Testing 3D printed biological platform for advancing simulated microgravity and space mechanobiology research.

The advancement of microgravity simulators is helping many researchers better understanding the impact of the mechanically unloaded space environment on cellular function and disfunction. However, performing microgravity experiments on Earth, using simulators such as the Random Positioning Machine, introduces some unique practical challenges, including air bubble formation and leakage of growth medium from tissue culture flask and plates, all of which limit research progress. Here, we developed an easy-to-use hybrid biological platform designed with the precision of 3D printing technologies combined with PDMS microfluidic fabrication processes to facilitate reliable and reproducible microgravity cellular experiments. The system has been characterized for applications in the contest of brain cancer research by exposing glioblastoma and endothelial cells to 24 h of simulated microgravity condition to investigate the triggered mechanosensing pathways involved in cellular adaptation to the new environment. The platform demonstrated compatibility with different biological assays, i.e., proliferation, viability, morphology, protein expression and imaging of molecular structures, showing advantages over the conventional usage of culture flask. Our results indicated that both cell types are susceptible when the gravitational vector is disrupted, confirming the impact that microgravity has on both cancer and healthy cells functionality. In particular, we observed deactivation of Yap-1 molecule in glioblastoma cells and the remodeling of VE-Cadherin junctional protein in endothelial cells. The study provides support for the application of the proposed biological platform for advancing space mechanobiology research, also highlighting perspectives and strategies for developing next generation of brain cancer molecular therapies, including targeted drug delivery strategies.

Introduction

The presence of extracellular mechanical forces are central to developmental biology, tissue homeostasis and disease onset in humans[1]. The process of mechanotransduction begins at the cellular level and is mediated by molecular mechanotrasducers complexes that upon sensation of mechanical cues induce activation and inhibition of biochemical signaling pathways that ultimately determine cell mechanical properties and functionality[2]. Whilst it has long been demonstrated and widely accepted that mechanical forces play a critical role in regulating human (patho)physiology, the reverse should also hold true, i.e., the absence or reduction of extracellular forces (known as mechanical unloading) also affects cell function and overall tissue homeostasis. Clear examples of the effects that mechanical unloading plays on human functions are the observed, apparent changes that occur when astronauts encounter the zero-gravity environment of space, including reduced bone and muscle density, increased risk of cardiovascular disease, vision problems and a compromised immune system[3-7]. As such, many scientists are putting great effort in understanding the underlying molecular mechanisms involved in mechanically unloaded diseases. However, conducting in vitro space research missions on the International Space Station is still in its infancy and is also often not a financially viable option. Hence, researchers have developed microgravity (µG) simulators capable of producing near zero-gravity environments, such as the random positioning machines (RPM) and clinostats[8,9]. In recent years, the advancement of µG simulators has helped and accelerated the discovery of several molecular candidates, which have been proposed as mechanosensing molecules involved in cell adaptation and survival to the μG environment, including adhesion receptors, extracellular ligands, ion channels, growth factor receptors, and membrane proteins[10]. Understanding how these molecules modulate, transduce, and integrate a “lack of gravitational force” represents a possible approach to reveal mechanotransduction processes driving not only space-related pathology but also severe terrestrial diseases that would go otherwise undetected in normal gravity condition. For instance, cancer cells have been found to be highly sensitive to the mechanical unloaded environment, responding by modulating malignancy, proliferation, chemoresistance, and cell survival[11-14]. Therefore, further investigation in the emerging field of space mechanobiology will bring meaningful information to be considered for next generation medical treatment and intervention on Earth.

However, performing microgravity experiments on Earth using simulators introduces some unique practical challenges for biological experimentation that limit the progress of the research[15]. In particular, the RPM simulator allows the study of cell behavior under disrupted gravitational force by randomly rotating the X, Y, and Z axis upon which a biological sample sits, thus inducing a constant change in the gravity vector calculated to be near zero gravity (10−3 g)[9]. Conventional use of RPM simulator utilizes cell culture plastic, like flasks, that need to be filled entirely with expensive media (~50 mL for the T25 flask) to avoid the addition of shear stress that may arise from air bubbles during rotation. Additionally, the large volume now covering the cells may limit gas and oxygen diffusion through the flask, resulting in confounding parameters that also need to be tested and appropriately accounted for. A final concern among μG researchers is the use of cell culture plastic welled plates, as aseptically sealing the plates to ensure RPM rotational experiments free of leakages, air bubbles, and contamination, is challenging. Thus, to further advance μG research and facilitate experiments in a laboratory setting, new economically accessible technologies and reliable tools are needed to provide alternatives for researchers.

