We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.
Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.
Palate development has been studied extensively as defects in palatogenesis lead to cleft palate-a common birth defect that occurs in isolated cases or as part of hundreds of syndromes1,2. The development of the embryonic palate is a dynamic process that involves movement and fusion of embryonic tissue. This process can be divided into four major steps: 1) induction of palatal shelves, 2) vertical growth of the palatal shelves next to the tongue, 3) elevation of the palatal shelves above the tongue, and 4) fusion of the palatal shelves at the midline1,3,4. Over the past several decades, many mouse mutants have been identified that manifest cleft palate5,6,7,8. Characterization of these models has indicated defects in palatal shelf induction, proliferation, and fusion steps; however, palatal shelf elevation defects have been rare. Thus, understanding the dynamics of palatal shelf elevation is an intriguing area of research.
Careful analysis of some mouse mutants with palatal shelf elevation defects has led to the current model showing that the very anterior region of the palatal shelf appears to flip up, while a vertical to horizontal movement or "remodeling" of the palatal shelves occurs in the middle to posterior regions of the palate1,3,4,9,10,11. The medial edge epithelium of the palatal shelf likely initiates the signaling required for this remodeling, which is then driven by the palatal shelf mesenchyme. Recently, many researchers have identified palatal shelf elevation delay in mouse models that showed transient oral adhesions involving palatal shelves12,13. The mesenchymal remodeling involves reorganization of the cells to create a bulge in the horizontal direction, while simultaneously retracting the palatal shelf in the vertical direction9,10,14. Among the several mechanisms proposed to affect palatal shelf elevation and the underlying mesenchymal remodeling are cell proliferation15,16,17, chemotactic gradients18, and extracellular matrix components19,20. An important question arose: is the palatal shelf elevation delay observed in Specc1l-deficient mice also partly due to a defect in the palatal shelf remodeling, and could this remodeling defect manifest in an intrinsic defect in behavior of primary MEPM cells21?
Primary MEPM cells have been used in the craniofacial field for many studies involving gene expression22,23,24,25,26,27,28,29, and a few involving proliferation30,31 and migration25,31,32, but none for collective cell behavior analysis. Time-lapse imaging of MEPM cells was performed in 2D culture and wound-repair assays to show that MEPM cells displayed directional movement and formed density-dependent cell streams-attributes of collective movement21. Furthermore, Specc1l mutant cells formed narrower cell streams and showed highly variable cell migration trajectories. This lack of coordinated motility is considered to contribute to the palate elevation delay in Specc1l mutant embryos13,21. Thus, these relatively simple assays using primary MEPM cells may serve as a proxy for studying mesenchymal remodeling during palatal shelf elevation. This paper describes the isolation and culture of primary MEPM cells, as well as the time-lapse imaging and analysis, for the 2D and wound-repair assays.
All experiments involving animals were carried out with a protocol approved by the KUMC Institutional Animal Care and Use Committee, in accordance with their guidelines and regulations (Protocol Number: 2018-2447).
1. Harvest E13.5 embryos
2. Dissection of palatal shelves from embryos (Figure 1)
NOTE: Sterilize the stainless steel dissection instruments (see the Table of Materials) after processing each embryo by placing the instruments first in a beaker of 100% ethyl alcohol (EtOH), then in an instrument sterilizer at 350 °C for 10 s, and then cooling them in a second beaker of 100% EtOH.
3. Culture of MEPM cells
NOTE: Under the conditions described here, the palate epithelial cells do not survive the first passage, resulting in a pure palate mesenchymal cell culture. Use sterile technique to perform all steps in a tissue culture hood.
4. Cryopreservation of MEPM cells
5. Live-imaging of MEPM cells – 2D collective migration assay (Figure 2)
6. Live-imaging of MEPM Cells in a wound-repair assay (Figure 3)
7. Computational analysis of time-lapse image sequences
NOTE: Perform the following procedures on a computer equipped with standard computational tools, such as the python interpreter, C compiler, and a shell (see the Table of Materials).
Figure 4: Analysis of individual cell trajectories. (A) Phase-contrast time-lapse micrographs are subjected to (B, C) a manual tracking procedure, which marks cells (green dots). (D) Cell positions (x,y) are stored for each cell distinguished by its ID and for each frame f. (E) Trajectories can be overlaid on the micrographs and color-coded to indicate temporal information. As an example, in each trajectory, a blue to red color palette indicates progressively later trajectory segments, with red and blue marking the initial and final cell locations, respectively. (F) Various statistical properties of trajectories, such as the mean square displacement, can be extracted and used to characterize the motility of various cell populations, which in this example include wildtype (wt, blue), and knockdown (kd, red) MEPM cells. The scalebars represent 100 µm. Please click here to view a larger version of this figure.
