Colloids and Surfaces A 572 (2019) 266–273

Contents lists available at ScienceDirect

Colloids and Surfaces A

journal homepage: www.elsevier.com/locate/colsurfa

Preparation of collagen/chitosan microspheres for 3D macrophage T

proliferation in vitro
Di Wanga,b,1, Meiyue Wangb,c,1, Anhe Wangb, Jieling Lib, Xin Lib,c, Honglei Jianb, Shuo Baib, ,
⁎

Jian Yina,
⁎

a
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 214122 Wuxi, China
b
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 100190 Beijing, China
c
University of Chinese Academy of Sciences, 100049 Beijing, China

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Keywords: Macrophages, as an important member of immune cells, have now become a research hotspot in the field of
Collagen immunotherapy, which requires prominent proliferation in vitro for clinical use. Herein, we reported the fab-
Microspheres rication of collagen/chitosan microspheres via emulsification for 3D macrophages proliferation. With glutar-
Emulsification aldehyde (GA) as cross-linker, the microspheres have an average diameter of 200 μm and positively charged
Macrophage
surfaces, which are of great stability and biocompatibility. Compared with 2D cell proliferation, the 3D mi-
3D proliferation
crospheres could dramatically enhance macrophages proliferation rate and promote their biological functions.
Furthermore, macrophages on the surface of microspheres could be easily collected by blowing and suction,
without the need of trypsin digesting or the degradation of microsphere. Hence the microspheres could be reused
for macrophage proliferation repeatedly. This study highlights opportunities for development of high-effective
cell proliferation approach and provides a great promise in immune cell based immunotherapy.

1. Introduction replace the damaged cells or have a stronger immune killing function. It
is regarded as a powerful clinical practice strategy and has gained more
Cytotherapy, which refers to the transplantation or importation of and more attention in disease treatment [1]. Cytotherapy can be typi-
normal or bioengineered human cells into the patients ' body, can cally divided into stem cell therapy by using bone marrow stem cells,

⁎
Corresponding authors.
E-mail addresses: baishuo@ipe.ac.cn (S. Bai), jianyin@jiangnan.edu.cn (J. Yin).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.colsurfa.2019.04.007
Received 18 March 2019; Received in revised form 4 April 2019; Accepted 6 April 2019
Available online 13 April 2019
0927-7757/ © 2019 Elsevier B.V. All rights reserved.
D. Wang, et al.
Scheme 1. Schematic illustration of collagen/chitosan microspheres fabricated via emulsification and their application in cell expansion.
skin stem cell, etc. and immune cell therapy such as T cells and mac-
rophage therapy [2,3]. Over decades, several novel diseases, including
cartilage lesions, burns or chronic wounds, cancers have been treated
effectively through the cellular therapy [1,4,5]. For example, macro-
phages play a key role in immune cell system and can be used to treat
diseases such as atherosclerosis and cancers [6–9]. Researchers have
found that macrophages in tumor tissues have a significant anti-cancer
effect after activation, which makes macrophages alternative antitumor
immune cells and have become a research hotspot in the field of tumor
immunotherapy [10]. Macrophages are a large group of immune cells
that are usually divided into two subgroups according to the specific
immune response involved, namely M1 and M2 [11]. Though M2 type
of macrophages are considered to be related to tumor formation and
growth [12], M1 type of macrophages, however, are recently found to
be capable of dissolving tumor cells, presenting tumor-associated an-
tigens to T cells, producing immune-stimulatory factors, promoting the
proliferation of T cells and NK cells, and finally strengthening their anti-
tumor effects [6,13,14]. Hence, great efforts have now been devoted to
employing macrophages for cellular anticancer immunotherapy. For
example, Daldrup-Link and co-workers employed ferumoxytol to pro-
mote the polarization of macrophages to M1 type for breast cancer
treatment [15,16]. Also, Johanna A Joyce and co-workers found that
CSF-1R inhibition could induce the transformation of macrophages
phenotype from tumor-promoting M2 type to anti-tumor M1 type, fa-
cilitating the treatment of proneural gliomas [17]. As well-known, the
number and functionality of macrophages are the most important in-
dicators during the cytotherapy. However, limited by their own char-
acteristics, macrophages cannot proliferate after injected into the body.
To obtain highly efficient treatments, the expansion of macrophages in
vitro is extremely important.
