Published: March 19th, 2021
A protocol for the culture and manipulation of mouse embryonic tissue on a microfluidic chip is provided. By applying pulses of pathway modulators, this system can be used to externally control signaling oscillations for the functional investigation of mouse somitogenesis.
Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and gradients are thought to control the timing and spacing of segment formation, respectively. While the involved signaling pathways have been studied extensively over the last decades, direct evidence for the function of signaling oscillations in controlling somitogenesis has been lacking. To enable the functional investigation of signaling dynamics, microfluidics is a previously established tool for the subtle modulation of these dynamics. With this microfluidics-based entrainment approach endogenous signaling oscillations are synchronized by pulses of pathway modulators. This enables modulation of, for instance, the oscillation period or the phase-relationship between two oscillating pathways. Furthermore, spatial gradients of pathway modulators can be established along the tissue to study how specific changes in the signaling gradients affect somitogenesis.
The present protocol is meant to help establish microfluidic approaches for the first-time users of microfluidics. The basic principles and equipment needed to set up a microfluidic system are described, and a chip design is provided, with which a mold for chip generation can conveniently be prepared using a 3D printer. Finally, how to culture primary mouse tissue on a microfluidic chip and how to entrain signaling oscillations to external pulses of pathway modulators are discussed.
This microfluidic system can also be adapted to harbor other in vivo and in vitro model systems such as gastruloids and organoids for functional investigation of signaling dynamics and morphogen gradients in other contexts.
Development is controlled by intercellular communication via signaling pathways. There is only a limited number of signaling pathways that orchestrate the complex formation of tissues and proper cell differentiation in space and time. To regulate this multitude of processes, information can be encoded in the dynamics of a signaling pathway, the change of a pathway over time, such as the frequency or duration of a signal1,2.
During somitogenesis, somitic tissue is periodically segmented off from the presomitic mesoderm (PSM)3. The PSM is spatially organized by gradients of Wnt, Fibroblast Growth Factor (FGF), and Retinoic acid signaling. In anterior PSM at the determination front, where Wnt and FGF signals are low, cells are primed for differentiation into somites. Differentiation occurs when a wave of transcriptional activation reaches this determination front. Within the PSM, Wnt, FGF, and Notch signaling oscillate. Neighboring cells oscillate slightly out of phase, which results in waves of oscillatory transcriptional activation downstream of the Wnt, FGF, and Notch pathways traveling from posterior to anterior PSM. In mouse embryos, a transcriptional wave reaches the determination front approximately every 2 h and initiates somite formation. Studying somitogenesis by perturbing or activating signaling pathways can illustrate the importance of these pathways4,5,6,7,8,9. However, to be able to investigate the function of signaling dynamics in the control of cellular behavior, it is essential to subtly modulate signaling pathways instead of permanently activating or inhibiting them.
To temporally modulate signaling pathway activity within the segmenting mouse embryo, Sonnen et al. have developed a microfluidic system10. This system allows the tight control of fluid flows within microchannels of a chip that contains the biological sample11. To study the importance of signaling dynamics for proper segmentation of PSM, this microfluidics setup is utilized to modulate signaling dynamics of the mouse segmentation clock ex vivo. By sequentially pulsing pathway activators or inhibitors into the culture chamber, external control of the dynamics of Wnt, FGF, and Notch signaling is achieved10. For instance, it is possible to modify the period of individual pathways and the phase relationship between multiple oscillatory signaling pathways. Using concomitant real-time imaging of dynamic signaling reporters, the effect of entrainment on the pathways themselves, on differentiation and somite formation can be analyzed. Using this level of control over signaling dynamics, the importance of the phase relationship between Wnt- and Notch-signaling pathways during somitogenesis was highlighted10.
Personalized chip designs allow for a plethora of options for spatiotemporal perturbations within the local environment, e.g., stable gradient formation12,13,14,15, pulsatile activation/inhibition10,16,17,18 or localized perturbations19,20. Microfluidics can also enable a more reproducible read-out and higher throughput due to automation of experimental handling21,22,23. The present protocol is meant to bring microfluidics and entrainment of endogenous signaling oscillations within tissues to every standard life sciences lab. Even in the absence of sophisticated equipment for chip generation, such as clean room and equipment for soft-lithography, microfluidic chips can be manufactured and used to address biological questions. Molds can be designed using freely available computer-aided design (CAD) software. A mold for the generation of microfluidic chips, usually consisting of polydimethylsiloxane (PDMS), can be printed with a 3D printer, or be ordered from printing companies. This way, microfluidic chips can be produced within one day without the requirement of expensive equipment24. Here, a chip design is provided, with which a mold for the entrainment of the mouse segmentation clock in two-dimensional (2D) ex vivo cultures25 can be printed with a 3D printer.
