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A Biomimetic Microfluidic Chip To Study The Circulation and Mechanical Retention of Red Blood Cells in The Spleen
A Biomimetic Microfluidic Chip To Study The Circulation and Mechanical Retention of Red Blood Cells in The Spleen
A JH
A biomimetic microfluidic chip to study the circulation and
mechanical retention of red blood cells in the spleen
Julien Picot,1,2,3,4 Papa Alioune Ndour,4,5 Sophie D. Lefevre,1,2,3,4 Wassim El Nemer,1,2,3,4 Harvey Tawfik,6
Julie Galimand,4,7 Lydie Da Costa,3,4,7,8 Jean-Antoine Ribeil,4,9,10,11 Mariane de Montalembert,10,12
Valentine Brousse,1,2,3,4,10,12 Bruno Le Pioufle,6 Pierre Buffet,4,5 Caroline Le Van Kim,1,2,3,4 and Olivier Français6*
Red blood cells (RBCs) are deformable and flow through vessels narrower than their own size. Their
deformability is most stringently challenged when they cross micrometer-wide slits in the spleen. In several
inherited or acquired RBC disorders, blockade of small vessels by stiff RBCs can trigger organ damage, but
a functional spleen is expected to clear these abnormal RBCs from the circulation before they induce such
complications. We analyzed flow behavior of RBCs in a microfluidic chip that replicates the mechanical
constraints imposed on RBCs as they cross the human spleen. Polymer microchannels obtained by soft
lithography with a hydraulic diameter of 25 lm drove flow into mechanical filtering units where RBCs flew
either slowly through 5- to 2-lm-wide slits or rapidly along 10-lm-wide channels, these parallel paths
mimicking the splenic microcirculation. Stiff heated RBCs accumulated in narrow slits seven times more
frequently than normal RBCs infused simultaneously. Stage-dependent retention of Plasmodium falciparum-
infected RBCs was also observed in these slits. We also analyzed RBCs from patients with hereditary
spherocytosis and observed retention for those having the most altered mechanical properties as
determined by ektacytometry. Thus, in keeping with previous observations in vivo and ex vivo, the chip
successfully discriminated poorly deformable RBCs based on their distinct mechanical properties and on
the intensity of the cell alteration. Applications to the exploration of the pathogenesis of malaria, hereditary
spherocytosis, sickle cell disease and other RBC disorders are envisioned.
Am. J. Hematol. 90:339–345, 2015. V
C 2015 Wiley Periodicals, Inc.
䊏 Introduction
Mature red blood cells (RBCs) are non-nucleated, biconcave disc-shaped cells of 7–8 lm in diameter, 2-lm-thick with an average volume of
80–100 lm3 [1]. Normal circulating RBCs are highly elastic and deformable and flow through capillaries narrower than their own size. When
RBC deformability is altered there is a risk of RBC blockade in small capillaries [2]. However, the most stringent mechanical challenge takes place
in the spleen when RBCs cross narrow inter-endothelial slits in the wall of red pulp sinuses [3,4]. In theory, a functional spleen clears the blood
from mechanically altered RBCs before they are trapped in microvessels, where they would create tissue damage. Altered mechanical properties
can be originated by multiple inherited RBC disorders, such as spherocytosis or sickle cell disease [5], or by infectious agents such as Plasmodium
falciparum [6]. Although these diseases result from very different molecular or cellular alterations they share the generic mechanism of poorly
deformable RBCs triggering complications, contributing for example to coma during severe Plasmodium falciparum malaria and anemia in heredi-
tary spherocytosis (HS) [7].
An important technical gap to study the pathophysiological impact of impaired RBC circulation is a device mimicking the dimensions of key
microcirculatory structures [8]. A physiologically relevant device should mimic 4- to 10-lm-wide small vessels (capillaries and small venules), and
0.5- to 3-lm-wide microcirculatory beds in the spleen [3,9]. Inter-endothelial slits in human spleens are typically in the 1-lm-wide range, as
measured by transmission electron microscopy [3]. To mimic the RBC retention rates encountered in the human spleen a device should thus
Additional Supporting Information may be found in the online version of this article.
