1. No category

Biomaterials 43 (2015) 23e31
Contents lists available at ScienceDirect
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
Cell selective chitosan microparticles as injectable cell carriers for
tissue regeneration
dio a, b, M.T. Cerqueira a, b, A.P. Marques a, b, R.L. Reis a, b, J.F. Mano a, b, *
C.A. Custo
a
udio do Barco,
3B's Research Group e Biomaterials, Biodegradables and Biomimetics, University of Minho, AvePark, Zona Industrial da Gandra, S. Cla
~es, Portugal
4806-909 Caldas das Taipas, Guimara
b
~es, Portugal
ICVS/3B's, PT Government Associated Laboratory, Braga, Guimara
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 September 2014
Accepted 24 November 2014
Available online
The detection, isolation and sorting of cells holds an important role in cell therapy and regenerative
medicine. Also, injectable systems have been explored for tissue regeneration in vivo, because it allows
repairing complex shaped tissue defects through minimally invasive surgical procedures. Here we report
the development of chitosan microparticles with a size of 115.8 mm able to capture and expand a specific
cell type that can also be regarded as an injectable biomaterial. Monoclonal antibodies against cell
surface antigens specific to endothelial cells and stem cells were immobilized on the surface of the
microparticles. Experimental results showed that particles bioconjugated with specific antibodies provide suitable surfaces to capture a target cell type and subsequent expansion of the captured cells.
Primarily designed for an application in tissue engineering, three main challenges are accomplished with
the herein presented microparticles: separation, scale-up expansion of specific cell type and successful
use as an injectable system to form small tissue constructs in situ.
© 2014 Elsevier Ltd. All rights reserved.
Keywords:
Cell separation
Antibody immobilization
Injectable materials
Functional materials
Chitosan microparticles
1. Introduction
The success of many cell therapies is highly dependent on the
development of techniques for isolation and selection of cells that
guarantee high yield and purity. One of the limitations in stem cell
isolation is the restricted quantity that can be isolated from a tissue
and the typical heterogeneity of the cell population. Obtain a pure
cell population is particularly challenging when the target cells are
rare in the total amount of cells in the sample. Methods for cell
separation based on a difference in physiochemical properties, such
as density and size have been reported, but the purity of the obtained population is generally low [1,2]. Thus the need for alternative methods to select subsets of cells with increased yield
decreasing cell manipulation and expansion time is a reality.
Moreover, the development of systems suitable for an efficient and
effective cell isolation/separation that can act as a cell expansion
platforms may result in a separation method that are much more
specific for the population of interest [3]. An efficient system for
* Corresponding author. 3B's Research Group e Biomaterials, Biodegradables and
udio do
Biomimetics, University of Minho, AvePark, Zona Industrial da Gandra, S. Cla
~es, Portugal.
Barco, 4806-909 Caldas das Taipas, Guimara
E-mail address: [email protected] (J.F. Mano).
http://dx.doi.org/10.1016/j.biomaterials.2014.11.047
0142-9612/© 2014 Elsevier Ltd. All rights reserved.
adhesion-based cell separation lies in the specificity of an immobilized biomolecule to the target cell population. Fluorescenceactivated cell sorting (FACS) effectively provides high efficiency in
cell sorting, but this technique has associated high cell manipulation and requires a demanding operational training [4,5]. Magnetic
activated cell sorting is another separation technique based in
surface markers, where cells bind to antibody labeled magnetic
particles [6,7]. Antibody-coated microchips have also been used to
successfully detect and isolate rare circulating tumor cells from
peripheral blood [8,9]. All of these techniques have been in use for
years and have shown some degree of success. Nevertheless, none
of the described techniques provide support for cell expansion or
proliferation. In an effort to overcome some of these limitations
Nguyen et al. have recently reported the use of multilayered magnetic microparticles as a novel strategy to isolate, expand and
detach endothelial progenitor cells [10]. Still, these particles are not
suitable for the implementation of an injectable system to form a
biodegradable construct in situ. Thus efforts are necessary to
develop implantable supports for specific cell populations [11]. To
overcome these issues while decreasing cell manipulation and time
consumption, we report a new system for cell separation and
expansion that may ultimately be injected to form a scaffold in situ
for tissue regeneration purposes.
