In this work package we develop novel label-free sorting schemes based on particle morphology, deformability and dielectric properties combining experimental
approaches with advanced modelling techniques.
Leading experimental groups provide the technological development of microfluidic label-free characterization, specifically Jonas Tegenfeldt at Lund University with expertise in deterministic lateral displacement (DLD), Thomas Laurell at Lund University with expertise in acoustophoresis, Jochen Guck at TU Dresden with expertise in cell mechanical characterization, Hywel Morgan at University of Southampton with expertise electrokinetics and impedance spectroscopy. For simulation and theory we have strong support from the leading group of Gerhard Gompper at Forschungszentrum Juelich.
Deterministic lateral displacement (DLD) is a highly powerful sorting mechanism that provides sorting based on size. The principle is simple. Small particles move along the fluid flow and particles larger than a certain threshold are deflected. In LAPASO our objective is to extend the usefulness of DLD by extending its scope beyond size-based sorting. We have demonstrated that combining it with dielectrophoresis renders it sensitive to the surface charge of the particles.
Acoustophoresis offers a non-contact mode of particle handling with minimal mechanical stress induced by acoustic forces. By applying an acoustic standing wave with a pressure node in the middle of a microchannel, most rigid particles and cells in suspension will be moved to the pressure node. The strength of the acoustic force is dependent on the physical properties of the cells in relation to the surrounding medium, enabling the separation of cells based on their size or density. In the framework of the LAPASO project we are targeting the isolation of rare cells from blood using acoustophoresis. One of the main objectives is the extraction and concentration of hematopoietic stem and progenitor cells for pre-clinical and research aims using acoustic forces in a microfluidic device. In addition, we are evaluating the separation of parasites from human blood using acoustophoresis to facilitate detection and diagnosis of infected patients. Besides, a clear need within the LAPASO project is the request for preprocessing whole blood; hence, we have initiated a research task to evaluate the possibility to use acoustophoresis as a preprocessing step of blood, removing the major fraction of red blood cells prior to taking on the task of separation and enrichment of rare cells in blood.
Figure: Schematic picture of an acoustophoretic chip design. A mixture of cells enters through the sample inlet in the pre-focusing channel with a standing
wave consisting of two pressure nodes. The cells are acoustically pre-focused into two parallel bands (A) before entering the main separation channel.
Here, acoustic forces in an ultrasonic standing wave field with a pressure node in the center of the channel, induce movement of cells depending on their
acoustophyscial properties (B). Larger cell complexes (indicated by the red color) experience a higher acoustic force and are moved quicker to the center
of the channel as compared to smaller cells (indicated by white circle). The separated cells can be collected either in the central outlet or the waste outlet
and used for further investigations.
Mechanical phenotyping and sorting of cells: the mechanical properties of cells are increasingly recognized as a potent inherent marker for cell functional changes. We have recently introduced real-time deformability cytometry (RT-DC) for the continuous mechanical screening of large cell populations with high throughput (1000 cells/sec). In several collaborations within LAPASO, we have already measured deformability and size for many cell types and pathogens of interest. Given the positive results in the first part of the project, there is now great impetus behind the goal to add sorting capabilities to RT-DC. Since RT-DC determines cell properties in real-time, this information will be used as a sorting trigger. We plan on realizing the sorting using Surface Acoustic Waves (SAW). Upon encountering the liquid flowing in the RT-DC microfluidic channel, the acoustic waves will leak into the liquid such that pressure nodes and anti-pressure nodes are formed within the channel. The acoustic radiation force will push the cell (in its wake) towards the pressure nodes, leading to cell sorting.
In future, mechanical phenotyping and sorting of cells could have significant impact on basic biological research, the diagnosis of cancer and blood-related disorders, and novel approaches in regenerative medicine and pharmacology.
Microfluidic Impedance Cytometry (MIC) is a cell characterization technique that measures the dielectric properties of cells as they flow between micro-electrodes in a microfluidic chip. In this project, MIC will be used to identify fundamental differences in rare stem cells as well as in cells infected with human pathogens. Skeletal stem cells from human bone marrow will be characterized in a label-free and non-invasive way with an anticipated impact on the development of new tools for rapid isolation of these cells for their clinical translation and application in bone regeneration therapies. Malaria-infected red blood cells will also be measured, and observed when compared to healthy erythrocytes. Further research aims to develop a point of care diagnostics tool for Malaria that can provide enhanced diagnostics at an early stage and without the need for complex sample preparation. Picture on the left: a Microfluidic Impedance Cytometry Chip.