1
Institut National De La Transfusion Sanguine, Paris, F-75739, France; 2InsermUMR_S1134, Paris, FranceF-75739; 3Universite Paris Diderot, Sorbonne Paris Cite, Paris,
France; 4Laboratory of Excellence GR-Ex, Paris, France; 5Inserm, U1135/Paris 6, Paris, FranceF-75634; 6SATIECNRS UMR8029, Ecole Normale Superieure De Cachan,
Cachan, FranceF-94235; 7AP-HP, Service Hematologie BiologiqueH^ opital RDebre, Paris, FranceF-75935; 8Inserm, U1149, Paris 7, Paris, FranceF-75018; 9Inserm,
UMR1163, Paris, FranceF-75743; 10Universite Paris Descartes, Sorbonne Paris Cite, Paris, France; 11Biotherapy DepartmentH^ opital Universitaire Necker Enfants
MaladesAPHP, Paris, France; 12Reference Centre for Sickle Cell Disease, Pediatric DepartmentH^ opital Universitaire Necker Enfants MaladesAPHP, Paris, France
Conflict of interest: Nothing to report
J.P., P.A.N., S.D.L., and W.E.N. contributed equally to this work.
Julien Picot is currently at GIP Genopole, Evry, F-91030, France
P.A.N. was supported by the laboratory of Excellence GR-Ex.
*Correspondence to: Olivier Français, SATIE, CNRS UMR8029, Ecole Normale Superieure de Cachan, 61 Avenue du President Wilson, 94235 Cachan, France.
E-mail: olivier.francais@satie.ens-cachan.fr
Contract grant sponsor: Laboratory of Excellence GR-Ex; Contract grant number: ANR-11-LABX-0051.
Contract grant sponsor: “Investissements d’avenir” of the French National Research Agency; Contract grant number: ANR-11-IDEX-0005-02.
Contract grant sponsor: Institute d’Alembert (Guidecells Project); LASIPS Labex (SIMULOS Project); DIM MalInf; the Gates Foundation; Follereau
Foundation.
Received for publication: 20 December 2014; Revised: 6 January 2015; Accepted: 8 January 2015
Am. J. Hematol. 90:339–345, 2015.
Published online: 12 January 2015 in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/ajh.23941
doi:10.1002/ajh.23941 American Journal of Hematology, Vol. 90, No. 4, April 2015 339
Picot et al. RESEARCH ARTICLE
Figure 1. Design (A), process flow (B), manufacture (C) of the spleen-like chip. Each filtering unit comprises of fifty-three 2-mm-wide slits between the 15-mm
pillars that form the boundaries of the lattice. The inner pillar lines comprise 12, 13, and 14 slits, the width of which is 5, 4, and 3 mm, from left to right. The U-
shaped filter is 275-mm-wide (including pillars) included a single 320 microscopic field captured by the camera sensor (18.6 3 6.7 mm2) Side channels are
10-mm-wide. The filtering unit is 5-mm-high. Microfluidic channels connecting the eight filtering units are 25-mm-high to minimize hydraulic resistance
between filtering units. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
contain narrow spaces of 2 lm. To replicate the specific shape the design, set-up and validation of a new biomimetic microfluidic
deformation of RBCs as they cross narrow splenic slits, in vitro device where the retention of poorly deformable human RBCs can be
obstacles should also be <10-lm long. With recent advances in robustly quantified and observed.
microtechnology and in particular in microfluidics [10–12], it is now
possible to envision “in vitro” experiments mimicking the microvas- 䊏 Methods
cular flow in vital organs [13]. Microchannels with dimensions of sev-
Patients. Small (0.5–1 ml) venous blood samples from 4 patients with hereditary
eral micrometers molded in PDMS have explored how RBC spherocytosis were retrieved with patient approval from EDTA tubes within 2 hr of
deformability may impact the pathogenesis of capillary obstruction collection in the context of normal medical care. The « Ile de France VI » Institu-
[14], and malaria [15]. Microfluidic devices with wider microchannels tional Review Board has approved this approach as a non-biomedical research pro-
(>30 lm) have opened the way to the real-time analysis of vaso- cess (Article L1121-1 of the French Code for Public Health) that does not affect the
medical care. Venous blood samples from three controls were also obtained using a
occlusion in sickle cell disease [16,17]. Most microfluidic approaches similar approach.