Figure 5: Characterization of stream formation of cultured cells. (A,D) Phase-contrast time-lapse images from Figure 4A are used to identify cell movements. For each moving cell, a frame of reference (blue) and spatial bins (white) were co-aligned to categorize adjacent cells as being in the front, rear, left, or right. (B,E) The velocity of adjacent cells (black vectors) was related to the same frame of reference (C,F). This procedure was repeated for each cell and time-point. (G) After pooling this local information, each bin will contain multiple velocity vectors (gray), which can be averaged to determine the average co-moving velocity (magenta arrows) at various locations relative to an average motile cell. (H) The average velocity map thus characterizes the typical cell velocities at various locations relative to a moving cell. (I,J) Finally, this field was sampled along the front-rear (parallel) axis and also along the left-right (perpendicular) axis. Please click here to view a larger version of this figure.
The dissection of palatal shelves is illustrated in Figure 1. The sequence of incisions is designed to minimize slippage of the tissue. Following the removal of the head (Figure 1A,B), the lower jaw is removed (Figure 1B,C). The incision of the upper part of the head (Figure 1C,D) is done to stabilize the tissue when placed upside down (Figure 1E) to visualize (Figure 1E, dotted lines), pinch (Figure 1F), and excise (Figure 1G) the palatal shelves.
The excised palatal shelf pair from a single embryo is trypsinized and cultured in a 35 mm dish or in a well of a 6-well dish. Larger dishes are not preferred as the cell density is too low for optimal growth. Upon confluence, the cells from each well are trypsinized and passaged into three 35-mm-equivalent wells (passage #1). Confluent cells from passage #1 can then be frozen down into aliquots of 1 × 106 cells/mL. Frozen aliquots are subsequently brought up in a 35-mm-equivalent dish and grown to confluence. The cells are then trypsinized (passage #2) and seeded according to the experiment. Creating and using frozen aliquots helped to normalize conditions, especially with respect to cell density. The use of fresh MEPM cells resulted in more variability in final cell density in experiments, which is believed to be due to a variable proportion of viable or sub-viable cells in fresh cultures. In addition, the number of MEPM cell passages were strictly limited to two (listed above) for these experiments.
Considering that a) cell density was critical, b) MEPM cells from a single embryo were limited, and c) imaging optics were better in a large dish, 2-well silicone inserts were used (Figure 2 and Figure 3). For wound-repair assays, MEPM cells were grown in the 2-well silicone inserts until high confluence, then the inserts were removed, and the wound imaged until closure (Figure 3). However, for 2D culture, the MEPM cells needed imaging as they grew, so the 2-well silicone inserts were simply trimmed to 1/3rd of their height to allow for clear imaging (Figure 2C). Small 3D printed rings40 in 35 mm dishes were also used for 2D motility analysis (Figure 2D).
MEPM trajectories (Figure 4E) are persistent: the direction of cell motility is maintained for several hours. The mean displacement vs. time analysis (Figure 4F) indicates the persistence in the form of displacement being proportional with elapsed time. The typical speed of MEPM cells on tissue culture plastic surface, 10 µm/h, can be also used for quality control of the cell culture. Flow analysis of the motility data reveals that the co-moving clusters of MEPM cells are ~300 µm in size (Figure 5I,J). The representative results also indicate a profound motility difference between wild-type and mutant MEPM cells. In addition to wildtype and mutant comparison, both 2D and wound-repair assays can be combined with various biochemical treatments. For example, PI3K-AKT pathway activators have been used with MEPM cells, as described previously 21.
Figure 1: Dissection of embryonic palatal shelves to isolate mesenchymal cells. (A) An E13.5 mouse embryo head is removed by cutting along the neck (red line). (B) Next, the lower jaw and tongue are removed with incisions along the oral cavity, between the upper and lower jaws (yellow line). (C) To stabilize the tissue for palatal shelf removal, the top of the head is excised (green line). (D) The resulting dissected upper jaw region is placed (E) upside down to visualize the palatal shelves (black dotted lines). (F) The excision of individual palatal shelves is depicted schematically, where individual protruding shelves (yellow) are pinched off from the maxilla (blue). (G) Excised pair of palatal shelves from a single embryo, which can then be trypsinized and cultured in a 35 mm dish or in a 6-well plate. Please click here to view a larger version of this figure.