For effective cell proliferation in vitro, methods of culturing cells in
three-dimensional (3D) environments are now drawing more atten-
tions, which can not only provides suitable spatial environment for cells
to distribute, but more importantly can provide natural ECM simulating
environment for cells to promote their bioactivities [18]. Among other
methods such as 3D scaffold and microcapsule encapsulation and so on,
microspheres used as carriers for cells proliferation on the 3D surfaces,
has been proved to be an ideal choice for cell expansion [19–23]. The
significant advantages of microspheres as cell proliferation carriers are
as follows: (i) it is relatively easy to fabricate and handle microspheres
[24]; (ii) microspheres can supply a large surface area due to their high-
volume ratio compared with other shapes of materials, facilitating cell
proliferation and connection [25]; (iii) expanded cells on the surfaces of
267
Colloids and Surfaces A 572 (2019) 266–273
microspheres are more easily to be collected with no destruction of
microsphere structure [23,26]. So far, various macromolecules such as
polyacrylamide [27] and polystyrene [28], have been used to fabricate
microspheres for cell expansion. Compared with the above synthetic
macromolecules, the endogenous biomaterials hold the advantages of
excellent biocompatibility, bioactivity and responsiveness and have
been widely used for the fabrication of biomedical nanomaterials
[29–31]. Particularly, the endogenous collagen, which is the most
abundant and widely distributed functional protein in mammals, has
long been used in fields of tissue engineering, protein delivery and
wound healing [32]. Yet the application of collagen as carriers for cell
expansion has rarely been reported.
Herein, we firstly reported the use of collagen-based microspheres
for 3D macrophage expansion (Scheme 1). The collagen-base micro-
spheres were fabricated by the cross-linking of collagen and chitosan
with glutaraldehyde (GA) as cross-linker via emulsification. The mi-
crospheres were positively charged, which was in favor of cell ab-
sorption and attachment. Compared with cell expansion in traditional
2D petri dish, 3D microspheres could effectively promote macrophage
cell proliferation rate and enhance their biological functions. Further,
macrophages on the surfaces of microspheres could be easily collected
by blowing and suction, without the need of trypsin digesting or de-
gradation of microsphere. Hence these microspheres could be reused
for more macrophage expansion.
2. Materials and methods
2.1. Materials
Type Ⅰ Collagen (extracted from bovine tendon, Mw: ˜ 0.3 millions)
was purchased from Yuanye Co., Shanghai, China. Glutaraldehyde (GA)
(50% aqueous solution) was purchased from Aladdin. Chitosan was
purchased from Sigma-Aldrich. Span 80 (≥97.5%), Glacial acetic acid
(≥99.5%), hexane and absolute ethyl alcohol (≥99.7%) were pur-
chased from Beijing Chemical Reagent Co., Beijing, China. CCK-8 was
purchased from Dojindo Laboratories. LIVE & DEAD viability kit and
1 × DPBS (pH = 7.2, 0.01 M) were purchased from Thermo Fisher
Scientific. RPMI-1640, FBS (foetal calf serum), 1 × PBS (pH = 7.2,
0.01 M) and 100 × Penicillin-Streptomycin Liquid (100 U/mL) were
purchased from Solarbio. All of them were used without further pur-
ification.
D. Wang, et al.
2.2. Methods
2.2.1. Preparation of collagen/chitosan microspheres
Collagen/chitosan microspheres were fabricated by emulsification.
Firstly, collagen (700 mg) and chitosan (700 mg) were dissolved in
100 mL acetic acid (0.1 M) aqueous solution at room temperature, re-
spectively. Meanwhile, 2 g span 80 was dissolved in 60 mL hexane at
room temperature as oil-phase. Then, 8 mL collagen solution was mixed
with 2 mL chitosan solution to form 10 mL blend solution regarded as
water-phase, which was added into the oil-phase dropwise under stir-
ring to form emulsion. 2 mL GA aqueous solution as cross-linker was
quickly injected into the emulsion, which was stirred for 8 h at
1000 rpm. Finally, the absolute ethyl alcohol was added to break the
emulsion. After centrifugation and washing the collagen/chitosan mi-
crospheres for three times with absolute ethyl alcohol, the microspheres
were obtained and dried under vacuum for further use.