On-chip cultures and precise perturbations, enabled by microfluidics, hold outstanding potential in unravelling the molecular mechanisms of how signaling pathways control multicellular behavior. Signaling dynamics and morphogen gradients are required for many processes in development. Previously, labs had cultured cells, tissues and whole organisms in microfluidic chips and protocols for spatiotemporal perturbation of primarily 2D cell culture are provided elsewhere12,26,27,28,29. Applying microfluidics to modulate local environments in multicellular systems opens new perspectives for high-throughput and precise spatiotemporal perturbations. The field of microfluidics has now reached a point that it has become a non-specialist, inexpensive, and easily applicable tool for developmental biologists.
Here, a protocol for the entrainment of the mouse segmentation clock to pulses of a Notch signaling inhibitor is provided. Such an experiment consists of the following steps: (1) generation of microfluidic chip, (2) preparation of tubing and coating of the chip, and (3) the microfluidic experiment itself (Figure 1A). Research involving vertebrate model systems requires prior ethical approval from the responsible committee.
The research presented here has been approved by the EMBL ethics committee10 and the Dutch committee for animal research (dierenexperimentencommissie, DEC), and follows the Hubrecht guidelines for animal care.
Wear gloves while working with liquid PDMS. Cover surfaces and equipment. Remove any spills immediately, as cleaning becomes difficult once it's hardened.
1. Generation of the chip
NOTE: Microfluidic chips are generated by casting PDMS (Polydimethylsiloxane) in a mold. Molds can be designed using CAD software (e.g., uFlow or 3DµF30). A repository of free designs is provided by the MIT (Metafluidics.org). Molds are either printed with a 3D printer or can be generated by soft lithography using a photomask that contains the desired design (for further information see, e.g., Qin et al., 201031). Here, a design for a mold is provided that can be printed with a 3D printer for the on-chip culture of mouse embryo tissue (Figure 1B,C, Supplementary Files 1 and 2). To allow printing with lower resolution 3D printers, the design has been modified compared to the previously published one10. A mold without air bubble traps and one with air bubble traps is provided (Supplementary Files 1 and 2, respectively). As the procedure for printing is dependent on the printer available, the details for printing will not be described. However, one must make sure that all unpolymerized resin is entirely removed. It is often required to perform extra washing with solvents to remove unpolymerized resin from the small <200 µm holes within the microfluidic chamber. These holes will form PDMS pillars to trap the embryonic tissue within the chip during the experiment. PDMS is generally used for microfluidics, as it is cheap, biocompatible, and transparent, and has low autofluorescence. After curing, the PDMS chip is cut out of the mold and bonded onto a glass slide. Plasma treatment of both the glass and the PDMS chip activates the surfaces and allows the formation of covalent bonds, when brought into contact.
2. Preparing microfluidics experiment
NOTE: The following preparatory steps are necessary to perform the microfluidics experiment: First, when performing the microfluidics experiment, a fluid flow is created from the inlets to the outlets. Any holes in the chip will function as outlets due to the pressure build-up from the inlets. Therefore, all holes that are not used must be closed; for instance, holes that are used to load tissue onto the chip. If these are not closed, the tissue might flow out with the fluid. To close these holes, PDMS-filled tubing is used. The PDMS-filled tubing can be kept indefinitely and, therefore, will not need to be prepared for each microfluidics experiment separately but can be made in larger batches. Second, before the start of the microfluidic experiment, tubing, PDMS-filled tubing, and the chip, required for the experiment, are prepared and UV-sterilized. Finally, the chip has to be coated with fibronectin, so that the embryonic tissue can attach to the glass25.
