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\begin{document}

\title{Development of a Novel In Vitro Co-Culture System for Discrete Subaortic
Stenosis}



\author[1]{Pengfei Ji}%
\author[1]{Sunita Brimmer}%
\author[1]{Swathi Balaji}%
\author[2]{Jane Grande-Allen}%
\author[1]{Ravi K. Birla}%
\author[1]{Sundeep G. Keswani}%
\affil[1]{Texas Children’s Hospital}%
\affil[2]{Rice University}%


\vspace{-1em}



  
  \date{June 07, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The aim of this study was to investigate the survival, distribution and
reaction of different cell types on a monolayer disk, as well as their
behavior under bioreactor treatment. Specifically, porcine EEC and
porcine fibroblasts (PCF) were labeled with GFT and Texas Red,
respectively, to track their viability and distribution. The experiments
involved monitoring the cells using various microscopy techniques and
comparing the results with controls. These findings have important
implications for understanding cell behavior and potential applications
for Discrete Subaortic Stenosis. This paper aims to discuss the
implications of the findings in the context of existing literature and
future research directions.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Children with Discrete Sub-Aortic Stenosis (DSS) experience a narrowing
of the left ventricular outflow tract (LVOT), caused by the formation of
a fibro membranous tissue. This blockage increases the pressure in the
LVOT, which is located just below the aortic valve {[}1,2{]}. DSS is an
uncommon occurrence in children with congenital heart defects, with a
prevalence rate of around 6\% {[}3\textasciitilde{}6{]}. The
pathogenesis of DSS is hypothesized to begin during the first 10 years
of life {[}6{]}. Symptoms include chest pain, heart failure, syncope,
with about 50\% of patients having a heart murmur that grows with age
{[}5,7{]}. The morphology of the lesions associated with DSS typically
consists of a fibromuscular tissue ring or a localized protrusion of
fibrous tissue {[}8{]}. It is believed that the condition is initiated
by geometric abnormalities in the LVOT, which generates turbulent blood
flow and altered shear forces that trigger a fibrotic response
{[}9\textasciitilde{}14{]}. Although surgical resection of the membranes
provides relief, there is a high risk of recurrence (20-30\%) in what is
defined as an aggressive DSS phenotype, with female sex being a risk
factor {[}15\textasciitilde{}17{]}. Without Intervention, DSS can lead
to left ventricle hypertrophy, aortic regurgitation, endocarditis,
arrhythmias, and in extreme cases, death {[}18\textasciitilde{}22{]}.

Surgical correction of the membrane has been demonstrated to be a viable
option for eliminating obstruction. The membrane will either be removed
via myomectomy, or just the fibrous tissue ring resected {[}123{]}.
However, it has the potential to pose numerous risks, such as those
associated with anesthesia, sternotomy, and heart bypass {[}4{]}. In
extreme cases of aggressive DSS, open-heart surgery is a higher risk,
thus patients undergo percutaneous balloon dilation {[}24{]}. This
procedure lasts months to a few years, and then surgery must be
performed {[}5{]}. These surgical approaches don't last, and most
patients will require reoperation, due to regrowth of the membrane, and
long-term follow up care {[}1, 5, 23, 25{]}.

It has been observed that DSS lesions are composed of 5 layers of
endothelial and fibroblast cells {[}26, 27{]}. Researchers have
hypothesized that the increased blood pressure, or turbulent jet due to
DSS lesions on the LVOT, narrows the subaortic tract, causing a
disturbance in cellular function in these layers {[}6, 28{]}.
Observations have shown that endothelial cells release growth factors
when subjected to shear stress above normal levels, inducing
proliferation of other cells, such as fibroblasts, that comprise the
blood vessel layers and influencing the mechanical properties of blood
vessels {[}1, 14, 29, 30, 31{]}. In a similar but separate study
conducted showed that endothelial cells, when exposed to shear stress,
induce the release of basic fibroblast growth factor (bFGF) that
stimulates the proliferation of fibroblasts {[}30{]}. Current studies
are still limited in understanding a consistent pattern of pathogenesis
of DSS that can be targeted for therapies and diagnostic options for
pediatric patients with this disease.

Although 2D cell culture studies allow researchers to understand the
biology and morphology of cells and tissues, it is limited in the
accuracy of how cells interact with the extracellular environment and
different cell types {[}32\textasciitilde{}34{]}. 3D models allow for a
more accurate study by mimicking disease conditions {[}35, 36{]}. With
3D models we can reproduce the authentic characteristics of the heart's
tissue organization and microenvironment {[}34\textasciitilde{}38{]}. As
previously determined, DSS is hypothesized to be caused by interactions
between 2 different cell types, EECs and Fibroblasts {[}39,40{]}.
Conventional cell culture systems and models are centered around a
single cell type, which is vastly different from the natural environment
of the cell {[}41, 42{]}. Thus, this limitation makes it difficult to
understand the mechanistic and biological properties of the disease.
Creation of a 3D co-culture system that eliminates the disadvantages of
a traditional culture system that is restricted in mimicking the natural
environment and interactions between cells and extracellular components
{[}42\textasciitilde{}45{]}.

