Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.
Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.
Cells and tissues in the body are subjected to various mechanical cues, including tensile strains. However, the effects of these cues on the biology of neural cells have not yet been studied extensively and understood fully. In the central nervous system, sources of mechanical strain include developmental growth1,2,3,4, physiological processes such as spinal cord bending, blood and cerebrospinal fluid pulsation, and pathological conditions such as trauma, axon swelling, glial scarring, or tumor growth5,6,7,8. It is worth investigating how tensile strain affects the differentiation of oligodendrocytes and the subsequent myelination of axons, which is a critical process in the vertebrate central nervous system. Using a custom-designed strain device and elastomeric multiwell plates, previous works9,10 have shown that static uniaxial strain can increase oligodendrocyte differentiation via global changes in gene expression10. To gain further understanding of the mechanisms of strain mechanotransduction in these cells, the previous experimental apparatus must be redesigned as described here, to enable high-resolution fluorescence imaging of nuclear dynamics in living cells under strain. Specifically, a single-well polydimethylsiloxane plate is developed, and the imaging configuration is redesigned to allow for the time-lapse imaging of live cells under strain using a 100x oil immersion lens. To eliminate the negative optical effects of polydimethylsiloxane in the light pathway, cells are imaged not through the polydimethylsiloxane plate, but in the inverted position, through the cover glass covering the cell compartment. Using this new imaging design, hundreds of high-resolution time-lapse movies are recorded, of individual cell nuclei within intact adherent cells, where chromatin is labeled by tagging histone H2B to green fluorescent protein. These movies demonstrate that tensile strain induces changes in chromatin structure and dynamics that are consistent with the progression of oligodendrocyte differentiation.
Live cell imaging under applied strain is technically challenging and requires a device design that is compatible with the microscope system. The custom design described here presents an inexpensive alternative to commercial solutions. Its dimensions enable its installation on microscope stages and live cell imaging at high spatial resolution during applied strain. The imaging setup is designed to facilitate live cell imaging using a 100x oil immersion lens with the highest clarity, through the cover glass, not through the layer of polydimethylsiloxane plate which otherwise decreases the image quality and is common in most imaging setups under strain. The device, with a mounted plate containing cells, can also be stored easily in the incubator. This device is designed to apply uniaxial strain to substrata that facilitate adherent cell culture and maintain a stable and uniform strain over multiple days. The setup described here can be used for the high-resolution imaging of various adherent cell types under strain, making it applicable to mechanotransduction studies in many fields of cell mechanobiology.
1. Design of the single-well polydimethylsiloxane mold for high-resolution imaging
NOTE: The mold for manufacturing polydimethylsiloxane plates is designed with the following features to enable imaging with a 100x oil immersion lens and a correct fit in the custom-build strain device (Figure 1A,B).
2. Fabrication of single-well polydimethylsiloxane plates and square compartments
3. Functionalization of polydimethylsiloxane plates
4. Functionalization of plastic dishes and flasks
5. Proliferation and differentiation medium for murine oligodendrocyte progenitor cells
6. Cell culture
7. Imaging
8. Data analysis
Recent work aimed at investigating the effect of tensile strain on oligodendrocytes10 showed that a 10% uniaxial tensile strain promotes the differentiation of oligodendrocyte progenitor cells by global changes in gene expression. The mechanism behind these changes in gene expression can be probed via the imaging of subcellular parameters, such as the cytoskeleton structure, transcription factor localization, nuclear dynamics, and chromatin organization. However, the previous geometry of the polydimethylsiloxane substratum did not allow high-resolution single-cell imaging. As described in this work, the redesigned geometry of the polydimethylsiloxane substratum and the imaging setup minimized the distance between the cells and the objective. This allows capturing time-lapse images of fluorescently labeled cell nuclei using a 100x oil immersion objective (Figure 6A).
Fluctuations in the nuclear projected area depend on the differentiation state of the cells11,12. A comparison of the nuclear fluctuations of oligodendrocyte progenitor cells and that of terminally differentiated oligodendrocytes showed that the latter have significantly lower fluctuations (Figure 6E). Next, the nuclear fluctuations of oligodendrocyte progenitor cells at 1 h, 24 h, and 48 h post-chemical induction of differentiation with and without a 10% tensile strain were compared. With chemical induction alone, the amplitude of nuclear fluctuations showed a significant decrease at 48 h but not at 24 h (Figure 6F). On the other hand, chemical induction together with a 10% tensile strain showed a significant decrease at 24 h, which remained constant, without further reduction at 48 h (Figure 6G).
