1 MARIE SKLODOWSKA-CURIE ACTIONS Co-funding of regional, national and international programmes (COFUND) DOC2AMU THESIS PROJECT 2018 CALL FOR APPLICATIONS Active response of Red Blood Cells to mechanical stress in splenic filtration 1. GENERAL INFORMATION Call 2018-23 Topic Nano-health Keywords Human red blood cells, splenic filtration, active volume regulation, microfluidics, live microscopy, computational modelling 2. THESIS DIRECTOR(S), RESEARCH UNITS AND DOCTORAL SCHOOLS Thesis director Emmanuèle HELFER Research Unit Centre Interdisciplinaire de Nanoscience de Marseille Doctoral school ED 352 - Physique et Sciences de la Matière Thesis co-director Catherine BADENS Research Unit Génétique Médicale et Génomique Fonctionnelle Doctoral school ED 062 - Sciences de la Vie et de la Santé
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MARIE SKLODOWSKA-CURIE ACTIONS
Co-funding of regional, national and international programmes (COFUND)
DOC2AMU THESIS PROJECT 2018 CALL FOR APPLICATIONS
Active response of Red Blood Cells to mechanical stress in splenic filtration
1. GENERAL INFORMATION
Call 2018-23
Topic Nano-health
Keywords Human red blood cells, splenic filtration, active volume regulation, microfluidics, live microscopy, computational modelling
2. THESIS DIRECTOR(S), RESEARCH UNITS AND DOCTORAL SCHOOLS
Thesis director Emmanuèle HELFER
Research Unit Centre Interdisciplinaire de Nanoscience de Marseille
Doctoral school ED 352 - Physique et Sciences de la Matière
Thesis co-director Catherine BADENS
Research Unit Génétique Médicale et Génomique Fonctionnelle
Doctoral school ED 062 - Sciences de la Vie et de la Santé
1
MARIE SKLODOWSKA-CURIE ACTIONS
Co-funding of regional, national and international programmes (COFUND)
DOC2AMU THESIS PROJECT 2018 CALL FOR APPLICATIONS
Active response of Red Blood Cells to mechanical stress in splenic filtration
(ActiveRed)
1. DESCRIPTION OF THE PHD THESIS PROJECT
1.1 OBJECTIVES OF THE PROJECT BASED ON THE CURRENT STATE OF THE ART
Blood consists in a highly concentrated suspension (45% in volume) composed mainly of red blood cells (RBCs,
99%) and few other blood cells (leukocytes and platelets, 1%). The efficient and sustainable circulation of RBCs
is an outstanding physical tour de force. During their 120-days lifespan, they continuously circulate through our
intricate microvascular network composed of slits, capillaries, bifurcations, etc. During such cycles they undergo
very strong deformations: for example, the RBC passes through blood vessels as small as 4 µm in diameter or
through submicron slits located in the spleen. The RBC being a biconcave disk around 2 µm in thickness and 8
µm in diameter, it cannot go through such constrictions if not highly deformable, and highly robust as well. This
is in part due to its complex double envelope that encloses the viscous haemoglobin solution. This double shell
is made of an incompressible fluid viscous lipid bilayer at the outside and a 2D elastic network of cross-linked
spectrin filaments at the inside, connected to the lipid bilayer by protein complexes. Additionally, a
mechanosensitive ion channel was recently discovered whose activation is triggered by a mechanical stress
applied on the RBC membrane [Coste 2012]. A second ion channel ion is then activated in cascade, leading to
water release out of the cell, as a way to control the RBC volume [Rapetti-Maus 2015]. A new hypothesis thus
arised that these ion channels could play an active role in RBC volume changes, in order to rapidly adjust the cell
deformability under a mechanical constraint.
The purpose of the ActiveRed project is to understand quantitatively the physical mechanisms of large
deformation and the molecular mechanisms of volume regulation in the specific case of the passage through
the splenic submicron slits. Recent studies indeed suggest that the spleen senses RBC deformability and
spheroidicity thus defining the size and shape of RBCs allowed to remain in the microcirculation [Pivkin 2016]. A
current hypothesis is that RBCs have to pass a ‘physical fitness test’ in the spleen, the submicron slits (Fig. 1A),
to be allowed to remain in the blood flow. Yet, no known physical mechanisms rationalize this hypothesis as
experiments are strongly lacking.
From a physics point of view, the process of splenic filtration raises basic physical questions: what is the link
between RBC mechanical parameters and passage/sequestration in splenic slits? What mechanical properties of
RBCs are most crucial to go through submicron splenic slits? Is the selection mechanism of RBCs by the spleen
based on mechanical criterions only? Or are indeed additional active phenomena, such as volume change,
needed to avoid RBC rupture under large deformation?