2.2.2. Morphology characterization and FTIR spectral measurement of the
microspheres
The light microscope images of collagen/chitosan microspheres
were measured by an XSP-37XC equipment (Teelen, Shanghai) with a
10 × objective and the scanning electron microscopy (SEM) images
were measured by an S-4800 equipment with 10 kV accelerating
(HITACHI, Japan). The confocal laser scanning microscope (CLSM)
graphs were obtained through a Zeiss LSM710 confocal system with an
excitation wavelength of 405 nm. The size distribution of collagen/
chitosan microspheres diameters was counted through a Nano Measurer
software where more than 50 microspheres were measured. Zeta po-
tential of microspheres was detected by a zeta potential analyzer and
FTIR spectra were measured on a JASCO FP-6500 spectrofluorometer
where collagen, chitosan and collagen/chitosan cross-linked micro-
spheres powder were ground with KBr and pressed into a round sheet
for FTIR spectra detection, respectively.
2.2.3. Stability test of collagen/chitosan microspheres
To test the stability of collagen/chitosan microspheres, the micro-
spheres were soaked in RPMI-1640 for 90 days. Then the RPMI-1640
treated microspheres were washed with ddH2O for three times to re-
move the adsorbed RPMI-1640 residues on surface and were dried
under vacuum. The microspheres were characterized with SEM, light
microscopy and infrared spectrometer with microspheres that were not
treated with RPMI-1640 as comparison.
2.2.4. Calculation of cell adsorption rate
Collagen/chitosan microspheres of different diameters were firstly
dispersed in the same volume of RPMI-1640 in 60 mm cell petri dishes.
Then equal volume of macrophage suspensions was added into the
above solutions respectively, to make sure the initial amounts of cells of
each dish were 5 × 105 (N0). After co-culturing for 1 and 2 h, we
counted the number of macrophages in the supernatant (N1) and on the
bottom (N2) of each dish with a cell counting plate. Same number of
macrophages cultured in a traditional 2D petri dish were used as con-
trol. The cell adsorption rate (R, %) was calculated as follows
R2D=N2/N0 (1)
R3D=(N0-N1-N2)/N0 (2)
Confocal microscopy was also employed to observe macrophage
attachment on microspheres. Before observation, macrophages were
stained by LIVE & DEAD viability kit with excitation wavelengths of
405 nm, 488 nm and 630 nm.
2.2.5. Cell cytotoxicity experiments
Macrophages were added into the 96-well plate mixed with dif-
ferent concentrations of collagen/chitosan microspheres. The cell con-
centration of each well was kept the same at 1 × 104 cell mL−1. After
268
Colloids and Surfaces A 572 (2019) 266–273
co-culturing for 24 h in constant temperature incubator at 37℃ in a
humidified atmosphere of 5% CO2, each well was added with 10 μL
CCK-8 solution and kept in the constant temperature incubator for 4 h.
After that, the 96-well plate was put into the Thermo Scientific
Microplate Reader to detect the OD value of each well with an excita-
tion wavelength of 405 nm.
2.2.6. Cell proliferation
For proliferation process, 1 mg collagen/chitosan microspheres
were firstly dispersed in 3 mL of RPMI-1640 in 60 mm cell petri dish.
Then macrophage suspension was added into the above solution to
make sure the initial number of macrophages was 5 × 105.
Macrophages cultured in the traditional 2D petri dish with the same
initial number were used as control. Light microscopy and CLSM were
both used to record macrophage proliferation process on microspheres
every day. For CLSM observation, macrophages were stained by the
LIVE & DEAD viability kit and the images were obtained with excitation
wavelengths of 405 nm, 488 nm and 630 nm.
Flow cytometry was also used to statistically record the proliferation
process of macrophages every other day both on 3D microspheres and
in 2D petri dish. For flow cytometryexperiment, macrophages with
different culture time were collected from 3D microspheres or 2D petri
dish and resuspended in 500 μL PBS. Before detection with flow cyto-
metry, the collected macrophages stained with LIVE & DEAD viability
kit. Then they were added into a 96-well plate and detected with a BD
LSRFortessa flow cytometry in combination with a statistics system.
2.2.7. Cytokine release
Macrophages with a concentration of 1 × 105 cells mL−1 were co-
cultured with 500 μg collagen/chitosan microspheres in 96-well plate
for 24 h. Then 10 μL of LPS with different concentrations were added to
the cells and incubated for another 24 h. Finally, the cell culture su-
pernatants were moved to another 96-well plate for ELISA detection
with an excitation wavelength of 450 nm.
