Droplet-Based Microfluidic Platforms for the Encapsulation and Screening of Mammalian Cells and Multicellular Organisms

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 describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.

Functional single-cell hybridoma screening using droplet-based microfluidics

Christoph Merten

Monoclonal antibodies can specifically bind or even inhibit drug targets and have hence become the fastest growing class of human therapeutics. Although they can be screened for binding affinities at very high throughput using systems such as phage display, screening for functional properties (e.g., the inhibition of a drug target) is much more challenging. Typically these screens require the generation of immortalized hybridoma cells, as well as clonal expansion in microtiter plates over several weeks, and the number of clones that can be assayed is typically no more than a few thousand. We present here a microfluidic platform allowing the functional screening of up to 300,000 individual hybridoma cell clones within less than a day. This approach should also be applicable to nonimmortalized primary B-cells, as no cell proliferation is required: Individual cells are encapsulated into aqueous microdroplets and assayed directly for the release of antibodies inhibiting a drug target based on fluorescence. We used this system to perform a model screen for antibodies that inhibit angiotensin converting enzyme 1, a target for hypertension and congestive heart failure drugs. When cells expressing these antibodies were spiked into an unrelated hybridoma cell population in a ratio of 1∶10,000 we observed a 9,400-fold enrichment after fluorescence activated droplet sorting. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be successfully sorted and recultivated.

2012

Microfluidic devices for the study of cell-cell, cell-particle and particle-particle interactions

Margaux Catherine Marie Duchamp

Microfluidics and microtechnologies are of great interest for biological applications. This interest is linked to the fact that microtechnologies enable the study of single cells at the cellular and sub-cellular level. One of many applications of such single cell assays is cell-cell interaction probing. Current cell-cell interaction tools used by biologists rely on manual cell manipulation. But in the last decade new microfluidic technologies have arisen to tackle this biological challenge. Despite this growing need, the developped microfluidic devices still force biologists to compromise between the throughput of the assay (number of cell pairs interrogated) and the control over the interaction parameters (contact force and contact time). This thesis proposes an engineering point of view of microfluidic tools for single cell and in particular cell-cell interaction studies. The developed microfluidic devices enable an advancement of the microfluidic technologies to leverage new tools for cell-cell interaction interrogation in a controlled manner and at a high throughput. In this work we report the development of a novel microfluidic device based on a “roll-over” mechanism. This new chip design enables the multiplexing of cell-cell interactions. This multiplexing is achieved by forcing into contact all cells from a sample of a first cell population with all cells from a sample of a second cell population. Using such a device, we characterized the interaction of cells expressing olfactory receptors. This chip enables the maximization of cell-cell interaction interrogation and thus is ideally suited for rare and nonredundant cell populations. A droplet microfluidic chip for controlled and reliable cell co-encapsulation was designed and implemented. Common droplet microfluidic devices rely either on random cell co-encapsulation in droplets, thus leading to high cell loss, or use multiple microfluidic devices sequentially to perform droplet manipulation steps and achieve cell co-encapsulation. Both cases lead to cell sample loss, first from the random cell co-encapsulation process and second from the possible droplet loss between different microfluidic devices. Therefore, the implementation of a single fully integrated device enabling controlled cell co-encapsulation in droplets is of high interest for the study of precious, large cell libraries interactions for example in the case of cancer immunotherapy. In this thesis we also developed new concepts, using already existing microfluidic designs. By reusing the microfabrication technologies and microfluidic principles of the previously designed devices we could develop a novel microfluidic chip for another biological application. The third device studied in this thesis enables to look further into cell membrane receptors by isolating plasma membrane fragments. This tool enables an in-depth study of receptors involved in cell-cell interactions. It can also be used for any other cell membrane receptor study as for example G protein coupled receptor screening for therapeutic drug discovery. The tools developed in this thesis set the grounds for the development of new generations of cell-cell interaction microfluidic devices, enabling high throughput and control over the cell-cell interaction parameters. The technological advancements presented in this work were validated for various biological applications.