Figure 2: Experimental setup of 2D MEPM cell culture. (A) Thaw a frozen aliquot of MEPM cells, and (B) culture the cells in a 35 mm dish or a 6-well plate. When confluent, trypsinize the cells and seed them as described in the protocol in a 35 mm dish either with (C) 2-well silicone inserts that have been trimmed or (D) with 3D-printed rings. A small culture space minimizes the need for large numbers of MEPM cells. The low profile of the trimmed silicone inserts or the 3D-printed rings allows for direct imaging without a halo effect. Time-lapse imaging can continue until the desired cell density is achieved, which can be up to 72 h. Representative images are shown at (E) 0 h, (F) 24 h, and (G) 45 h timepoints. The images were taken using a 4x objective. The scalebars for E, F, G = 300 µm. Abbreviations: 2D = two-dimensional; MEPM = mouse embryonic palatal mesenchyme; 3D = three-dimensional. Please click here to view a larger version of this figure.
Figure 3: Experimental setup of wound-closure assay using MEPM cells. (A) Thaw a frozen aliquot of MEPM cells, and (B) culture cells in a 35 mm dish or a 6-well plate. When confluent, trypsinize the cells and (C) seed them in the 2-well silicone inserts in a 35 mm dish. Culture the cells in the insert until the desired confluence is achieved (~48 h), then remove the silicone insert and image. Representative images are shown (D) immediately after removal of the insert, (E) after 24 h, and (F) after 40 h. Wound closure takes around 36 h. The images were taken using a 10x objective. The scalebar represents 300 µm. Abbreviations: MEPM = mouse embryonic palatal mesenchyme. Please click here to view a larger version of this figure.
Palatal shelf elevation constitutes a vertical to horizontal remodeling event1,3,4,9,11. It is postulated that this remodeling process requires palatal shelf mesenchymal cells to behave coordinately. The analyses with wildtype MEPM cells show that this cell behavior is intrinsic and can be quantitated21. Thus, these assays can be used to uncover primary palatal shelf elevation defects in new and existing mouse models of cleft palate. The methods outlined in sections 1 and 2 should allow investigators to isolate, culture, and freeze aliquots of primary MEPM cells. These cells can then be used for a wide variety of applications, including the 2D culture and wound-repair assays described here. The 2D culture, when combined with time-lapse imaging, presents a simple method to determine basic cell attributes over a range of cell densities. Presented here is a method to assess cell alignment and cell stream formation, which are attributes of collective cell movement (Figure 4). Wound-closure assays are commonly used to assess cell migration41,42,43. The cell-free region of the "wound" provides a cue for directional movement of cells at the periphery. Time-lapse imaging of this process allowed the determination and assessment of cell trajectories during migration. These cell trajectories, in turn, determine cell migration speed and directionality (Figure 5).
It is important to first optimize conditions for culture and assays using only wildtype MEPM cells. After the palatal shelves have been successfully trypsinized and the resulting single-cell suspension plated overnight, the vast majority (~90%) of cells will readily attach to the growth-surface of the dish. If the cells do not readily adhere, then there may be something wrong with the culture conditions. Initially, the adhered cells will look fairly homogeneous, with a triangular or slightly elongated shape, but as they grow to a high density, they become more spindle-shaped and elongated (Figure 2). If the cells become dramatically large in size or multinucleated, they should not be used for experiments. This optimization will also establish basic wildtype parameters for successful growth (time to confluence) and wound-repair (time to closure). The cells should proliferate by doubling in number almost daily and in time-lapse images, show motility of ~5-10 µm/h. Similarly, if in any subsequent experiment involving a wildtype-mutant comparison, these basic parameters are not met by wildtype cells, the experiment may need to be excluded from analysis. For example, wildtype MEPM cells take 36-40 h to completely close the wound using the 2-well silicone inserts. If in an experiment, wildtype cells take significantly longer than 40 h to close the wound, the entire experiment would be suspect. Poor performance from MEPM cells may be due to 1) poor frozen aliquot quality, 2) poor revival, or 3) excessive differentiation. Differentiated or senescent cells can be detected visually as they grow very large and do not divide. A culture with a large number of such cells should be avoided. In general, there should be no abnormal cells in the field of view of a 10x objective (Figure 3); a cell or two outside the field of view should not materially affect the analysis. Restricting analysis to only two cell passages (above) greatly helps maintain the quality of MEPM cells.
