Diosangeles
Soto Veliz
*a,
Hongbo
Zhang
bc and
Martti
Toivakka
a
aLaboratory of Paper Coating and Converting, Åbo Akademi University, Porthaninkatu 3, 20500 Turku, Finland. E-mail: diosangeles.sotoveliz@abo.fi
bDepartment of Pharmaceutical Science Laboratory, Åbo Akademi University, Tykistökatu 4, 20520 Turku, Finland
cTurku Center for Biotechnology, University of Turku and Åbo Akademi University, Tykistökatu 4, 20520 Turku, Finland
First published on 4th June 2019
Traditional cell culture relies mostly on flat plastic surfaces, such as Petri dishes and multiwell plates. These commercial surfaces provide limited flexibility for experimental design. In contrast, cell biology increasingly demands surface customisation, functionalisation, and cell monitoring in order to obtain data that is relevant in vivo. The development of research areas such as microfluidics and electrochemical detection methods greatly promoted the customised design of cell culture platforms. However, the challenges for mass production and material limitations prevent their widespread usage and commercialisation. This article presents a new cell culture platform based on stacks of a transparent flexible printable substrate. The arrangement introduces multi-layered stacks for possible manipulation and access to the cells. The platform is highly compatible with current technologies, such as colorimetric imaging and fluorescence microscopy. In addition, it can potentially integrate, e.g., biomaterials, patterning, microfluidics, electrochemical detection and other techniques to influence, monitor, and assess cell behaviour in a multitude of different settings. More importantly, the platform is a low-cost alternative customisable through functional printing and coating technologies. The device shown in this manuscript represents a prototype for more sophisticated variations that will expand the relevance of in vitro studies in cell biology.
Programmable and customised platforms for cell culture could potentially influence, manipulate, and monitor cell behaviour simultaneously. Such a device would overcome the fundamental limitations of conventional cell cultures. This ambition has led to the development of cell culture approaches capable to integrate functional biomaterials, printing, traditional microfluidics and electrochemical sensing.
Microfluidic research leads in applications such as high throughput screening,5 single cell analysis,6 organ-on-a-chip,7 and radiation biology.8 Polydimethylsiloxane (PDMS) is currently the material of choice for microfluidic devices, and a favourite to engineers due to its optical clarity, low cost, reproducibility, ease of use, and rapid prototyping. However, the major limitations of PDMS in cell culture are the permeability to water vapour and small hydrophobic molecules, unstable surface treatment or functionalisation, and uncrosslinked oligomers, all of which affects the resulting cell behaviour.9,10 In addition, some of the devices are not compatible with the technologies commonly used in cell studies,11 and the manufacturing methods are difficult to scale-up for mass production.12,13 Therefore, despite the advances and advantages, cell culture platform design remains a challenge.14 Other approaches include the use of hydrogels,15 paper,16 and other polymeric systems.17
Recently, wax printing has emerged as an alternative approach to develop cell culture platforms. The method is largely used in paper-based microfluidics due to its low cost, simplicity, and speed of production.18–20 In 2011, Derda et al. used wax printing to design a high-throughput paper-based 3D cell culture platform based on Cells-in-Gel-in-Paper (CiGiP).21 The principle was to use the hydrophobic wax as a barrier to create hydrophilic zones for cells to grow. Since then, wax printing is extensively used in paper-based 3D cell culture studies. The next step is to exploit the benefits of using wax printing methods for cell culture and expand its applications to cell patterning,22 and to create microwells for cell studies.23
This article shows an easy to manufacture, scalable, customisable alternative to current cell culture platforms. The device takes advantage of commercialised techniques such as wax printing, and desktop cutting. Once assembled, the platform is compatible with current protocols and methods like microscopy and fluorescence detection. More importantly, it has the means to integrate numerous state-of-art technologies through large-scale fabrication techniques, such as functional printing and coating, thereby improving their accessibility into research.