24
dio et al. / Biomaterials 43 (2015) 23e31
C.A. Custo
Chitosan is well known as a biodegradable and biocompatible
material [12e14]. In addition, the amino and hydroxyl chemical
groups along chitosan chains enable this polysaccharide to form
stable covalent bonds with many molecules of interest [14e16]. In
our previous work, we demonstrated that chitosan films grafted
with antibodies were able to promote selective cell attachment and
growth [15]. Additionally to cell isolation and expansion, we
envisage the development of a system for directly deliver cells in
vivo, decreasing cell manipulation. The goal of this work was to
explore the use of functional chitosan microparticles, as a strategy
for cell separation and expansion that may be used as an injectable
system to form tissue constructs at the lesion site for tissue
regeneration purposes.
Chitosan microparticles were firstly functionalized with biotin.
Such modification allows engineering the surface of microparticles
with a variety of biotinylated biomolecules via streptavidin (SaV),
increasing its versatility and yield due to the multiple binding sites
for biotinylated molecules. We tested the immobilization of biotinylated antibodies to target endothelial cells and human adipose
stem cells (ASCs). Biotinylated antibody anti-CD31 was used to
target human umbilical vein endothelial cells (HUVECs) cells while
biotinylated antibody anti-CD90 was used to capture ASCs.
2. Materials and methods
2.1. Preparation of chitosan microparticles
Medical grade chitosan (150e300 kDa and a deacetylation degree of 95%)
(Heppe Medical Chitosan GmbH, Germany) was dissolved in a 2% v/v aqueous
acetic acid (VWR, UK) solution to a final concentration of 2% w/v. Subsequently,
the chitosan solution was passed through an aerodynamically-assisted jetting
equipment (Nisco Encapsulation Units VAR J30, Switzerland) at a speed of 1 ml/
min. The injected air led the chitosan solution to break up into a spray at the outlet
of the nozzle. The generated microdroplets were hardened into a 1.0 M sodium
hydroxide solution (Panreac, Spain) that resulted in the production of chitosan
microparticles. After solidification, microparticles were thoroughly washed in
distilled water and sieved to the desired particle size of z120 mm. Microparticles
were then sterilized in 70% ethanol and stored in phosphate buffered saline (PBS)
(Sigma, USA) solution at 4 C until further use. The produced particles were
imaged using an Axiocam MRC-5 camera on a Axio Imager Z1M microscope (Zeiss,
Germany). Analysis of microparticle size was performed using ImageJ software
(Image processing and analysis in Java). Scanning electron microscopy (SEM)
(JSM-6010LV, JEOL, Japan) was performed to characterize the surface of the particles. Samples were dehydrated in graded series of ethanol solutions and sputtered with gold prior analysis.
2.2. Functionalization with biotin
Microparticles were resuspended in a solution of 1 mg/ml (þ)-Biotin Nhydroxysuccinimide ester (Biotin-NHS) (Sigma, USA) in PBS:DMSO (3:1) and stirred
for 3 h at room temperature. Microparticles were then washed thoroughly with PBS,
to remove the unlinked biotin. To assess the effective functionalization with NHSBiotin, modified particles were incubated with DyLight 488 SaV (BioLegend, Germany) (10 mg/ml) for 15 min at room temperature and finally washed with PBS. As
control, plain particles were similarly incubated with the fluorescent labeled SaV.
Images were acquired using an Axiocam MRm camera on a Axio Imager Z1M
microscope.
2.3. Bioconjugation with biotinylated antibodies
Particles were incubated with purified SaV (Promega, USA) (50 mg/ml) in PBS
under constant stirring. After 15 min incubation at room temperature, the particles
were washed to remove unbound SaV. Biotinylated antibodies were then used to
functionalize the modified particles. The functionalization was performed with both
biotin-anti-CD31 and biotin-anti-CD90 antibodies (10 mg/ml) (eBioscience, UK)
under constant stirring for 15 min at room temperature. Microparticles were
washed with PBS at the end of the process to remove all unbound antibodies. The
detection of the immobilized biotinylated antibody was assessed by conjugation
with a secondary labeled antibody Alexa Fluor 594 (Invitrogen, USA). Images were
acquired using an Axiocam MRm camera on a Axio Imager Z1M microscope. SEM
analysis (JSM-6010LV, JEOL, Japan) was performed to evaluate the surface of the
particles after modification.
a collaboration protocol with 3B's Research Group approved by the ethical committees of both institutions. hASCs were isolated as previously described [17].