have quantified RBC deformability based on velocity through devices Microfluidic device fabrication. The spleen-like chip includes polymer micro-
that mimic microcirculatory structures [15]. An experimental system channels with an average hydraulic diameter of 25 lm, including local mechanical
that quantifies retention rather than velocity may better reflect patho- filtering units with an equivalent behavior of apertures from 5 to 2 lm (Fig. 1A).
Side flow, which is observable in the spleen, is rendered possible in the device, with
physiological processes occurring in malaria or other RBC diseases side channels added in the filter zone (Fig. 1A). The microfluidic shape that defines
that are associated with marked clearance of altered RBCs in the the filtering functions of the device is casted in polydimethylsiloxane (PDMS, Syl-
spleen, a process that contributes to the enlargement of the spleen. gard), a silicone elastomer [23]. The procedure to design the device is based on
A microfiltration device has been recently developed that uses standard microfabrication techniques associated to molding techniques [24]. We
first fabricated a master using SU-8 photoresist (Microchem, Newton, MA) to
microspheres to mimic the mechanical sensing of RBCs by the obtain a negative mold on a 4 in. Silicon substrate (Fig. 1B). A two-step phase of
human spleen [18]. This device provides relevant retention rates for photolithography with UV exposure had been developed. The first photolithogra-
poorly deformable RBCs [19,20] but does not allow a direct observa- phy defines the microchannels with a uniform thickness of 5 lm and a geometry
tion of RBCs as they cross narrow slits. By contrast, most microflui- corresponding to the repeated mechanical solicitation and filter design. The second
dic devices allow a direct morphological and dynamic exploration of photolithography gives the fluidic accesses with a uniform thickness of 25 lm in
order to reduce the access fluidic resistance. From the SU-8 mold, the casting of
RBCs as they navigate in the chip. RBC deformability is a complex PDMS (ratio 10:1) channels is realized. The chip obtained is then irreversibly
function involving membrane stiffness, cytoplasmic viscosity and bounded to a microscope cover slide using plasma 02 activation (30 W, 300 mT,
surface-to-volume ratio [21]. The deformability of P. falciparum- 20 s) on both surfaces. Before sealing, access holes were done in the PDMS with a
infected RBCs is even more complex [22]. Fine analysis of the event homemade punch giving female Luer (TM) connection compatibility.
The design is based on eight mechanical filtering units associated in parallel and
as it occurs is likely the most powerful way to decipher the complex connected together with a microchannel network as seen on the mold (Fig. 1C). To
cellular processes with a filtering capacity large enough to allow reduce the hydraulic resistance of the full design, the microchannel network is 25-
robust quantification of subpopulation enrichment. We present here lm-high compared to the 5-lm height of each filtering unit. The obtained
340 American Journal of Hematology, Vol. 90, No. 4, April 2015 doi:10.1002/ajh.23941
RESEARCH ARTICLE Red blood cells deformability in a spleen-like-microfluidic-chip
microfluidic device is fully transparent which makes possible the use of conven- From in silico to in vitro validation of physiological
tional inverted microscopes.
