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\begin{document}

\title{Fabrication of oxygen carrying microparticles functionalized with liver
ECM-proteins to improve phenotypic three-dimensional in vitro liver
assembly, function, and responses}



\author[1]{Nic Leipzig}%
\author[1]{Mona Mansouri}%
\author[2]{Imes W.D}%
\author[1]{Roberts O.S}%
\affil[1]{University of Akron College of Engineering}%
\affil[2]{University of Akron Department of Chemistry}%


\vspace{-1em}



  
  \date{March 14, 2025}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Oxygen and extracellular matrix (ECM)-derived biopolymers play vital
roles in regulating many cellular functions in both the healthy and
diseased liver. This study reveals the importance of synergistically
tuning the internal microenvironment to enhance oxygen availability
alongside phenotypic ECM ligand presentation to promote native metabolic
functions of human liver three-dimensional (3D) cell aggregates. First,
fluorinated (PFC) chitosan microparticles (MPs) were generated with a
microfluidic chip, then their oxygen transport properties were studied
using a custom ruthenium-based oxygen sensing approach. Next, to allow
for integrin engagements the surfaces of these MPs were functionalized
using liver ECM proteins including fibronectin, laminin-111,
laminin-511, and laminin-521. These MPs were used to assemble
heterogeneous composite spheroids composed of human hepatocytes and
human hepatic stellate cells. After \emph{in vitro} culture,
liver-specific functions and cell adhesion patterns were compared
between groups and cells showed enhanced liver phenotypic responses in
response to laminin-511 and 521 as evidenced via enhanced E-cadherin and
vinculin expression as well as albumin and urea secretion. Furthermore,
hepatocytes and stellate cells arranged in more phenotypic arrangements
when cocultured with laminin-511 and 521 modified MPs providing clear
evidence that specific ECM proteins have distinctive roles in the
phenotypic regulation of liver cells in engineering 3D spheroids. This
study advances efforts to create more physiologically relevant organ
models allowing for well-defined conditions and phenotypic cell
signaling which together improve the relevance of 3D spheroid and
organoid models.%
\end{abstract}%



\sloppy


\textbf{Introduction:}

The need to create physiologically realistic \emph{in vitro} liver
models has resulted in the emergence of technologies such as organ on a
chip, 3D printing and rotational culture methods, yielding protocols to
create improved human preclinical
models.\textsuperscript{{[}1{]}}However, 3D cellular aggregates present
certain drawbacks. First, there is no vascular network formation
therefore nutrient and waste transport limits the maximum size of 3D
tissues grown \emph{in vitro.} \textsuperscript{{[}2{]}} Second,
\emph{in vivo} niche-based cues such as ECM components are missing,
which slows initial aggregation and cell activation resulting in loss of
function and phenotype in long term cultures.\textsuperscript{{[}3{]}}
Unlike other organs, the liver has a unique makeup with a
proportionately small ECM in relation to its volume, mainly consisting
of collagen, fibronectin, and laminin.\textsuperscript{{[}4{]}} Collagen
represents 60\% of human liver ECM molecules and constitutes mostly
fibrillar collagens such as type I and III
collagen,\textsuperscript{{[}5{]}} providing tensile strength to the
organ. Whereas non-collagenous proteins such as fibronectin and laminin
are vital to maintaining basement membrane and functional
integrity.\textsuperscript{{[}6{]}} Fibronectin is a multifunctional
adhesive glycoprotein that originally synthesized by liver cells and
abundantly present in liver tissue. This protein is directly involved in
regulating cellular behavior such as cell survival and
proliferation.\textsuperscript{{[}7{]}} Laminin is another major
non-collagenous adhesive glycoprotein presents in the hepatic
perisinusoidal space (space of Disse). It includes specific combinations
of \selectlanguage{greek}α, β, \selectlanguage{english}and \selectlanguage{greek}γ \selectlanguage{english}chains giving rise to functional diversity within a
common structural framework. Within this family the distribution and
expression of \selectlanguage{greek}α\selectlanguage{english}5, \selectlanguage{greek}β\selectlanguage{english}1 and \selectlanguage{greek}β\selectlanguage{english}2 laminin chains in mammalian liver have been
widely reported.\textsuperscript{{[}8,9{]}}

ECM-based molecules (peptides or whole proteins) have been widely
incorporated into biomaterials to promote the formation and function of
various types of spheroids and
organoids.\textsuperscript{{[}10,11{]}}For example, human intestinal
organoids were generated from pluripotent stem cells in a synthetic
hydrogel based on a four-armed, maleimide-terminated poly(ethylene
glycol) macromer functionalized with arginine-glycine-aspartate (RGD)
adhesive peptides.\textsuperscript{{[}12{]}} The authors showed that
organoids encapsulated in this scaffold show high viability as well as
expression levels of pluripotency, endoderm, and epithelial junction
markers compared to non-modified gels. Similarly, Lin et
al.\textsuperscript{{[}13{]}} prepared a hydrogel based on poly
(ethylene glycol)-tetra-norbornene (PEG4NB) functionalized by bioactive
peptides (e.g., fibronectin-derived arginine-glycine-aspartate-serotonin
(RGDS)) to improve cell--matrix interactions. They observed elevated
urea secretion and Cytochrome P450 3A4 (CYP3A4) enzymatic activities, as
well as upregulated mRNA levels of multiple hepatocyte genes (e.g.,
CYP3A4, bile salt export pump (BESP) and sodium-taurocholate
cotransporting polypeptide (NTCP)) of two human hepatoma-derived cell
lines (Huh7 and HepG2) encapsulated in these gels.