3. Microfluidics experiment
NOTE: Here, a protocol for the entrainment of the mouse segmentation clock in 2D ex vivo cultures by applying pulses of the Notch signaling inhibitor DAPT is presented. Applying pulses with a period of 130 min, close to the natural period of the mouse segmentation clock (137 min25), allows efficient entrainment. Pulses of 100 min of medium and 30 min of 2 µM DAPT are applied (Figure 2A). The presence of drug within the chip is monitored using a fluorescent dye. The excitation and emission spectra of this dye and the fluorescent signaling reporter must be different enough to prevent bleed-through during fluorescence real-time imaging. When yellow fluorescent proteins are used as signaling reporter, such as the Notch signaling reporter LuVeLu5 (Lunatic fringe-Venus-Lunatic fringe), the dye, Cascade Blue, can be applied. To identify other possible combinations of fluorescent dye and reporter, freely available spectra viewer can be used. The effect of entrainment can be detected by real-time imaging of dynamic signaling reporters5,10,32. Each chip has two incubation chambers. This allows the direct comparison of drug pulses (DAPT) to control pulses (DMSO).
With this protocol, a method for the external entrainment of signaling oscillations of the mouse segmentation clock using microfluidics is presented. By applying pulses of Notch signaling inhibitor, signaling oscillations in independent embryo cultures get synchronized to each other10. A prerequisite for the application of this system to study the functionality of the segmentation clock is that signaling dynamics and segmentation are still present on-chip. It was shown previously that both Wnt and Notch signaling dynamics are maintained despite constant medium flow and physical boundary formation persists in peripheral 2D cultures, representing anterior PSM10.
To confirm entrainment of signaling oscillations to external drug pulses, real-time imaging of, for instance, the Notch signaling reporter LuVeLu5, expressing the yellow fluorescent protein Venus, during the microfluidic experiment is performed. Since orientation of the 2D cultures on chip is difficult to control, signaling oscillations in anterior tissue are analyzed. The periphery of the 2D culture can reproducibly be detected. Orientation of the tissue sections on-chip cannot be controlled at will and the whole inner surface of the chip gets coated with Fibronectin. Therefore, it occasionally happens that cultures attach to the sides or ceiling of the chip. In case the samples move out of the field of view (in x, y, and z direction) or they attach with the posterior end of the tail facing downwards, generally these samples are excluded.
For further analysis, multiple of such entrainment experiments are combined. Independent experiments can be aligned to each other using the timing of the drug pulses visualized by the dye, Cascade Blue, at approximately 400 nm. To analyze and visualize synchronization, quantified oscillations can be detrended (Figure 2B,D) and then either displayed as mean and standard deviation or phases of the oscillations can be calculated. This allows the analysis of the phase-relationship between oscillations of independent posterior embryo cultures to each other and to the external drug pulses (Figure 2C,E). The python-based program pyBOAT33 is a straightforward and user-friendly tool to determine period, phase, and amplitude of such signaling oscillations. To confirm entrainment, one can, for instance, determine the period of the endogenous signaling oscillations. When applying pulses with a period of 130 min, Notch signaling oscillations also show a period of 130 min (Figure 2F)10. In addition, using Cascade Blue pulses, independent experiments with different signaling reporters can be aligned to each other. This way the phase-relationship between oscillations of multiple signaling reporters can indirectly be determined10.
Thus, the presented microfluidic system allows the control of signaling oscillations in ex vivo cultures of the developing mouse embryo. In combination with imaging of markers for segment formation and differentiation, this system can now be applied to dissect how signaling pathways of the segmentation clock interact and how they control somite formation.
Figure 1: Schematic overview of the microfluidics protocol. (A) Chip molds can be designed using computer-aided-design (CAD) software. A chip design to entrain signaling oscillations in ex vivo models of mouse somitogenesis is provided. Molds can, for instance, be generated by 3D printing or ordered. Microfluidic chips are produced by molding of PDMS. A hardened PDMS chip is cut out and covalently bonded to a glass slide using a plasma oven. The glass surface within the chip is coated with fibronectin to allow dissected tissue to attach. Embryonic tissue is loaded onto the chip via loading inlets, which are subsequently closed. Pumps with different media conditions are attached to the inlets of the chip and a flow program is initialized. Tissue can be imaged using a confocal microscope during the experiment. (Kymographs adapted from Sonnen et al., 201810. Reprinted and modiﬁed from Cell according to Creative Commons Attribution CC BY-NC-ND 4.0.) (B) Schematic drawing of a mold design for on-chip culture of posterior mouse embryo tissue. The height of the microfluidic channels is 500 µm, sufficient for the culture of posterior embryonic mouse tails. The file to print this mold is provided in Supplementary File 1, and an alternative is given in Supplementary File 2. (C) Brightfield image of an E10.5 sectioned mouse tail enclosed by PDMS pillars on-chip. Please click here to view a larger version of this figure.