Briefly, after digestion with 0.05% Collagenase type II (Sigma, USA), a filtration and
centrifugation at 800 g were performed and the stromal vascular fraction (SVF)
obtained. The red blood cells were lysed with a 155 mM of ammonium chloride,
12 mM of potassium bicarbonate and 0.1 M of ethylenediaminetetraacetic acid buffer
(all from SigmaeAldrich, Germany). The red blood cells-free SFV was resuspended in
alpha-MEM medium (Invitrogen, USA), supplemented with 10% fetal bovine serum
(FBS) (Invitrogen, USA) and 1% Antibiotic/Antimycotic (Invitrogen, USA). Cell culture
medium was changed, 48 h after initial plating and every 3 days thereafter. ASCs
were used between passages 2 and 5.
The HUVECs (Gibco, USA) were maintained in M-199 medium (Sigma, USA)
supplemented with sodium bicarbonate, 1% antibiotic/antimycotic, 20% FBS (Invitrogen, USA), 0.34% glutamax (Gibco, USA), 50 mg/ml Endothelial Cell Growth Supplement (ECGS) (BD Biosciences, USA) and 50 mg/ml Heparin (Sigma, USA). Cell
culture medium was changed every 3 days. HUVECs were used between passages 2
and 6.
2.5. Cell capture from a homotypic cell population and expansion
The specificity of the functionalized particles was evaluated by analyzing their
capacity to capture the cells from a homotypic cell suspension. hASCs and HUVECS
were seeded separately at a density of 10 cells per particle on anti-CD31, anti-CD90
and SaV-bioconjugated particles. A total of 5 103 particles per well were placed in a
non-adherent 24-well plate. The interaction between the cells and the functionalized particles was monitored within the first 6 h by time-lapse live imaging in Axio
Observer (Zeiss, Germany).
After 6 h of incubation at 37 C, 5% CO2, the suspension of cells and particles was
passed through a 37 mm strainer (StemCell Technologies, France) in order to remove
non-attached cells. The cell-particles complexes were then cultured up to 7 days, in
the respective cell media. To assess the capacity of the particles to support cell
expansion, the DNA quantification was performed after 1, 3 and 7 days in culture. A
set of samples was analyzed for cell morphology. Samples were fixed with 10%
formalin and stained with phalloidin-TRITC (Sigma, USA) for the cytoskeleton
visualization, and DAPI (4,6-diamidino-2-phenylindole, dilactate) (Invitrogen, USA)
to stain the nuclei and analyzed under fluorescence microscopy using an Axiocam
MRm camera on Axio Imager Z1M microscope.
2.6. Cell selection/isolation from a heterotypic cell suspension
As a cell separation system model, the specific selection of ASCs and HUVECs
from a heterotypic cell suspension was examined using anti-CD31 and anti-CD90
particles. hASCs and HUVECs were mixed at a 1:1 ratio and a sequentially incubated with biofunctionalized CD31 and CD90 microparticles. ASCs and HUVECs
were pre-stained with 20 mM 1,10 -Dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate (Dil) (Sigma, USA) and 3,30 -Dioctadecyloxacarbocyanine
perchlorate (Dio) (Sigma, USA) respectively. Cells were trypsinized and resuspended in 2 mM cell dye in serum-free medium for 10 min at 37 C and washed with
PBS. Each batch of particles (anti-CD31 and anti-CD90 particles) was incubated
with the heterotypic cell culture. After 1 h of incubation, cell-particle complexes
were separated from non-attached cells by passing the mixture through a 37 mm
strainer. Particles with attached cells were then cultured up to 24 h prior analysis
by fluorescence microscopy. The culture of the cell-particle complexes after cell
separation, was performed in complete alpha-MEM medium for the anti-CD90
particles and in complete M-199 for the anti-CD31 particles. For flow cytometry
analysis the mixture of cells (without pre-staining) was seeded on the functional
anti-CD31 and anti-CD90 particles. After 1 h in culture, non-attached cells were
collected and analyzed by flow cytometry.