3. Results and discussion
Collagen/chitosan microspheres were fabricated by emulsification
with GA as cross-linker. The collagen/chitosan mixed solution as water
phase (W) was firstly added dropwise into hexane as oil phase (O) with
stirring to form W/O emulsion. Then 1.7% GA aqueous solution was
added to the emulsions for cross-linking. After 8 h, stable collagen/
chitosan microspheres could be obtained by centrifugation. Light mi-
croscope, SEM and CLSM images (Fig. 1A–C) all showed that the mi-
crospheres had an average diameter of 200 μm, which was in consistent
with statistical result (Fig. 1D). The inserted SEM image in Fig. 1B
further showed a cristae-like micro-rough surfaces of the microspheres,
which may facilitate cells attachment on the microspheres.
The cross-linking between collagen and chitosan was based on the
Schiff base reaction with GA as cross-linker where amino group (eNH2)
in collagen and chitosan could react with one of the two aldehyde
groups in GA, respectively, forming Schiff base and realizing the cross-
linking between collagen and chitosan [33]. To confirm the successful
cross-linking between collagen and chitosan, FTIR spectra were re-
corded (Fig. 2A). The absorptions from 3300 to 3500 cm−1 and that at
1530 cm-1 represented the functional group of -NH2 in collagen and
chitosan, and the significant decrease in peak intensity after the cross-
linking reaction indicated the reduce of -NH2 number in the system
[33,34]. Meanwhile, the intensity of the absorption peak at 1617 cm−1
which stood for the OC]NH group showed an obvious increase com-
pared with that of collagen and chitosan, suggesting the successful
formation of Schiff base. The above results demonstrated the successful
cross-linking between collagen and chitosan by GA. The cross-linked
microspheres were tested to have great stability when immersed in cell
culture (RPMI-1640). As shown in Fig. 2B, after soaking microspheres
in RPMI-1640 for 90 days, no obvious changes were observed in FTIR
D. Wang, et al. Colloids and Surfaces A 572 (2019) 266–273

Fig. 1. Characterizations of collagen/chitosan microspheres. (A) Light microscopy images of collagen/chitosan microspheres. (B) SEM images of the collagen/
chitosan microspheres. (C) CLSM images with an excitation wavelength of 405 nm. (D) The size distribution of collagen/chitosan microspheres.

Fig. 2. (A) FTIR spectra of collagen (a), chitosan (b) and cross-linked collagen/chitosan microspheres (c) and (B) FTIR spectra of collagen/chitosan microspheres with
(a) and without (b) RPMI-1640 treatment. (C) SEM and (D) light microscopy images of collagen/chitosan microspheres after soaking in RPMI-1640 for 90 days.

269
D. Wang, et al.
Fig. 3. (A) Zeta potential of collagen/chitosan microspheres with different ratios of collagen and chitosan. (B) CLSM images of collagen/chitosan microspheres with
macrophage adhesion for 4 h. (C) Macrophage attachment rate on microspheres with different diameters. (D) Viability of macrophage mixed with different con-
centrations of collagen/chitosan microspheres for 24, 48, 72 and 96 h.
spectra compared with the freshly fabricated ones, suggesting little
structural changes in RPMI-1640 treated microspheres. SEM and light
microscope images (Fig. 2 C and D) also showed that there were hardly
any changes in morphology and size of microspheres before and after
RPMI-1640 treatment. All the above results demonstrated the great
stability of GA cross-linked microspheres, which permitted their ap-
plication in the long-term cell proliferation.
Fig. 3A demonstrated the zeta potential of microspheres with dif-
ferent collagen/chitosan ratios. When the molar ratio of collagen/
chitosan was 2:1, the microspheres were negatively charged. However,
when increasing the ratio of collagen to 3:1, there was a charge shift
from negative to positive. The charge of microspheres increased with
the increase of collagen ratio. Positively charged surface of the micro-
spheres may facilitate the cells attachment.