Red blood cell preparation. Cell suspensions: Packed RBCs from the French flow through the chip
blood bank (Etablissement Français du Sang, Rungis, France) were washed twice
The whole dimensions of the microfluidic biomimetic filter were
with 4 volumes of RMPI 1640 (Life technologies, France) and used for further
preparation. optimized using 3D numerical calculation based on finite element
Heated RBCs: Washed RBCs were suspended in PBS 1% AlbuMAX II (Life analysis (ComsolV C ), with the goal to obtain 80% of the cell flow pass-
Technologies, France) and then heated for 15 min at 50 C in a water bath fol- ing in the slit-shaped central zone that mimics the slow open micro-
lowed by two washes with PBS 1% AlbuMAX II (Life technologies, France).
circulation of the human spleen; the 10-mm-wide side channels
PKH labeling: RBCs were stained with PKH 67 fluorescent Cell Linker Kit
(heated or HS RBCs) or PKH 26 fluorescent Cell Linker Kit (control non-heated mimic the closed fast microcirculatory pathways (Supporting Infor-
RBCs) according to the manufacturer’s instructions (Sigma–Aldrich, France). mation Fig. S2A).
Infected RBCs: FUP line Plasmodium falciparum parasites (FCR3) was used for The microfilter hydraulic resistance, calculated from the numerical
all experiments. Parasites were cultured following Worldwide Antimalarial Resist- simulation, was 6.13 3 1014 Pa m23 s21. Such a high resistance value
ance Network (WARN) procedures in 5% hematocrit, at 37 C, with gas mixture:
5% CO2, 10% O2 and 85% N2, and RPMI 1640 media containing Hepes 25 affects considerably the fluidic properties within the device. Indeed, a
mmol l21, NaHCO3 25 mmol l21, Glutamine 0.3 g l21, Gentamycin 10 mg l21, cell solution flowing at 1 ll mn21 within the device will induce 100
and Serum 10%. Cultures were synchronized by sorbitol method (Lambros C, Van- mbar pressure drop, while the average cell velocity will be 1.1
derberg JP. 1979) and then by VarioMACS Separator (Miltenyi Biotec, France) to cm s21. The complete microfluidic structure, including eight filtering
obtain different, tightly synchronized stages of maturation.
Parasites staining: Synchronized ring-infected RBCs (8–16 hr) were stained with units set in parallel and access channel, presents 2.4 3 1014
Hoechst dye while trophozoites (26-34h) were stained with SYBR Green (Hoechst Pa m23 s21 as hydraulic resistance value [29].
and SYBR Green stain the nucleic acid of DNA, Life technologies, France). RBC On Supporting Information Fig. S2B, the velocity flow behavior
suspensions were prepared with the following composition: 5% of labeled ring- inside the chip is represented, close to the filter. It had been obtained
infected RBCs, 5% of labeled trophozoite-infected RBCs and 90% of control unla-
beled RBCs. A 0.1% hematocrit RBC suspension in PBS 1% AlbuMAX II was pre-
with an applied pressure of 100 Pa. The fluid velocity inside the filtering
pared with this cell mixture before being perfused in the microfluidic device. unit is different in the inner slit-shaped zone and in the side channels.
The microfluidic system setup. The microfluidic system consists of the microflui- The velocity profile (perpendicular to the flow, Supporting Informa-
dic device, tubing, fluid connectors, syringe and syringe pump (Syramed SP6000, tion Fig. S2C) obtained in the filter highlights the difference of velocity
Arcomed’Ag, Regensdorf, Switzerland) or MFCSTM-EZ microfluidic flow control value between the side channels and the inner zone of the filtering unit.
system (Fluigent, France). Tubes and fluid connectors were used to connect the
syringe to the chip. The syringe was filled with RBC suspensions and RBCs were The average ratio between these velocities was calculated to be 4.5 with
perfused through the microchannel network at a specified flow rate (0.2 ml h21 the chosen geometry, which was further confirmed by following experi-
and a mean of 100 mBar). The device was placed in an inverted Zeiss AxioOb- ments. Such velocity ratio between fast and slow circulations is reminis-
server Z1 microscope for monitoring RBC movements. Pictures of the eight filter- cent of observations in isolated-perfused spleens [3,4].
ing units were taken sequentially and at regular time intervals with a high
resolution AxioCam MRm Rev.3 camera and AxioVision 4 analysis software (Carl Mechanical retention of rigid heated red blood cells
Zeiss). Pictures of the whole filtering unit were taken using the 203/0.45 Plan-
Apochromate objective; smaller zones are imaged at higher magnification using the The capacity of the microfluidic chip to retain poorly deformable
633/1.40-Oil Plan-Apochromate objective (Carl Zeiss). Blue, green and red fluores- RBCs was tested. RBC rigidity was induced by heating the cells at 50
cent cells were visualized using the Colibri LED 365, 470, and 555 nm modules
C for 15 min. This reduces their size, induces their sphericity and
(Carl Zeiss), respectively.
