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. 2020 Nov 30;15(11):e0242627. doi: 10.1371/journal.pone.0242627

Use of dual-flow bioreactor to develop a simplified model of nervous-cardiovascular systems crosstalk: A preliminary assessment

Nicoletta Marchesi 1,#, Annalisa Barbieri 1,#, Foroogh Fahmideh 1, Stefano Govoni 1, Alice Ghidoni 2, Gianfranco Parati 3,4, Emilio Vanoli 5,6, Alessia Pascale 1,‡,*, Laura Calvillo 3,‡,*
Editor: Gaetano Santulli7
PMCID: PMC7703955  PMID: 33253266

Abstract

Chronic conditions requiring long-term rehabilitation therapies, such as hypertension, stroke, or cancer, involve complex interactions between various systems/organs of the body and mutual influences, thus implicating a multiorgan approach. The dual-flow IVTech LiveBox2 bioreactor is a recently developed inter-connected dynamic cell culture model able to mimic organ crosstalk, since cells belonging to different organs can be connected and grown under flow conditions in a more physiological environment. This study aims to setup for the first time a 2-way connected culture of human neuroblastoma cells, SH-SY5Y, and Human Coronary Artery Smooth Muscle Cells, HCASMC through a dual-flow IVTech LiveBox2 bioreactor, in order to represent a simplified model of nervous-cardiovascular systems crosstalk, possibly relevant for the above-mentioned diseases. The system was tested by treating the cells with 10nM angiotensin II (AngII) inducing PKCβII/HuR/VEGF pathway activation, since AngII and PKCβII/HuR/VEGF pathway are relevant in cardiovascular and neuroscience research. Three different conditions were applied: 1- HCASMC and SH-SY5Y separately seeded in petri dishes (static condition); 2- the two cell lines separately seeded under flow (dynamic condition); 3- the two lines, seeded in dynamic conditions, connected, each maintaining its own medium, with a membrane as interface for biohumoral changes between the two mediums, and then treated. We detected that only in condition 3 there was a synergic AngII-dependent VEGF production in SH-SY5Y cells coupled to an AngII-dependent PKCβII/HuR/VEGF pathway activation in HCASMC, consistent with the observed physiological response in vivo. HCASMC response to AngII seems therefore to be generated by/derived from the reciprocal cell crosstalk under the dynamic inter-connection ensured by the dual flow LiveBox 2 bioreactor. This system can represent a useful tool for studying the crosstalk between organs, helpful for instance in rehabilitation research or when investigating chronic diseases; further, it offers the advantageous opportunity of cultivating each cell line in its own medium, thus mimicking, at least in part, distinct tissue milieu.

Introduction

Biomedical molecular research aimed to the study of complex relationships between various tissues, as it happens in chronic diseases or when investigating resilience from a traumatic event, such as for example in rehabilitation or during aging, needs translationally relevant experimental models. In vivo/ex vivo models using laboratory animals or classic in vitro cultures on petri dishes have been extensively employed so far allowing researchers to achieve many important findings. However, cells growing in a petri dish do not behave like the original cells belonging to organs in living organisms. They are not connected with the whole complex environment and, furthermore, they are seeded on a hard matrix without being subjected to flow conditions [1]. Overall, this setup is far from properly reproducing the physio-mechanical characteristics of the organ of interest, and cells do not communicate with other different cell types neither bioelectrically nor through biohumoral exchange. Of note, though, a static system is useful to study reactions to specific stimuli, at both biochemical and molecular level. On the other side, such a system is not representative of the exchange of information through various mechanisms occurring physiologically between the various cell types located in different organs. These complex interactions are better modelled in in vivo models that, on the other hand, may be hard to study at molecular level. The recent development of Next-Generation In Vitro Testing Tools, engineered within the 3Rs research, opens a new scenario to explore living systems providing a bridge between the use of cultured cells on a petri dish and in vivo/ex vivo experiments on small laboratory animals. An example of such a tool are IVTech Bioreactors [1], where, within the tissue engineering field, a bioreactor is defined as a device able to simulate a physiological environment allowing cell or tissue growth. Inside IVTech Bioreactors, cells are subjected to the physiological shear stress and nutrient absorption typical of the blood stream [1, 2], and two cell types can be connected to study their reciprocal crosstalk under physiological or pathological conditions. This system is modular, with culture chambers designed to be added sequentially or in parallel, thus simulating a multiple organ system [1, 3–5]. Further, it is designed to be consistent with plates or transwells, thus allowing the use of standard protocols for in vitro procedures.

The primary goal of the present study was to create for the first time an in vitro model able to connect two distinct cell types, namely human neuroblastoma cells (SH-SY5Y; widely used as a neuronal-like cellular model [6]) and Human Coronary Artery Smooth Muscle Cells (HCASMC), in a dual-flow IVTech LiveBox2, thus representing a simplified model of the nervous-cardiovascular systems crosstalk. Since bioreactor technology is relatively recent, information on a number of methodological aspects is still lacking (i.e. culture conditions, growing condition for several types of cells, flow parameters etc.). Moreover, with respect to the classic in vitro models, extensively used within the last decades and for which several procedures are available, only a few tried-and-tested experimental protocols are available for researchers to work under dynamic conditions, especially when considering specific interconnected cell lines, as SH-SY5Y cells and HCASMC. Therefore, our primary aim was to develop and share with the scientific community a new co-culture set-up involving specifically SH-SY5Y cells and HCASMC, where each cell type is seeded in its own medium, to avoid a forced adaptation to a different culture medium, and under flow conditions, thus mimicking a more physiological environment.

Secondly, we aimed to explore some aspects of the nervous-cardiovascular systems crosstalk. The dynamic reciprocal relationship between brain and heart is important both in acute, second by second regulation, and in chronic derailment situations when either organ is suffering because of a disease or of an improper drug use (see [7] for an extensive analysis of these relationships). In particular, within this context, we chose to explore some of the relationships involving AngII treatment effects on the two cell types, seeded inside IVTech Bioreactor LiveBox 2 and connected under flow conditions, and to study AngII-dependent PKCβII/HuR/VEGF (vascular endothelial growth factor) cascade activation in different experimental settings.

Material and methods

IVTech LiveFlow® and LiveBox2

The system (IVTech Srl., Massarosa, LU, Italy) consisted of a peristaltic pump (IVTech LiveFlow®), which creates the flow, connected with modular and transparent double flow bioreactors named LiveBox2 (LB2), where cells are seeded. Cell medium, in the supplied 25 ml plastic bottle, is connected to the IVTech LiveFlow® and to the LB2 by silicon tubes. LB2 is a dual-flow IVTech bioreactor formed by two chambers, upper and lower, developed to model physiological barriers in vitro (Fig 1). In particular, LB2 consists of three parts (Fig 1C):

Fig 1. The IVTech system settings.

Fig 1

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A) Example of basic system size with two LB2, measuring stick in cm. B) The complete setting with four LB2 (for AngII vs control treatments) under laminar flow hood. C) Example of LB2 with outlet silicon tubes, in the figure the lower chamber is hidden by the blue support. D) The complete setting with four LB2 inside humidified incubator at 37°C with 5% CO2.

  1. an apical chamber with a wet volume of 1.5 mL, equipped with an inlet and an outlet tube;

  2. a basal chamber with a wet volume of 1 mL, equipped with an inlet and an outlet tube;

  3. a membrane holder, placed between the two chambers.

All the components of the IVTech bioreactor were autoclaved and the entire experiment was performed under a laminar flow hood (Fig 1B). Membranes were conditioned keeping them in ethanol 70% for two hours and exposed to UV light for 15 min, before cell seeding. The hood surface was cleaned with the same detergents normally used to sterilize materials employed in cell cultures. The removable transparent glass bottom allows live imaging during culture and enables sample processing (Fig 2).

Fig 2.

Fig 2

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SH-SY5Y cells (left) and HCASMCs (right) seeded under flow condition in two different LB2. SH-SY5Y were seeded on the glass in the bottom of LB2, HCASMC were seeded onto the membrane within the other LB2.

The possibility of having two independent circuits, one for the apical and one for the basal chamber, is an advantage that allows solving the problem of having two different media. Chambers are made in a biocompatible silicone polymer with self-sealing properties [4]. The chambers were connected by tubes, and the circuit dimensions were calculated using allometric laws [3]. The membrane is a polyester 0.45 μm-pore membrane, optically transparent and treated to permit cell adhesion; the pores allow the passage of cell metabolism products, preventing the translocation of the cells between the two environments of the bioreactor. Modules can be configured in different ways to obtain in series or in parallel circuits. The whole system is designed to be compatible with the most common laboratory instruments, like microscope and incubators, having the connected bioreactors the typical size of a multi-well plate (Fig 1A, 1B and 1D).