For the application of cell proliferation, we firstly test macrophage
adsorption rate on microspheres with different diameters. The cell ad-
sorption rate (R3D) was calculated as follow: R3D = (N0 - N1 - N2) / N0,
where N0, N1, N2 represented the initial total number of cells, the
number of cells in the medium and on the bottom of petri dish, re-
spectively. As Fig. 3B and C shown, macrophages could quickly attach
to microspheres and almost half of cell could attach onto microspheres
within one hour. After two hours, cell adsorption rate could reach as
high as 70%, which suggested that microspheres were suitable carriers
for supporting cell proliferation. Also, we found that microspheres with
larger curvature had a higher cell adsorption rate. As shown in Fig. 3C,
cells on the surfaces of microspheres with a diameter of 200 μm, which
were of the largest curvature among the microspheres, exhibiting the
highest adsorption rate. Hence, to better support macrophage expan-
sion in vitro, we carried out our subsequent experiments with micro-
spheres with a diameter of 200 μm.
As the biocompatibility is one of the important characters for mi-
crospheres to be used as cell proliferation carriers, we next performed
CCK-8 experiment to confirm the biocompatibility of microspheres. As
shown in Fig. 3D, cells co-cultured with different concentrations of
270
Colloids and Surfaces A 572 (2019) 266–273
microspheres showed high viability even when the co-culture time was
prolonged to 96 h. The cell viability could still reach up to 90% when
the concentration of microspheres was as high as 30 μg mL−1, sug-
gesting the great biocompatibility of microspheres. The slight toxicity
with high microspheres was probable caused by the increased dosage of
GA.
After proving the great biocompatibility of microspheres, we next
tested their ability of facilitating cell proliferation. For this experiment,
macrophages were co-cultured with microspheres and their prolifera-
tion process was first recorded with light microscope. As mentioned
above, macrophages started to attach to the microspheres within one
hour and their numbers kept increasing with time. After co-cultured
with microspheres for 1 day, more than half surfaces of microspheres
were covered by macrophages (Fig. 4A and B). On the fifth day, the cell
coverage could reach up to 80% (Fig. 4C) and over 10 days, macro-
phages have almost fully covered the surfaces of microspheres
(Fig. 4D). Fig. 4E was the light microscope image of an individual mi-
crosphere co-cultured with macrophages for 10 days. As can be seen,
the microsphere was fully covered by macrophages, suggesting the
ability of microspheres to support cell proliferation. The flow cytometry
test was next carried out to statistical demonstrate the ability of mi-
crospheres to promote cells proliferation with cells cultured in 2D petri
dish as a control. Macrophage proliferation process in both the ex-
perimental and control group were observed for 10 days. The number of
cells was recorded through flow cytometry every other day. As shown
in Fig. 4F, the numbers of macrophages both on 3D microspheres and in
2D petri dish kept increasing with time. In the first two days, macro-
phages in 2D petri dish grew a little faster than those on 3D micro-
spheres. However, after two days, macrophages on microspheres ex-
hibited an explosive growth tendency, which was much faster than that
in the 2D petri dish. After four days, macrophages proliferation in 2D
petri dish almost reached its stationary phase and the number of cells
slowly increased. While macrophages on 3D microspheres kept growing
in an exponential manner and the total cell number began to exceeded
D. Wang, et al.
Fig. 4. (A–D) Light microscopy images of macrophage proliferation on collagen/chitosan microspheres with time. (E) The image of an individual collagen/chitosan
microspheres with macrophages proliferation for 10 days. (F) Numerical statistics of cell proliferation over time by flow cytometry: (a) 2D petri dish; (b) 3D collagen/
chitosan microspheres.
that in 2D petri dish. On the tenth day, the total cell number on 3D
microspheres was at least 2 times as high as that in control group. The
above comparison suggested the high efficiency of collagen/chitosan
cross-linked microspheres in promoting cell proliferation and the mi-
crospheres may become excellent carrier candidates for cell expansion.
Macrophage proliferation process was also recorded by confocal
microscopy (Fig. 5). Before observation, macrophages were stained
with LIVE & DEAD viability kit, by which only living cells could be
stained by Calcein-AM to emit green fluorescence while dead cells
could be stained by PI to emit red fluorescence. Consistent with the
microscopic result, the number of macrophages on the microsphere
kept increased with time, and the microsphere had almost covered by
macrophages on the tenth day. Especially, no red fluorescent signal
could be observed from CLSM image and all cells could emit green
fluorescence, suggesting that all the macrophages on microsphere were
alive. It further demonstrated the low cytotoxicity and the great ability
of microspheres to support cell proliferation.