Despite these reports, new methodologies are needed for synthetic
cellular microenvironments to reduce mass transport limitations,
especially for metabolically active cell types/tissues such as the
liver. Various methodologies have been formulated to promote oxygen
transport and maintain viability as well as the function of cells in 3D
culture.\textsuperscript{{[}14,15{]}} In our recent work, we created
PFC-MPs to overcome common limitations of spheroids such as inadequate
oxygen supply and ultimate loss of cell/organ specific functions over
long-term cultures.\textsuperscript{{[}16{]}} Our data suggested that
PFC- conjugated MPs offer a simple, affordable, and direct approach for
improving mass transport of nutrients within spheroids and other
engineered tissues. In the present study, we extended our PFC-MP
approach to yield improved hepatic 3D cell culture models using ECM
components. First, we examined if PFC- MPs play a role in driving oxygen
into spheroidal aggregates using optical sensing. Then, we covalently
tethered ECM proteins including laminin-111, laminin-511, laminin-521,
and fibronectin on the surface of PFC- MPs followed by co-culturing
these particles with two immortalized human liver cells including human
hepatoma (HepG2) and hepatic stellate cells (HSCs). We characterized
liver-specific synthetic functions and cell adhesion patterns, which
allowed us to collect evidence that different ECM proteins presented
from PFC-MPs have distinctive roles in the physiological regulation of
liver cells \emph{in vitro} 3D culture.

\textbf{Experimental Section}

\textbf{Microparticle synthesis and characterization}

PFC-MPs were synthesized using a T-shaped polydimethylsiloxane (PDMS)
droplet generator as previously
described.\textsuperscript{{[}16{]}}Briefly, using oil (span80, Sigma
Aldrich, cat\# 1338-43-8) as a continuous phase in the two side channels
and polymer solution as the dispersed phase in the middle channel, we
produced a steady stream of homogenous PFC- MPs. These MPs were passed
through a glass tube and polymerized using the photoinitiator Lithium
phenyl-2,4,6-trimethylbenzoylphosphinate (LAP,0.1wt\% in distilled
water, Sigma-Aldrich, cat\# 85073-19-4) included in the polymer phase,
under the irradiation of a lamp (600-700 nm). The composition of MPs was
characterized via X-ray photoelectron spectroscopy (XPS) with a scanning
monochromated Al K\selectlanguage{greek}α \selectlanguage{english}(117.40 eV; 25.0~W; spot size, 100 \selectlanguage{greek}μ\selectlanguage{english}m). The take-off
angle between the sample surface and analyzer was 45.0\selectlanguage{ngerman}°, and the X-ray
beam collected C1s, N1s, F1s, and O1s elemental information. We also
tested the MPs using thermogravimetric analysis (TGA) to study their
thermal stability. 5 mg of PFC- MPs were weighed and placed in an open
pan (platinum 100 \selectlanguage{greek}µ\selectlanguage{english}L) attached to a microbalance (TGA550, TA
Instruments). The sample was heated at 10 \selectlanguage{ngerman}°C.min\textsuperscript{-1}
from 25 to 700 °C under dry nitrogen in standard mode with a ramp test
type.

\textbf{Studying oxygen levels in MPs}

We used the ruthenium complex Tris(4,7-diphenyl-1,10-phenanthroline)
ruthenium (II) dichloride (Santa Cruz, cat\#sc-216023B), or RuDPP, as an
oxygen sensing molecule. RuDPP shows a large Stokes shift by emitting
orange light (610 nm peak) when excited with blue light (455 nm peak).
We used a custom-made chamber with two inlets for sample and
gas\textbf{(Figure S1)} , a CCD camera (Hamamatsu ORCA-R2/model:
C10600-10B), and HCImage Live software (version 4.8.3.0, Hamamatsu
Corporation, NJ) to measure light intensity. 1 mg of RuDPP was dispersed
in 1X phosphate buffered saline (PBS) solution (pH=7.4) with or without
PFC-MPs (10 mg.ml\textsuperscript{-1}) and sonicated thoroughly before
getting injected into the channel. A light emitting diode (LED,
4\selectlanguage{greek}µ\selectlanguage{english}W.cm\textsuperscript{-2}) was used as the excitation source (peak
wavelength 455nm) and a long wave pass filter (Thorlabs, FMP1, cutoff
wavelength 600 nm) was used to minimize unwanted wavelengths. We
compared the oxygen level within the chamber using two gases including
nitrogen (N\textsubscript{2}) and oxygen (O\textsubscript{2}) to
represent 0\% and 100\% oxygen, respectively. N\textsubscript{2} gas was
initially injected into the channel followed by imaging and then the gas
source was switched to O\textsubscript{2} to present a saturated state.
For each condition, 8 frames per second were obtained for up to 60s. The
fluorescent intensity of unquenched images (I\textsubscript{0}) was
divided by quenched images (I) on a frame-by-frame basis to obtain a
Stern-Volmer image.