Static cell cultures

Human neuroblastoma SH-SY5Y cells were obtained from ATCC (Manassas, VA) and cultured in T75 flasks in a humidified incubator at 37°C with 5% CO2. SH-SY5Y cells were grown in Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, L-glutamine (2 mM), non-essential amino acids (1 mM), and sodium pyruvate (1 mM). HCASMC were obtained from Gibco and were cultured in a humidified incubator as SH-SY5Y cells (at 37°C with 5% CO2). HCASMC were grown in Medium 231 supplemented with Smooth Muscle Growth Supplement and 1% penicillin–streptomycin. In MTT experiments, the cells were exposed to 1, 10 and 100 nM AngII (Sigma, A925) for 6, 24 and 48 hours. For Western blotting experiments, the cells were exposed to 10 nM AngII for 6 hours. The entire experiment was performed under a laminar flow hood.

Dynamic cell cultures

HCASMC and SH-SY5Y cell types were seeded in two different LB2. The LB2 allows to monitor what happens in the first and second compartment independently, both for observations under the microscope (Fig 2), and for any sampling. In the connected setting, HCASMC were in contact with mediators eventually released by SH-SY5Y cells, thus simulating the crosstalk between tissues (Fig 3). SH-SY5Y cells were seeded at 2x105 cells/mL, on the glass in the bottom of LB2, with a tangential configuration, whereas HCASMC were seeded at 8x104 cells/mL onto the membrane within the other LB2 (Figs 2 and 3). LiveFlow® system together with the two LB2 were placed in the cell culture incubator at 37°C with 5% CO2, to keep an aseptic condition (Fig 1D). Both cells lines were stimulated with 10 nM AngII. The entire experiment was performed under a laminar flow hood.

Fig 3. The experimental setting enables to connect, under flow conditions, the two cell lines exposed to their own medium (orange: HCASMC; green: SH-SY5Y).

Fig 3

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The mediators released from SH-SY5Y cells can interact with HCASMC cells through the membrane interface. This setting avoids the potential confounding effect of a common culture media, thus allowing the identification of specific factors released by each cell type. As indicated in Fig 1B, the bioreactor consists of two modules, one setting used for control and the other for treatment with AngII (10nM).

Experimental design

The experimental design was developed according to the following steps:

  1. Assessment of SH-SY5Y and HCASMC viability after AngII treatment in static conditions performed by MTT, after exposing for 6, 24 and 48 hours both cell types, seeded in petri dishes, to increasing concentrations of AngII (1-10-100nM).

  2. Assessment of AngII-dependent PKCβII/HuR/VEGF activation in static conditions, in SH-SY5Y and HCASMC cell types, performed by Western blot.

  3. Assessment of AngII-dependent PKCβII/HuR/VEGF activation evaluated, under dynamic conditions, in separately seeded SH-SY5Y and HCASMC cell types without any biohumoral exchange between them, and performed by Western blot.

  4. Assessment of: a) AngII-dependent PKCβII/HuR/VEGF activation investigated, under dynamic conditions, in connected SH-SY5Y and HCASMC cell types, and performed by Western blot; b) VEGF release examined in SH-SY5Y and HCASMC respective medium evaluated by ELISA. Biohumoral exchange was possible through the porous membrane.

MTT assay

Mitochondrial enzymatic activity was estimated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] assay (Sigma). A cell suspension of 5x103 cells/mL (for HCASMC cell line) and 2x104 cells/mL (for SH-SY5Y cell line) was seeded into 96-well plates. Following each treatment of 6, 24 and 48 hours, 50 μL of MTT (concentration equal to 2.5 mg/mL) were added to each well. After incubation at 37° C for 3 hours, the purple formazan crystals were formed. The formed crystals were solubilized in dimethylsulfoxide (DMSO; Sigma-Aldrich). Specifically, after removing the MTT from the wells, 100 μL of DMSO were added in order to lyse the cellular and mitochondrial membranes, and solubilize the formazan crystals. After 10 minutes, absorbance values were measured at 595 nm using a Synergy HT microplate reader (BioTek Instruments) and the results expressed as % with respect to control.

Western blotting

Proteins were measured according to Bradford’s method, using bovine albumin as internal standard. Proteins were diluted in 2xSDS protein gel loading solution, boiled for 5 min and separated on 12% SDS-PAGE. The anti-PKCβII rabbit polyclonal antibody (Santa Cruz), anti-HuR mouse monoclonal antibody (Santa Cruz) and the anti-VEGF rabbit monoclonal antibody (Abcam) were diluted based on each datasheet instructions. The nitrocellulose membrane signals were detected by chemiluminescence. The same membranes were re-probed with α-tubulin antibody and used to normalize the data. Statistical analysis of Western blot data was performed on the densitometric values obtained with the ImageJ image-processing program (https://imagej.nih.gov/ij).

ELISA assay

The VEGF protein levels in SH-SY5Y and HCASMC cells were estimated in the respective medium with a specific ELISA kit (R&D Systems Inc.), according to the manufacturer’s instructions.

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for VEGF is already pre-coated onto a microplate. Standards and samples were pipetted into the wells and any VEGF present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for VEGF was added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added and the colour developed in proportion to the amount of VEGF bound in the initial step. The colour development was stopped and the intensity of the colour measured (570/450 nm).

Statistics

For statistical analysis the GraphPad Instat statistical package (GraphPad software, San Diego, CA, USA) was used. The data were analysed by analysis of variance (ANOVA) followed, when significant, by an appropriate post hoc comparison test, as detailed in the legends. Differences were considered statistically significant when p values ≤ 0.05.

Results

Static conditions

Cell viability after AngII treatment

The cell viability was studied after exposing both SH-SY5Y and HCASMC cell types for 6, 24 and 48 hours to increasing concentrations of AngII (1nM, 10nM and 100nM). SH-SY5Y cell viability was unaffected after 6 and 24 hours of AngII exposure (at all the tested concentrations). Instead, after 48 hours treatment, consistent with previously published data [8], a significant decrease in SH-SY5Y viability was observed at all AngII concentrations (85.5%±2.1, 78.1%±2.1 and 74.9% ±1.9 at 1nM, 10nM and 100nM respectively). On the contrary, and as expected [9], MTT assay carried out on HCASMC did not show a significant decrease in cell viability following 6, 24 or 48 hours, at any AngII concentration (Fig 4).

Fig 4. Cell viability of SH-SY5Y cells after 6, 24- and 48-hours following Angiotensin II (ANG II) exposure at 1 nM, 10 nM, 100 nM.

Fig 4

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The values are expressed as mean MTT [in %±S.E.M.]. ***p < 0.001, *p<0.05 vs control (CTR), Dunnett's multiple comparisons test (n = 16).

Based on this evidence, the 10 nM AngII treatment for 6 hours was chosen, as it did not affect viability and it was well tolerated by both cell lines, and specifically by SH-SY5Y.

AngII treatment and PKCβII/HuR/VEGF cascade

There was no significant difference in PKCβII/HuR/VEGF cascade activation after 10 nM AngII treatments in both cellular types when seeded in static conditions (Fig 5).

Fig 5.

Fig 5

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Densitometric analysis and representative Western blotting of PKCβII, HuR and VEGF protein levels in the total homogenate of HCASMC (upper) and SH-SY5Y cells (lower) exposed to solvent (CTR) or Angiotensin II (ANG II) at 10 nM for 6 hours, in static condition. PKCβII, HuR and VEGF bands were normalized to α-tubulin, and the results are expressed in percentage ± S.E.M. with respect to the control value (100%), n = 8–10.

Dynamic conditions

Parameters

The following parameters have resulted to be the most suitable for cell growth in LB2:

  1. Cell density: 8x104 cells/mL for HCASMC and 2x105 cells/mL for SH-SY5Y.

  2. Flow Rate: 200 μL per minute in both circuits.

AngII treatment and PKCβII/HuR/VEGF cascade in non-connected cells

When cell types were separately seeded in dynamic conditions, the results confirmed what seen in static cultures, with no difference in PKCβII/HuR/VEGF cascade activation after AngII treatments in both cellular types (Fig 6).

Fig 6.

Fig 6

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Densitometric analysis and representative Western blotting of PKCβII, HuR and VEGF protein levels in the total homogenate of HCASMC (upper) and SH-SY5Y cells (lower) exposed to solvent (CTR) or Angiotensin II (ANG II) at 10 nM for 6 hours under flow conditions, not connected. PKCβII, HuR and VEGF bands were normalized to α-tubulin, and the results are expressed in percentage ± S.E.M. with respect to the control value (100%), n = 3.