Fig. 1b shows a summary of the design and fabrication of the cell growth platform. Initially, patterns are designed in Adobe Illustrator CC (Adobe Systems) and wax printed (ColorQube 8570, Xerox®). The layer designs used in this research are shown in Fig. A.1 of Appendix A.† However, the platform geometry can be modified to suit the intended investigation or analysis technique. It is possible, e.g., to mimic 96-well plate geometry. In the case of larger designs, the device requires additional points of alignment since maintaining only the corners together will result in bending at the centre due to the film flexibility. After printing, the spacer layers are cut with a desktop cutter (Silhouette Curio™, Silhouette) according to the desired design (Silhouette Studio® Software, Silhouette). Before cell culture, the spacers are rinsed with 70% ethanol, and dried. Subsequently, all layers are sterilised with UV-C radiation in a laminar flow hood. Stitch pins are used (8 mm, platinum) to align and keep all layers together, but can be exchanged with other methods for alignment, such as a 3D printed case.
The ATR-FTIR spectra confirms the composition of the Melinex® OD and the wax (Fig. 2b).27 Peaks from Melinex® OD are representative of polyester films including C–O stretching (1097–1244 cm−1), C–H bending (1340–1409 cm−1), CC aromatic stretching (1505–1578 cm−1), C
O stretching (1715 cm−1), and C–H stretching (2907–2969 cm−1). Peaks from the black wax are similar to those of paraffin. The peaks include the CH2 in plane rocking band (719–730 cm−1), C–H bending vibration (both asymmetric and symmetric) from CH2 groups (1472 cm−1), C–H bending vibrations from CH3 groups (1378–1462 cm−1), strong C–H stretching vibrations (both asymmetric and symmetric) from the CH2 groups (2847–2866 cm−1), and CH3 stretching vibrations (2955 cm−1). Other bands present in the wax spectra are possibly from the black pigment compound of the ink and weaker methylene bands at 1350–1150 cm−1.
The ATR-FTIR spectra shows changes in the surface chemistry after contact with both cell culture media (Fig. 2b). Despite the differences between Melinex® OD and the black wax ink, both materials have similar spectra after exposure to the cell culture media. The change is not permanent, and it is possible to reverse it through multiple washes by deionised water (Appendix A, Fig. A.2†). Since the changes occur for DMEM with and without serum, the adsorption of molecules is not just the proteins in the serum but also other compounds present in the serum free DMEM. Serum free DMEM contains inorganic salts, amino acids, vitamins and other additives such as phenol red, glucose and penicillin–streptomycin. Therefore, it is difficult to single out specific adsorbed compounds from the complex mixture.
Contact angle measurements help further understand the changes to the surface chemistry induced by the cell culture media. Table 1 shows the contact angles at 10 seconds after droplet placement for the Melinex® OD and black wax printed surfaces before and after contact with the liquids. In this case, the liquids considered were deionised water (DI), phosphate buffered solution (PBS), serum free DMEM (SF), and complete DMEM (COMP).
Sample | DI | PBS | SF | COMP |
---|---|---|---|---|
Melinex® OD | 88 ± 0.9 | 89 ± 0.3 | 88 ± 1 | 91 ± 2 |
Melinex® OD after liquid | 89 ± 2 | 85 ± 2 | 80 ± 3 | 8 ± 2.8 |
Black wax | 112 ± 1.3 | 110 ± 0.4 | 110 ± 2 | 105 ± 0.8 |
Black wax after liquid | 110 ± 1 | 102 ± 0.8 | 12 ± 2 | 14 ± 2 |
The contact angle of both surfaces decreases significantly after contact with the cell culture media. This change confirms the interaction seen in the ATR-FTIR spectra. In the implementation of the stacked platform, the cell culture media does not cover the black wax. Therefore, despite the possible interactions between the wax and the media, the black wax can still form the boundaries of the wells. In the case of the Melinex® OD, the initial contact angle is not hydrophilic enough to promote spontaneous wetting and spreading of the liquid on the surface. Functional coatings on the film surface before wax printing are possibilities to enhance the wetting. In this case, it was enough to pre-wet the wells with cell culture media before cell seeding.