\textbf{Preparation of liver spheroids incorporated with PFC- MPs}

Creation of spheroids and incorporation with MP followed procedures
recently published.\textsuperscript{{[}16{]}} Briefly, HepG2 and HSC
cells were used for \emph{in vitro} experimentation. Low glucose
Dulbecco's Modified Eagle's Medium with L-glutamine and sodium
bicarbonate (DMEM; Sigma Aldrich, cat\#D6046) supplemented with fetal
bovine serum (10\%v/v, FBS, R\&D systems, cat\# S11150) and
penicillin/streptomycin (1\%v/v, P/S, Thermofisher, cat\# 15070-063) was
used for expansion of HepG2 cells. For HSC cells, High glucose DMEM
containing L-glutamine, sodium pyruvate, sodium bicarbonate (Sigma
Aldrich, cat\#D6029), 10\% v/v FBS and 1\% v/v P/S was used. The
fabrication of cell spheroids with MPs utilized previous
findings.\textsuperscript{{[}16{]}} Briefly, HepG2 and HSC cells were
mixed at a 4:1 ratio, then added to MPs to achieve a desired final cell
to particle ratio (150:1) and transferred to the 96-well cell repellant
plates (CELLSTAR\selectlanguage{ngerman}® Plates with Cell-Repellent Surface, cat\#: 650970) at
a seeding density of 20,000 cells/well. Cells were overlaid with a
serum-free and defined hepatocyte culture media (Corning®, cat\#
355056). To form cell aggregates, the well plate was centrifuged at 300
× \emph{g} for 5 min.

\textbf{Measurement of oxygen concentration within spheroids}

For oxygen characterization, RuDPP was added to cell suspensions (2
\selectlanguage{greek}µ\selectlanguage{english}g.ml\textsuperscript{-1}) before spheroid formation. After 5 days of
culture the spheroids were live imaged using a laser scanning confocal
microscope (Olympus FV1000) to capture Z-stack images throughout the
spheroids. \selectlanguage{greek}µ-\selectlanguage{english}Slide 8 well glass bottom slides from ibidi (cat\# 80827)
were used to image spheroids (excitation: 455nm, emission: 610nm). The
imaging was performed by taking z-stacks at 10 \selectlanguage{greek}µ\selectlanguage{english}m intervals using 4X
magnification. The fluorescent signals for spheroids cultured
with/without PFC-MPs were quantitatively and spatially assessed using
ImageJ (National Institutes of Health, Bethesda, MD, USA). To correlate
fluorescent intensity to concentration of oxygen, a calibration curve
was generated for RuDPP relating fluorescent intensity versus various
oxygen concentrations (\%O\textsubscript{2}).

\textbf{Surface functionalization of MPs using liver adhesive ligand}

The protocol for covalent bonding of proteins on PFC- MPs was similar to
that reported in previous studies.\textsuperscript{{[}17{]}} The
activation of proteins was achieved by mixing 10
\selectlanguage{greek}μ\selectlanguage{english}g.ml\textsuperscript{-1} of each respective ECM protein solution for 30
minutes at room temperature in a solution containing 2mM\emph{N}
-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) (Sigma Aldrich,
cat\# E7750) in ultrapure water (18.2 M\selectlanguage{greek}Ω\selectlanguage{english}) and 5mM N-hydroxysuccinimide
esters (NHS) (Sigma Aldrich, cat\#130672) in 0.1M 2-(N-morpholino)
ethanesulfonic acid hemisodium salt (MES) buffer (Sigma Aldrich,
cat\#M3671. PFC- MPs were also dispersed in 0.1M MES and their pH was
adjusted to four different values including 5.90, 6.20, 6.50, and 6.80
by addition of 1M NaOH. Afterward, activated proteins were added to the
particles to react with the free amino groups available on MP surfaces.
The mixture was kept under mild shaking for 5 h at room temperature,
dialyzed for 48h using SnakeSkin Dialysis Tubing (3.5K MWCO, Fisher
scientific, cat\#PI68035) against distilled water included 0.05\% triton
100X (Sigma Aldrich, cat\#X100-100ML) and then freeze-dried. The amount
of protein conjugated to MPs was measured by Bicinchoninic acid (BCA)
assay (Thermofisher, cat\# PI23235) using manufacturer's directions, as
well as via XPS surface analysis, as described above. For simplicity, we
picked MPs decorated with laminin-111 as a model to study efficiency of
protein grafting.

\textbf{Functional characterization of liver spheroids}

After spheroids were cultured with functionalized MPs, levels of albumin
were determined using a commercially available kit (QuantiChrom BCP
Albumin, BioAssay Systems, cat\# DIAP-250) as a measure of liver cells'
function. The experiment was performed according to the manufacturer's
instructions. Briefly, the medium was collected on days 5 and 10. A 20
\selectlanguage{greek}µ\selectlanguage{english}l aliquot was used to measure albumin secretion into the medium. 200 \selectlanguage{greek}μ\selectlanguage{english}L
of working reagent was added and after 5 min incubation at room
temperature, the optical density at 590-630 nm (peak absorbance at 610
nm) was read by a plate reader (Tecan Infinite M200, Tecan Austria GmbH,
Austria). Urea was also measured by the QuantiChrom Urea Assay Kit
(BioAssay Systems, cat\# DIUR-100,) using a similar procedure to the
albumin kit. 5 \selectlanguage{greek}μ\selectlanguage{english}L of sample was mixed with 200 \selectlanguage{greek}μ\selectlanguage{english}L of working reagent,
incubated for 20 min, and absorbance was read at 520 nm by plate reader.