Microfluidics and 3D lab-on-a-chip (LOC) approaches have been developed as highly valuable miniaturized platforms that improve high-throughput analysis and allow cost-effective, reproducible, disposable chips that can be fabricated in mass production[16]. LOC systems are typically fabricated by polydimethylsiloxane (PDMS) replica molding[17] and are suitable for applications in the biomedical field, including cell analysis, cell manipulation and drug discovery, as they demonstrated to be highly biocompatible and gas permeable[18-21]. With the advancement of 3D printing technology, LOCs systems are further expanding their versatility by offering precision and high-resolution design to establish a fast and cheaper alternative approach to conventional lithography processes[22-24].

Here, we showcase the hybrid production of a biological platform that utilizes 3D printing technology, using sacrificial material, coupled with PDMS microfluidic fabrication process to develop reusable user’s friendly device, henceforth called microgravity-on-a-chip (MOC), that facilitates reliable and reproducible RPM-simulated µG cellular experiments. The aim of the present study was then to demonstrate the versatility and compatibility of MOC with a vast array of biological assays, i.e., proliferation, viability, morphology, protein expression and imaging of molecular structures and to characterize the system with two cell lines of different phenotype and functionality in the contest of brain cancer research. We characterized the system using A-172 Glioblastoma cells and HUVEC cells to respectively model the tumor and the endothelial monolayer, mimicking the vascular barrier observed in vivo. Indeed, in addition to the direct impact on tumor on set and progression, μG may also advance our understanding in treatment development and drug delivery strategies, as the main challenge in most cancer therapies is the ineffective delivery of chemotherapeutics across blood vessels[25-28]. Lining the blood vasculature are endothelial cells that form a continuous physical and biological barrier via intercellular adherent junctions that regulate both vascular integrity and solute permeability, including chemotherapeutics. Importantly, these junctional structures have been shown to be highly sensitive to changes in the mechanical environment with recent μG studies identifying that a mechanically unloaded environment affected endothelial cell function, viscoelastic properties, and proteome[29-32]. Thus, using the proposed MOC system, we questioned if μG can be exploited as an approach to facilitate the discovery of molecular targets that determine Glioblastoma aggressiveness, including vascular junctional architecture, to ultimately develop brain cancer molecular therapies and targeted drug delivery strategies.

Methods

Fabrication of microgravity-on-a-chip (MOC)

The MOC was designed using CAD software (Solidworks, Dassault Systèmes) to generate a.STL file consisting of four circular chambers (6 mm in diameter) connected in series by a microfluidic channel. To realize different MOCs, 4 CAD designs were prepared with different chamber heights, namely 0.8, 2, 2.7 and 3.5 mm. A Cellink Biox 3D bioprinter (Cellink, Sweden) with a 27 G nozzle was used to extrude Pluronic F127 40% in a standard 100 mm petri dish (Corning, Australia) at 5 mm/s under 160 kPa at 26 °C. The printing parameters include a grid infill pattern with 60% density with printed fibers of 0.1 mm each. After the designed construct was printed, PDMS at a ratio of 10:1 was casted into the petri dish carefully covering the entire printed structure. The petri dish was then placed overnight in an oven at 35 °C to let the PDMS polymerized and subsequently peeled off and cut to separate each device. The mold was then washed carefully with distilled water to remove residual Pluronic F127. Inlet and outlet holes were punched before the device was plasma bonded to a glass slide. The MOC was sterilized with UV light for 30 min and washed twice with Dulbecco Phosphate Buffered Saline (PBS, Sigma Aldrich, Australia) using 1 mL syringe and tygon tubing (John Morris Scientific, Cat. NO ND-100-80, Australia).