Electronic microscopy of a human spleen. For the sizing of the microfluidic chip, increases their irreversible stiffness [30]. Heated and control RBCs
samples of human spleen were processed as described [25] then examined and photo- were then fluorescently labeled with PKH67 (green) or PKH26 (red),
graphed with a JEOL JSM 6700F field emission scanning electron microscope operat- respectively. A RBC suspension at 0.1% hematocrit, containing equal
ing at 5 or 7 kV (images were acquired with the upper SE detector on the lower
amounts of heated and control RBCs (Fig. 2A), was perfused through
secondary sector), or with a JEOL 1200 EX electron microscope operating at 80 kV.
Ektacytometry analysis. The ektacytometer [26] (LoRRca MaxSis, the device. RBC retention was monitored over time in the eight filter-
R
MechatronicsV, Hoorn, Netherlands) is a laser diffraction viscometer, in which the ing units. Pictures were taken at different pre-set time points during
deformation of red cells suspended in a viscous PVP solution at defined values of the experiment. For each filtering unit images were captured in the
applied shear stress of 30 Pa and at a constant temperature measurement of 37 C
green, red and brightfield channels and merged. A picture was also
are monitored as a continuous function of suspending medium osmolarity. Blood
samples from 4 HS patients and three healthy controls were exposed to increasing taken before perfusing the RBC suspension in order to confirm that
shear stress and an osmotic gradient. Three distinct features of the osmotic gradient the filtering units where not blocked by debris (Fig. 2B-T0). Early pic-
ektacytometry profiles are the Omin, the DImax and the O’hyper points. The tures showed that stiff heated RBCs (green-labeled) but not normal
Omin point corresponds to the osmolarity at the minimal deformability in hypoos-
(red-labeled) RBCs were retained preferentially in the 2 lm-wide slits
molar area or at the osmolarity when 50% of the red cells hemolyzed during the
regular osmotic resistance test. It reflects the surface to area ratio. DImax corre- (Fig. 2B-T1; T2; T3; T4 and C). The number of stiff heated RBCs
sponds to the maximal deformability index or elongation index (EI). The hyper retained in slits increased over perfusion time, with the vast majority
point or O’ corresponds to the osmolarity at half of the DImax and reflects the of the 2-lm-wide slits being clogged by these RBCs at the end of the
hydration state of the cells. In case of HS [27,28], the constant and characteristic assay (Fig. 2B-T4).
features are a decrease in the DImax, in conjunction with a shift of the Omin point
to the right (reduced surface to volume ratio) and a shift of the O’hyper point to
At the experiment endpoint, retained RBCs present in each zone
left (increased dehydration of the red cells). The severity is correlated to the level of were counted and sorted depending on their fluorescence (Fig. 2D).
the DImax. At the end of the assay, poorly deformable heated RBCs were seven
times more abundant in the filtering unit than control RBCs (Fig.
䊏 Results 2D, right panel) and often had a quasi-spherical aspect (Fig. 2C). Fil-
tering units could thus discriminate between deformable and non-
Size of human spleen microcirculation deformable RBCs, and could reveal the presence of subpopulations of
The dimensions of an interendothelial slit were determined from a rigid RBCs in a suspension.
transmission electron microscopy picture (Supporting Information Fig.