As M1 macrophages played a key role in macrophage-based cell-
immunotherapy, we next tested the polarization ability of macrophages
grew on microspheres into M1 type. Also, macrophages grew in 2D
petri dish were used as a control. For the detection of M1 macrophages
polarization, we here detected the release of IL-6 and TNF-α, which are
the symbol cytokines of the M1 macrophage and could be released
under the stimulation of the LPS, as a reference of the biological
functional M1 macrophages [15,35]. As shown in Fig. 6A and B, under
271
Colloids and Surfaces A 572 (2019) 266–273
the stimulation of the LPS, macrophages, grew both on 3D microspheres
and in 2D petri dish could release IL-6 and TNF-α. However, macro-
phages grew on 3D microspheres exhibited a more powerful ability for
the release of cytokine and could release much more IL-6 and TNF-α
than those in 2D petri dish. With the increase of LPS concentration, the
advantages of macrophages grew on 3D microspheres in releasing cy-
tokine became more and more distinct and when the concentration of
LPS was 10 μg mL−1, the amount of IL-6 released by 3D cultured
macrophages was almost 3 times as much as that of 2D cultured mac-
rophages, suggesting 3D culture environment was more conducive to
macrophages for their biological function.
4. In conclusion
In conclusion, we reported the fabrication of collagen/chitosan
microspheres via emulsification with GA as cross-linker for 3D macro-
phages proliferation. The microspheres have an average diameter of
200 μm and a positive charged, cristae-like micro-rough surface, facil-
itating cells attachment. Besides, these microspheres were tested to be
stable for at least 90 days in cell culture medium with little cytotoxicity,
which permit their long-term cell proliferation application. Cell pro-
liferation experiment showed that microspheres could effectively sup-
port macrophages proliferation and compared with 2D cell prolifera-
tion, microspheres could dramatically enhance macrophages
proliferation rate with high cell viability and promote bio-functions of
D. Wang, et al.
Fig. 6. ELISA kit for detection of two inflammatory cytokines (A) IL-6 and (B) TNF-α released by microphages growing on 2D petri dish and 3D collagen/chitosan
microspheres stimulated by different concentrations of lipopolysaccharide (LPS).
macrophages. Our result should shed light on the fabrication of bio-
materials for high-effective cell proliferation and hold great promise in
macrophage-based cell-immunotherapy.
Acknowledgements
We are grateful for financial support from National Natural Science
Foundation of China (21703253, 21774132, 21877052, 31700706),
Natural Science Foundation for Distinguished Young Scholars of
Jiangsu Province (BK20180030), the Fundamental Research Funds for
the Central Universities (JUSRP51712B), the Max Planck Society
International Partner Group Program.
References
[1] N. Cuende, J.E.J. Rasko, M.B.C. Koh, M. Dominici, L. Ikonomou, Cell, tissue and
gene products with marketing authorization in 2018 worldwide, Cytotherapy 20
(11) (2018) 1401–1413.
272
Colloids and Surfaces A 572 (2019) 266–273
Fig. 5. CLSM images of the growth of macrophages on collagen/
chitosan microspheres with time: (A) 1 day, (B) 3 days, (C) 5 days
and (D) 10 days with excitation wavelengths of 405 nm for col-
lagen/chitosan microspheres, 488 nm for live cells and 630 nm for
dead cells. (Macrophages were stained by LIVE & DEAD viability
kit before observation).
[2] B.E. Strauer, R. Kornowski, Stem cell therapy in perspective, Circulation 107 (7)
(2003) 929–934.
[3] M. Najafi, B. Farhood, K. Mortezaee, Contribution of regulatory T cells to cancer: a
review, J. Cell. Physiol. 234 (6) (2019) 7983–7993.
[4] M. Dominici, K. Nichols, A. Srivastava, D.J. Weiss, P. Eldridge, N. Cuende,
R.J. Deans, J.E.J. Rasko, A.D. Levine, L. Turner, D.L. Griffin, L. O’Donnell, M. Forte,
C. Mason, E. Wagena, W. Janssen, R. Nordon, D. Wall, H. Ho, M.A. Ruiz, S. Wilton,
E.M. Horwitz, K.C. Gunter, Positioning a scientific community on unproven cellular
therapies: the 2015 international society for cellular therapy perspective,
Cytotherapy 17 (12) (2015) 1663–1666.