We aim to replicate the functional response of fibroblasts in DSS
patients and transition from a monolayer 2D culture to a 3D co-culture
system. To gain an understanding of DSS progression, several aspects of
the tissue must be considered. First, the tissue is 3D in nature, i.e.,
the fibroblasts are organized within a complex 3D ECM matrix. Second,
the matrix will become stiffer over time, i.e., dynamically changing
stiffness. Third, the relative spatial positions of the EECs and the
fibroblasts are more complex than can be replicated in simple 2D
monolayer cultures. In this article we will show the development and
optimization of a 3D co-culture system with porcine cardiac fibroblasts
and endocardial endothelial cells and subject the model to shear stress
conditions as observed in DSS.

\textbf{Materials and Methods}

Gelatin from bovine skin powder (9000-70-8), Calcium Chloride pellets
(10043-52-4), and sodium alginate (9005-38-3) obtained from Sigma
Aldrich. Commercial Cellink BIOX Printer (Model: S-10001-001; S/N:
202041). ECM media (Sciencell, 1001), Fibroblast growth media 3
(PromoCell Inc. C-23025). 0.05\% Trypsin EDTA (Gibco 25300054).

Research Design

4 different types of disks were fabricated. The
1\textsuperscript{st}group contained only EECs in all 3 layers, adding
\(3x10^{5}\) EECs per layer. The 2\textsuperscript{nd} group
contained only Porcine Fibroblasts in all 3 layers, \(3x10^{5}\)
Porcine Fibroblasts per layer. The 3\textsuperscript{rd} group of disks
were comprised of 2 layers of Porcine Fibroblasts and 1 layer of EECs as
the top layer, with same ratio of cells per layer as the first 2 groups.
Lastly, the 4\textsuperscript{th} group of disks contained 3 layers of
contained Porcine Fibroblasts and EECs dispersed throughout all 3
layers,\(3x10^{5}\) EECs and \(3x10^{5}\) Porcine
Fibroblasts in each layer for a total of \(6x10^{5}\) cells per
layer. Cells were cultured in the disk for 48 hours and then each group
subjected to shear stress in the cone-and-plate bioreactor at 6
dynes/cm2 for each group for 24 hours. Before and after inducing shear
stress, confocal images were collected of each layer of each disk.

Cell culture

Porcine Cardiac Fibroblasts and Endocardial Endothelial Cells were
cultured each cell type on an individual monolayer of 0.1\% gelatin,
seeding \(5x10^{5}\) cells. EECs and Porcine Fibroblasts were
maintained in ECM media and Fibroblast Growth Media 3, respectively.
Once cells reach confluency and before trypsinization, each cell type
was labelled using CellTrackers. To be more specific, the Porcine EECs
were labelled by GFP (ThermoFisher, Cat \#Q25041MP), and Porcine Cardiac
Fibroblasts were labelled by Texas Red (ThermoFisher, Cat \# Q25021MP).
We then detached them using 0.05\% Trypsin EDTA. We then cultured the
Celltracker-labelled cells as previously mentioned.

Optimization of Bioink with Cells --

In previous studies {[}35{]}, we established that 8\% alginate produced
the best results of reproducibility, ease of crosslinking, and
appropriate stiffness. We established the appropriate seeding density of
cells in alginate hydrogel by using a 3-way luer lock to combine one
syringe containing 1mL of 8\% alginate with a separate syringe
containing 200uL of 1x10\^{}6 cell suspension. After printing the disks,
confocal images were collected and analyzed to determine that 1 million
cells in 200uL of media is optimal cell concentration to print the
disks. Then, printed under sterile conditions using cellink.

Disk Fabrication

From the Fig 1., the disks were developed through a 4-step process.
Firstly, we created an STL file using bioprinter that formulates a set
of instructions on how to build a disk with 3 dimensions through
programmed printhead movements in the x, y, and z planes {[}35{]}. The
disks were 55 mm in diameter. The file was then transferred to the 3D
printer, where it is sliced into layers and printed as a grid pattern.
In order to ensure the disk can withstand the cone-and-plate bioreactor,
we utilized the printers' customizable features to modify the number of
layers of the disk, inserting 2 additional layers, 0.1mm each.

