Université
de Strasbourg


Par
JENIFER CLAUSELL-TORMOS

Strasbourg 2010







SOUTENUE PUBLIQUEMENT LE 18 MARS 2010 DEVANT LA
COMMISSION D’EXAMEN :





Directeur de Thèse: Prof. Andrew Griffiths
Institut de Science et d’Ingénierie Supramoléculaire, Strasbourg, France




Rapporteur: Prof. Josep Esteve Romero
Universitat de la UJI, Castelló, Espagne


Examinateur: Directeur de Recherche Roland Marquet
Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France


Rapporteur: Prof. Andrew de Mello
Imperial College, London, Royaume-Uni


Rapporteur: Directeur de Recherche Jean-Louis Viovy
Institut Curie, Paris, France




















Solo se que no se nada
Je sais que je ne sais rien
I know that I know nothing
Ich weiß, daß ich nichts weiß


Socrates






























Dedicat als meus pares, als meus iaios i als meus abuelos.


















ABSTRACT

High-throughput cell-based assays require small sample volumes to reduce assay costs
and to allow for rapid sample manipulation. However, further miniaturization of
conventional microtiter plate technology is problematic due to evaporation and capillary
action. To overcome these limitations, we have developed a two-phase microfluidic
platform in which human cells and multicellular organisms can be cultivated for several
days in aqueous microcomparments separated by an inert perfluorocarbon carrier oil.
Furthermore, we focussed on the automated generation of chemically-dictinct
microcompartment to exploit the technology for screening purposes. In particular, we
interfaced an autosampler with our microfluidic platform sequentially loading compounds
from microtiter plates into a length of tubing. All compounds are loaded in form of
aqueous plugs (nanoliter volumes) separated by fluorinated oil. The resulting array of
plugs can be split into multiple small volume copies which can be used as replicates for
the same assay as well as for different assays. Moreover, each array of plugs can be
injected into a microfluidic chip for further manipulation (such as the addition of reagents
or the detection of fluorescence signals). Since the order of the compounds and thus
their identity is known throughout the whole screening procedure, the system does not
require direct compound labelling. Furthermore, each individual plug can be monitored
over time, thus allowing the recording of kinetic data. In the last part of the work we
focussed on the development of a novel assay coupling a positive fluorescence signal
with the inhibition of viral transduction. This should ultimately allow the screening of
antivirals in the previously developed microfluidic systems.






Table of contents


Table of contents

1. Introduction................................................................................................................... 1
1.1 Current High-Throughput Screening (HTS) technologies ...................................... 1
1.1.1 Microtiter plate technologies .......................................................................... 1
1.1.2 Microarrays technologies ................................................................................ 2
1.2 Novel High-Throughput Screening (HTS) technologies ......................................... 4
1.2.1 Microfluidics .................................................................................................... 4
1.2.1.1 Continuous flow-based microfluidics ....................................................... 5
1.2.1.1.1 Working principle ................................................................................ 5
1.2.1.1.2 Applications ........................................................................................ 7
1.2.1.2 Two-phase microfluidics ......................................................................... 14
1.2.1.2.1 Working principle .............................................................................. 14
1.2.1.2.2 Applications ...................................................................................... 21
1.2.2 Viral-inhibition assays compatible with on-chip readouts systems............... 31
1.2.2.1 Retroviruses ........................................................................................... 32
1.2.2.1.1 Structure of retroviruses .................................................................. 32
1.2.2.1.2 Replication cycle of retroviruses ...................................................... 34
1.2.2.1.3 Retroviral pseudotyped vectors ........................................................ 36
1.2.2.1.4 RNAi systems .................................................................................... 38
1.3 Aim of the thesis ................................................................................................. 41
2. Material and methods ................................................................................................ 42
2.1 Molecular biology ................................................................................................ 42
2.1.1 Plasmids and vectors .................................................................................... 42
2.1.2 Oligonucleotides ............................................................................................ 43
2.1.3 Cloning of the vector pSIREN-puro ................................................................ 43
2.1.4 Agarose gel electrophoresis and DNA extraction from gels .......................... 47
2.1.5 Generation of competent bacteria and transformation thereof ................... 48
2.1.6 Plasmid preparation ...................................................................................... 49
2.1.7 Nucleic acid sequencing ............................................................................... 50
2.2 Cell biology .......................................................................................................... 50
2.2.1 Cells ............................................................................................................... 50
2.2.2 Freezing and thawing of cultured cells ......................................................... 51

Table of contents


2.2.3 Generation and purification of viral pseudotype particles ............................ 52
2.2.4 Viral transduction .......................................................................................... 53
2.2.4.1 Generation of stable cell lines ............................................................... 53
2.2.4.2 Determination of viral titers ................................................................... 53
2.3 Assays ................................................................................................................. 54
2.3.1 Fluorescence analysis of single cells on-chip ............................................... 54
2.3.2 Fluorescence analysis of plugs on chip ........................................................ 55
2.3.3 Cell-based fluorescence assays in bulk ........................................................ 56
2.3.4 Determination of Z factors ............................................................................ 57
2.4 Surfactants .......................................................................................................... 58
2.4.1 Synthesis of the surfactans ........................................................................... 58
2.4.2 Assay for the biocompatibility of surfactants ............................................... 60
2.5 Encapsulation and cultivation of cells and multicellular organisms .................. 60
2.5.1 Droplet-based systems .................................................................................. 60
2.5.1.1 Cell encapsulation .................................................................................. 60
2.5.1.2 Live/dead staining of cells recovered from drops ................................. 61
2.5.1.3 Determination of the cell recovery ......................................................... 62
2.5.2 Plug-based systems....................................................................................... 63
2.5.2.1 Cell encapsulation ..............................................................................