Figure 2: Representative results from a microfluidics experiment. (A) Schematic representation of entrainment experiment with medium and drug pump. Pulses of signaling pathway modulator are applied to ex vivo cultures of mouse embryos and signaling oscillations can be visualized by real-time imaging of dynamic signaling reporters (for instance, the Notch signaling reporter LuVeLu5 and Wnt signaling reporter Axin2T2A10). Dashed lines indicate the region that is used for analyses over time. (B-F) LuVeLu reporter oscillations are entrained by periodic pulses of the Notch signaling inhibitor DAPT. (B,D) Quantifications of Notch signaling oscillations in anterior PSM upon entrainment using DAPT or a DMSO control. (C,E) Phase-phase relation plots between the phase of Notch signaling oscillations and the timing of external DAPT/DMSO pulses. In case of entrainment, a stable phase-relationship is established. (F) Quantification of the mean period and SEM of reporter oscillations comparing samples entrained with DMSO and DAPT pulses. (Figure adapted from Sonnenet al., 201810. Reprinted and modiﬁed from Cell according to Creative Commons Attribution CC BY-NC-ND 4.0.). Please click here to view a larger version of this figure.
|The PDMS does not bond to the glass.
|- Make sure both the PDMS and glass are clean.
|- Make sure there is enough PDMS around the chamber for bonding.
|- Optimize the settings of the plasma oven.
|There are air bubbles on my chip.
|- Check if the pump was turned on.
|- Degas the chip before loading tissue onto the chip.
|- Degas the medium before start of the experiment.
|- Make sure the humidity in the incubation chamber is high enough.
|The tissue dies at the beginning of the experiment.
|- Add HEPES to the medium when loading and assembling the chip.
|- Do not flush the tissue in and out too often during loading.
|- Check that the flow rate is not too high.
|The tissue dies during imaging.
|- Make sure there is no phototoxicity from the imaging
|- Make sure there is no contamination.
|- The flow rate should not be too high.
|The focus changes during imaging.
|- Use autofocus during imaging to adjust for drift of the imaging slide.
|- Let the chip equilibrate to the temperature within the microscope for at least 30 minutes.
|- Push all tubing completely down to the bottom.
|The glass of the chip breaks.
|- Be more careful, the glass is very fragile.
|- The thin glass is only required for imaging. If imaging is not performed, a normal thicker glass slide can be used.
|- If the break is outside of the chip, it might not be a problem. If the glass seal to the chip is broken, liquid will leak out and air will come in.
|There is a contamination on my chip.
|- Add antibiotics to the culture medium.
|- Sterilize all equipment and tubing.
|- Work fast and clean.
Table 1: Troubleshooting
Supplementary File 1: STL file for printing a mold with a 3D printer. This mold is used to generate a microfluidic chip for the on-chip culture of posterior embryonic tissue (design shown in Figure 1). Please click here to download this File.
Supplementary File 2: STL file for printing a mold with a 3D printer to generate a microfluidic chip for the on-chip culture of posterior embryonic tissue. In contrast to Supplementary File 1 and the design shown in Figure 1, this design contains bubble traps at all inlets to prevent small amounts of air from entering the main microfluidic chamber. Please click here to download this File.
Supplementary File 3: Design file for the generation of a holder to place the chip into the microscope. This holder has the dimensions of a 96-well plate and should fit into standard holders of most microscopes. Please click here to download this File.
How signaling dynamics control multicellular systems has been a longstanding question in the field. Functional investigation poses a key challenge, because these dynamics have to be subtly modulated to allow this11. Such temporal control over pathway perturbations can in principle be achieved with optogenetics, which also enables a high spatial control34. However, optogenetics require the establishment of sophisticated genetic tools for the analysis of each signaling pathway in question. The current protocol described here provides a highly versatile tool for the perturbation of any signaling pathway. The microfluidics experiment allows the application of temporally controlled drug pulses to functionally investigate the dynamics of signaling pathways in tissue cultures. Here, the focus is on the study of somitogenesis in ex vivo cultures, but the protocol can be adapted to fit any other model systems.