3g of gelatin was combined with 30mL of 0.2\% CaCl2, sodium alginate
crosslinking agent. After 10 minutes, the gelatin mixture was
centrifuged and plated to fill the entire volume of the Nunclon  dish.
Next, the cartidridge containing 1mL of alginate with 200uL of cell
suspension is attached to a stainless steel dispense tip (Nordson EFD,
Cat \#7018345), which is calibrated to a position 10mm deep into the
gelatin support bath. Once printing is complete, the dish is placed in
the incubator for 2 hours, to allow the gelatin to completely melt.
Lastly, the gelatin is aspirated and 20mL of media with 1\% CaCl2 is
added to supply nutrients to the cells imbedded in the disk and allow
the disk to achieve maximum stiffness over 24 hours. The disk is then
stored in the incubator.

\textbf{Application of Shear Forces}

This study uses the cone and plate bioreactors (CPB) to investigate the
fluid shear stress experienced by endothelial cells (EECs) in the left
ventricular outflow tract (LVOT) of patients with DSS. The CPB system
operates based on the following principle: an inverted cone is
positioned above a stationary flat plate, creating a narrow gap through
which fluid flows. This arrangement ensures the distribution of uniform
shear stress across the surface {[}46{]}.

To control the fluid flow, a magnetic stir plate is employed, which
enables adjustment of the cone's angular velocity by modifying the
revolutions per minute (RPM) setting on the control unit. This mechanism
allows for precise manipulation of the shear stress experienced by the
EECs in the CPB system {[}46{]}.

\textbf{Fixing the Disks and Confocal Microscopy imaging}

The Disks containing cells were initially cut to half or small pieces
and transferred onto a 6-well plate and then fixed using a 4\%
paraformaldehyde solution for 24 hours. Following the fixation, the
paraformaldehyde solution was removed, and the disks were washed three
times with PBS. The Disks were subsequently imaged and processed using a
CellVoyager CV8000 Yokogawa confocal scanner microscope. For imaging, a
z-depth range of -150\selectlanguage{greek}μ\selectlanguage{english}m to 550\selectlanguage{greek}μ\selectlanguage{english}m was selected, with a scanner layer
thickness of 6.5\selectlanguage{greek}μ\selectlanguage{english}m. The lasers used were 640nm for PCF labeled with
Texas Red and 488nm for EEC labeled with GFP. The objective lens chosen
for observing the Purkinjean was the 4x lens. The acquired images were
then merged and analyzed using CellPathfinder (a high-content analysis
software developed by Yokogawa) to generate 2D or 3D aligned images.

\textbf{Results}

The Cells are alive on the Monolayer Disks with the labelling system.

To detect the cells whether or not survive after using celltrackers, as
mentioned above again, the Porcine EEC were labeled with GFT and PCF
were labeled with Texas Red. From the Fig. 2, the cells are observed by
Lecia microscope with 10x and 20x lens after 72 hours. Thus, different
types of the cells are alive after using celltrackers. Then, the cells
are transferred to the Monolayers (Disks), using the ECM medium for the
Porcine EEC and Porcine Fibroblasts were maintained in Fibroblast Growth
Media 3. After culturing for 72 hours, the Monolayers were fixed and the
images were taken by Yokogawa CellVoyager CV8000 under the 4x objective
in the Fig 3. The images were from different z-depth such as -50, 100,
350 and 500 \selectlanguage{greek}μ\selectlanguage{english}m, and cells are widely distributed in the Monolayers.

The mixed cells survived on the multiple layers

To investigate that whether or not the PCF and Porcine EEC survive in
the multiple layers (Disks), we mixed the cells and transferred them to
the multiplex layers. After fixing and washing the Disks, we still
utilized the Yokogawa CellVoyager CV8000 to monitor the cells. From the
Fig 4, we found that the Porcine fibroblast- Texas Red and EEC- GFP were
mixed and survived after 48 hours in the Disks. Also, from the images,
the cell numbers of PCF and Porcine EEC were different at the different
z-depth of 100 and 200 \selectlanguage{greek}μ\selectlanguage{english}m. From the images, we confirmed that the PCF
and Porcine EECs were alive after bioprinting and mixed together with
conditioning mediums.

The mixed cells on the Disks were alive after bioreactor treatment.

To further detect the mixed cells on the Disks whether can be potential
facilitated for DSS project, we utilized the cone and plate bioreactors
since it can imitate the heart fluids as Fig.1 \& Fig. 6 showed the
schematic and model diagrams. In addition, we designed two groups to
conducted for experimental comparing, one group is control group without
bioreactor treatment, and the other one is the experimental group within
bioreactor treatment for 24 hours. During the bioreactor treatment, we
also used the 1:1 ratio conditioning medium as mentioned above. In
addition, the Disks were all fixed by 4\% PFA and sectioned for Yokogawa
CellVoyager CV8000 to observe the cells. From the Fig 5, the Disks under
bioreactor treatment seems to be widely spread or distributed comparing
to the 0-hour control group. Also, Disks after bioreactor treatment had
less cells comparing to control groups, especially the PCF labeled with
Texas-Red. Additionally, cells are still alive and stayed on the Disks
after bioreactor treatment.