Manufacture of the stacked cell culture platform for laboratory scale was straightforward and successful. See Appendix A, Video A.† for a demonstration of the platform. The design of the layers changed according to the purpose of the platform as explained in the Experimental section. The size of the cell staining wells meant for fixation was equivalent to a coverslip 13 mm, No. 1 in order to use the latter one as a control, except for the hypoxia studies. Higher magnifications of the wax printed designs showed uneven coverage by the fused wax ink toner particles (Appendix A, Fig. A.3†). The toner particle size and spreading during fusing partially define the resolution limitations of the used printing method to approximately 100 μm.
In the device, the cell culture media is pinned to the base and cover layer by the hydrophobic boundaries, and further kept in place by surface tension, as shown in Fig. 1a. This defines the volume of the well which depends on the ratio between the radius of the well and the gap between the cover and base layers. If the gap is too large, the surface tension is insufficient to keep the liquid between the layers. If the gap is too small, then the amount of liquid might not be enough to counter or delay the drying promoted by the airflow between the layers. Additionally, the shape of the hydrophobic boundaries, or wells, will affect the successful containment of the liquid.
Alignment of all the layers depend on the cutting resolution of the used cutter. The positioning of the layers is important since any contact between the inner layers and the cell culture media results in a fast lateral spreading of the liquid. In this study, a 1 mm distance between the printed well edges and the holes from the inner layers was enough to restrain the liquid within the hydrophobic boundaries, prevent any contact with the spacers, and correct any misalignments from the cutting process. An alternative approach could utilise hydrophobic material as spacer layers.
Fig. 3 summarises the results obtained from implementing the platform for cell culture. Pre-wetting of the surface prior to the addition of cells resulted in an evenly distributed cell seeding. The distribution is visible in both the imaging and scanning of the HDFs stained with Calcein AM. Fig. 3a shows images after one and three days of cell culture. Cells have the characteristic spindle-shape of HDFs and proliferate towards confluency by day three. This is suggested by the increased coverage of cells in the wells. Therefore, the device is suitable for the cell culture of HDFs. The method sufficed to maintain a contamination-free environment. It was possible to observe minor scratches and defects on the plastic substrate (not shown), yet they were not detrimental for the imaging. Images of cisplatin-treated HDFs can be found in Fig. A.4 of Appendix A.†
Quantification of the scanned surface revealed an increasing mean intensity proportional to the increase in cell amount (Fig. 3b). The linear increase makes it possible to create a standard curve to measure cell proliferation in future drug-screening studies. In the current setup and used liquid volumes, cell culture is possible up to three days before significant drying in the outer wells (Appendix A, Fig. A.5†). This period is enough for a wide range of assays that study cell behaviour. Longer cell culture times require manual change of cell culture media or the future design of an (semi)automatic fluid handling method.
Immunofluorescence microscopy is a method widely used by researchers. Therefore, it is important to ensure that the proposed device is compatible with the technique. Imaging of cells grown on Melinex® OD showed lower resolution than imaging of cells on coverslips when using the traditional sample preparation (Fig. 3c). Traditional sample preparation for imaging includes mounting fixed/stained cells directly to the microscope slide. The location of the cells is between the growth substrate and the microscope slide; therefore, in an inverted microscope, the imaging is done through the substrate. Microscope objectives and immersion oils are mostly designed to improve imaging through glass. Coverslip glass has a thickness between 130–160 μm, high spectral transmission, and a refractive index of approximately 1.5230. In contrast, Melinex® OD has a thickness of 125 μm, slightly lower spectral transmission, and a refractive index over 1.6, and haze of 0.4%, according to the manufacturer. The differences between the materials result in the loss of imaging resolution when imaging through the substrate.