\textbf{Histologic and immunohistochemical examinations}

For immunofluorescence staining of 3D liver spheroids, spheroids were
fixed for 30 min in 4\% w/v paraformaldehyde in PBS. Then whole samples
were permeabilized with 0.1\% v/v Triton X-100 in PBS for 5 min and
pre-blocked for 30 min with 10\% FBS diluted in PBS. Next, spheroids
were incubated with mouse glial fibrillary acidic protein (GFAP; 1:50,
Developmental Studies Hybridoma Bank (DSHB), cat \# 8-1E7) for HSCs,
rabbit CYP3A4 antibody (1:200, Fisher Scientific, cat\# 50-173-2058) for
HepG2 cells, anti-human E-cadherin monoclonal antibody (1:200, R\&D,
cat\# MAB1838) and anti-vinculin antibody (1:200, Santa Cruz, cat\#
sc-25336) overnight in the fridge. The next day we stained the spheroids
with Alexa Fluor 546 goat anti-mouse IgG (H+L) highly cross-adsorbed
secondary antibody (Thermofisher, cat\# A11030) and Alexa Fluor 546
donkey anti Sheep IgG (H+L) Secondary Antibody (Thermofisher, cat \#
A21098) for 90 min, as appropriate. The spheroids were then incubated
with 10 \selectlanguage{greek}µ\selectlanguage{english}M Hoechst 33342 (Thermofisher, cat\# H3570) diluted in 1\% BSA
for nuclei detection for 5 min before collecting confocal microscope
images (Nikon Corporation confocal microscope, inverted). The step size
of Z-stacks was set to 10 \selectlanguage{greek}µ\selectlanguage{english}m thickness and 30-40 slices per image were
taken to image entire spheroids.

\textbf{Data analysis}

Data processing and displaying were performed using GraphPad Prism 5
(GraphPad Software, USA). Mean and standard deviation were displayed for
all data sets. Statistical significance was determined using either
Student's \emph{t} -test or two-way ANOVA followed by Tukey's \emph{post
hoc} test (\emph{p} \textless{} 0.05). Groups with significant
differences are shown with alphabetic letters to signify ranking of
means. Groups indicated with the same letters were not found to be
significantly different.

\textbf{Results and Discussion:}

\textbf{Characterization of PFC- MPs}

It has been well established that oxygen carrying properties of PFC-MPs
stem from the PFC substitutions, \textsuperscript{{[}18,19{]}} therefore
in this report we focused on studying the composition of microfluidic
generated MPs while confirming the presence of PFC
molecules\textbf{(Figure 1). Figure 1B} shows the TGA spectrum of
crosslinked non-PFC-MPs and PFC-MPs. The weight loss in both spectra
begins at approximately 90\selectlanguage{ngerman}°C and continues until 130°C which is likely
associated with loss of absorbed water in the crosslinked MPs. Based on
these data both MPs were able to absorb water correlating to
\textasciitilde{}25\% via hydrophilic moieties in
chitosan.\textsuperscript{{[}20{]}} Another major mass loss stage is
observed between 280-400°C, which is related to thermal decomposition of
chitosan.\textsuperscript{{[}21{]}} Overall, around 43.1\% w/w of
PFC-MPs and 52.82\% w/w of non-PFC-MPs were lost when heated above
400°C, suggesting higher thermal stability of PFC-MPs compared to
non-PFC-MPs. Carbon--fluorine (C-F) bonds in PFC molecules are strongly
polarized, which accounts for their thermal
stability.\textsuperscript{{[}22{]}} Next, XPS was used to provide a
quantitative analysis from the outer 10 nm of MP surfaces. XPS spectra
showed carbon (C1s at 284.7 eV) and oxygen (O1s at 531.5 eV) as well as
fluorine (F1s at 688.6 eV) on surface of PFC-MPs \textbf{(Figure
1C),}suggesting the presence of PFC molecules on the surface of MPs.
This is expected since the particles were crosslinked while still in the
oil phase. We tried Fourier-transform infrared spectroscopy (FTIR) as
well\textbf{(Figure S2)} ; however, due to PFC abundance being lower
than the detection threshold (2 wt\%), we could not detect C-F bonds.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure} \textbf{1:} A) Studying the arrangement of PFC molecules
after MP formulation B) Thermal degradation study of MPs using TGA to
assess their stability. The shaded box show\emph{s} temperature rand of
interest (100-150\selectlanguage{ngerman}°C). C) Characterization of PFC modified and non-PFC
modified MPs using XPS. The resulting spectrums showed a sharp peak
related to PFCs, suggesting availability of PFC groups on the surface. *
refers to fluorine spectrum located at 688.6 eV.