2.7. Injection of cell-particle complexes
To test the ability of the developed cell-particle complexes to be used as an
injectable system and form constructs in situ, cultured particles were injected into a
tubular silicone mold. The silicone mold was prepared with Sylgard 184 silicone
elastomer kit (Dow Corning, USA) with a needle as a template to acquire the desired
geometry dimension and shape. The obtained cylindrical cavity was about 1.0 mm
diameter.
CD90 functionalized particles were seeded with hASCs at a density of 10 cells
per particle and then cultured up to 6 h at 37 C, 5% CO2. After removal of nonattached cells, the seeded particles were injected into the tubular mold using a
25G needle and cultured for 3 days, at 37 C, 5% CO2 in complete alpha-MEM
medium. Samples were fixed with 10% formalin and stained with phalloidinTRITC for the cytoskeleton visualization, and DAPI to stain the nuclei. Samples
were analyzed under fluorescence microscopy using an Axiocam MRm camera on
Axio Imager Z1M microscope.
2.8. DNA quantification
2.4. Cell isolation and culture
Human subcutaneous adipose tissue was obtained from liposuction procedures,
provided by Hospital da Prelada (Porto), after patient's informed consent and under
Cell proliferation was determined by DNA quantification using a fluorimetric
dsDNA quantification kit (PicoGreen) (Invitrogen, USA). Samples collected after 1, 3
and 7 days were washed with PBS and immersed in 1 ml of ultrapure water, frozen
dio et al. / Biomaterials 43 (2015) 23e31
C.A. Custo
at 80 C, thawed at room temperature, and sonicated for 30 min. Protocol was
followed, according to manufacturer's indications. Fluorescence was measured using an excitation wavelength of 480 nm and an emission wavelength of 528 nm on a
microplate reader (Sinergy HT, Bio-Tek Instruments, USA).
2.9. Flow cytometry analysis
Cell suspensions of the initial mixed cell population, and the cells that do not
attached to each of the functional particles (anti-CD31 and anti-CD90) were incubated at room temperature for 15 min with CD90 (FITC) (eBioscience, UK), CD73 (PE)
and CD31 (APC) (BD Biosciences, USA) fluorescent-labeled monoclonal antibodies.
Samples were analyzed on a FACSCalibur Flow cytometer and the resulting data was
processed using CellQuest software V3.3 (both BD Biosciences, USA).
2.10. Statistical analysis
All the experiments were performed at least 4 times with at least three replicates each. Results were expressed as mean ± standard deviation. Differences between the experimental results were analyzed using the Student t-test.
3. Results
3.1. Fabrication of microparticles
Chitosan-based microparticles were generated by forming small
droplets using a coaxial air-flow that were hardened in a NaOH
solution. The size and morphology of the chitosan microparticles
was determined using optical microscopy. The obtained particles
exhibited rounded shape and the size ranged from 80 to 140 mm
(Fig. 2A). The average diameter of the microparticles was
115.8 ± 10.61 mm (Fig. 2B) and they exhibited a rough surface
(Fig. 2C and D).
Chitosan
microparticle
Chemical
modification
with biotin
In situ scaffold
formation
- NHS-biotin
Cell expansion
Streptavidin
25
3.2. Bioconjugated microparticles
The chitosan particles were modified with NHS-biotin thought
the binding of their succinimide groups and the amine groups at
the surface of the particles, which allowed further conjugation of
SaV (Fig. 3A). The effective conjugation of the chitosan particles
with NHS-biotin was indirectly verified by assessing the fluorescence of the fluorescent-labeled SaV and the unmodified (control)
microparticles. Results revealed the specific binding of fluorescent
SaV on NHS-Biotin modified microparticles (Fig. 3B and C). After
confirming the modification of the particles, the biotinylated antibodies biotin-anti-CD90 and biotin-anti-CD31 were tethered to
NHS-Biotin microparticles via pure SaV. This conjugation was
confirmed by immunostaining, after combining the modified particles with a secondary fluorescently labeled antibody (Fig. 3D). A
high fluorescent signal can be seen on the bioconjugated microparticles, indicating the presence of antibody (Fig. 3E), whereas no
fluorescence was detected on the control (biotin-SaV particles)
(Fig. 3F). Images from optical microscopy and SEM analysis revealed
that the bioconjugation process did not alter particle size or modify
the surface of the particles (Supplementary Information S1).