From a clinical point of view, there is a strong need for understanding the clearance process by the spleen. It
normally occurs as RBCs age and lose their deformability, to eliminate older RBCs and renew the RBC pool in the
blood stream. However, the spleen is also a major player in a number of diseases, whether infectious (malaria)
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or genetic (sickle cell disease (SCD), hereditary spherocytosis (HS), hereditary xerocytosis (HX)…). In all these
diseases, the RBC deformability is altered and RBCs get massively sequestered in the spleen. It thus causes severe
hemolytic anemia due to an accelerated splenic destruction of RBCs. Therefore, we expect that physics
experiments leading to understanding how an RBC passes the spleen fitness test and how the process is affected
in RBC genetic disorders will have a major impact in haematology.
Figure 1. A) An RBC (highlighted in red) squeezing through a splenic slit (≈0.5 x 2 x 5 µm3). B) SEM image of
the silicon master of a typical microfluidic device with a series of slits. C) Optical images of RBCs squeezing
through 0.8 x 1.9 x 5 µm3 biomimetic slits. The bottom one displays a tip while exiting the slit. Scale bar: 5 µm.
The ActiveRed project is based on the recent technological breakthrough we made in 2017, by fabricating a
microfluidic device with slits of physiological splenic dimensions (Fig. 1B) [Gambhire 2017]. This device allowed
us to observe human RBCs passing through these biomimetic slits and revealed new modes of deformation due
to high confinement (Fig. 1C, bottom). This is the first device that reproduces the dimensions of splenic slits (≈0.5
x 2 x 5 µm3) in comparison with previous biomimetic devices that could not reach such small dimensions [Rigat-
Brugarolas 2014, Deplaine 2011]. We will study the RBC behavior as they are submitted to controlled mechanical
stress (the slit dimensions and the flow pushing the RBCs). To investigate whether the RBC volume actively
changes in response to the stress, we will target the two ion channels which are thought to act together, the
mechanosensitive Piezo1 and the Ca2+-sensitive Gardos (Fig. 2A).
Figure 2. A) Schematics of Piezo 1 and Gardos interplay that controls ion fluxes: mechanical stress, e.g. RBC
stretching, activates Piezo1 which become permeable to cations, including calcium. The Ca2+ influx activates
Gardos leading to K+ and Cl- exit concomitantly with water. It results in a decrease in RBC volume, thus in an
increase in area-to-volume ratio, and presumably an increase in healthy RBC deformability. B) Activators
(arrows) and inhibitors of the two channels that will be used in the study.
Most studies on RBC ion channels are done on non human cells, mostly murine ones [Cahalan 2015], neglect
physiological flow and usually focus on one or the other channel. Here, modulation of Piezo1 and Gardos channel
activities will be studied in combination and not separately, in human RBCs, and in the physiological situation of
RBCs flowing though splenic slits. A recent work by the group of Kaestner studied their interplay using 3-µm wide
constrictions, they observed a response even at such small RBC deformation [Danielczok 2017]. We thus expect
a stronger response by using our biomimetic splenic slits.
The channels’ activity and interplay will be modulated via various combinations of known inhibitors and
activators (Fig. 2B). Healthy RBCs as wells as RBCs with disordered channels will be studied. Indeed, HX disease,
A B
ℓ= 1.9 µm 𝓌 = 0.8 µm
C
A B
Ca2+
Piezo 1 Gardos K
+
RBC
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due to various mutations in either Piezo1 or Gardos channels, is characterized by abnormal cation leak and cell
dehydration, leading to cell fragility and hemolytic anemia [Badens 2016]. We have access to a pool of patients
with some of these mutations. Their RBCs will be assayed in the biomimetic slits, untreated and treated with the
biochemical blocking/activating agents.
Our quantitative results will be combined with 3D computations (from international collaborator) that take into
account the RBC dynamics and the channels’ activity to derive the physical mechanisms responsible for RBC
active response to mechanical stress applied. Our findings will highlight which physical parameters can be used
as a new read out to follow disease evolution or treatment effect, and potentially lead to novel therapeutic
targets.
References:
Badens C and Guizouarn H. Advances in understanding the pathogenesis of the red cell volume disorders. Review.
Brit J Haematology 174:674-685 (2016)
Cahalan SM, Lukacs V, Ranade SS, Chien S, Bandell M, Patapoutian A. Piezo1 links mechanical forces to red blood
cell volume. eLife 4:e07370 (2015)
Coste B et al. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.