Retention of red blood cells parasited by plasmodium
S1A). The length and width of this slit were graphically estimated as 1.8–
2 lm and 4.5 lm (Supporting Information Fig. S1B), respectively, i.e., falciparum
similar to previous measurements [3,4]. We used these values for the The ability of the chips to retain poorly deformable RBCs was fur-
width and length of the narrowest slits in the filtering part of the chip. ther tested using RBCs infected with Plasmodium falciparum.
doi:10.1002/ajh.23941 American Journal of Hematology, Vol. 90, No. 4, April 2015 341
Picot et al. RESEARCH ARTICLE
Figure 2. Preferential retention of poorly deformable heated RBCs in slits of the filtering unit: (A) Equal concentrations of PKH26-stained normal (red) and
PKH67-stained poorly deformable heated RBCs (green) in the RBC suspension perfused through the chip. (B) T1!T4 progressive accumulation of poorly
deformable heated RBCs in slits: 4 (T1), 8 (T2), 12 (T3), 16 (T4) minutes after initiation of RBC circulation through the filtering unit (flow is from left to right).
(C) Ellipsoid or quasi spherical aspect of heated RBCs retained in 2-mm-wide slits (flow is from left to right, red arrows). (D) Number of poorly deformable
heated RBCs (green) and normal RBCs (red) retained in narrow slits of each of the eight filtering units in a chip, and mean values (right panel). [Color figure
can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
P. falciparum proteins (RESA, KAHRP, PfEMP3) exported at the green-labeled trophozoites and 15% (38/253) of blue-labeled ring-
membrane of infected RBCs contribute, among other factors [22,25] infected RBCs (Fig. 3D). Furthermore, during the first minutes of
to their stage-dependent decrease in deformability [31,32]. To test the perfusion, the ring-infected RBCs were preferentially retained in the
differential retention of infected RBCs, we perfused the biochip with 2-mm slits while trophozoites were retained in slits of all sizes (from
a cell suspension at 0.1% hematocrit containing 3 different RBC pop- 2 to 5 mm), consistent with the differences in the RBC mechanical
ulations: 5% of blue-labeled ring-infected RBCs, 5% of green-labeled properties between these two infection stages.
trophozoite-infected RBCs and 90% of control non-infected unlabeled Thus, despite the relatively low proportion of infected RBCs in the
RBCs (Fig. 3A). initial RBC suspension, the spleen-like microfluidic device successfully
RBC retention was monitored every 4 min in the eight filtering discriminated infected RBCs from non-infected RBCs, as well as
units of the chip. For each filtering unit images were captured in the young-infected from the late-infected RBCs, based on their distinct
blue, green and brightfield channels and merged. They showed that mechanical properties and on the intensity of the mechanical
infected RBCs (blue- and green-labeled RBCs) were preferentially alteration.
retained in the slits as compared to non-infected control RBCs (unla-
Retention of red blood cells from patients with
beled) (Fig. 3B,C). We analyzed the retained infected RBC sub-
populations and found that the mature trophozoites (26–34 hr of hereditary spherocytosis
maturation) accounted for a higher proportion of retained RBCs than We next explored another pathophysiological model using RBCs
young ring-infected RBCs (8–16 hr of maturation after reinvasion). from patients with hereditary spherocytosis (HS). In HS, RBCs are
Indeed, the retained RBCs were composed of 85% (215/253) of poorly deformable because of mutations in RBC membrane proteins
342 American Journal of Hematology, Vol. 90, No. 4, April 2015 doi:10.1002/ajh.23941
RESEARCH ARTICLE Red blood cells deformability in a spleen-like-microfluidic-chip
Figure 3. Stage-dependent retention of Plasmodium falciparum-infected RBCs in narrow slits. (A) Image capture of RBC suspension at 0.1% hematocrit con-
taining subpopulations of young ring-infected RBCs (rings) and late trophozoite-infected RBC, each at 5% parasitemia. Ring-infected RBCs were stained
with Hoechst dye (blue) and triphozoites were stained with SYBR Green (green). (B) Retention pattern of infected RBCs in the chip at the end of the experi-
ment. (C) High magnification of infected RBCs trapped in 2-mm-slits (flow in the chip is from bottom to top, red arrows). (D) Number of rings- or trophozoite-
infected RBC trapped according to the size of the slits at the end of experiment. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
that affect the vertical interactions between the lipid bilayer and the than the two others and that the microfluidic chip only retained HS
membrane skeleton, negatively impacting membrane cohesion and RBCs displaying the greatest phenotypic alterations.
leading to surface loss.