\textbf{Discussion:}

Preliminary experiments showed that labeled Porcine EEC and PCF cells
remained viable after being labeled with a cell tracker and transferred
to a single-layer disk. Observation of living cells with a Leica
microscope confirmed the successful labeling and survival of different
cell types. In addition, images of Yokogawa CellVoyager CV8000 at
different z depths show that the cells are widely distributed within a
single-layer disk, indicating their ability to adhere and proliferate on
the disk surface. This finding is consistent with previous studies that
have shown that monolayer cultures are suitable for supporting cell
growth and maintaining viability {[}47{]}.

The survival of mixed PCF and porcine EEC cells in multilayer disks was
investigated with encouraging results. Yokogawa CellVoyager CV8000
images showed that Porcine fibroblasts - Texas Red and EEC-GFP cells
co-existed and were viable inside the disks after 48 hours. Importantly,
the different cell numbers observed at different Z-depths indicate a
pattern of differential potential distribution within the multilayer.
This finding is consistent with research highlighting the importance of
spatial organization and cell-cell interactions in mixed cell cultures
{[}48{]}. Future research should focus on characterizing the spatial
arrangement and intercellular communication between different cell types
within the composite layer.

The simulation of cardiac fluid using cone and plate bioreactors has
provided valuable insights into the mixed cell populations under
conditioning medium. There were significant differences in cell
distribution between the control group and the experimental group after
24 hours of bioreactor treatment. Yokogawa CellVoyager CV8000 images
showed that disks exposed to bioreactor processing showed a wider
distribution of cells compared to controls. This observation suggests
that the dynamic fluid environment promotes the dispersion of cells
within the disk. In addition, a reduction in the number of cells in the
petri dish after bioreactor treatment, particularly PCF labeled with
Texas red, may indicate potential cell detachment or altered cell
survival ratio under the shear stress conditions. Future research could
explore the specific mechanisms of cell detachment under bioreactor
conditions and potential differences in cell viability and function.

In this study, the bioreactor provided the shear stress was used as a
model to study the DSS. The present study investigated the co-cultural
system under bioreactor treatment. The results showed that the mixed
cell population successfully survived, distributed, and reacted on a
monolayer disk, highlighting the behavior of these cells under
bioreactor treatment. The findings of this study provide valuable
insights into cell behavior in DSS.

The use of bioreactors in co-culture systems can significantly affect
cell distribution from the results. The decrease in the number of cells
observed in the bioreactor treated samples suggests that shear stress
caused by the flow dynamics of the bioreactor may lead to cell
detachment. This finding is consistent with previous research and
highlights the importance of optimizing bioreactor conditions to
maintain cell adhesion and prevent cell loss during culture.
Understanding the effects of shear stress on cell behavior is critical
to successfully implementing a co-culture system in DSS.

Also, the successful survival and distribution of a mixed cell
population in a co-culture system including single layer and multiple
layers, which demonstrates the potential for a more holistic approach in
DSS applications. By combining multiple cell types, such as endothelial
cells, and fibroblasts, it is possible to develop more efficient tissue
structures for DSS repair or replacement {[}49{]}.

The co-culture system is also designed to address the limitations of
single-cell type therapy. By integrating multiple cell types that are
critical to the formation and function of target tissues, researchers
can create tissue structures that more closely resemble natural tissues
{[}50{]}. For instance, the addition of endothelial cells helps promote
blood vessel formation, smooth muscle cells support mechanical
properties, and fibroblasts contribute to extracellular matrix
production and tissue stability in DSS {[}51{]}.

By providing a more comprehensive and versatile approach, co-culture
systems have the potential to create organizational structures that
better mimic the complexity and function of native tissues {[}52{]}.
This approach allows researchers to study cell-cell interactions,
signaling pathways, and tissue development in a more physiologically
relevant way. Ultimately, it provides more efficient and functional
tissue structures to improve the treatment for DSS patients.

\textbf{Conclusion:}

The results of this study demonstrate the successful survival,
distribution, and reaction of mixed cell populations on a monolayer
disk, as well as their behavior under bioreactor treatment. The results
showed the viability of the labeled cells and their ability to
proliferate in single - and multilayer (Disks) cultures. The use of
bioreactors affects cell distribution and results in a decrease in cell
numbers, which may be due to detachment caused by shear stress. These
findings contribute to the understanding of cell behavior in engineered
systems and have implications for DSS applications.

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\verb`Figures for Manuscript Co-culture system.pptx`  available at \url{https://authorea.com/users/650961/articles/659221-development-of-a-novel-in-vitro-co-culture-system-for-discrete-subaortic-stenosis}

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