3.3. hASCs and HUVECs capture and expansion on functionalized
particles
The interaction between the cells and the functionalized particles was first monitored with homotypic cell suspensions of hASCs
and HUVECs respectively taking advantage of their specific
expression of CD90 and CD31 markers.
Conjugation with
streptavidin
Conjugation with
biotinylated
antibodies
Selection and
attachment of target
cells
- Biotinylated antibody
- Cell type I (target cell)
- Cell type II
Fig. 1. Schematic representation of the strategy of preparation and functionalization of the cell-instructive particles. Chitosan particles are chemically modified with biotin that
allows the conjugation with biotinylated antibodies via streptavidin. The functionalized particles are used for specific cell isolation/separation from an heterogeneous cell population and for further cell expansion representing in situ forming construct that can be injected at a lesion site.
dio et al. / Biomaterials 43 (2015) 23e31
C.A. Custo
26
A
B
15
N=66
Mean: 115.8 µm
SV:10.61
Frequency
10
100µm
5
0
70
80
90 100 110 120 130 140 150
Particle size (µm)
C
D
10 µm
5 µm
Fig. 2. A) Optical microscopy image of the fabricated microparticles. B) Histogram of the distribution of microparticles size (n ¼ 66). C) SEM image of a chitosan microparticle. D)
SEM image of the surface of a chitosan microparticle.
Time-lapse imaging showed that less than twenty minutes of
incubation time were sufficient for capturing hASCs by the antiCD90 particles (Supplementary Information S2). When hASCs
were seeded on anti-CD31 or SaV-terminated particles, few cells
attach even after 2 h incubation (Supplementary Information S3
and S4). The same results were observed for HUVECs. In that case
anti-CD31 particles were capable to capture the cells in a short
period of time (less than 20 min) whereas anti-CD90 and SaVterminated particles were not able to bind a significant number
of cells after 2 h of incubation (Supplementary Information S5, S6
and S7). Furthermore hASCs and HUVECs showed good attachment after 1 h of culture in CD90 particles and CD31 particles,
respectively (Fig. 4A and B). Apparently, longer incubation times do
not significantly increase cell attachment to the particles. Indeed,
cells that are not yet captured started to adhere to the bottom of the
well plate.
Supplementary video related to this article can be found at
http://dx.doi.org/10.1016/j.biomaterials.2014.11.047.
To further evaluate the ability of the functionalized microparticles to support cell expansion, cell-seeded particles were cultured
up to 7 days. Results showed that cells were able to attach and
organize their cytoskeleton, early in culture, on the specific
antibody-coated particles (Fig. 5A and E). Unlike functional particles, non-modified particles did not support cell growth over the
time (data not shown). Moreover, both cell types were able to
proliferate along the culture time as observed by microscopy
analysis (Fig. 5BeD and FeH). In addition it was noted that after 7
days in culture, seeded particles started aggregating. Cell proliferation on the antibody-coated particles was confirmed by the DNA
quantification results. Anti-CD90 particles were able to support
hASCs growth up to 7 days in culture (Fig. 5I) such as anti-CD31
particles supported the growth of HUVECs (Fig. 5J). Additionally,
our results suggest that the chitosan particles although allowing
the attachment of few cells due to non-specific cell recognition,
were not able to support their growth.
3.4. hASCs and HUVECs specific separation from heterotypic cell
populations
The capacity of the functionalized microparticles to specifically
select different cell subsets from a heterotypic cell population by
varying the type of antibody bond to their surface was assessed in a
1:1 mixture of HUVECs and hASCs (46.94% CD31þ/CD90 and
48.70% were CD31/CD90þ) (Fig. 6A). The pre-labeling of the cells
allowed to see that 1 h after seeding particles had attached to their
surface only one cell type, HUVECs on the anti-CD31 particles as
indicated by the green fluorescent signal (Fig. 6B) and the red
corresponding to the hASCs on the anti-CD90 particles (Fig. 6C).