\textbf{Assessing oxygen carrying properties of MPs using RuDPP}

In our previous studies, PFC conjugated chitosan hydrogels were tested
for oxygen transport properties using commercial phosphorescent
dots\textsuperscript{{[}23{]}} and needle
sensors.\textsuperscript{{[}24{]}}Our investigations showed these
hydrogels enhanced oxygen transport in aqueous conditions and allowed
oxygen tensions to reach higher equilibrium states compared to controls
not integrating PFCs. In this study, we were interested in understanding
the impact of our PFC-MPs on oxygen tensions using RuDPP in finer detail
and at a cellular level. RuDPP has a long fluorescence lifetime and a
long Stokes-shift. It quenches in the presence of oxygen and thus is a
suitable probe dye for determining dissolved
oxygen.\textsuperscript{{[}25{]}} A mixture of MP solution and RuDPP in
PBS was injected into a sealed, custom-built chamber and exposed to
gases (\textbf{Figure 2A)} . We measured the fluorescence intensity
after flowing pure N\textsubscript{2} and pure O\textsubscript{2} gasses
to create oxygen partial pressures at the extremes of 0\% oxygen and
100\% oxygen in solution. A solution without any MPs was used as a
control. As shown in \textbf{Figure 2B} , when switching from pure
N\textsubscript{2} to pure O\textsubscript{2}, the fluorescent intensity
decreased due to oxygen quenching of RuDPP. We also observed a decrease
in fluorescence intensity in the presence of MPs which can be attributed
to increased dissolved oxygen concentration. The sensitivity of the
optical oxygen sensor with (\emph{I)} and without (\emph{I}
\textsubscript{0}) a quencher (here oxygen) was quantified in term of
the ratio \emph{I} \textsubscript{0}/\emph{I} \textsubscript{100}, as
shown in \textbf{Figure 2C} at equilibrium. Our results indicated that
the solution containing PFC-MPs exhibited the highest sensitivity to
oxygen with the maximum quenching ratio of\emph{I}
\textsubscript{0}/\emph{I} \textsubscript{100}\selectlanguage{english} =1.1. Following
saturation, we then stopped oxygen flow and studied the release
kinetics. We observed that the control group showed complete RUDPP
recovery within 3 min whereas MPs slowed the RuDPP recovery process to
10 min \textbf{(Figure 2D)} . This can be explained by MPs' ability to
uptake oxygen and then release it gradually into a low oxygen tension
aqueous environment. These results confirm that PFC-MPs can act as both
an oxygen absorbing material as well as a reservoir to release oxygen
gradually in an aqueous environment. This finding is in agreement with
our earlier work with PFC-modified hydrogels where we examined their
ability to deliver and sustain biological levels oxygen for enhancing
cellular functions essential in wound
healing.\textsuperscript{{[}26,27{]}} Most recent evidence from another
research group shows the potential of PFC nanoemulsions as artificial
oxygen carriers.\textsuperscript{{[}28{]}} In this study, PFC
nanoemulsions exhibited five times higher dissolved
O\textsubscript{2}~concentration compared to water, as measured by a
submerged O\textsubscript{2} probe.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2:} A) Illustration of custom-built oxygen sensing
measurement setup with CCD camera, excitation light, selective filter,
and flow chamber. B) Fluorescence emission spectra at different oxygen
(0\% and 100\%) concentrations excited at 455nm measured by CCD camera
equipped with a telecentric lens. C) Relative fluorescent intensity of
Stern-Volmer equation over time obtained by dividing values of
unquenched and quenched fluorescence intensities (I\textsubscript{0}/I)
representing the oxygen concentration in the chamber. D) MPs oxygen
release kinetics containing following saturation with 100\% oxygen. The
intensity of each condition was obtained by averaging the intensity over
the channel.

\textbf{Oxygen levels within live spheroids cultured with/without
PFC-MPs}

To better understand MP effects on oxygen levels in our spheroid model,
we used RuDPP and fluorescent microscopy building upon our earlier
validation experiments without cells. For the spheroid system, we mixed
cell suspension with different amounts of RuDPP to find an optimum
concentration required for the formation of spheroids with
RuDPP\textbf{(Figure S1A).} Although the working concentration of dye in
our system is low, we were able to utilize this concentration to
directly image intracellular oxygen levels. To study cellular oxygen
levels throughout spheroids we made 3D images of z-stacks and looked at
the fluorescence intensity throughout the whole spheroid as shown
in\textbf{Figure 3A} . We observed cells at higher oxygen tensions
(shown in black) in spheroids cultured with PFC-MPs, demonstrating that
MPs improve oxygen levels in the spheroids. Quantitative cellular
measurements corresponding O\textsubscript{2}\% were performed via a
calibration curve based on RuDPP in a controlled oxygenated
environment\textbf{(Figure S1C).} To perform this, we focused on
individual cells and the intensity of those cells along a line drawn in
a central confocal plane of spheroids. As shown in \textbf{Figure 3B,}
the addition of MPs increased oxygen levels from 7 to 10\%, emphasizing
the validity of our approach. In related work, partial pressure of
oxygen (PO\textsubscript{2}) levels in human colorectal spheroids with a
diameter of ~600 \selectlanguage{greek}μ\selectlanguage{english}m were measured using electron spin resonance (ESR)
microscopy, and PO\textsubscript{2} values were reported in the range of
50-60 mmHg which is equivalent to 6.6-7.9\% oxygen at inner
regions.\textsuperscript{{[}29{]}} \emph{In vitro} experiments are
usually performed in incubators that maintain a PO\textsubscript{2} of
\textasciitilde{}142 mmHg (18.7\% oxygen), whereas cells in our body do
not experience a PO\textsubscript{2} greater than 100 mmHg (13\%
oxygen). Despite lower oxygen tensions \emph{in vivo} , cells can
tolerate these levels and easily survive because of short transport
distances from supply via blood vessels.\textsuperscript{{[}30{]}} When
it comes to avascular 3D cell aggregates, especially larger ones, we
expect lower PO\textsubscript{2} due to greater distances and mass
transport limitations. The Hypoxyprobe (pimonidazole hydrochloride)
method is often used to study hypoxia responses in
spheroids;\textsuperscript{{[}31{]}} however, the method is an indirect
endpoint test with no quantitative analysis in terms of oxygen tensions.
Using RuDPP is advantageous for live and kinetic imaging since the
fluorescence lifetime of RuDPP is largely insensitive to pH, ion
concentrations, and cellular contents, making it suitable for cell
culture applications.\textsuperscript{{[}32{]}} Previous work has shown
that MCF-7 cells labeled with RuDPP at a concentration of 20
\selectlanguage{greek}µ\selectlanguage{english}g.mL\textsuperscript{-1} maintained 90\% viability after
24h,\textsuperscript{{[}33{]}} confirming its biocompatibility.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3:} Visualizing oxygen within liver spheroids using RuDPP
as an oxygen sensing dye, which quenches fluorescence emission in the
presence of sufficient oxygen. A) Reconstruction of a 3D spheroid and
intensity variance throughout the spheroid cultured for 5 days. PFC- MPs
resulted in lower fluorescence intensity compared to the control which
did not have any MPs. B) Measurement of intensity of individual cells
along a circular line in a central plane of spheroids with quantitative
data. The average intensity of bright dots around each line was
quantified and converted to O\textsubscript{2}\% via a calibration
curve. Data represented as mean \selectlanguage{ngerman}± SD. n = 3 independent spheroids,
one-way ANOVA and Tukey's \emph{post hoc} .