[5] W.L. Ng, S. Wang, W.Y. Yeong, M.W. Naing, Skin bioprinting: impending reality or
fantasy, Trends Biotechnol. 34 (9) (2016) 689–699.
[6] G. Chinetti-Gbaguidi, S. Colin, B. Staels, Macrophage subsets in atherosclerosis, Nat.
Rev. Cardiol. 12 (1) (2015) 10–17.
[7] K.J. Moore, F.J. Sheedy, E.A. Fisher, Macrophages in atherosclerosis: a dynamic
balance, Nat. Rev. Immunol. 13 (10) (2013) 709–721.
[8] M. Feng, J.Y. Chen, R. Weissman-Tsukamoto, J. Volkmer, P. Ho, K.M. McKenna,
S. Cheshiera, M. Zhang, N. Guo, P. Gip, S.S. Mitra, I.L. Weissman, Macrophages eat
cancer cells using their own calreticulin as a guide: Roles of TLR and Btk, PNAS 112
(7) (2015) 2145–2150.
[9] R.S. Riley, C.H. June, R. Langer, M.J. Mitchell, Delivery technologies for cancer
immunotherapy, Nat. Rev. Drug Discov. 18 (3) (2019) 175–196.
[10] A. Mantovani, F. Marchesi, A. Malesci, L. Laghi, P. Allavena, Tumour-associated
D. Wang, et al.
macrophages as treatment targets in oncology, Nat. Rev. Clin. Oncol. 14 (7) (2017)
399–416.
[11] J.W. Pollard, Trophic macrophages in development and disease, Nat. Rev. Immunol.
9 (4) (2009) 259–270.
[12] M. Falleni, F. Savi, D. Tosi, E. Agape, A. Cerri, L. Moneghini, G.P. Bulfamante, M1
and M2 macrophages’ clinicopathological significance in cutaneous melanoma,
Melanoma Res. 27 (3) (2017) 200–210.
[13] D.M. Mosser, The many faces of macrophage activation, J. Leukocyte Biol. 73 (2)
(2003) 209–212.
[14] A. Sindrilaru, T. Peters, S. Wieschalka, C. Baican, A. Baican, H. Peter, A. Hainzl,
S. Schatz, Y. Qi, A. Schlecht, J.M. Weiss, M. Wlaschek, C. Sunderkotter,
K. Scharffetter-Kochanek, An unrestrained proinflammatory M1 macrophage po-
pulation induced by iron impairs wound healing in humans and mice, J. Clin.
Invest. 121 (3) (2011) 985–997.
[15] S. Zanganeh, G. Hutter, R. Spitler, O. Lenkov, M. Mahmoudi, A. Shaw,
J.S. Pajarinen, H. Nejadnik, S. Goodman, M. Moseley, L.M. Coussens, H.E. Daldrup-
Link, Iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory
macrophage polarization in tumour tissues, Nat. Nanotechnol. 11 (11) (2016)
986–994.
[16] H.E. Daldrup-Link, D. Golovko, B. Ruffell, D.G. DeNardo, R. Castaneda, C. Ansari,
J.H. Rao, G.A. Tikhomirov, M.F. Wendland, C. Corot, L.M. Coussens, MRI of tumor-
associated macrophages with clinically applicable iron oxide nanoparticles, Clin.
Cancer Res. 17 (2011) 5695–5704.
[17] S.M. Pyonteck, L. Akkari, A.J. Schuhmacher, R.L. Bowman, L. Sevenich, D.F. Quail,
O.C. Olson, M.L. Quick, J.T. Huse, V. Teijeiro, M. Setty, C.S. Leslie, Y. Oei,
A. Pedraza, J. Zhang, C.W. Brennan, J.C. Sutton, E.C. Holland, D. Daniel, J.A. Joyce,
CSF-1R inhibition alters macrophage polarization and blocks glioma progression,
Nat. Med. 19 (10) (2013) 1264–1272.
[18] M.E. Rodrigues, A.R. Costa, P. Fernandes, M. Henriques, P. Cunnah, D.W. Melton,
J. Azeredo, R. Oliveira, Evaluation of macroporous and microporous carriers for
CHO-K1 cell growth and monoclonal antibody production, J. Microbiol. Biotechnol.