Cell lines in connection: AngII treatment and PKCβII/HuR/VEGF cascade

Once connected each other as shown in Fig 3, following AngII exposure at 10 nM for 6 hours under flow conditions, there was a statistically significant increase of PKCβII/HuR/VEGF pathway activation in HCASMC (VEGF: +215.2% ±58.3 vs control, PKCβII: +85.8% ±34.5 vs control, HuR: +100.4% ±32.3 vs control), while no change was observed in SH-SY5Y cells (Fig 7). Nevertheless, we observed an AngII-dependent increase in VEGF protein release in SH-SY5Y medium (control: 80.5 pg/mL ± 8.8, AngII: 138.0 pg/mL ± 6.7, control vs treatment p = 0.002) while no changes were found in VEGF protein release in HCASMC medium (control: 93.5 pg/mL ± 33.5, AngII: 80.5 pg/mL ± 15.5, control vs treatment p = N.S.) (Fig 8).

Fig 7. Densitometric analysis and representative Western blotting of PKCβII, HuR and VEGF protein levels in the total homogenate from HCASMC and SH-SY5Y (SH) cells connected and exposed to solvent (CTR) or Angiotensin II (ANG II) at 10 nM under flow conditions for 6 hours.

Fig 7

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Optical densities of PKCβII, HuR and VEGF bands were normalized to α-tubulin, and the results are expressed in percentage ± SEM with respect to the relative control value (100%). *p<0.05; **p<0.01; Unpaired t test, n = 5.

Fig 8. Released VEGFA protein in the medium of cells, following treatment with Angiotensin II for 6 hours under flow conditions when cells were connected.

Fig 8

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The release of VEGFA (pg/mL) was measured by ELISA and expressed as the means ± S.E.M. **p < 0.01, Student’s t-test, n = 5.

Discussion

Our group has been involved for years in studying the interactions between the nervous and cardiovascular systems [10–13], and the evidence that stress [11], pain [14] or peptides acting at central level [10, 15] might affect cardiovascular functions has pushed us to deepen the study of the crosstalk between the two systems. The main novelty of this study was to create an in vitro model able to explore the in vitro dialogue between two distinct human cell lines (SH-SY5Y; HCASMC), grown and connected in a bioreactor, representing a simplified model of the nervous-cardiovascular systems crosstalk.

The innovative feature of this work is represented by the possibility to have two different environments in LiveBox2, where the conditions can be set up by the user. In particular, two different environmental conditions were used: the apical side of the LiveBox2 chamber was filled with the SH-SY5Y medium, whereas the basal compartment was filled with the HCASMC medium. This is an innovative feature since, in general, a multi-organ approach needs to previously characterize the potential common medium, which has to be compatible with all the tissues within the pathway [16]. This process forces the cells to adapt to a novel environmental condition that is not the best option for them; therefore, this could cause a change in their behaviour. In this work, we solved the problem by using the native media developed for that specific cell type. Another innovative contribution consists in providing an experimental set up useful to culture cells belonging to different organs, connected under flow conditions and seeded in a dual flow bioreactor. Specific guidelines on flow rate and speed to use, or volume and type of culture medium were lacking and, in this regard, we have improved technical knowledge on bioreactors use for connecting cells growing in a different medium.

This also represented an improvement within the 3Rs research (Replacement, Reduction, Refinement of animal models), which develops new methods to enhance the quality standards of preclinical experimental models. To test the system, we explored AngII treatment effects on the two cell types, seeded inside IVTech Bioreactor LiveBox 2 and connected under flow conditions, and we investigated AngII-dependent PKCβII/HuR/VEGF cascade activation in different experimental settings.

In mammal organisms, AngII has important roles in both cardiovascular and nervous systems: the renin-AngII system (RAS) constitutes one of the most important systems in the physiological regulation of blood pressure, and an inappropriate level of AngII is considered as a major risk factor in the development of cardiovascular diseases. Furthermore, several studies have demonstrated that an altered RAS may cause both neurodegeneration [17] and cardiovascular complications, often affecting each other’ [18–21]. Moreover, although controversial [22], the existence of a so-called brain renin–angiotensin system [23–28], which might play a role in the regulation of neuroinflammation and progression to potential rehabilitation problems, is gaining interest, with concerns on societal and individual patient costs [11, 29–32]. Since Ang II is a potent stimulator of VEGF [33, 34] and its stimulus is able to favour the shuttling of HuR protein from the nucleus to the cytoplasm [35–37], a possible AngII-dependent activation of PKCβII/HuR/VEGF pathway was investigated in HCASMC and in SH-SY5Y, both in static and under flow conditions. The PKCβII/HuR/VEGF pathway is a molecular cascade, first described by our group in retinal bovine pericytes, which controls VEGF expression also under hypoxic conditions. In particular, PKCβII is able to increase VEGF protein expression through the RNA-binding protein ELAV/HuR [37–39].This in vitro model was settled through four main steps: first, in preliminary experiments, the cells under flow with respect to static conditions were observed, also selecting the more appropriate parameters for the dynamic setting. Second, AngII-dependent PKCβII/HuR/VEGF pathway activation in SH-SY5Y and HCASMC, both under static and dynamic conditions, were separately studied. Third, the best growing conditions to put the two cell lines in connection were identified. Finally, the two cell lines subjected to flow were connected and exposed to AngII treatment, evaluating the PKCβII/HuR/VEGF pathway in a condition of potential crosstalk.

We think we have reached the goal to culture and study different cell types in connection, mainly thanks to the use of a dual flow IVTech LiveBox 2 bioreactor system, which allows cells to grow in their own medium, enabling physio-pathological phenomena to be simulated in vitro. This system represents an innovative model, developed according to the 3Rs research objectives, that meets the necessary requirements of a bioreactor apparatus [40]. Finally, unlike the classical co-culture, dual-flow bioreactor made possible communication among different cell types, without the potential confounding effect of a common culture medium, thus allowing the identification of specific factors released by each cell type.

From the data collected during the setting of the system, and following the AngII treatment, we could make some preliminary considerations on cell response: according to literature [8], data on SH-SY5Y showed a weak decrease in viability (93.5%±1.8) after 24 hours of treatment with AngII 100 nM. MTT assay carried out on HCASMC did not show a significant decrease in cell viability following 6, 24 or 48 hours, at any AngII tested concentration. This is consistent with literature data showing that HCASMC treated at the same concentrations of angiotensin used in this work do not lose their ability to divide and migrate [9]. After the set-up of the system, we verified the feasibility of seeding and connecting HCASMC and SH-SY5Y cell types inside the dual flow model of IVTech LiveBox2 bioreactor under flow conditions, thus allowing biochemical communication between the two cell types, each grown in its culture medium. According to literature and to our preliminary experiments, a flow of 200 μL/min was applied in both circuits. In fact, in this model, higher flow rates caused cells detachment from the membranes and some evidence [41–43] suggested possible DNA damage following excessive shear stress. The behaviour displayed by the cells, once put in communication, has generated some critical information not otherwise collectable with previous methodologies:

1) In HCASMC connected to SH-SY5Y cells, AngII treatment caused an increased intracellular expression of VEGF through the activation of the PKCβII/HuR cascade, with no change in VEGF release in HCASMC medium. The absence of a change in VEGF in HCASMC medium, despite its increase in SH-SY5Y medium (Fig 8), indicates the good separation of the two media ensured by the membrane interface. However, the results suggest that VEGF released by SH-SY5Y was able to stimulate HCASMC laying onto the membrane, thus possibly activating the PKCβII/HuR cascade. Nevertheless, we cannot exclude that the duration of the experiment (6 hours) was not enough to allow HCASMC producing a measurable VEGF amount in the medium.

While VEGF is a pivotal factor for vascular development and angiogenesis [44] and its production is a well-known cell response to hypoxic conditions [45–50], HuR belongs to a small family of evolutionarily conserved RNA-binding proteins, named ELAV, which act at post-transcriptional level and are able to influence virtually any aspect of the post-synthesis fate of the targeted mRNAs [51]. Of interest, VEGF is a target of HuR and we previously demonstrated that, in the rat retina, diabetes-activated PKCβII/HuR cascade induces VEGF overexpression [37]. Further, the upstream inhibition of this cascade blunts these effects, thus supporting the concept that the PKCβII/HuR cascade can modulate, post-transcriptionally, VEGF expression through a route that is independent from the classic VEGF transcriptional control [37]. Notably, in hypoxic conditions, such as those also observed within the context of diabetic retinopathy, both the transcriptional and post-transcriptional pathways may be operant in controlling VEGF expression.

2) A significant increase of VEGF protein was observed in SH-SY5Y medium after AngII cell exposure and connection of the two cell lines.