\subsubsection*{Covalent immobilization of liver ECM adhesive ligands to
PFC-MPs}

{\label{covalent-immobilization-of-liver-ecm-adhesive-ligands-to-pfc-mps}}

Next, we modified MP surfaces by covalently binding ECM proteins to
overcome the limitations associated with simple physical adsorption of
ECM proteins, including their undesired conformational changes and
release from surfaces over time.\textsuperscript{{[}34{]}} In this
study, biofunctionalization with the ECM proteins plasma fibronectin,
laminin-111, 511, and 521 to the surface of PFC-MPs was accomplished by
a coupling reaction between the carboxyl residues of proteins to the
free amine residues of chitosan using EDC as crosslinker and NHS ester
as an enhancer of coupling \textbf{(Figure. 4A)} . The EDC/NHS coupling
reaction is a selective method for preservation of biological activity
of the protein and has previously been applied in the production of
protein functionalized chitosan
derivatives.\textsuperscript{{[}35--37{]}} Since the pH of the reaction
solution is critical for maximizing the amination reaction, we tested
different pH levels keeping all other conditions the same, to determine
the favored pH to maximize attachment. The recommended pH conditions for
the carbodiimide reaction via EDC/NHS is in the range of pH
4.5--5.5.\textsuperscript{{[}38{]}} However, due to the presence of
amine groups, chitosan based materials become protonated at a pH below
pK\textsubscript{a} 6.0.\textsuperscript{{[}39{]}} Between a pH of 5.9
and 6.8 we achieved ligand conjugation on MPs ranging from 2 to 3.5
\selectlanguage{greek}µ\selectlanguage{english}g.ml\textsuperscript{-1}, as quantified by BCA kit \textbf{(Figure 4B)}
. We can attribute the poorer yield at the lowest pH (5.9) to high
chitosan protonation, resulting in a slower reaction. Poor yield at the
highest pH (6.8) might be due to the formation of macroscopic aggregates
resulting in reduced availability of surface area for reactions. The
formation of aggregates at pH \textgreater{}6.5 where chitosan particles
are weakly charged and less stable was reported
previously.\textsuperscript{{[}40,41{]}} We further characterized the
uncoated and coated MPs modified at different pH levels via
XPS.\textbf{Figure 4C} presents atomic compositions of MPs modified at
different pHs. This result shows an increase in atomic nitrogen
percentages (N1s\%) as a reliable marker to detect additional amide
groups (N--C=O) from amino acids in added ECM proteins compared to
non-modified MPs. Interestingly, we observed an increase from 0.9\% to
6.0\% at pH=6.2 which confirms the data obtained from colorimetric
quantification. Similarly, Huang and coworkers evaluated the grafting
efficiency of laminin (derived from Engelbreth-Holm-Swarm mouse sarcoma
basement membrane) on poly(lactic-co-glycolic acid) (PLGA) film using
XPS and observed an increase in N1s from 0.5 to 1.1\% after modification
with laminin.\textsuperscript{{[}42{]}} We opted to focus on only N1s
because C1s and O1s peaks can originate from the MP composition itself
and might lead to misleading data.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4:} EDC/NHS coupling reaction for bioconjugation of ECM
proteins to MPs. A) Schematic of EDC/NHS reaction between protein
carboxyl residues and free amine groups from PFC- oxygenating MPs in
varied pHs. B) Quantification of covalently tethered protein (laminin
111) via BCA assay kit. Data represented as mean \selectlanguage{ngerman}± SD, n = 4.
Statistical analysis via one-way ANOVA with Tukey's \emph{post
hoc}testing with A as the highest mean and C with the lowest mean. C)
Atomic composition of modified and non-modified PFC- MPs measured via
XPS. The N1S percentage correlates to the concentration of proteins
attached to the surface.

\textbf{Influence of specific ECM proteins on liver-specific functions}

After confirming successful covalent ECM tethering to MPS, we moved to
assess biological responses in spheroids by combining hepatocytes and
stellate cells with modified MPs \textbf{(Figure 5A)} , specifically to
study potential impact of different ECM proteins on liver-specific
functions. In culture, viable hepatocytes synthesize and excrete albumin
and urea into the surrounding medium, replicating important functions
they perform \emph{in vivo} in the liver. To characterize these critical
cellular functions, we collected culture medium and measured albumin and
urea production at specific time points.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5:} Schematic of experimental procedure for formation of
enhanced spheroids using ECM modified oxygenating MPs and two liver cell
lines. B) Quantification of albumin and urea secretion into the media
over the culture period (10 days). Data represented as mean \selectlanguage{ngerman}± SD. n = 3
independent spheroids. Statistics via two-way ANOVA followed by a
Tukey's \emph{post hoc} testing.