It is possible to increase the compatibility of the stacked cell culture platform for high-resolution imaging by modifying the traditional sample preparation method. The substrate is mounted on the microscope slide with the fixed/stained cells facing outwards. Then, a coverslip is mounted on top of the cells. Consequently, the cells are located between the substrate and a coverslip, but the imaging occurs through the coverslip. The result are images with a resolution comparable to the ones obtained by growing cells on coverslips and with no interferences from the substrate even when using 100× objectives and immersion oil. Another future alternative is to replace the plastic film in the device with thin flexible glass substrate, which both maximizes the optical imaging resolution and maintains modification options through printing and solution processing techniques.
At last, it is important to consider the oxygen availability to the cells inside the device. At the moment, the layers are not sealed together and there is access for oxygen in between them, as shown in Fig. 1a. The separation between the layers is possibly due to the elevated edges created during the cutting step of manufacture. The effect is also noticeable by the increased and faster drying of the wells close to the edges (Appendix A, Fig. A.5†). The uneven drying raises a concern of hypoxic condition to the cells in the inner wells.
Fig. 4 shows the immunofluorescence staining of cells in a side well, and at the centre of the stacked cell culture platform. As a control, hypoxia was chemically induced in another device at the same locations. The images show that under hypoxic conditions HIF-1 alpha relocates to the nuclei, as reported in previous literature.28 In comparison, cells cultured normally in the device show no accumulation of the protein in the nuclei. This indicates that the cells do not undergo hypoxia.
Parameter | Traditional cell culture | PDMS devices | Stacked cell culture |
---|---|---|---|
Cell number | Thousands to tens of millions | Single cells to tens of thousands | Single cells to tens of thousands |
Gas transport | Irregular and governed by free convection (uncontrollable due to large liquid volumes) | Customisable through modifications to the design | Potentially customisable through modifications to spacer geometries and dynamic control due to low liquid volumes |
Geometry | Predefined mould design | Predefined mould design | Size and shape can be altered for each design |
Growth area | Limited to the bottom of the wells and the media suspension | Depends on the design | Both cover and base layer, in addition to the media suspension |
Manufacture | Defined geometries mostly through injection moulding | Cross-linking of liquid PDMS into moulds and heated to replicate the mould geometry | Customisable through cutting/printing of flexible films |
Material | Stiff polystyrene/glass | PDMS | Any flexible printable film |
Scalability | Each design requires an investment for a different mould | Each design requires the making of a different mould | Compatible to a wide range of roll-to-roll process techniques, such as laser cutting and in-line printing. However, challenging due to assembly |
Surface modifications | Possible through drop casting | Unstable modifications due to hydrophobic recovery | Possible through traditional and commercial techniques suitable for flexible films, such as coating and printing |
Volume | High volume to area of cell growth ratio | Low volume to area of cell growth ratio | Low volume to area of cell growth ratio |
The printability of the platform is another benefit over traditional and PDMS cell culture, since it allows for the modification and functionalisation of the cell growth surface. Printed patterns inside the wells can separate the cells into smaller clusters or be used to study cellular processes like migration. Printed functional biomaterials or molecules can be assessed in the device or be used to regulate cell behaviour. Inclusion of electrochemical detection, such as thin, organic and large area electronics (TOLAE) technologies, is also possible through printing of solution processable materials. This can be particularly useful to monitor cells, and assess responses to different types of stimulations such as currents and lasers. In summary, the potential applications for the stacked platform include co-cultures, functional printing, patterning, electrochemical detection, and biofluidics.
Considering the future prospects, the proposed platform is an attractive alternative to current methods. Further studies and modifications are needed to exploit the potential of the device. However, the simplistic approach together with the ease of production and scalability make it a feasible new approach to cell culture studies.
The proposed device represents an alternative to current platforms for cell culture. Its fabrication is potentially scalable to large volumes while still maintaining the freedom of design brought about by digital printing and converting technologies. From a research perspective, the device provides the means to include state-of-art technologies and expand their access to researchers in biological sciences.
Footnote |
† Electronic supplementary information (ESI) available: Appendix A, video A. See DOI: 10.1039/c8bm01694a |
This journal is © The Royal Society of Chemistry 2019 |