We analyzed RBC samples from 4 HS patients. HS and control 䊏 Discussion
RBCs were fluorescently labeled with PKH67 (green) and PKH26
(red), respectively, and perfused in the microfluidic chip as described We developed a simple microfluidic device to study and quantify
above for heated RBCs. RBCs of two patients (P1 and P2) were the mechanical ability of RBCs to cross the human spleen. We
retained in the chip, preferentially in 2-mm slits (Fig. 4A), while those favored a simple chip design that would reproduce key microcircula-
from the 2 other patients (P3 and P4) were not. We hypothesized tory structures of the human spleen and display a biomimetic behav-
that this difference resulted from the known heterogeneity among HS ior. These include unique, narrow filtering slits localized at the exit of
patients. As a matter of fact, HS patients exhibit heterogeneous phe- a slow circulatory pathway, and a wider fast circulatory pathway into
notypes depending on their mutated protein(s), the disease severity which RBCs are derived when slits are saturated. This fluidic architec-
being primarily dependent on the extent of membrane surface area ture was initially designed with numerical simulations using 3D finite
loss [33,34]. We evaluated the deformability and the surface area loss element analysis. Soft-lithography molding techniques were precise
of the 4 HS RBC samples by osmotic gradient ektacytometry profiles, enough to generate PDMS pillars delineating slits as narrow as 2 mm,
measuring three parameters: [1] the maximal deformability index the channel height being 5 mm. Relatively large slits (3- to 5-lm
(DImax) value, which represents the maximal cellular deformability wide) are predicted to exert mechanical constraints on RBCs similar
of the RBC population and [2] the Omin point, which corresponds to to those generated by the tortuous structures of the cords in the sple-
the osmolarity at which 50% of the RBCs are lysed in the classical nic red pulp (also called reticular meshwork [35]). The narrowest 2
osmotic fragility test and reflects the reduction in surface area to vol- mm-wide slits mimic the last and most stringent mechanical obstacle
ume ratio of the RBC population being analyzed (a shift to the right that RBCs must cross on their way from the cords back to the venous
of the Omin corresponding to a reduced surface-to-volume ratio), circulation [18]. The width of side channels mimicking the fast circu-
and [3] the O’ or the hyper point, which reflects the hydration state lation were set at 10 mm to allow RBC circulation with minimal
of the red cells. As expected, in conjunction with the presence of mechanical constraints. A biomimetic spleen-like device has been
spherocytes on the blood smears, all HS RBC samples exhibited the recently reported, beautifully mimicking the closed-fast and the open-
classical features of HS when compared with control RBCs (Fig. 4B). slow microcirculations, the latter ending by a row of a dozen of paral-
In addition, the two RBC samples from P1 and P2, that were retained lel 2-lm constrictions resembling the inter-endothelial slits. Dynamic
in the microfluidic chip, showed lower DImax and higher Omin than crossing could be observed but the number of slits was too limited to
those from P3 and P4 (Fig. 4B), indicating that these RBCs were less robustly quantify enrichment of poorly deformable subpopulations
deformable and more spherical (reduced surface-to-volume ratio) [36]. Our device includes eight filtering units per chip, each
doi:10.1002/ajh.23941 American Journal of Hematology, Vol. 90, No. 4, April 2015 343
Picot et al. RESEARCH ARTICLE
344 American Journal of Hematology, Vol. 90, No. 4, April 2015 doi:10.1002/ajh.23941
RESEARCH ARTICLE Red blood cells deformability in a spleen-like-microfluidic-chip
might partly result from mechanical constraints and this could be 䊏 Acknowledgments
evaluated and measured using our microfluidic device.
The authors thank Selin Topçu for her help during the fabrication
phase of the biodevices.
doi:10.1002/ajh.23941 American Journal of Hematology, Vol. 90, No. 4, April 2015 345