The substratum geometry and the imaging configuration described in this paper enabled the recording of high-resolution movies of strained cells. Subsequent analysis of these movies demonstrated that strain hastens the dampening of nuclear fluctuations, which is a marker of differentiation. These results give insight into the mechanism by which strain promotes oligodendrocyte differentiation. Further discussion on the interpretation of these results and future experiments are described in Makhija et al.13.
Figure 1: Design and geometry of the polydimethylsiloxane mold. (A) Sketch of the mold showing all dimensions in millimeters (this sketch was generated by Whits Technologies, Singapore). (B) Three-dimensional view of the mold (adapted from Makhija et al.11). Inset shows a photo of the mold. (C) Three-dimensional view of the polydimethylsiloxane plate fabricated using the mold (adapted from Makhija et al.11). Inset shows a photo of the polydimethylsiloxane plate. Please click here to view a larger version of this figure.
Figure 2: Sample preparation. (A) Photo of a polydimethylsiloxane plate and a square compartment. (B) The square compartment is placed on the raised square on the polydimethylsiloxane plate. Its purpose is to contain medium for the cells. (C) Polydimethylsiloxane plates are stored in 150 mm-diameter plastic dishes using parafilm paper to prevent the polydimethylsiloxane from sticking onto the plastic. (D) While mounting the plate onto the strain device, support it from the bottom using two fingers to prevent any sagging of the plate. If the plate still sags a little bit after mounting, increase the distance between the strain device arms by turning the translation stage. At this step, the translation of the stage should not induce strain in the polydimethylsiloxane plate. Please click here to view a larger version of this figure.
Figure 3: Cell stretching. (A) Measure the initial length of the plate between the clamps, using a ruler. (B) Stretch the plate by turning the screw on the translation stage to increase the initial plate length by the desired percentage. X% increase in length corresponds to X% of strain. Note that, to generate the desired strain (X%) in the raised cell culture compartment, the main plate must be strained by a higher amount (approximately 2X%) because of the difference in their thickness in the described plate geometry. Please click here to view a larger version of this figure.
Figure 4: Preparation for imaging. (A) Bring the 100x objective to the central position in the turret, unscrew the objective, and screw it back together with the objective ring. (B) Sketch of the holder showing all dimensions in millimeters. (C) Spread vacuum grease at the periphery of the window in the holder, using a pipette tip, and place a glass coverslip on top. Use a cover glass with thickness #0 or #1, to enable focusing with the 100x oil immersion lens. (D) Place the holder (1) on the microscope stage (2). Bring the oil (6) objective (3) closer to the coverslip (5) that is stuck to the holder, using vacuum grease (4). Please click here to view a larger version of this figure.
Figure 5: Imaging setup on the microscope. (A) Assemble the z-translation stage (yellow arrow) and the holder (red arrow) onto the microscope stage. (B) Tilt the strain device onto the microscope stage so as to let any medium drop onto the glass coverslip of the holder. Stick a double-sided tape (blue arrow) on the strain device that will stick onto the z-translation stage. (C) Place the strain device in an inverted position, supporting it on the z-translation stage. The cells must be aligned with the glass coverslip of the plastic holder. (D) Inverted geometry of the strain device minimizes the distance between the cells and the objective, thereby facilitating high-resolution imaging (adapted from Makhija et al.11). (E) Side view before bringing the strain device down (1); side view after bringing the strain device down (2); top-view after bringing the strain device down (3). Please click here to view a larger version of this figure.
Figure 6: Representative images and area fluctuations data of the nucleus. This figure is adapted from Makhija et al.11. (A) Typical image of a nucleus labeled with histone H2B tagged to green fluorescent protein, and a differentiating oligodendrocyte progenitor cell. The nucleus is in focus, while the cell processes are out of focus. (B) Typical time series of a nuclear area (in pixels). (C) Third order polynomial fitted to the data. (D) Percentage of the residual area fluctuation time series. (E) Nuclear edge fluctuations of proliferating oligodendrocyte progenitor cells and terminally differentiated oligodendrocytes. (F) Nuclear edge fluctuations at 1 h, 24 h, and 48 h postinduction of chemical differentiation without strain. (G) Nuclear area fluctuations at 1 h, 24 h, and 48 h postinduction of chemical differentiation with 10% tensile strain. Please click here to view a larger version of this figure.