Cell separation efficiency was then indirectly measured by assessing the phenotype of the cells that did not attach to the functionalized particles. CD90 and CD31, within the hetereptypic
population specifically expressed by hASCs and HUVECs respectively (Supporting information S8), allowed to independently
quantify the selected subsets. As shown in Fig. 6D after incubation
with anti-CD31 the remaining cellular fraction showed a decrease
(46.94% to 22.30%) of cells expressing CD31, along with an increase
of CD90þ cells (48% to 68.67%). Such results suggest that the antiCD31 functionalized particles specifically bind to CD31 positive
cells. When using anti-CD90 functionalized particles (Fig. 6E), a
decrease of CD90þ cells (48.70% to 7.14%) and an increase of CD31þ
dio et al. / Biomaterials 43 (2015) 23e31
C.A. Custo
A
27
Biotin
tin
NH2 +
Streptavidin
NHS
BIOTIN
B
C
50μm
D
F
E
50μm
- Fluorescently labeled streptavidin
- Streptavidin
- Biotinylated antibody
- Fluorescently labeled secondary antibody
Fig. 3. A) Schematic representation of the chemical modification of chitosan microparticles with NHS-biotin and functionalization with SaV. B) Micrographs indirectly showing
biotin-modified particles after incubation with fluorescently labeled SaV and respective. C) negative control, i.e. plain particles incubated with a fluorescently labeled SaV. D)
Schematic representation of the bioconjugation with biotinylated antibodies. E) Micrographs showing functionalized particles incubated with a secondary antibody Alexa Fluor 594
and respective. F) negative control, i.e. particles terminated with a SaV layer incubated with a secondary antibody Alexa Fluor 594.
cells (46.94% to 89.05%) was observed in the remaining fraction,
indicating a higher efficiency in the separation of the CD90þ cell
subset, the hASCs.
3.5. In situ construct formation
As a proof of concept, the possibility of using the bioconjugated
particles as an injectable system to fill defects, forming small tissue
constructs in situ was investigated. The CD90 particles coated with
hASCs were injected into a mold containing a cylindrical cavity
with about 1.0 mm diameter (Fig. 7A). After 3 days of culture, the
previously observed tendency for aggregation was confirmed by
the 3D tubular structure formed by particles tightly connected by
the cells (Fig. 7B).
4. Discussion
In this study the goal was to develop polymeric microparticles
that along with cell selection/isolation allow cell expansion i.e., that
may work as selective microcarriers to expand a target cell type
(Fig. 1). Additionally we hypothesize that the developed bioconjugated microparticles may be used as an injectable system for
in situ formation of small tissue constructs for regeneration
purposes.
Cell microcarriers are typically submillimeter-sized polymeric
particles that provide sites for initial cell attachment [18,19]. In the
current set of experiments, chitosan microparticles with a mean
diameter of 115.8 mm were modified with antibodies, mimicking
the magnetic particles commonly used for cell separation purposes
[6,7,20,21]. Additionally, due to the large surface area, the particles
here developed support cell adhesion and expansion on their surface. Such approach may eliminate the multiple trypsinization
steps required for the sub-cultivation of the selected cells,
providing a versatile, cost effective, and easy-to-operate combined
isolation and expansion approach.
Chitosan materials have been widely used in tissue regeneration, owing to their low immunogenicity, biocompatibility,
biodegradability, low toxicity and facilitated chemical modification [12,13,15,16,22]. In this study, free amine reactive groups on
the surface of the particles were used to tether biotin. Because of
its strength and specificity, the SaV-biotin pair is often used for
chemical conjugation of biomolecules [23e25]. The ready to use
strategy herein proposed could be particularly interesting as
several biomolecules of interest are often biotin-conjugated and
can be easily used to functionalize the chitosan particles via SaV.
In the present work the focus was the immobilization of biotinylated antibodies for specific cell attachment. Sulfo-NHS-Biotin
was used to chemically functionalize the surface of chitosan
dio et al. / Biomaterials 43 (2015) 23e31
C.A. Custo
28
SaV
CD90
CD31
ASCs
A
HUVECs
B
100μm
Fig. 4. Optical micrographs showing hASCs (A) and HUVECs (B) attachment on SaV, CD31 and CD90 terminated particles after 1 h in culture.