The release of VEGF in the medium by SH-SY5Y cells may be interpreted as a reaction to hypoxia, this is in accordance with previously published data showing that oxygen deprivation caused VEGF production in SH-SY5Y at both mRNA and protein level, and with the evidence of a correlation between AngII and hypoxic conditions [47, 48, 52–55]. SH-SY5Y cells have AngII type 1 receptor localized in the membrane [56], and when treated with AngII they displayed a stressed behaviour with subsequent upregulation of factors responsible for VEGF-mediated angiogenesis [8, 57]. Considering that we observed in HCASMC an increased intracellular VEGF expression only when HCASMC were connected with SH-SY5Y, it is tempting to speculate that a factor released by SH-SY5Y, perhaps VEGF itself [48], might have given to HCASMC a signal for a risk of hypoxia, thus promoting the activation of PKCβII/HuR/VEGF cascade in HCASMC.

Interestingly, the preliminary observations obtained after the connection of the two cell types through the bioreactor resemble more closely what seen in vivo. In particular, AngII administration in several in vivo models was reported to increase VEGF production, also via HuR activation, and to promote angiogenesis. In mice treated with Ang II infusion there was an increased VEGF expression inside the cells of the aortic wall [58]. In a model of cardiac hypertrophy induced by AngII administration, a significant increase in HuR cytoplasmic translocation, indicative of its activation, was observed in neonatal rat hypertrophic cardiomyocytes [59], thus emphasizing the involvement of HuR in the AngII-mediated increase of VEGF protein expression, as observed in our model.

AngII was also reported to enhance angiogenesis associated with tissue ischemia, via VEGF production. Indeed, in a murine model of myocardial ischemia [60], Ang II-pretreated rat mesenchymal stem cells showed enhanced VEGF synthesis, tube formation and angiogenesis in vivo, and in a model of femoral artery occlusion, AngII significantly increased VEGF protein content in ischemic hindlimb [61]. The same group described an AngII-dependent VEGF expression within the neovascular stromal interface in the Matrigel model in mice [62].

Study limitation

This project was designed as a preliminary exploration of a system to culture cells belonging to different organs, connected under flow conditions and seeded in a dual flow bioreactor. The novelty of this work relies on the use of dual flow bioreactor system to culture and study the crosstalk between HCASMC and SH-SY5Y cell lines in vitro, each maintaining its own medium. Specific guidelines on flow rate and speed to use, or volume and type of culture medium were lacking [63] and, in this regard, we have improved technical knowledge on bioreactors use for connecting cells growing in a different medium. Further applications by other investigators should validate the reliability and robustness of the system. Biochemical studies were used to test the system and not as main goal of our paper. Although we reported and discussed some preliminary observations, a complete biochemical assessment of mediators released by each of the connected cells and a comprehensive study on their eventual proliferation, migration and differentiation were not made. Nevertheless, considering the scope of this project, which was to test new technologies potentially useful in experimental medical research, we feel to have reached the goal of giving useful information on an innovative tool with a great potential in preclinical studies.

Perspectives

Chronic disorders requiring rehabilitation therapies are difficult to investigate due to the extreme complexity of the interaction between the involved systems, e.g. nervous, cardiovascular and/or immune. A comprehensive understanding of the crosstalk among them is still lacking and studies with appropriate methodological approaches are needed to explore in detail the reciprocal role of mediators and cell communication. In this context, this new model is able to be alongside the existing in vivo, ex-vivo and in vitro tools given its unique property of being simultaneously enough simple to allow specific observations and reasonably complex to more closely reflect the physiological conditions of the cell’s environment. This work does open new perspectives in future investigation on biochemical crosstalk between cells belonging to different organs and different systems, and describes a new model that can be combined with in vivo and classic in vitro models, supporting a global approach to 3R’s in preclinical research.

Acknowledgments

Authors are grateful to Dr. Lidia Cova for the precious suggestions.

Data Availability

All relevant data are within the manuscript. Supplement data (S1 raw images) and bioreactor sterilizing protocol are held in the public repository: https://doi.org/10.5281/zenodo.4106227https://doi.org/10.5281/zenodo.4246966.