Cell maintenance and cell seeding into the MOC

Human Glioblastoma cells (A-172) were purchased from Sigma Aldrich, Australia (Cat. No. 88 062 428) and cultured with high-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Thermofisher, Cat. No. 11 965 084, Australia) supplemented with 10% fetal bovine serum (Thermofisher, Australia) and 5% penicillin-Streptomycin (Thermofisher, Cat. No. 15 140 122, Australia) and kept in humidified incubator at 37 °C with 5% CO2. Media was changed every 3–4 days and the cells were passaged upon reaching 90–95% confluence. Cells were tested for mycoplasma contamination and found negative. HUVECs were purchased from Lonza, Australia (Cat. No. CC-2517) and grown in humidified incubator at 37 °C and 5% CO2 using basal medium-2 (EBM-2) supplemented with endothelial growth medium (EGM-2) BulletKit from Lonza, Australia (Cat. No. CC- 3162). To ensure the expression of key endothelial proteins, HUVEC cells were only used between passage numbers 2–5. Once the cells reached 80–90% confluency, they were washed twice with PBS and detached using Trypsin-EDTA solution (Sigma Aldrich, Australia). Cell suspension was collected and centrifuged at 180 × g for 2 mins and the supernatant was discarded. Cells were then resuspended in the corresponding culture medium at an average concentration of 1 × 105 cells/mL for both cell types. Prior to cell seeding, MOCs were washed with PBS and functionalized with either Collagen Type I (Thermofisher Scientific, Cat. No. A1048301, diluted in PBS 1:20) for Glioblastoma cells (A-172) or fibronectin (1:50 in PBS, Sigma Aldrich, Cat. NoF1141, Australia) for HUVEC. Cell suspensions were injected into the MOC using tygon tubes. Cells were then incubated overnight under static condition, and media changed prior to μG experiments. Importantly, for junction opening evaluation HUVECs were grown into the MOC platform to confluency to ensure the complete maturation of junction and barrier functionality.

Microgravity assays

A Random Position Machine (RPM) (EXPLOR Space Technologies, Australia) was used to simulate the μG condition experience in space. The system was a desktop-size 3D clinostat placed in a incubator and worked by changing the X, Y, and Z axis of the arm, inducing random changes to the gravity vector orientation thus resulting in an average gravity zero vector over time. The MOC containing the cells was carefully placed in the center of the arm to avoid any points of the sample being affected by residual centrifugal acceleration. Relative static control (1 G) was placed in the same incubator. All μG experiments lasted maximum 24 h unless otherwise specified.

Viability and counting cells procedures

To verify both the biocompatibility of MOC platform and assess viability of Human Glioblastoma A-172 and HUVEC cells after μG experiments, live imaging using LIVE/DEAD Viability/Cytotoxicity Kit (Sigma-Aldrich, Cat. No. L3224) was performed as per manufactures instructions. Briefly, fluorescent calcein-AM (2 μM) and red-fluorescent ethidium homodimer-1 (EthD-1) (4 μM) were gently injected into the MOC and incubated for 15 min at 37 °C and humidify atmosphere. MOCs were then washed with PBS and imaged using EVOS M5000 microscope (Invitrogen). To evaluate proliferation, cells were detached from the MOC using Trypsin-EDTA, collected, centrifugated and counted using a Hemocytometer. Trypan Blue 0.4% (Thermofisher Scientific, Cat. No. 15250061) was used to determine the percentage of viable cells present in the suspension.

Fluorescence staining and imaging

Visualization of actin cytoskeleton filaments and endothelial adherent junctions was assessed by immunofluorescence imaging with EVOS M5000 microscope. Media from the MOC was aspirated and cells were washed with PBS prior to being fixed with 4% paraformaldehyde (PFA) for 20 min at RT. Cells were washed to remove residual PFA and permeabilized using 0.1% Triton X-100 for 10 min. Cells were then blocked for 1 h with 1% Goat serum (Sigma Aldrich, Cat. No. G9023) and immunostained for VE-Cadherin (1:1000; 1% Goat serum; Abcam, Cat. No. ab33168) and incubated overnight at 4 °C. Cells were then co-incubated with Goat anti-rabbit Alexa fluor 488, (1:200; PBS; Abcam, Cat. No. ab150077) and TRITC-Phalloidin (1:10000; Sigma- Aldrich, Cat. No. P1951) for 1 h in the dark. Nuclei were stained with DAPI.

Cell morphological analysis

Morphological analysis was performed on Human Glioblastoma A-172 and HUVEC cells to evaluate parameters such as area (A), shape index (SI), and Tortuosity index (TI) as described previously[33]. The cell outline was manually extracted using ImageJ software by selecting the peripheral actin filaments in fluorescence images. A binary image was created and used for automatic quantification of morphological parameters, such as A, SI and perimeter. Also, the major and minor axes of the equivalent ellipse for the cell outline were determined. The SI value is a measurement of cell roundness, from a perfect spherical shape (SI = 1) to an elongated shape (SI = 0) and is defined as follows:where A is the cell area and P the cell perimeter. Another parameter, known as tortuosity index (TI), assesses and quantifies whether cell approaches a smooth circular or elliptical profile (TI = 1) or an irregular star shape profile (TI > 1). The TI is defined as follows:where P is the cell perimeter and P′ the equivalent ellipse perimeter of the cell. Morphological parameters were measured from 50 cells in 8 different fluorescence images for each experimental condition.