Day 1
Day 3
B
C
D
hASCs
A
Day 7
50μm
100μm
10
00μm
0
0μm
m
F
G
H
HUVECs
E
50μm
100μm
I
DNA ( g/ml)
1.40
*
*
0.60
0.20
HUVECs
1.80
*
*
1.00
-0.20
J
1.40
DNA ( g/ml)
1.80
hASCs
*
Day 1
Day 3
*
Day 7
1.00
0.60
0.20
ASCs 90particles anti-CD31
ASCs 31 particles
anti-CD90
HUVECs 90
HUVECs 31
-0.20 anti-CD90 particles anti-CD31 particles
Fig. 5. Optical micrographs of the (AeD) hASCs- and (EeH) HUVECs-particle cultures up to day 7, demonstrating that cells were able to attach and organize their cytoskeleton as
shown by the phalloidin staining (red). Nuclei were stained with DAPI (blue). DNA quantification confirming the proliferation of I) hASCs on the anti-CD90 and J) HUVECs on antiCD31 up to 7 days of culture. Results are expressed as mean ± standard deviation with n ¼ 9 for each bar. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
dio et al. / Biomaterials 43 (2015) 23e31
C.A. Custo
A
Cell mixture
29
anti-CD90 particles
anti- CD31 particles
46.94%
HUVECs
B
CD31
C
48.70%
50μm
ASCs
D
CD90
Negative fraction of
anti- CD31 particles
E
89.05%
22.30%
CD31
CD31
Negative fraction of
anti- CD90 particles
7.14%
68.67%
CD90
CD90
Fig. 6. A) Representative dot plots after flow cytometry analysis of 1:1 HUVECs/hASCs cell mixture. BeC) hASCs were stained red with Dil, HUVECs were stained green with Dio; a
50:50 mixture of both cells was seeded in CD90 and CD31 particles. Fluorescent images after 24 h seeding show good adherence of ASCs on CD90 particles. HUVECs readily attach on
CD31 particles. D) Representative dot plots of the remaining cellular fraction after incubation with CD31 functional particles (negative fraction CD31). E) Representative dot plots of
the remaining cellular fraction after selection with CD90 functional particles (negative fraction from CD90). (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
microparticles with biotin. Sulfo-NHS-Biotin displays a spacer arm
with 13.5 Å length that reduces steric hindrances associated with
SaV binding. This allow for efficient capturing of the biotinylated
antibody in order to accomplish favorable orientation, long-term
stability for a high capture efficiency. A similar strategy, aiming
to promote the adhesion of stem cells to a decellularized heart
valve, was already proposed with biotinylated anti-CD90 antibody
[26]. Nonetheless we aimed to go further developing a system to
select/isolate specific cell sub-sets from heterogeneous populations. Different cells are known to express specific antigens on
their surfaces thus, by coating the chitosan microparticles with
different antibodies directed against specific markers we expect to
obtain from example from a single sample more than one subpopulation of cells.
CD90 microparticles seeded with ASCs
A
B
Culture for 3 days
500μm
Fig. 7. A) Tube-shaped PDMS mold chamber. B) Microscopy image of the obtained cell-particle constructs after 3 days in culture, demonstrating the aggregation of the particles
through the hASCs that retain an organized cytoskeleton as shown by the phalloidin staining (red). Nuclei were stained with DAPI (blue). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
30
dio et al. / Biomaterials 43 (2015) 23e31
C.A. Custo
Adipose tissue is a rich and very convenient source of cells for
regenerative medicine therapeutic approaches [27,28]. Recent
studies have confirmed within the SVF of the adipose tissue the coexistence of different cell sub-populations, among which endothelial and stem cells that are of significant relevance in the tissue
engineering and regenerative medicine field [29,30]. As a proof of
concept of our system we have explored the use of antibodies
against endothelial and stem cell markers, CD31 and CD90
respectively, that may be thereafter used to target those subpopulations within the SVF. To validate the hypothesis that the
herein developed platform is suitable for cell selection and
expansion, microparticles were functionalized with anti-CD31 and
anti-CD90 antibodies and used to select and expand HUVECs and
ASCs respectively. CD31 is a 130-kDa membrane-spanning glycoprotein and is part of the panel of endothelial celleassociated
markers [31,32], while it has showed a residual expression in
mesenchymal stem cells [33]. CD90 is a 25e37-kDa N-glycosylated
anchored cell surface protein, highly expressed by mesenchymal
stem cells but not by endothelial cells [33]. Live imaging results
showed that cell attachment to the functional microparticles is fast,
occurring within 20 min for both cell types (Supplementary information S2 and S5). Nonetheless the kinetics of adhesion was
dependent on the cell type. The recognition and attachment of
hASCs to CD90 modified particles occurred within an interval of
5 min. In fact, a limitative parameter of all the cell selection
antibody-based methods is not only the identification of a marker
that is exclusively expressed by the population of interest, most of
the times only preferentially expressed, but also the amount of
epitopes displayed on the surface of the cells that varies with the
cell type and their environment/condition. The mean fluorescent
intensity observed for CD90 for hASCs (x ¼ 2046.28) and CD31 for
HUVECs (y ¼ 1144.66) that can be correlated with the number of
free epitopes for antibody targeting, justify our results regarding
the kinetics of adhesion (Supplementary Information S8).