Morphology-sensitive sorting to learn about bacterial pathogenesis and to develop devices that provide improved diagnostics of bacterial disease and information
about subpopulations to assist targeted treatment. Specifically, this individual sub-project is currently being carried out:
Bacterial extraction from whole blood
The goal of the project is to work towards a facilitation and acceleration of sepsis diagnosis. We identified two angles to approach the objective involving two different techniques available in the consortium. One approach is to concentrate bacteria from whole blood using acoustic trapping in collaboration with the group of Prof. Thomas Laurell. We aim to develop a diagnostic procedure including sample preparation, bacterial enrichment using acoustic trapping and detection/ identification. We have been working on different sample preparation protocols in the laboratories at the Karolinska Institute, Stockholm and continuously evaluate their performance in the acoustic trapping device with secondments at Lund University. Moreover we are exploiting the possibility to measure a differential mechanical response of white blood cells to an infection using Real-time Deformability Cytometry (RT-DC) in collaboration with the group of Prof. Jochen Guck. We got promising results from a two weeks secondment at Dresden Biotechnology Center and currently plan further experiments.
Morphology and deformability sensitive sorting to enrich parasites and parasite-infected cells for simpler and quicker diagnostics of severe disease.
Based on a fundamental understanding of the physical characteristics of rare cells we will develop optimum sorting schemes to extract rare to extract rare stem cells from bone marrow.
In the context of the work package 4 - "Rare Cells" - Walter Minnella, an ESR working at Elvesys, developed an "all purposes" microfluidic-based perfusion system for live cell imaging, which allows seamless media switches in hundreds of milliseconds. The system is presented on the left.
The core techniques for label free sorting, such as DLD and DEP, rely on the relative properties of the sample in comparison to the surrounding media. Therefore, being able to quickly and seamlessly change the buffer enables higher contrast and thus better sorting capabilities.
How does it work?
The system utilizes a pressure controller coupled to a rotative valve which allows the perfusion system to have high control over the flow rate and manage up to 10 different media at once. Furthermore, this design allows the user to use both microfluidic chips or standard perfusion chambers for cell culture and imaging. The advantages on using a rotative valve rely on the fact that it allows to have really low dead volumes (7 µL), thus minimizing sample waste, and eliminates the possibility of having a cross contamination between the reagents used. Furthermore, the high response time (120 ns) of the rotative valve permits extremely fast flow switches, of the order of 200 ms (see graph below).
The pressure controller allows to have a huge range of flow rates, spanning from 0.1 µL/min up to 5 mL/min. Furthermore, such precise control over the pressure enables to induce low shear stress on the cultured cell. These operations are remotely controlled via software, where the user can define programmed injections and sequences depending of his needs. Moreover, the design of complex injection profile allows to better mimic in vivo conditions, such as oscillating flows due to muscles contractions.
For more details and many proposed applications of this system please visit http://www.elveflow.com/ .
System integration and mass production combining different types of particle isolation schemes.
The purpose of the training is to prepare the fellows for future careers in academia, industry and other sectors of society as well as make them attractive on the job market as a whole. The training in LAPASO is structured into 5 components.
The results of the work in the project will be disseminated through publications in scientific journals, presentations at international conferences, at workshops and conferences organized by the project.
Outreach is a natural part of the fellow's dissemination. Picture below: Bao demonstrates microfluidic separation to high school students.
Picture below: Bao demonstrates microfluidic separation to high school students. Spherical Image - RICOH THETA
The LAPASO project is coordinated by Prof. Jonas Tegenfeldt, Lund University.
In addition, there are two committees, which share responsibility for decision-making in this project.
The Steering Committee is the main decision-making body of the project and is responsible for the general administrative and financial management. The Steering Committee consists of the representatives of each full partner, and a representative of the fellows.
The Supervisory Board co-ordinates the network-wide training activities and monitors the individual research projects. It consists of all of the primary supervisors, representatives of the Associated Partners, and a representative of the fellows.
The representative of the fellows in both committees during the first half of the project was Anke Urbansky, Lund University (nominated until June 2016). Since June 2016, Dr. Ahmad Ahsan Nawaz from TU Dresden has served as representative of the fellows.
© Stefan Holm 2014; last updated 28/10/2016 by GRE