Funding Statement

This work was financially supported by Association I-CARE Europe Onlus. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Vozzi F, Mazzei D, Vinci B, Vozzi G, Sbrana T, Ricotti L, et al. A flexible bioreactor system for constructing in vitro tissue and organ models. Biotechnol Bioeng. 2011;108(9):2129–40. 10.1002/bit.23164 [DOI] [PubMed] [Google Scholar]
  • 2.Vandrangi P, Sosa M, Shyy JYJ, Rodgers VGJ. Flow-dependent mass transfer may trigger endothelial signaling cascades. PLoS One. 2012;7(4):e35260 10.1371/journal.pone.0035260 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Mazzei D, Guzzardi MA, Giusti S, Ahluwalia A. A low shear stress modular bioreactor for connected cell culture under high flow rates. Biotechnol Bioeng. 2010;106(1):127–37. 10.1002/bit.22671 [DOI] [PubMed] [Google Scholar]
  • 4.Giusti S, Sbrana T, La Marca M, Di Patria V, Martinucci V, Tirella A, et al. A novel dual-flow bioreactor simulates increased fluorescein permeability in epithelial tissue barriers. Biotechnol J. 2014;9(9):1175–84. 10.1002/biot.201400004 [DOI] [PubMed] [Google Scholar]
  • 5.Iori E, Vinci B, Murphy E, Marescotti MC, Avogaro A, Ahluwalia A. Glucose and fatty acid metabolism in a 3 tissue in-vitro model challenged with normo- and hyperglycaemia. PLoS One. 2012;7(4):1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Kovalevich J, Langford D. Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology. Methods Mol Biol. 2013;1078:9–21. 10.1007/978-1-62703-640-5_2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Govoni S. (2020) Psychiatric and Neurological Effects of Cardiovascular Drugs. In: Govoni S., Politi P., Vanoli E. (eds) Brain and Heart Dynamics Springer, Cham: 10.1007/978-3-319-90305-7_46-1. [DOI] [Google Scholar]
  • 8.Parga JA, Rodriguez-Perez AI, Garcia-Garrote M, Rodriguez-Pallares J, Labandeira-Garcia JL. Angiotensin II induces oxidative stress and upregulates neuroprotective signaling from the NRF2 and KLF9 pathway in dopaminergic cells. Free Radic Biol Med. 2018;129:394–406. 10.1016/j.freeradbiomed.2018.10.409 [DOI] [PubMed] [Google Scholar]
  • 9.Kohno M, Ohmori K, Nozaki S, Mizushige K, Yasunari K, Kano H, et al. Effects of valsartan on angiotensin II-induced migration of human coronary artery smooth muscle cells. Hypertens Res. 2000;23(6):677–81. 10.1291/hypres.23.677 [DOI] [PubMed] [Google Scholar]
  • 10.Govoni S, Pascale A, Amadio M, Calvillo L, D’Elia E, Cereda C, et al. NGF and heart: Is there a role in heart disease? Pharmacol Res. 2011;63(4):266–77. 10.1016/j.phrs.2010.12.017 [DOI] [PubMed] [Google Scholar]
  • 11.Calvillo L., Gironacci M.M., Crotti L., Meroni P.L., Parati G. Neuroimmune crosstalk in the pathophysiology of hypertension. Nat Rev Cardiol. 2019;16:476–490. 10.1038/s41569-019-0178-1 [DOI] [PubMed] [Google Scholar]
  • 12.Govoni S., Politi P., Vanoli E. Edr. Brain and Heart Dynamics. Springer; 2020, ISBN: 978-3-030 28007–9; [Google Scholar]
  • 13.Calvillo L, Vanoli E, Andreoli E, Besana A, Omodeo E, Gnecchi M, et al. Vagal Stimulation, Through its Nicotinic Action, Limits Infarct Size and the Inflammatory Response to Myocardial Ischemia and Reperfusion. J Cardiovasc Pharmacol. 2011;58(5):500–7. 10.1097/FJC.0b013e31822b7204 [DOI] [PubMed] [Google Scholar]
  • 14.Gemes G., Rigaud M. Dean C., Hopp F.A., Hogan Q.H., Seagard J. Baroreceptor Reflex is Suppressed in Rats that Develop Hyperalgesia Behavior after Nerve Injury. Pain. 2009;146(3):293–300. 10.1016/j.pain.2009.07.040 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zubcevic J, Santisteban MM, Pitts T, Baekey DM, Perez PD, Bolser DC, et al. Functional neural-bone marrow pathways: Implications in hypertension and cardiovascular disease. Hypertension. 2014;63(6):129–40. 10.1161/HYPERTENSIONAHA.114.02440 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ahluwalia A, Misto A, Vozzi F, Magliaro C, Mattei G, Marescotti MC, et al. Systemic and vascular inflammation in an in-vitro model of central obesity. PLoS One. 2018;13(2):1–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Abiodun OA, Ola MS. Role of brain renin angiotensin system in neurodegeneration: An update. Saudi J Biol Sci. 2020;27:905–12. 10.1016/j.sjbs.2020.01.026 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Roger VL. The heart-brain connection: From evidence to action. Eur Heart J. 2017;38:3229–31. 10.1093/eurheartj/ehx387 [DOI] [PubMed] [Google Scholar]
  • 19.Goldston K., Baillie AJ. Depression and coronary heart disease: a review of the epidemiological evidence, explanatory mechanisms and management approaches. Clin Psychol Rev. 2008;28:288–306. 10.1016/j.cpr.2007.05.005 [DOI] [PubMed] [Google Scholar]
  • 20.Felder R.B., Francis J., Zhang Z.H., Wei S.G., Weiss R.M., Johnson AK, et al. Heart failure and the brain: new perspectives. Am J Physiol Regul Integr Comp Physiol. 2003;284(2):R259–R276. 10.1152/ajpregu.00317.2002 [DOI] [PubMed] [Google Scholar]
  • 21.Calvillo L, Parati G. Immune System and Mind-Body Medicine–An Overview. In: Brain and Heart Dynamics (Govoni S, Politi P, Vanoli E. Eds) Springer 2020, ISBN: 978-3-030 28007–9, pp. 1–19.
  • 22.Uijl E, Ren L, Danser AHJ. Angiotensin generation in the brain: A re-evaluation. Clin Sci. 2018;132:839–850. 10.1042/CS20180236 [DOI] [PubMed] [Google Scholar]
  • 23.Huber G, Schuster F, Raasch W. Brain renin-angiotensin system in the pathophysiology of cardiovascular diseases. Pharmacol Res. 2017;125:72–90. 10.1016/j.phrs.2017.06.016 [DOI] [PubMed] [Google Scholar]
  • 24.Johns EJ. Angiotensin II in the brain and the autonomic control of the kidney. Exp Physiol. 2005;90(2):163–8. 10.1113/expphysiol.2004.029025 [DOI] [PubMed] [Google Scholar]
  • 25.Bali A, Jaggi AS. Angiotensin II-triggered kinase signaling cascade in the central nervous system. Rev Neurosci. 2016;27(3):301–15. 10.1515/revneuro-2015-0041 [DOI] [PubMed] [Google Scholar]
  • 26.McKinley MJ, Albiston AL, Allen AM, Mathai ML, May CN, McAllen RM, et al. The brain renin-angiotensin system: Location and physiological roles. Int J Biochem Cell Biol. 2003;35(6):901–18. 10.1016/s1357-2725(02)00306-0 [DOI] [PubMed] [Google Scholar]
  • 27.Grobe JL, Xu D, Sigmund CD. An Intracellular Renin-Angiotensin System in Neurons: Fact, Hypothesis or Fantasy. Physiol. 2008;23:187–93. 10.1152/physiol.00002.2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Xu P, Sriramula S, Lazartigues E. ACE2/ANG-(1–7)/Mas pathway in the brain: The axis of good. Am J Physiol—Regul Integr Comp Physiol. 2011;300(4):804–17. 10.1152/ajpregu.00222.2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Lehn A, Gelauff J, Hoeritzauer I, Ludwig L, McWhirter L, Williams S, et al. Functional neurological disorders: mechanisms and treatment. J Neurol. 2016;263(3):611–20. 10.1007/s00415-015-7893-2 [DOI] [PubMed] [Google Scholar]
  • 30.Särkämö T, Sihvonen AJ. Golden oldies and silver brains: Deficits, preservation, learning, and rehabilitation effects of music in ageing-related neurological disorders. Cortex. 2018;109:104–23. 10.1016/j.cortex.2018.08.034 [DOI] [PubMed] [Google Scholar]
  • 31.Abbruzzese G, Marchese R, Avanzino L, Pelosin E. Rehabilitation for Parkinson’s disease: Current outlook and future challenges. Park Relat Disord. 2016;22:Suppl. 1: S60–S64. 10.1016/j.parkreldis.2015.09.005 [DOI] [PubMed] [Google Scholar]
  • 32.Winstein CJ, Stein J, Arena R, Bates B, Cherney LR, Cramer SC, et al. Guidelines for Adult Stroke Rehabilitation and Recovery: A Guideline for Healthcare Professionals from the American Heart Association/American Stroke Association. Stroke. 2016;47(6):e98–169. 10.1161/STR.0000000000000098 [DOI] [PubMed] [Google Scholar]
  • 33.Walter A, Etienne-Selloum N, Sarr M, Kane MO, Beretz A S-KV. Angiotensin II induces the vascular expression of VEGF and MMP-2 in vivo: preventive effect of red wine polyphenols. J Vasc Res. 2008;45:386‐394. 10.1159/000121408 [DOI] [PubMed] [Google Scholar]
  • 34.Imanishi T, Hano T, Nishio I. Angiotensin II potentiates vascular endothelial growth factor-induced proliferation and network formation of endothelial progenitor cells. Hypertens Res. 2004;27(2):101–8. 10.1291/hypres.27.101 [DOI] [PubMed] [Google Scholar]
  • 35.Doller A, Schlepckow K, Schwalbe H, Pfeilschifter J, Eberhardt W. Tandem Phosphorylation of Serines 221 and 318 by Protein Kinase Cδ Coordinates mRNA Binding and Nucleocytoplasmic Shuttling of HuR. Mol Cell Biol. 2010;30(6):1397–410. 10.1128/MCB.01373-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Parihar SP, Ozturk M, Marakalala MJ, Loots DT, Hurdayal R, Maasdorp DB, et al. Protein kinase C-delta (PKCδ), a marker of inflammation and tuberculosis disease progression in humans, is important for optimal macrophage killing effector functions and survival in mice. Mucosal Immunol. 2018;11(2):496–511. 10.1038/mi.2017.68 [DOI] [PubMed] [Google Scholar]
  • 37.Amadio M, Bucolo C, Leggio GM, Drago F, Govoni S, Pascale A. The PKCβ/HuR/VEGF pathway in diabetic retinopathy. Biochem Pharmacol. 2010;80(8):1230–7. 10.1016/j.bcp.2010.06.033 [DOI] [PubMed] [Google Scholar]
  • 38.Amadio M, Scapagnini G, Lupo G, Drago F, Govoni S, Pascale A. PKCβII/HuR/VEGF: A new molecular cascade in retinal pericytes for the regulation of VEGF gene expression. Pharmacol Res. 2008;57(1):60–6. 10.1016/j.phrs.2007.11.006 [DOI] [PubMed] [Google Scholar]
  • 39.Amadio M, Osera C, Lupo G, Motta C, Drago F, Govoni S, et al. Protein kinase C activation affects, via the mRNA-binding Huantigen R/ELAV protein, vascular endothelial growth factor expression in a pericytic/endothelial coculture model. Mol Vis. 2012;18:2153–64. [PMC free article] [PubMed] [Google Scholar]
  • 40.Ahmed S, Chauhan VM, Ghaemmaghami AM, Aylott JW. New generation of bioreactors that advance extracellular matrix modelling and tissue engineering. Biotechnol Lett. 2019;41(1):1–25. 10.1007/s10529-018-2611-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Yu Y, Shamsi MH, Krastev DL, Dryden MD, Leung Y WA. A microfluidic method for dopamine uptake measurements in dopaminergic neurons. Lab Chip. 2016;16:543–52. 10.1039/c5lc01515d [DOI] [PubMed] [Google Scholar]
  • 42.Edwards M.E., Good TA. Use of a mathematical model to estimate stress and strain during elevated pressure induced lamina cribrosa deformation. Curr Eye Res. 2001;23:215–25. 10.1076/ceyr.23.3.215.5460 [DOI] [PubMed] [Google Scholar]
  • 43.Triyoso DH, Good TA. Pulsatile shear stress leads to DNA fragmentation in human SH-SY5Y neuroblastoma cell line. J Physiol. 1999;515(2):355–65. 10.1111/j.1469-7793.1999.355ac.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Liao XH, Xiang Y, Li H, Zheng DL, Xu Y, Xi Yu C, et al. VEGF-A Stimulates STAT3 Activity via Nitrosylation of Myocardin to Regulate the Expression of Vascular Smooth Muscle Cell Differentiation Markers. Sci Rep. 2017;7(1):1–11. 10.1038/s41598-016-0028-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Ramakrishnan S, Anand V, Roy S. Vascular endothelial growth factor signaling in hypoxia and inflammation. J Neuroimmune Pharmacol. 2014;9(2):142–60. 10.1007/s11481-014-9531-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Adair TH, Gay WJ MJ. Growth regulation of the vascular system: evidence for a metabolic hypothesis. Am J Physiol. 1990;259(3 Pt 2):R393‐R404. 10.1152/ajpregu.1990.259.3.R393 [DOI] [PubMed] [Google Scholar]
  • 47.Prabhakaran K, Sampson DA, Hoehner JC. Neuroblastoma survival and death: An in vitro model of hypoxia and metabolic stress. J Surg Res. 2004;116(2):288–96. 10.1016/j.jss.2003.08.008 [DOI] [PubMed] [Google Scholar]
  • 48.González A, González-González A, Alonso-González C, Menéndez-Menéndez J, Mart Ínez-Campa C, Cos S. Melatonin inhibits angiogenesis in SH-SY5Y human neuroblastoma cells by downregulation of VEGF. Oncol Rep. 2017;37(4):2433–40. 10.3892/or.2017.5446 [DOI] [PubMed] [Google Scholar]
  • 49.Osada-Oka M, Ikeda T, Imaoka S, Akiba S, Sato T. VEGF-enhanced proliferation under hypoxia by an autocrine mechanism in human vascular smooth muscle cells. J Atheroscler Thromb. 2008;15(1):26–33. 10.5551/jat.e533 [DOI] [PubMed] [Google Scholar]
  • 50.Miura SI, Fujino M, Matsuo Y, Tanigawa H, Saku K. Nifedipine-induces vascular endothelial growth factor secretion from coronary smooth muscle cells promotes endothelial-containing receptor/fetal liver kinase-1/NO pathway. Hypertens Res. 2005;28(2):147–53. 10.1291/hypres.28.147 [DOI] [PubMed] [Google Scholar]
  • 51.Pascale A, Govoni S. The complex world of post-transcriptional mechanisms: Is their deregulation a common link for diseases? Focus on ELAV-like RNA-binding proteins. Cell Mol Life Sci. 2012;69(4):501–17. 10.1007/s00018-011-0810-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Wang J, Zhou X, Lu H, Song M, Zhao J, Wang Q. Fluoxetine induces vascular endothelial growth factor/Netrin over-expression via the mediation of hypoxia-inducible factor 1-alpha in SH-SY5Y cells. J Neurochem. 2016;136(6):1186–95. 10.1111/jnc.13521 [DOI] [PubMed] [Google Scholar]
  • 53.Maugeri G, D’Amico AG, Rasà DM, Saccone S, Federico C, Cavallaro S, et al. PACAP and VIP regulate hypoxia-inducible factors in neuroblastoma cells exposed to hypoxia. Neuropeptides. 2018;69:84–91. 10.1016/j.npep.2018.04.009 [DOI] [PubMed] [Google Scholar]
  • 54.Nangaku M, Inagi R, Miyata T, Fujita T. Angiotensin-Induced Hypoxia in the Kidney: Functional and Structural Changes of the Renal Circulation. In: Hypoxia and the Circulation (Roach RC, Wagner PD, Hackett PH, Editors), Springer US, Boston, MA, ISBN: 978–0. [DOI] [PubMed]
  • 55.Wolf G, Schroeder R, Stahl RAK. Angiotensin II induces hypoxia-inducible factor-1α in PC 12 cells through a posttranscriptional mechanism: Role of AT2 receptors. Am J Nephrol. 2004;24(4):415–21. 10.1159/000080086 [DOI] [PubMed] [Google Scholar]
  • 56.Jiang L, Zhu R, Bu Q, Li Y, Shao X, Gu H, et al. Brain Renin–Angiotensin System Blockade Attenuates Methamphetamine-Induced Hyperlocomotion and Neurotoxicity. Neurotherapeutics. 2018;15(2):500–10. 10.1007/s13311-018-0613-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Huang Y, Mao Y, Li H, Shen G NG. Knockdown of Nrf2 inhibits angiogenesis by downregulating VEGF expression through PI3K/Akt signaling pathway in cerebral microvascular endothelial cells under hypoxic conditions. Biochem Cell Biol. 2018;96(4):475‐482. 10.1139/bcb-2017-0291 [DOI] [PubMed] [Google Scholar]
  • 58.Zhao Q, Ishibashi M, Hiasa K, Tan C, Takeshita A, Egashira K. Essential Role of Vascular Endothelial Growth Factor in Angiotensin II–Induced Vascular Inflammation and Remodeling. Hypertension. 2004;44(3):264–70. 10.1161/01.HYP.0000138688.78906.6b [DOI] [PubMed] [Google Scholar]
  • 59.Slone S, Anthony SR, Wu X, Benoit JB, Aube J, Xu L, et al. Activation of HuR downstream of p38 MAPK promotes cardiomyocyte hypertrophy. Cell Signal. 2016;28(11):1735–41. 10.1016/j.cellsig.2016.08.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Liu C, Fan Y, Zhou L, Zhu HY, Song YC, Hu L, et al. Pretreatment of mesenchymal stem cells with angiotensin II enhances paracrine effects, angiogenesis, gap junction formation and therapeutic efficacy for myocardial infarction. Int J Cardiol. 2015;188(1):22–32. 10.1016/j.ijcard.2015.03.425 [DOI] [PubMed] [Google Scholar]
  • 61.Tamarat R, Silvestre JS, Kubis N, Benessiano J, Duriez M, DeGasparo M, et al. Endothelial nitric oxide synthase lies downstream from angiotensin II-induced angiogenesis in ischemic hindlimb. Hypertension. 2002;39(3):830–5. 10.1161/hy0302.104671 [DOI] [PubMed] [Google Scholar]
  • 62.Tamarat R, Silvestre JS, Duriez M, Levy BI. Angiotensin II angiogenic effect in vivo involves vascular endothelial growth factor- and inflammation-related pathways. Lab Investig. 2002;82(6):747–56. 10.1097/01.lab.0000017372.76297.eb [DOI] [PubMed] [Google Scholar]
  • 63.Ismadi MZ, Gupta P, Fouras A, Verma P, Jadhav S, Bellare J, et al. Flow characterization of a spinner flask for induced pluripotent stem cell culture application. PLoS One. 2014;9(10):e106493 10.1371/journal.pone.0106493 [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Gaetano Santulli