Science 330:55–60 (2010)
Danielczok et al. Red Blood Cell Passage of Small Capillaries Is Associated with Transient Ca2+-mediated
Adaptations. Front Physiol 8:979 (2017)
Deplaine et al. The sensing of poorly deformable red blood cells by the human spleen can be mimicked in vitro.
Blood 117:e88–e95 (2011)
Gambhire et al. High aspect ratio sub-micron channels using wet etching: Biomimetic spleen slits for red blood
cell studies. Small 13:1700967 (2017)
Pivkin IV et al. Biomechanics of red blood cells in human spleen and consequences for physiology and disease.
PNAS 113:7804–7809 (2016)
Rapetti-Maus et al. A mutation in the Gardos channel is associated with hereditary xerocytosis. Blood 126:1273-
1280 (2015)
Rigat-Brugarolas et al. A functional microengineered model of the human splenon-on-a-chip. Lab Chip 14:1715
(2014)
1.2 METHODOLOGY
The PhD program is pluridisciplinary and the student will learn the different required skills in the groups of the
two supervisors. The different tasks and milestones of the ActiveRed project are:
Task 0 (UMR_S910): Blood collection – Healthy and pathological blood sample collection will be obtained in the
context of regular clinical follow up at Hospital La Timone, and characterized.
Task 1 (UMR_S910): Characterization of Piezo1 and Gardos channel activity – Inhibitors and activators of Piezo1
and Gardos will be used to modulate RBC permeability (healthy and mutated). RBC ion content will be measured
under the various treatments to define the optimal combinations that will be assayed in the physics experiments.
Task 2 (CINaM): Relationship between RBC mechanical properties, RBC volume and channel activity as a function
of the applied mechanical stress – Prior to the microfluidic experiments, RBCs will be submitted to
meso/macroscopic deformations using atomic force microscopy (AFM) and optical tweezers. These techniques
allow applying forces in the pN-µN range. The resulting Ca2+ influx will be tracked using the commercial Fluo-4
calcium probe. Healthy and channel-deficient RBCs will be studied. From these measurements the
mechanosensitive characteristic response times will be extracted for healthy and patients’ RBCs.
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Task 3 (CINaM): Design and fabrication of new biomimetic devices – In the current device both the main wide
channel and the thin slits have the same height, thus RBCs are horizontally constrained before reaching the slits.
To get a geometry closer to that of the spleen, the device will be improved to have a main channel of larger
height (> 10 µm) with slits of 5 µm height.
Task 4 (CINaM + UMR_S910): Physical fitness test: relation between RBC deformation, transit time, flow rate,
channel activity, and slit size – Microfluidic experiments on individual RBCs flowing through biomimetic slits
(current and future devices) will be performed using ultrafast-videomicroscopy (> 1,500 fps) to observe RBC
deformation combined with standard fluorescence videomicroscopy (25 fps) to track calcium influx.
Experimental conditions (flow rate, slit dimensions) will be optimized so that the transit time is higher than the
response time derived from mesoscopic experiments and to be able to observe the calcium entry. The passage
of RBCs from healthy donors and from patients will be studied in absence and presence of the channel’s
modulators. The goal is to establish the laws of behaviour between the severity of the fitness test (given by slit
dimensions and flow rate) and RBC dynamics (shape deformation and velocity, ion fluxes).
Task 5 (CINaM + UMR_S910): PhD thesis writing and defense
Task 6 (Collaborator, Univ Notre Dame, USA): Modelling of the RBC active deformation under mechanical stress
– During the total duration of the project, we will communicate with our collaborator in the USA who will develop
a 3D model of the RBC that integrates the double envelope components, the channels, the resulting ion
transport, and the effect of the channel regulators. The coupling of experimental and numerical approaches will
lead to identification of the critical factors that cause RBC deformation, entrapment or damage, and investigate
how molecular mutations influence these critical factors. We expect to provide a complete physical
understanding of the dynamics of RBCs in spleen-like slits that will allow to predict the RBC behavior and the risk
of damage in case of altered mechanical properties. Moreover, we will conclude on the existence of an active
volume regulation in response to applied stress during spleen filtration.
1.3 WORK PLAN
1.4 SUPERVISORS AND RESEARCH GROUPS DESCRIPTION
Supervisor 1 – Centre Interdisciplinaire de Nanoscience de Marseille (AMU/CNRS, UMR7325, Marseille)
Emmanuèle HELFER joined CINaM in 2014, and now belongs to the newly created Physics and Engineering of
Living Systems (PIV) Department. The CINaM is a multidisciplinary structure, composed of approximately 180
persons, which hosts a nano/micro-fabrication platform that allows design and fabrication of complex