For both the 2D culture and wound-repair assays using primary MEPM cells, the key requirement for success was a high initial cell density. This requirement was first determined during optimization of wound-repair assay in which cells were seeded at >1400 cells/mm2. The density of cells migrating into the wound was then used as the seeding density in 2D culture assays. A seeding density of ~300 cells/mm2 formed cell streams, while still allowing for automated analysis of cell trajectories. Densities higher than 300 cells/mm2 tend to form more vivid streams that can be visualized by eye, but make individual cell tracking difficult. When comparing primary MEPM cells from wildtype and mutant embryos, although wound-closure delay can be assessed without computational analysis, stream formation and directionality differences may not be discernable by eye. If the cell density is too high or image quality is poor/blurred, for example due to loss of focus, automated cell tracking may be difficult. In such a case, it is possible to use manual cell tracking to determine cell trajectories in wound-repair assays using ImageJ. A very low concentration of Hoechst nuclear stain (3 µL/mL of 20 mM solution in MEPM culture medium) can also be used to facilitate cell tracking; however, fluorescent laser toxicity can be an issue with extended use.
For manual tracking, it was easier to track cells in reverse from the end of wound-closure backwards as far as possible until high cell density obscured further tracking. Even partial cell trajectories, when combined for a cell population, were informative. In contrast to wound-repair assays, automated cell tracking is required for 2D cell culture analysis, which is one reason why intermediate cell densities were chosen. Lastly, sensitive primary MEPM-based analyses can be used to identify compounds and pathways that may affect palate elevation. Previous studies indicated that activation of the PI3K-AKT pathway improved both cell speed and directionality of Specc1l mutant MEPM cells21. Other MEPM studies have also used various growth factor or drug treatments to stimulate downstream signaling cascades or to assess proliferation22,29,30,44,45. Thus, MEPM-based analyses offer a quick method to identify many more positive or negative regulatory factors, which can then be validated in vivo.
The authors have nothing to disclose.
This project was supported in part by the National Institutes of Health grants DE026172 (I.S.), and GM102801 (A.C.). I.S. was also supported in part by the Center of Biomedical Research Excellence (COBRE) grant (National Institute of General Medical Sciences P20 GM104936), Kansas IDeA Network for Biomedical Research Excellence grant (National Institute of General Medical Sciences P20 GM103418), and Kansas Intellectual and Developmental Disabilities Research Center (KIDDRC) grant (U54 Eunice Kennedy Shriver National Institute of Child Health and Human Development, HD090216).
Beaker, 250 mL (x2) | Fisher Scientific | FB-100-250 | |
CO2 | Matheson Gas | UN1013 | |
Conical tubes, 15 mL (x1) | Midwest Scientific | C15B | |
Debian operating system | computational analysis of time-lapse images | ||
Dulbecco's Modified Eagles Medium/High Glucose with 4 mM L-Glutamine and Sodium Pyruvate | Cytiva Life Sciences | SH30243.01 | |
EtOH, 100% | Decon Laboratories | 2701 | |
EVOS FL Auto | ThermoFisher Scientific | AMAFD1000 | |
EVOS Onstage Incubator | ThermoFisher Scientific | AMC1000 | |
EVOS Onstage Vessel Holder, Multi-Well Plates | ThermoFisher Scientific | AMEPVH028 | |
Fetal Bovine Serum | Corning | 35-010-CV | |
Fine point #5 Stainless Steel Forceps (x2) | Fine Science Tools | 11295-10 | Dissection |
Instrument sterilizer bead bath | Fine Science Tools | 18000-45 | |
Microcetrifuge tubes, 1.5mL | Avant | 2925 | |
Micro-Dissecting Stainless Steel Scissors, Straight | Roboz | RS-5910 | Dissection |
NucBlue (Hoechst) Live Ready Probes | ThermoFisher Scientific | R37605 | |
Penicillin Streptomycin Solution, 100x | Corning | 30-002-CI | |
Silicone Insert, 2-well | Ibidi | 80209 | |
Small Perforated Stainless Steel Spoon | Fine Science Tools | MC17C | Dissection |
Spring Scissors, 4 mm | Fine Science Tools | 15018-10 | |
Sterile 10 cm dishe(s) | Corning | 430293 | |
Sterile 12-well plate(s) | PR1MA | 667512 | |
Sterile 6-well plate(s) | Thermo Fisher Scientific | 140675 | |
Sterile PBS | Corning | 21-031-CV | |
Sterile plastic bulb transfer pipette | ThermoFisher Scientific | 202-1S | |
Trypsin, 0.25% | ThermoFisher Scientific | 25200056 |