10 Aug 2020

PONE-D-20-22750

Use of dual-flow bioreactor to develop a simplified model of nervous-cardiovascular systems crosstalk

PLOS ONE

Dear Dr. Calvillo,

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Reviewer #1: Yes

Reviewer #2: Partly

**********

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Reviewer #1: Yes

Reviewer #2: N/A

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: This manuscript uses a commercial, two chamber bioreactor to grow two different cell lines (a smooth muscle cell line and a neuroblastoma cell line) in vitro. Metabolites are exchanged between the two chambers so the two cell lines can chemically interact. Chemical interactions between the two cell lines have been studied extensively. The two chamber bioreactor is shown to allow chemical interactions in the manuscript. It is not clear to me, however, what is new and novel in this manuscript. The two chamber, commercial bioreactor has been demonstrated previously on other cell lines. Likewise, interactions between the cell lines studied here have been examine and measured previously. Starting on page 9, the manuscript identifies two, new 'critical' observations with this system: an increased expression of VEGF (this has been observed previously as referenced in the manuscript), and a significant increase of VEGF production. The manuscript does not offer "a more complete biochemical assessment" of the connection between these two cell lines, which would have been valuable. It is also difficult to evaluate the novelty of this manuscript because some of the authors are publishing a book (reference 7 in the manuscript) titled, "Brain and Heart Dynamics" that is not available until this fall.

Reviewer #2: Although the manuscript seemed to be written carefully, several crucial factors were missing owing to the nature of exp design. Therefore, the ms is far from mature at its current status.

1. How did the authors keeping the system in an aseptic condition?

2. Did the real crosstalk b/w nervous-cardiovascular really happened? More evidence should be provided.

3. Fig 1 and 2. Provide real photo with cells on the dish and/or container. We should know the growth condition of the two cell cultures.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Nov 30;15(11):e0242627. doi: 10.1371/journal.pone.0242627.r002

Author response to Decision Letter 0

19 Oct 2020

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

5. Review Comments to the Author

Reviewer #1: This manuscript uses a commercial, two chamber bioreactor to grow two different cell lines (a smooth muscle cell line and a neuroblastoma cell line) in vitro. Metabolites are exchanged between the two chambers so the two cell lines can chemically interact. Chemical interactions between the two cell lines have been studied extensively. The two chamber bioreactor is shown to allow chemical interactions in the manuscript. It is not clear to me, however, what is new and novel in this manuscript. The two chamber, commercial bioreactor has been demonstrated previously on other cell lines. Likewise, interactions between the cell lines studied here have been examine and measured previously. Starting on page 9, the manuscript identifies two, new 'critical' observations with this system: an increased expression of VEGF (this has been observed previously as referenced in the manuscript), and a significant increase of VEGF production. The manuscript does not offer "a more complete biochemical assessment" of the connection between these two cell lines, which would have been valuable. It is also difficult to evaluate the novelty of this manuscript because some of the authors are publishing a book (reference 7 in the manuscript) titled, "Brain and Heart Dynamics" that is not available until this fall.