As \textbf{Figure 5B} shows, the more mature laminin isoforms 511 and
521 boosted albumin and urea production compared to the other groups
(\emph{p} \selectlanguage{english} \textless{}\selectlanguage{english} 0.01), providing evidence that these two isoforms
can more closely reflect the \emph{in vivo} composition of cells present
in the liver. Consistent with this interpretation laminin-511 and -521
coated surfaces were proved to improve culture matrices for hepatic
specification and differentiation of human pluripotent stem cells
(hPSCs),\textsuperscript{{[}43{]}} as well as human embryonic stem cells
(hESC).\textsuperscript{{[}44{]}} We also considered laminin-111 to
represent a variation of laminin not found abundantly in the adult liver
to compare the difference between liver specific isotypes to
non-specific ones. Laminin-111 is expressed during embryogenesis and
plays an important role in assembling early basement
membranes.\textsuperscript{{[}45{]}} We did not find any significant
differences (p\textgreater{} 0.05) between laminin-111 conjugated and
non-coated MPs in terms of liver specific functions probed, highlighting
the importance of proteins native to the mature \emph{in vivo} liver
environment for benefitting cell culture \emph{in vitro} . Our result is
consistent with an experiment conducted by Klaas et.
al\textsuperscript{{[}46{]}} where they cultured HepG2 cells on 3D nano-
and microfiber structures based on gelatin, preincubated with
laminin-111 and observed no significant changes in cell morphology,
viability and production of proteins between coated with laminin-111 and
uncoated 3D gelatin scaffolds.

\textbf{Visualizing cell-cell and cell-ECM interactions}

Improved function in response to laminin modified MPs, further
strengthened our hypothesis that effects exerted on cells by various ECM
ligands can be differently sensed and translated by cells. To
investigate ECM activation events, we used immunofluorescence staining
to probe for vinculin and cadherin expression in spheroids. Vinculin is
a common component of activated focal adhesion complex when cells are
actively engaging ECM with integrin receptors. Cadherins are essential
constituents of adherens junctions, important in the formation and
maintenance of cell-cell contacts.\textsuperscript{{[}47{]}}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Figure 6:} Immunofluorescent staining of fixed spheroids
composed of HepG2s:HSCs (4:1), 20k cells initially per spheroid cultured
with different oxygenating MPs for 10 d stained with A) Anti-Vinculin
(for focal adhesions, yellow), B) E-Cadherin (for adherens, purple), and
Hoechst 33342 (for nuclei, blue) using confocal microscopy on central
plane. C) Quantitative analysis of images using ImageJ. Statistics via
one-way ANOVA and Tukey's \emph{post hoc} testing with A as the highest
mean and D with the lowest mean, p\textless{} 0.05. Mean \selectlanguage{ngerman}± SD, n =2
spheroids with S=3.

\textbf{Figure 6A} shows representative confocal microscope images of
liver spheroids cultured with various modified MPs and stained for
vinculin. We observed high levels of vinculin expression in the
laminin-511 group suggesting better integrin mediated focal adhesion
kinase (FAK) activation and focal adhesion formation. In connection to
our findings, the Mitaka group investigated the deposition of laminins
containing the \selectlanguage{greek}α\selectlanguage{english}5 chain in the liver cancer environment and concluded
that laminin-511 and -521 are major isoforms in liver tumors. They
showed that cancer cells could specifically interact with these ligands
through integrin \selectlanguage{greek}α\selectlanguage{english}3\selectlanguage{greek}β\selectlanguage{english}1 and \selectlanguage{greek}α\selectlanguage{english}6\selectlanguage{greek}β\selectlanguage{english}4 which are widely expressed in
hepatocellular carcinoma.\textsuperscript{{[}48,49{]}} Laminin-111
primary binding integrins are \selectlanguage{greek}α\selectlanguage{english}6\selectlanguage{greek}β\selectlanguage{english}1\textsuperscript{{[}50{]}} and
\selectlanguage{greek}α\selectlanguage{english}7\selectlanguage{greek}β\selectlanguage{english}1\textsuperscript{{[}49{]}} while fibronectins main integrin
receptors are \selectlanguage{greek}α\selectlanguage{english}5\selectlanguage{greek}β\selectlanguage{english}1, \selectlanguage{greek}α\selectlanguage{english}8\selectlanguage{greek}β\selectlanguage{english}1, \selectlanguage{greek}α\selectlanguage{english}V\selectlanguage{greek}β\selectlanguage{english}1.\textsuperscript{{[}51{]}} HepG2 cells
are known to express \selectlanguage{greek}α\selectlanguage{english}2, \selectlanguage{greek}α\selectlanguage{english}6, \selectlanguage{greek}β\selectlanguage{english}1, and \selectlanguage{greek}β\selectlanguage{english}4 integrin
subunits\textsuperscript{{[}52{]}} and very low levels of the
fibronectin receptors, presenting the possibility that they may respond
to laminin-511 and 521 but not fibronectin and laminin-111 simply
because they do not express the appropriate integrin receptors.