Quantitative in-cell western assay using MOC

Quantitative analysis of proteins expression was carried out using the rapid and high-throughput In-Cell Western™ (Odyssey®) as per manufactures instructions. Briefly, cells were seeded in MOCs at a concentration of 1 × 105 cells/mL and cultured overnight. The cells were then subjected to μG for 24 h and immediately fixed as described above. Cells were permeabilized with 5 washes of 0.1% Triton X-100 (v/v)/PBS for 5 min per wash. Cells were then blocked in Odyssey Blocking Buffer for 90 min at RT and incubated overnight at 4 °C with primary antibodies against VE-Cadherin (1:1000) or active (non-phosphorylated) Yap-1 (1:200; Abcam, Cat. No. ab205270) and Vinculin (1:400; Sigma-Aldrich, Cat. No. V9131). Cells were washed with 0.1% Tween-20 (v/v)/PBS for 5 min and stained with IRDye secondary antibodies (1:200), 1 hr at RT. Finally, the MOCs were scanned with the Odyssey CLX system (Li-Cor Biosciences) equipped with a near-infrared light technology for signal detection, adapting the scanning process for sample on glass slide with at a distance of 1 mm. Signal intensity was quantified with Image Studio software (Version 4.0; Li-Cor) according to the manufacturer’s instructions.

Image analysis

Junction protein and gap formation evaluation

To evaluate the effect of μG at the molecular level in HUVECs, VE-Cadherin pattern was followed and analyze using MATLAB software (MathWorks) at 2 different conditions: sub-confluent and confluent condition. For junction protein development evaluation, a sub-confluent monolayer of HUVECs was subjected to 24 h of μG and then imaged following fluorescence staining procedures. The raw intensity profile of the junction along the entire cell perimeter and the average intensity of multiple random regions within cell membrane were determined for 50 cells. The intensity profile of junction protein was calculated as the VE-Cadherin intensity subtracted by the average intensity of cytoplasm and then normalized pixel by pixel by the intensity along the cell border. The corrected intensity profile was then plotted with the positive intensity indicating junctional VE-Cadherin formation and the negative intensity representing gap between cells. The reported staining percentage of VE-Cadherin was calculated as the percentage of pixels with positive intensity values along the entire cell border. For junction protein opening quantification, a matured confluent monolayer of HUVECs was subjected to μG condition for 24 and 48 h and imaged following fluorescence staining procedures for VE-Cadherin protein at each time point. From the cell membrane staining, the space between cells were circled, counted, and measured by area using ImageJ for 30 different fluorescence images for each experimental condition. Results were presented as normalized percentage of the opened area and as distribution of gaps in number and dimension.

Actin stress fibers remodeling

Changes in actin stress fibers organization were evaluated by performing line scans using ImageJ and analyzing the resulting fluorescence profile, as already described[34]. Briefly, lines were drawn within 50 individual cells chosen randomly within the MOC chamber, along the smaller axis, perpendicular to stress fibers. After image correction for background, the resulting fluorescence intensity profiles were filtered and analyzed for the number of peaks above an arbitrary baseline and at a defined distance from neighbors. In this way, two neighboring top-values were considered as two separate peaks only when the distance between them was equal or higher than 3 μm. For each cell, the number of peaks was divided by the length of the scan line, resulting in the density of actin stress fibres. A chart box is then plotted, showing the median density, scatter data points and error bars for each experimental conditions and cell lines.

Statistical analysis

The statistical analysis of the data was performed using one-way analysis of variance (ANOVA) with Turkey’s test for multiple comparisons, using GraphPad Prism (v 7.04). Average values of at least three independent experiments ± SEM are showed for each of the assays. Morphological parameters and junction evaluation were measured from 50 cells in 8 different fluorescence images for each experimental condition while gaps quantification has been performed on 30 different fluorescence images for each experimental condition. P value is reported for statistical significance. Comparisons between samples were considered to be statistically significant if the p value was *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.