We thank the Reviewer for these comments and for highlighting issues in need of better clarification in our paper.

• In a section of the comments, the Reviewer wrote: “It is not clear to me, however, what is new and novel in this manuscript” and “The two chamber, commercial bioreactor has been demonstrated previously on other cell lines”

We thank the Reviewer for this relevant comment which allows us to improve the manuscript by clarifying an important point. From this comment we realized that the novelty of this work, that is the setup and use of dual flow bioreactor system to culture and study the crosstalk between HCASMC and SH-SY5Y cell lines in vitro, each grown in its own medium, was not described clearly enough. Therefore, we modified a few sentences in the text to better describe the contribution given by our work. In particular:

The innovative feature of LiveBox2 is represented by the possibility to have two different environments where the conditions can be set up by the user. In particular, we used two different environmental conditions: the apical side of the LiveBox2 chamber was filled with the SH-SY5Y medium, whereas the basal compartment was filled with HCASMC medium. As we have now specified in the new version of our paper, this is an innovative feature of the work since, in general, a multi-organ approach needs to previously characterize a potential common medium, which has to be compatible with all the tissues in the pathway [1]. This process forces the cells to adapt to a novel environmental condition that is not the best option for them; therefore, this could cause a change in their behaviour. In this work, we solved the problem by using the native media developed for that specific cell type.

Another innovative contribution of our paper consists in the description of an experimental set up useful to culture cells belonging to different organs, connected under flow conditions and seeded in a dual flow bioreactor. Specific guidelines on flow rate and speed to use, or volume and type of culture medium are not available, and, in this regard, we believe that with our work we have improved technical knowledge on bioreactors use for connecting cells growing in a different medium.

The initial part of the discussion is now updated with a clearer statement about the innovative aspects of our work.

Regarding the comment on commercial bioreactors, it is true that the use of bioreactors has already been validated for the study of several cell lines, including neuroblastoma cells or HCASMC. Nevertheless, the studies available in PubMed after searching the keywords: “neuroblastoma AND bioreactor”-“bioreactor AND coronary artery”- “human coronary artery smooth muscle cell AND bioreactor”, gave no information regarding a crosstalk between the two cell lines seeded in co-culture, under flow conditions. Considering the importance of the reviewer comment, we briefly report here below the most interesting papers published in this field.

A co-culture under flow of neuroblastoma SK-N-BE(2) with HUVECcells is reported by Villasante et al (Vascularized Tissue-Engineered Model for Studying Drug Resistance in Neuroblastoma Theranostics. 2017; 7(17): 4099–4117. doi: 10.7150/thno.20730), who investigated the effects of retinoid therapy on tumor vasculature and drug-resistance.

Izzo et al. (Biomed Microdevices. 2019; 21(1): 29. Influence of the static magnetic field on cell response in a miniaturized optically accessible bioreactor for 3D cell culture) investigated the influence of the static magnetic field on SH-SY5Y neuroblastoma cells response in a bioreactor for 3D cell culture, measuring heat-shock protein 70 (Hsp-70), Bcl-2 and Bax.

An interesting work (Yahya Elsayed et al. Modeling, simulations, and optimization of smooth muscle cell tissue engineering for the production of vascular grafts. Biotechnol Bioeng 2019 Jun;116(6):1509-1522) describes a smooth muscle cell tissue engineering, useful for the production of vascular grafts, by using a scaffold in a dynamic bioreactor with a rotating shaft. Mechanical properties and oxygenation level were studied, without investigating metabolic pathways. Again, no co-culture was present.

Sharifpoor and colleagues (Functional characterization of human coronary artery smooth muscle cells under cyclic mechanical strain in a degradable polyurethane scaffold -Biomaterials. 2011 Jul;32(21):4816-29) describe a degradable polyurethane scaffold to grow HCASMC, useful for eventual use in bioreactor.

Balcells et al. (Smooth muscle cells orchestrate the endothelial cell response to flow and injury Circulation. 2010 May 25;121(20):2192-9) exposed a co-culture of smooth muscle cells and endothelial cells to coronary artery flow in a perfusion bioreactor, but they studied the mTOR pathway and the expression of phospho-S6 ribosomal protein in both cells.

Kural and colleagues described an in vitro human arterial injury model in bioreactor, helpful in the study of smooth muscle cells and endothelial cells interactions, without any involvement of neuroblastoma cell line.

As clearly shown by the above list of published works, there are no studies using dual-flow bioreactors, and specifically IVTech bioreactors, which are focussed on a co-culture of SH-SY5Y cells and HCASMC, and their crosstalk. In this regard, our work offers novel information, also considering the fact that this system allows two different cell types to grow and to communicate while being cultured in their own medium, thus allowing the identification of specific factors released by each cell type without the potential confounding effect of a common culture medium.

• In another section, the Reviewer wrote: Likewise, interactions between the cell lines studied here have been examined and measured previously. Starting on page 9, the manuscript identifies two, new 'critical' observations with this system: an increased expression of VEGF (this has been observed previously as referenced in the manuscript), and a significant increase of VEGF production.

These cell lines were previously extensively studied and mentioned in several papers, however, they have never been studied in connection under flow condition, and in the context of a bioreactor tool. The co-culture of these cell lines in the bioreactor, and exploration of their crosstalk, are completely novel.

Considering this important point of criticism, we have repeated the literature search, and we provide here below a summary of the results of such a search. When we searched in PubMed the combined keywords “SH-SY5Y and HCASMC” and “SH-SY5Y and coronary artery”, we were not able to find papers reporting a complete pattern of biochemical interactions between the two lines, nor methodological works describing a SH-SY5Y and HCASMC co-culture in a dual flow bioreactor.

Below, we briefly report the most interesting papers published in this field among which, as mentioned above, there are no studies investigating in depth the crosstalk between SH-SY5Y and HCASMC. Moreover, there are no methodological works describing a SH-SY5Y and HCASMC co-culture system in a dual flow bioreactor.

The following paper:

Srivastava, S., Blower, P.J., Aubdool, A.A.et al.Cardioprotective effects of Cu(II)ATSM in human vascular smooth muscle cells and cardiomyocytes mediated by Nrf2 and DJ-1.Sci Rep6,7 (2016). https://doi.org/10.1038/s41598-016-0012-5

where HCASMC are used, cite neuroblastoma cells only as reference to state that “Excess intracellular Cu(II) levels are known to cause mitochondrial toxicity and dysfunction” in SH-SY5Y.

A published thesis partly available at https://search.proquest.com/openview/82d8708084462075a51e84c9dfafd7f7/1?cbl=18750&diss=y&pq-origsite=gscholar

investigated the “effects of Mg ions on human coronary artery smooth muscle cells (HCASMCs)” in a “In vitro Biocompatibility Evaluation of Biodegradable Metals for Cardiovascular Stent Application”. In this work, SH-SY5Y cells are cited as test used for assessment of in vitro corrosion and cytotoxicity of a magnesium alloy.

When the keywords inserted in PubMed were: “human coronary artery smooth muscle cell AND neuroblastoma”, the only result was J B Smith et al. Lowering extracellular pH evokes inositol polyphosphate formation and calcium mobilization J Biol Chem. 1989 May 25;264(15):8723-8

where authors found that “a decrease in pH, triggers cell Ca2+ mobilization in fibroblasts, endothelial, smooth muscle, and neuroblastoma cells.

When the keywords inserted in PubMed were: “SH-SY5Y AND coronary artery”, the result was Shi et al. Panax notoginseng saponins provide neuroprotection by regulating NgR1/RhoA/ROCK2 pathway expression, in vitro and in vivo. J Ethnopharmacol. 2016 Aug 22; 190:301-12. doi: 10.1016/j.jep.2016.06.017. Epub 2016 Jun 8.

In this paper, the investigators showed that Panax notoginseng saponins provided neuroprotective effects in a rat model of cerebral ischemia and in SH-SY5Y cells exposed to oxygen/glucose deprivation injury. The term “coronary” is related to the used of Panax notoginseng in the context of coronary heart disease.

In the work by Sugimoto et al. (Neuroblastoma Cell Lines Showing Smooth Muscle Cell Phenotypes-Diagnostic Molecular Pathology: December 2000 - Volume 9 - Issue 4 - pp 221-228), authors found that “S-type cells have either the immature or mature smooth muscle cell phenotype, and neural crest cells very likely have the ability of to differentiate into smooth muscle cells in the human system”. In this paper, the expressions of smooth muscle-specific proteins were described in several neuroblastoma parents and clones, and no chemical interactions between cells were described.