Equally important in the liver to integrin activation is cell-cell
adherins formation. Hepatocytes in the liver arrange with one surface in
direct contact with blood and the other surface forming tight junctions
with adjacent cells. For every eight hepatocytes, one HSC helps to
maintain the storage of vitamin A and protects hepatocytes in case of
injury.\textsuperscript{{[}53{]}} Thus, we studied physical linkage
between cells to explore the effect of modified MPs on E-cadherin
expression levels. E-cadherin, a cell-cell adhesion molecule in
epithelial tissues, is localized on the surfaces of cells and allows
physical linkage between neighboring cells.
\textsuperscript{{[}54{]}}\textbf{Figure 6B} shows increased expression
of E-cadherin in spheroids cultured with laminin-511 and -521 modified
MPs as compared with other groups (\emph{p}  \textless{} 0.01).
Importantly, we observed high expression of E-cadherin at the edges,
which is associated with more heterotypic cell-cell contacts. The degree
of E-cadherin contact has been shown to affect proliferation and
liver-specific function.\textsuperscript{{[}55{]}} Taken together,
analysis of vinculin and E-cadherin expression suggest that the
phenotype of cells was maintained better in groups cultured with
laminin-511 and -521 and may indeed play a role in modulating
hepatocytes functions. Another important aspect of adherens-type
junctions is stabilization of the interactions between adjacent cells,
which is a crucial factor in forming and maintaining 3D cellular
aggregates. In our previous study with liver
spheroids,\textsuperscript{{[}16{]}} we showed that HSCs play a central
role in compactness and formation of stable liver spheroids. Improved
E-cadherin expression prompted us to determine if we could use MPs
modified with laminin 511 or 521 instead of HSCs to induce the cells
form compact spheroids. Our data suggested that although these MPs lead
to smaller size spheroids and more cell-cell junctions compared to HepG2
only spheroids \textbf{(Figure S3),} they failed to achieve the
compactness caused by HSCs. This is likely because HSC secrete many
biomolecules, cytokines, and growth factors leading to better shape and
stability. In the liver, HSCs do not constitute direct cell adhesions
with liver sinusoidal endothelial cells (LSECs), whereas they establish
adherens junctions with hepatocytes through thorn-like microprojections
or spines \textsuperscript{{[}56{]}} with E-cadherin as the main
adhesion molecule.

\textbf{Visualizing cell arrangements}

\emph{In vivo} , ECM molecules including fibronectins, collagens, and
laminins not only affect signaling properties and cell function but also
modulate overall tissue architecture with direct consequences on cell
arrangements and tissue organization.\textsuperscript{{[}57{]}} To
better understand the effects of specific ECM proteins on the cell
organization, we used immunohistochemistry staining for HepG2 and HSC
cells in whole mount spheroids to visualize the impact of ECM modified
MPs on arrangements \textbf{(Figure 7)} . Although HepG2 cell
distribution was homogenous, we observed distinct localization of HSCs
toward the periphery of spheroids, generating a distinct outer layer of
similar thickness in all groups. This also correlates with E-cadherin
staining results (\textbf{Figure 6B} ). The contacting area of the
hepatocytes contains collagen fibrils,
\textsuperscript{{[}58{]}}resulting in a membrane thickening which we
also observed in our images (the purple ring at the edge of all groups).
We observed that groups treated with laminin-511 and 521 resulted in
localization of some HSCs toward the center of spheroids, perhaps
because PFC-laminin 511/521 MPs stimulate cells to change position
relative to their neighbors toward contact strengthening. This finding
lends support to prior findings and confirms that interactions between
cells and the ECM at integrin-based adhesion sites allow cells to sense
their physical surroundings and adjust mechanisms of migration and
anchorage.\textsuperscript{{[}59{]}}Thus, besides organization, our co
culture system provides a similar physicochemical structure that more
closely mimics \emph{in vivo} tissue counterparts.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\textbf{Figure 7:} Cell heterogeneity in spheroids using HepG2: HSC
(4:1), 20k cells initially per spheroid cultured with different
oxygenating MPs. A) Representative confocal microscopic images of
central plane of spheroids stained with CYP3A4 (for HepG2 cells, green),
GFAP (for HSC cells, purple), and Hoechst 33342 (for nuclei, blue).
Arrows highlight the localization of HSCs. B) Quantitative analysis of
images using ImageJ. Statistics via one-way ANOVA and Tukey's \emph{post
hoc} testing with A as the highest mean and C with the lowest mean,
p\textless{} 0.05. Mean \selectlanguage{ngerman}± SD, n =2 with S=3.

\textbf{Conclusions}

In this study, we demonstrated a new strategy for improving cellular
assembly and functions inside human cell-based spheroids via
synergistically promoting biophysical and biochemical cues using
engineered MPs. Our MPs integrated covalently attached PFC groups and
ECM adhesive ligands on the surface to enhance oxygen tensions
internally while improving cell/tissue architecture and functions,
respectively. We confirmed the value of PFC-MPs for dissolving and
releasing oxygen to enhance oxygen tensions at the cellular level using
a RuDPP oxygen sensitive dye with microscopy-based fluorescence sensing.
We next observed that cells in assembled liver spheroids responded best
to MPs presenting laminin-511 and -521 ECM proteins, which are more
prevalent in the mature liver as compared to laminin-111. These laminin
isoforms encouraged enhanced phenotypic liver spheroid formation with
up-regulation of E-cadherin and vinculin expression, as well as greater
albumin and urea secretion as compared to MPs presenting other ECMs and
the controls. HSCs also arranged in native liver type arrangements when
laminin 511/521 conjugated MPs were used as compared to laminin-111,
fibronectin, and control groups; providing evidence that ECM proteins
have distinct roles in the phenotypic regulation of mature liver derived
cells. In conclusion, via synergistic enhancement of internal oxygen
availability as well as presentation of specific ECM cell adhesion
ligands, our engineered MP approach provides a unique tool to help cells
to assemble in a more native 3D microenvironment within spheroids,
thereby improving \emph{in vitro} modeling applications such as in the
first stages of drug development or for personalized drug screening
applications.

\textbf{Acknowledgments}

We would like to acknowledge partial funding from the University of
Akron. We are also grateful to Dr. Ali Dhinojwala for his valuable
suggestions and discussions regarding MP characterization and
Chinnapatch Tantisuwanno for technical assistance in conducting
Thermogravimetric testing and analysis.

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