A device has previously1 been designed for the application of extracellular tensile strain on adherent cells. The design of the polydimethylsiloxane substratum in that work was sufficient for biochemical assays, as well as the low-resolution imaging of stretched cells. In this work, the substratum was redesigned, and a novel imaging configuration that facilitates high-resolution subcellular live cell imaging was introduced. The advantages of this system are numerous: it can be built in-lab using simple components, it is inexpensive compared to commercial strain devices (500 USD per cell strain device), and it is small enough to fit inside tissue culture incubators, as well as onto microscopes. Moreover, although the imaging system has been described here with the inverted microscope setup, it can be readily adjusted for the upright microscope configuration.
There are a few critical steps involved in the sample preparation and imaging. First, the polydimethylsiloxane plates and square compartments have to be thoroughly cleaned before cell seeding (protocol step 2.8) to ensure cell survival (as uncured polydimethylsiloxane is toxic to cells). Second, since the volume of fluid medium that can fit inside the square compartment is less than 1 mL, it may evaporate if the sample is in the incubator for a few days. Hence, the medium must be checked every day and replenished when required. Additionally, the raised area on the polydimethylsiloxane plate can be covered with an inverted 60 mm-diameter plastic dish to minimize evaporation. Third, extreme caution must be exercised while moving the strain device on the microscope down via the z-translation stage to bring the cells closer to the glass coverslip. Compressing the cells (even for a moment) between the polydimethylsiloxane substratum and the glass coverslip may cause cell death. Fourth, the dead cells and cell debris may remain stuck to the glass coverslip after the polydimethylsiloxane sample has been removed. Hence, after imaging, the glass coverslip must be pulled off from the vacuum grease and a fresh coverslip should be attached prior to mounting a new sample.
The limitations of the strain device are that the application of stage lateral displacement cannot be programmed to perform cyclic or ramped strain and that it can only apply uniaxial strain. The limitation of the polydimethylsiloxane substratum in the described geometry is that a strain higher than 25% may cause a fracture of the polydimethylsiloxane.
A future modification to improve the design of the polydimethylsiloxane substratum could be the incorporation of a grid on the cell culture surface. This would allow for tracking the same cell before and after the application of tensile strain.
The authors have nothing to disclose.
All authors gratefully acknowledge funding support from the National Research Foundation of Singapore through the Singapore-MIT Alliance for Research and Technology (SMART) BioSystems and Micromechanics (BioSyM) interdisciplinary research group. Authors Dr. Jagielska and Dr. Van Vliet also gratefully acknowledge funding and support from the Saks-Kavanaugh Foundation. The authors thank William Ong and Dr. Sing Yian Chew from Nanyang Technological University, Singapore, for providing rat oligodendrocyte progenitor cells for some experiments described in this work, and the authors thank Dr. G. V. Shivashankar from Mechanobiology Institute, National University of Singapore, Singapore, for discussions about nuclear area fluctuations.
Primary cells | Primary Oligodendrocyte Progenitor Cells isolated from Neonatal Rats were provided by Dr. S. Y. Chew's lab at Nanyang Technological University |
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Media | |||
DMEM high glucose | Gibco | 11996-065-500ml | |
Pen/Strep | Gibco | 15070-063-100ml | |
FGF | Prospec | CYT-608-50ug | |
PDGF | Prospec | CYT-776-10ug | |
Media Stock components | |||
BSA | Sigma | A9418-10G | |
Progesterone | Sigma | P8783-1G | |
Putrescine | Sigma | P5780-5G | |
Sodium Selenite | Sigma | S5261-10G | |
Tri-iodothyronine | Sigma | T6397-100MG | |
Thyroxine | Sigma | T1775-100MG | |
Holo-Transferrin | Sigma | T0665-500MG | |
Insulin | Sigma | I9278 | |
Mold and PDMS plate | |||
PDMS – Dow Corning Sylgard 184 | Ellsworth | 184 SIL ELAST KIT 0.5KG | |
Mold – Liquid Plastic | Smooth-On | Smooth Cast 310 – Trail Size | |
Substrate Coatings | |||
poly-D-lysine | Sigma | P6407-5MG | |
Fibronectin | Sigma | F1141-2MG | |
Histone staining | |||
CellLigt H2B-GFP BacMam | Molecular Probes | C10594 | |
Surface functionalization | |||
APTES | Sigma | A3648-100ml | |
BS3 | Covachem | 13306-100mg | |
HEPES buffer pH 8.0 | Alfa Aesar | J63578-AK-250ml | |
Cell detachment | |||
Accutase | Invitrogen | A1110501-100ml |