For the rest of the experiments we decided to use 1 h of incubation for cell capturing in order to guarantee efficient cell
attachment and avoid non-specific cell attachment. As it was expected, particles that were modified with antibodies that recognize
antigens that are not expressed or low expressed by the cells show
relatively low cell attachment.
The ability of the bioconjugated particles to support cell
spreading and proliferation up to 7 days in culture was then
examined. After 3 days in culture cells proliferated covering the
surface of the particles. Results suggest that the presence of the
antibody had no inhibitory effect on cell proliferation. After 7 days
in culture small clusters of cultured particles were observed. Such
ability to aggregate is in accordance with several reports regarding
cell culture on the commonly used microcarriers used for cell
expansion [18,19,34,35]. The formation of small cell-microparticle
aggregates may be an advantage for bead-to-bead migration and
microtissue formation. Furthermore, as our final objective is to
create a system that leads to the formation of a 3D construct, the
presence of small aggregates may be an advantage. The use of
agglomerated polymeric particles to produce constructs for tissue
engineering applications has been already exploited [16,36,37]. An
innovation in this study is the use of cell selective micro-fabricated
modules. Microparticles conjugated with CD90 antibody were
seeded with ASCs, incubated 24 h in cell culture conditions to
guarantee a good cell attachment to the particles, and then injected
into a tubular mold. After three days in culture, a robust tubular
structure of cells entrapped within the bioconjugated microparticles was obtained. We believe that there is clinical potential in the
use of the herein developed bioinstructive microparticles for the
fabrication of small tissue constructs by layering combinations of
particles seeded with different cell types. From a clinical point of
view our strategy may offer several advantages as a significant
decrease in time consumption from the biopsy to the implantation
of the constructs. By this method, it may be also possible to
assemble a combination of different cells obtained from one isolated biopsy performed to a patient.
The bioconjugated microparticles were then analyzed on the
ability to effectively separate a target cell type from a heterogeneous cell population. The obtained results showed the ability of
the developed microparticles to separate ASCs from endothelial
cells. The present system may be used as a simple technique that
uses antibody coated microparticles, suitable for isolation and
expansion of different cell types present in the same tissue sample.
This could allow for instance, the selection and culture of endothelial cells, which yield is normally limited and insufficient in a
clinical setting. Therefore, the proposed technique allows the boost
of relevant cell types, particularly useful when their availability is
limited.
5. Conclusions
Here was demonstrated the ability of biofunctionalized particles
to select specific cell types from mixed cell populations and to
promote cell expansion, by using hASCs and HUVECs as examples.
The versatility of this method allows the combination of the biotinSaV conjugated microparticles with any biotinylated molecule as
antibodies, growth factors or peptides of interest. It was shown that
biodegradable and biocompatible particles functionalized with
antibodies presented selective affinity to cells, making them
potentially suitable for separating subpopulations of cells from
complex mixtures. Besides the ability for cell separation, the
cultured particles proved to be also suitable for cell expansion. A
versatile, cost effective, and easy-to-operate system with the
capability to simultaneously separate and expand different cells
sub-sets in vitro was developed. Moreover, the aggregation of the
functionalized microparticles has been also shown to successfully
form 3D robust structures upon injection into a mold. Thus the
herein developed microparticles demonstrated that might be
potentially used for further studies accomplishing the formation of
a construct in situ upon implantation using minimally invasive
procedures.
Acknowledgments
This work was supported by European Research Council grant
agreement ERC-2012-ADG 20120216-321266 for project ComplexiTE and by the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement n REGPOTCT2012-316331-POLARIS. The authors acknowledge the FCT for the
fellowship SFRH/BD/61390/2009 (C.A.C.) for the financial support.
We are grateful to Hospital da Prelada for the lipoaspirates
donations.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biomaterials.2014.11.047.
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