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.
Streptococcus pneumoniae is a gram positive human pathogen that causes invasive diseases such as pneumonia, meningitis and septicemia. The objectives of the project were to gain better understanding of pneumococcal pathogenesis and to participate in the development of microfluidic devices that can be used for sorting bacterial populations based on their physical properties such as size, cell arrangement (single cocci, diplococcic, chains) and presence of capsule.
Our research has led to advancements in the understanding of the pathogenesis of pneumococcal meningitis. We have shown that a pilus, expressed in a subset of strains, increases the ability of pneumococci to invade the brain to cause meningitis. The expression of this pilus could be correlated with the ability of pneumococci to form small single cocci in the brain. Strains which express this pilus are significantly better at entering the brain and causing meningitis than their non-piliated counterparts. These results were published in the Journal of Clinical Investigations (see Publications). Our work has also allowed advancements in the optimization of microfluidic devices and sorting parameters for the study of bacterial populations, especially in their use for sorting pathogenic bacterial strains in subpopulations that have different physical characteristics.
In the course of this project many opportunities were provided for hands-on training in the fields of microfluidics, such as casting devices, working with microfluidic devices (sample preparation, running the device, troubleshooting) and with the associated instruments such as microscopes and pressure controllers. Other training activities included workshops and access to national and international conferences in the fields of infection biology, bacterial genetics and microfluidics.
The results of this project will provide added knowledge to understand the pathogenesis of the pneumococcus, one of the major human pathogens, as well as providing new tools to study different aspect of pneumococcal biology and virulence.
The project has allowed the fellows to interact and learn in a multidisciplinary network of very high scientific quality. The interdisciplinary context of the project has expanded the fellows’ knowledge in other research areas; have provided them with new approaches to solve problems as well as providing them with opportunities for collaborations beyond the immediate project. This project has, and will, generate publications in high impact factor journals.
Morphology and deformability sensitive sorting to enrich parasites and parasite-infected cells for simpler and quicker
diagnostics of severe disease.
Towards diagnosis of parasitic diseases (leishmaniosis, sleeping sickness and malaria) we have used the three technologies (1) deterministic lateral displacement (DLD), (2) real time-deformability cytometry (RT-DC) and (3) microfluidic impedance cytometry (MIC). DLD was successfully applied to the separation of Leishmania mexicana promastigotes from red blood cells. RT-DC measurements demonstrated that the changes in mechanical properties of infected macrophages are correlated to the infection progress culture. Also, the effect of L. mexicana infection on the dielectric properties of macrophages was studied by microfluidic impedance cytometry (MIC). Especially the design of deterministic lateral displacement (DLD) devices for separation of parasites (leishmania/trypanosomes) or malaria infected red blood cells from healthy erythrocytes was supported by modelling. Development of 2D and 3D simulation methods were developed for predicting particle trajectories through devices.
Our work on the enrichment of parasites and parasite-infected cells, as well as on biomarker discovery, has the potential to lead to the development of novel diagnostic techniques which are affordable, easy to use in the field, rapid, sensitive and specific. This could therefore have an important impact on the control of leishmaniosis as well as malaria.
Based on a fundamental understanding of the physical characteristics of rare cells, this WP developed optimum sorting schemes to extract rare stem cells
The cell size and dielectric properties, and the mechanical properties of skeletal stem cells (SSC) were investigated using a
custom-designed microfluidic impedance cytometer and real-time deformability cytometry respectively. These studies helped define
the requirements for designing a microfluidic device for label-free SSC enrichment from human bone marrow based on the principle
of deterministic lateral displacement (DLD), a fundamentally size-based cell sorting technique that is also sensitive to
differences in cell mechanical properties. DLD devices were designed and fabricated, and validated using cell lines.
On-going studies beyond the end of the project aim to assess the potential and functionality of sub-populations of SSCs
fractionated using DLD.
Acoustophoresis as an initial step for further enrichment of rare cells was investigated. We tested the separation of mononuclear cells (MNC), a subpopulation of white blood cells accounting 0.6% of all blood cells, from whole blood. By optimizing the buffer conditions and thereby changing the acoustophoretic mobilities of the different cell types we were able to achieve efficient enrichment of MNCs. Further we designed an acoustophoresis microchip for multiparameter sorting allowing high separation efficiency and purity for up to three different populations. The system was successfully tested with different sized beads as well as separation of unlabeled white blood cell populations.
Extraction and concentration of hematopoietic stem and progenitor cells using acoustophoresis was challenging in a label-free manner due to similar acoustophysical properties of the different cell types in the starting sample. We therefore worked on an acoustophoretic affinity-bead mediated system and showed a proof-of-principle for both CD4 and CD8 cells from peripheral blood. We are currently working on improving binding of the affinity-beads to the stem cells to be able to acoustically isolate the desired cell population.
This research has been a success mainly due to the highly multi-disciplinary expertise offered by the different project partners. PhD students and post-doctoral fellows were able to benefit from collaborative sub-projects between partners and accelerate their learning process by interacting directly with experts from different scientific areas.
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 were disseminated through publications in scientific journals (see Publications), 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 until the end of the project, Dr. Ahmad Ahsan Nawaz from TU Dresden served as representative of the fellows.
© Stefan Holm 2014; last updated January 2019 by GRE