Certain steps in the protocol are critical to perform a successful microfluidics experiment (summarized in Table 1). Key points are discussed as follows. Due to medium flow during the course of the experiment, the tissue culture experiences shear stress. Exposure of the model system to continuous flow can cause cell stress and in extreme cases cell death due to shearing effect. For ex vivo tissue cultures of mouse tailbuds and the current chip design, a flowrate of 60 µL/h (maximum of 100 µL/h) was found to work well with minimal shearing effect. When multiple pumps are turned on simultaneously for the same chip, flow rate of individual pumps needs to be adjusted accordingly to not cause an increase in total flow rate. When the current protocol is adapted to other model systems and chip designs, the flow rate should be optimized. Alternatively, it is possible to not flush medium continuously, but periodically change medium on the chip35. Such a setup can also be applied to control signaling oscillations in mouse somitogenesis (unpublished, data not shown). Moreover, a setup can also be envisioned, in which tissue cultures are not exposed to direct fluid flow, but drugs reach the tissue by diffusion from a neighboring channel. Such systems have been used successfully to apply gradients of growth factors to in vitro models of ESC differentiation14,36.
One major issue of microfluidic experiments is the occurrence of air bubbles on-chip during the experiment. Air bubbles within the chip or tubing can interfere with uniform liquid flow on the chip and can lead to removal of the embryo culture from its location. To prevent the presence of air bubbles, various steps of the protocol are indispensable. First, culture medium within syringes and the microfluidic chip itself must be degassed using a desiccator (step 3.1.3). Second, when loading samples into the loading inlets, one must be careful not to pipet air into the chip together with the sample (step 3.2.3). Third, when filling the tubing with medium, all air bubbles must be pumped out before attaching the chip (step 3.3.2). Otherwise, these bubbles will be pushed into the main chip during the experiment. Lastly, during the experiment, the medium is equilibrated within the imaging chamber or incubator due to the semi-permeable tubing. The permeability of the tubing also allows for evaporation of the medium, so make sure that the humidity is regulated during the experiment to prevent the formation of new bubbles in the tubing.
In general, microfluidics is a highly versatile tool that can be adapted to the researcher's specific question. The current design approach allows for imaging up to 12 ex vivo cultures in parallel per chip. This number is mainly limited by the number of embryos per experiment and the time it takes to image each embryo culture with sufficient resolution. If multiple conditions need to be compared in a single experiment, multiple chips can be mounted on a single glass slide. A chip design is provided, which is ideal for imaging of multiple tissue explants in parallel, but personalized designs can overcome limitations in sample number to allow high throughput analysis and increase the combination of more conditions within a single experiment16,17. Microfluidics is limited to models that can be cultured on a chip and on-chip culture will have to be optimized for each model system individually27,37,38.
Over the last 5-10 years, microfluidics has been applied to address various biological questions due to its potential for high throughput approaches and subtle modulations of signaling dynamics and gradients. Nowadays, microfluidics has become an easily applicable, cheap, and versatile tool that laboratories can establish with ease. Here, the current protocol is optimized for a specific application, namely, studying signaling dynamics governing somitogenesis. It is straightforward to adapt this protocol and design to suit individual research questions in tissue biology.
The authors have nothing to disclose.
We are grateful to Yang Li and Jos Malda from the UMC Utrecht for help with 3D printing of molds, Karen van den Anker from the Sonnen group for very useful feedback on the protocol, and Tjeerd Faase from the mechanical workshop at the Hubrecht Institute for the holder for microfluidic chips within the microscope. We would like to thank the entire Sonnen group for critical reading of the manuscript and the reviewers for their constructive feedback. This work received funding from the European Research Council under an ERC starting grant agreement no. 850554 to K.F.S.
|10 mL syringe
|Becton, Dickinson and company
|184 Silicon Elastomer curing agent
|184 Silicon Elastomer PDMS
|3 ml syringes
|Becton, Dickinson and company
|Scotch Magic tape or similar
|Blunt-tip forceps Bochem 18/10 Stainless steel
|Bovine Serum albumin Lyophilised pH~7
|Compressed air system
|Cell culture technologies
|DMEM/F-12, no phenol red, no L-glutamine, no Glucose
|Flow hood BioVanguard Greenline
|CleanAir by Baker
|For UV light source
|Glass slide No 1.5H high precision, 70x70 mm
|Leica SP8 MP
|Becton, dickinson and company
|Plasma oven Femto
|Punch Ø 1mm
|Tubing Ø 1mm
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