EPFL2020

Microtechnologies to Map the Fate of Single Stem Cells and their Progeny

Stefan Kobel

Since stem cells have the unique ability to produce more of themselves (i.e. to "self-renew") and to generate specialized tissue cells, they are an ideal source of cells for regenerative medicine and in vitro tissue models. In order to fully exploit this potential, we have to better understand the mechanisms that regulate stem cell behavior at a single cell level. However, current in vitro methodologies to study single cells are limited by their throughput, do not afford to distinguish the fate of daughter cells or track the progeny of a single cell over long time periods. This thesis addresses these shortcomings by developing miniaturized devices to handle and analyze single live stem cells. To this end, a micro-structured platform was first established that consisted of miniaturized wells with diameters of 100 µm that were arranged in a regular grid. Single hematopoietic stem cells (HSCs) were then seeded into these "microwell arrays" and, since the HSCs could not leave the microwells, they could be tracked over many days, and their dynamics and proliferation rates could be studied. However, in order to analyze the daughter cells of these single HSCs individually, the daughter cells have to be physically separated by micromanipulation. For this purpose, a microdevice was developed to reliably manipulate single cells using hydrodynamic single cell trapping. This platform was thoroughly characterized using computational simulations combined with particle flow velocimetry and live-cell time-lapse microscopy. The optimization of design parameters towards increasing trapping efficiency ensured a nearly perfect efficiency of single cell trapping (97%) and a successful re-capture of daughter cells generated by dividing mother cells. Furthermore, it was demonstrated that cell trapping under very low flow is sufficiently gentle to enable long-term cell trapping in the microfluidic environment. To identify and track captured cells in these microfluidic single cell traps, automated image analysis tools were developed. A reliable and fast algorithm was generated to identify the borders of microchannels that were indicative for the position of single cell traps. These detected edges were used to individualize the single cell traps with a sub-pixel resolution such that it was possible to segment single HSCs on the chip by thresholding. Accordingly, single HSCs were discovered with efficiencies as high as >95%, allowing the automated tracking of microfluidic single cell trapping, as well as the detection of cell cycle phases of trapped single HSCs engineered with dual fluorescence reporter system marking G1 and S/G2-M. To actively micromanipulate single cells for downstream cell-fate analyses, on-chip valves were integrated on the microfluidic platform to precisely control the direction of medium flow in the microfluidic channels. This approach enabled a proof-of-concept study to demonstrate a more complex single cell manipulation: Cells were first captured in single cell traps and transferred into a culture chamber for clonal expansion. Subsequently, the progeny of these single cells was removed from the culture chamber and trapped in a series of single cells traps. Importantly, since these traps could be addressed individually, a reliable physical separation of the daughter cells was achieved. To enable individual analyses of these separated daughter cells, the last aim of this thesis was to interface a microfluidic chip with standard multititer plates in order to efficiently recover the cells or the cDNA of the single cell generated on-chip from extracted mRNA. For this purpose, the bottom of the chip was equipped with an array of access holes that matched the layout of a 1536-well plate. These access holes were linked to a single cell trap via an analysis chamber, where the HSCs were lysed and their mRNA was reverse transcribed using bead-coupled primers. In this vain, the obtained cDNA was coupled to these beads that could then removed from the chip into a 1536-well by centrifugation. These beads could then be analyzed using standard bench-top PCR machines. In conclusion, this thesis presented several microfluidic modules to handle and analyze single stem cells in high-throughput. In a proof-of-concept study, these modules were applied to automatically capture single cells, culture them, and subsequently separate the daughter cells for downstream cell-fate analyses, thereby greatly enhancing the throughput compared to manual micromanipulation. During the development of this system, it became apparent how crucial reliable and gentle cell handling is to perform complex experimental tasks with live cells on microfluidic chips. The further integration of such microfluidic modules into multi-functional lab-on-a-chip devices will open the door to experimental paradigms previously not imaginable and promises novel insights in stem cell biology.