When the keywords “human coronary artery smooth muscle cells AND neuroblastoma” were inserted in Google Scholar, the reported papers described the behaviour of either cell separately, citing the other cell line as reference, e.g. as test used for various assessments. We were not able to find works describing co-culture of HCASMC and SH-SY5Y cells, neither in classic petri dishes nor under flow conditions.

In case the Reviewer is aware of other papers on these issues that we might have missed, we would be grateful if he/she could share them with us, so that we might refer to them in relation to our findings.

Finally, the increase of VEGF expression in the context of AngII infusion has been previously reported, and has been replicated in different and heterogeneous experimental settings. We used and cited these published data to reinforce our results obtained in the bioreactor system.

• The Reviewer commented that “The manuscript does not offer "a more complete biochemical assessment" of the connection between these two cell lines, which would have been valuable”.

We are grateful for this stimulating suggestion. Following this comment, we have performed a further experiment aimed indeed at improving the biochemical assessment. We measured VEGF release in the medium of HCASMC to have more biochemical information. This new result has been integrated in the manuscript (results and discussion sections), and a new figure describing these results is now added (the new figure 8).

As the Reviewer can notice, our paper does not provide a complete biochemical assessment of the co-culture, that is beyond the scope of the work. Biochemical studies were used to test the system and their performance was not the main aim of our study. This methodological issue is now clearly stated in the study limitations section.

• Finally, the Reviewer noted that the publication "Brain and Heart Dynamics" is not available until this fall.

We thank the Reviewer for reporting this problem. A copy of the cited book chapter is now provided for the Reviewer as attachment.

Reviewer #2: Although the manuscript seemed to be written carefully, several crucial factors were missing owing to the nature of exp design. Therefore, the ms is far from mature at its current status.

The Reviewer made important comments and we thank him/her for the opportunity of pointing out some critical aspects, thus offering us the possibility to better specify the main goal of this research work and to improve its presentation.

Since bioreactor technology is relatively recent, information on a number of basic aspects is still lacking (e.g. culture conditions, grown condition for several type of cells, flow parameters etc.). Moreover, with respect to the classic in vitro models, extensively used within the last decades, for which several procedures are available, only a few tried-and-tested experimental protocols are available for researchers to work under dynamic conditions, especially when considering specific interconnected cell lines. Therefore, our primary endpoint was to develop and share with the scientific community a new co-culture set-up involving specifically SH-SY5Y cells and HCASMC, where each cell type is seeded in its own medium and under flow conditions, in a more physiological environment. Therefore, the experimental design was developed accordingly, giving priority to technical information over biomedical information. Our idea was to accelerate the diffusion of these innovative experimental models, allowing others to more deeply study the topics we approached. Moreover, our work is characterized by an innovative feature: usually a multi-organ approach needs to previously characterize the potential common medium that has to be compatible with all the cells involved [1]. This process forces the cells to adapt to a different medium, potentially causing changes in their behaviour. In this work, we solved this problem by using the native medium developed for that specific cell type. These aspects are now described in the text, in the introduction and discussion sections.

1. How did the authors keeping the system in an aseptic condition?

We thank the Reviewer for this comment which allows us to add more details to our material and methods paragraph. All the components of the IVTech bioreactor are autoclaved and the entire experiment is performed under a laminar flow hood. The surface is cleaned with the same detergents normally used to sterilize materials used in cell cultures. Membranes were conditioned keeping them in ethanol 70% for two hours and exposed to UV light for 15 min, before cell seeding. Figure 1 describing this setting has been improved, and the material and methods section has been updated accordingly.

2. Did the real crosstalk b/w nervous-cardiovascular really happened? More evidence should be provided.

To better describe the crosstalk between nervous/cardiovascular cells, we have performed a further experiment, measuring VEGF release in the medium of HCASMC. These new data are now added in the text and illustrated in an additional figure (Figure 8). As stated in the paper, the main endpoint of this work was to test new technologies potentially useful in preclinical research, and to present reliable setup data on the co-culture of HCASMC and SH-SY5Y cell lines in a dual flow bioreactor.

Admittedly, all the observations/results we reported on the nervous/cardiovascular crosstalk should be considered exploratory data obtained with the use of this novel system. Our findings would now need to be confirmed and validated by additional studies. A complete biochemical assessment was not provided since was beyond the scope of this work. This point is now more clearly addressed in the “Study limitation” section.

3. Fig 1 and 2. Provide real photo with cells on the dish and/or container. We should know the growth condition of the two cell cultures.

We thank the Reviewer for this suggestion. A new figure (Figure 2) including a real photo with cells on the container and a short sentence giving more information on the growth condition of the two cell cultures are now provided.

Bibliography

1. Ahluwalia A, Misto A, Vozzi F, Magliaro C, Mattei G, Marescotti MC, et al. Systemic and vascular inflammation in an in-vitro model of central obesity. PLoS One. 2018;13(2):1–15.

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Decision Letter 1

Gaetano Santulli

4 Nov 2020

PONE-D-20-22750R1

Use of dual-flow bioreactor to develop a simplified model of nervous-cardiovascular systems crosstalk:a preliminary assessment

PLOS ONE

Dear Dr. Calvillo,

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We look forward to receiving your revised manuscript.

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #2: All comments have been addressed

**********

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Reviewer #2: Yes

**********

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Reviewer #2: Yes

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Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: I am happy with the revision and only two minors should be revised.

Please improve the quality of Fig 3.

Please explain how the reactor was kept aseptic.

**********

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PLoS One. 2020 Nov 30;15(11):e0242627. doi: 10.1371/journal.pone.0242627.r004

Author response to Decision Letter 1

5 Nov 2020

Response to Reviewers

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

We thank the Reviewer for the time dedicated to our work

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: I am happy with the revision and only two minors should be revised.

We thank the Reviewer for her/his approval

Please improve the quality of Fig 3.

We thank the Reviewer for her/his comment. We have improved the quality of Fig. 3 by uploading the file to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool (https://pacev2.apexcovantage.com/). PACE helps ensure that figures meet PLOS requirements. It is possible to download the corrected figure by following the link in the pdf sent for approval.

Please explain how the reactor was kept aseptic.

Thank you for this suggestion. We report the complete sterilizing techniques protocol:

Cell Culture & IVTECH Bioreactor

The staff who worked on this project always followed all the good standard procedures:

• wash hands thoroughly before and after working with cell cultures;

• wear a lab coat, gloves and safety glasses to protect themselves from any hazardous materials and to prevent contamination of the cell cultures from microbes present on skin and clothes.

Work area

• Static and dynamic cell cultures (IVTECH Bioreactor) were carried out in a biosafety cabinet, under a laminar flow, located in an ad hoc cell culture room.

• The inside of the biosafety cabinet was wiped with disinfectant (benzalkonium chloride solution) followed by 70% ethanol before and after use. Further, UV light was turned on to sterilize the biosafety cabinet when unused.

• Areas of work only contained the items required for the current procedures.

Handling & sterilizing techniques

• 70% ethanol was sprayed on gloved hands before starting the work in the biosafety cabinet.

• All the equipment and reagents (cell culture media or other reagents) utilized into the biosafety cabinet were sprayed beforehand with 70% ethanol.

• Disposable plastic pipettes were used to manipulate solutions, Pasteur glass pipettes were autoclaved as well as each single component of the bioreactor (connector, holder, upper and lower cylinders, top and bottom cylinders, thin glasses reservoirs). Specifically, each component was wrapped in aluminum foil and, after autoclaving, all the packages were opened into the hood and the bioreactor circuit was assembled in the hood itself under laminar flow.

• The sterilization of the polyester membranes was achieved by keeping them in ethanol 70% for two hours followed by UV light exposure for 15 min.

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Decision Letter 2

Gaetano Santulli

6 Nov 2020

Use of dual-flow bioreactor to develop a simplified model of nervous-cardiovascular systems crosstalk:a preliminary assessment

PONE-D-20-22750R2

Dear Dr. Calvillo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Gaetano Santulli, MD

Academic Editor

PLOS ONE

Acceptance letter

Gaetano Santulli

12 Nov 2020

PONE-D-20-22750R2

Use of dual-flow bioreactor to develop a simplified model of nervous-cardiovascular systems crosstalk: a preliminary assessment.

Dear Dr. Calvillo:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Gaetano Santulli

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

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    Data Availability Statement

    All relevant data are within the manuscript. Supplement data (S1 raw images) and bioreactor sterilizing protocol are held in the public repository: https://doi.org/10.5281/zenodo.4106227https://doi.org/10.5281/zenodo.4246966.

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