23 (9) (2013) 1308–1321.
[19] M.S. Ferreira, W. Jahnen-Dechent, N. Labude, M. Bovi, T. Hieronymus, M. Zenke,
R.K. Schneider, S. Neuss, Cord blood-hematopoietic stem cell expansion in 3D fibrin
scaffolds with stromal support, Biomaterials 33 (29) (2012) 6987–6997.
[20] A. Wilson, A. Trumpp, Bone-marrow haematopoietic-stem-cell niches, Nat. Rev.
Immunol. 6 (2) (2006) 93–106.
[21] D.M. Headen, G. Aubry, H. Lu, A.J. Garcia, Microfluidic-based generation of size-
controlled, biofunctionalized synthetic polymer microgels for cell encapsulation,
273
Colloids and Surfaces A 572 (2019) 266–273
Adv. Mater. 26 (19) (2014) 3003–3008.
[22] A. Batorsky, J. Liao, A.W. Lund, G.E. Plopper, J.P. Stegemann, Encapsulation of
adult human mesenchymal stem cells within collagen-agarose microenvironments,
Biotechnol. Bioeng. 92 (4) (2005) 492–500.
[23] W. Leong, D.A. Wang, Cell-laden polymeric microspheres for biomedical applica-
tions, Trends Biotechnol. 33 (11) (2015) 653–666.
[24] M.E. Rodrigues, A.R. Costa, P. Fernandes, M. Henriques, P. Cunnah, D.W. Melton,
J. Azeredo, R. Oliveira, Evaluation of macroporous and microporous carriers for
CHO-K1 cell growth and monoclonal antibody production, J. Microbiol. Biotechnol.
23 (2013) 1308–1321.
[25] A.R. Costa, J. Withers, M.E. Rodrigues, N. McLoughlin, M. Henriques, R. Oliveira,
P.M. Rudd, J. Azeredo, The impact of microcarrier culture optimization on the
glycosylation profile of a monoclonal antibody, Springer Plus 2 (2013) 25.
[26] Y. Park, Y. Chen, L. Ordovas, C.M. Verfaillie, Hepatic differentiation of human
embryonic stem cells on microcarriers, J. Biotechnol. 174 (2014) 39–48.
[27] S. Reuveny, A. Mizrahi, M. Kotler, A. Freeman, Factors affecting cell attachment,
spreading, and growth on derivatized microcarriers. I. Establishment of working
system and effect of the type of the amino-charged groups, Biotechnol. Bioeng. 25
(1983) 469–480.
[28] A. Zuhlke, B. Roder, H. Widdecke, J. Klein, Synthesis and application of new mi-
crocarriers for animal cell culture. Part I: design of polystyrene based microcarriers,
J. Biomater. Sci. Polym. Ed. 5 (1993) 65–78.
[29] S. Li, Q. Zou, Y. Li, C. Yuan, R. Xing, X. Yan, Smart peptide-based supramolecular
photodynamic metallonanodrugs designed by multicomponent coordination self-
assembly, J. Am. Chem. Soc. 140 (34) (2018) 10794–10802.
[30] R. Xing, S. Li, N. Zhang, G. Shen, H. Mohwald, X. Yan, Self-assembled injectable
peptide hydrogels capable of triggering antitumor immune response,
Biomacromolecules 18 (11) (2017) 3514–3523.
[31] J. Li, A. Wang, P. Ren, X. Yan, S. Bai, One-step co-assembly method to fabricate
photosensitive peptide nanoparticles for two-photon photodynamic therapy, Chem.
Commun. 55 (2019) 3191–3194.
[32] W. Friess, Collagen – biomaterial for drug delivery, Eur. J. Pharm. Biopharm. 45
(1998) 113–136.
[33] Y. Jia, J. Li, Molecular assembly of Schiff Base interactions: construction and ap-
plication, Chem. Rev. 115 (3) (2015) 1597–1621.
[34] J. Xia, B. Sun, Y. Yang, J. Li, Y. Jia, W. Dong, J. Li, Controlled movement of kinesin-
driven microtubule along a directional track, Colloids Surf. A 550 (2018) 186–192.
[35] T. Calandra, T. Roger, Macrophage migration inhibitory factor: a regulator of innate
immunity, Nat. Rev. Immunol. 3 (10) (2003) 791–800.