Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.
The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.
The preferred movement of cells towards a concentration gradient, known as chemotaxis, plays an important role in pathological and physiological processes in the body. Such examples are skin and mucosa wound healing1, morphogenesis2, inflammation3, and tumor growth4,5. It has also been shown that cancer cells can migrate through both individual and collective cell-migration strategies6. Moreover, diffusional instability mechanisms can induce the separation of single or clustered cells from a tumorous body/object and then can immigrate towards a source of nutrients and thus invade wider areas and tissues7.
Furthermore, it has been shown that diverse migration mechanisms can be active in 2D and in 3D, due to different roles of adhesion molecules8. Therefore, a move to physiologically relevant in vitro assays to investigate cell motility in a measureable and simple way is of significance in understanding cell movement phenomena9. Unfortunately, the difficulty in analyzing cell migration, a comprehensive quantifiable chemotaxis assay usually requires a long laborious method, founded on the measurement of impartial cell motility and transport phenomena models.
Past experimental approaches to investigate cell chemotaxis include the Boyden chamber10 and the under agarose assay11. However, within these early assays, cell migration experiments did not monitor the movement in respect to time. More, importantly, the concentration gradients used for the experiments were not well defined or completely understood while only sustaining the signaling for no more than a few hr. Furthermore, early chemotaxis chamber attempts restricted cell migration to two dimensions and did not allow one to monitor the kinetics of migration12. Looking at the Boyden chamber, an endpoint assay would not allow the researcher to observe migration visually and could not directly differentiate chemotaxis (directional movement) from chemokinesis (random movement). Additionally, several variables—differences in the pore size and thickness of membranes—made the chamber very difficult to easily reproduce and concealed the migrant reaction of cells to chemokines13,14.
With the new understanding of microfluidics, new chambers and micro-devices have been investigated as an instrument to investigate cell locomotion under interstitial flow conditions or chemotaxis15,16. Under these new devices, new cell metrics were introduced and investigated, like the effect of shear stress on a cell17,18. Unfortunately, past and current microfluidic chemotaxis chambers limited studies of cell migration to 2D substrates—an important setback since many biological processes, including tumor cell invasion and metastasis, and immune cell migration, involve 3D migration.
Direct observation chambers—where a chemoattractant solution is in contact with a 3D gel containing cells have also been also reported19,20. These chambers have two compartments, one containing a chemoattractant and one containing cells, are joined beside one another horizontally21 or as concentric rings22. These systems are pointed in the right direction, but do not keep a chemotaxis system for an extended period of time.
Furthermore, researchers have also examined diffusivity through collagen membranes in dialysis cells, as well as the diffusion of tracer molecules through collagen samples subjected to hydrostatic pressure23-25. Some diffusion experiments in collagen gels rely on physical and chemical modifications of the gel using magnetic fields and chemical incorporation26. A popular method for modeling diffusivity in collagenous tissues relies on the fluorescence imaging of continuous point photobleaching. This method has revealed anisotropy in the diffusion coefficients of macromolecules in oriented collagenous tissues. Yet, photobleaching has been used in articular cartilage and not collagen matrices. While similar, the necessary modeling experiments must be carried through specifically understanding the diffusion coefficient of collagen gels. More importantly, the systems do not utilize a method for measuring cell force generation.
Unfortunately, most systems seem to be missing one or two key elements for an ideal system: the allowing of cell tracking, a diffusion gradient understanding with a chemotactic factor through the matrix, a relatively simple set up with an ease of reproducibility, the minimization of cell-cell interactions, and the ability to measure dimensional units for quantification (i.e., velocity, force, specific concentration). Moghe et al.27 proposed a system that fulfilled most of these requirements in which cells were initially dispersed throughout the gel rather than concentrated on the filter surface, but was difficult to measure forces that the cell generates.
To this purpose, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays, which is based on time-lapse microscopy, coupled with image analysis techniques to measure cell forces in a 3D environment. This protocol provides a simple, yet innovative way of creating a simple 3D diffusion chamber that can be used to investigate 3D chemotaxis in different cells.
1. 3D Mold Design and Parts
2. Mold Assembly
3. Collagen Mixture and 3D Matrix
4. Imaging and Diffusion Modeling
5. Experimental Measurements
6. Tracking Cell Migration Utilizing TFM
The ability of this assay to accurately assess the migration of the cell relies upon a good setup of the system. Therefore, it is critical for make sure to design the diffusion system mold accurately and take great care in placing both hydrophobic and hydrophilic coverslips, as illustrated in Figure 1. If the system is properly designed and during the diffusion modeling stage ensuring to find a very good linear starting line, one is able to achieve nice fluoresces images, as depicted in Figure 2, for analyzing diffusion of the system.
The key to a successful cell chemotaxis analysis is modeling the diffusion and solving the general diffusion equation to find the coefficient of diffusion and specific concentration equation through a computational software generated code. After importing the images into computational software and running analysis (Figure 3), the coefficient of diffusion can be assessed (Table 2). Figure 4 represents the plots of the diffusion of rhodamine through the five tested collagen concentrations. The shapes of the diffusion plot match the traditional shape of a linear diffusion model. It can be seen that diffusion of rhodamine is inversely proportional to the concentration of collagen—the lower the collagen density the faster the diffusion. This is to be expected, as this concentration of collagen has a higher porosity. The horizontal lines in the collagen diffusion plot at a concentration of 1.5 mg/ml (Figure 4B) are simply the result of over saturation of the tracer signal. More importantly, as shown in Figure 4 each line represents a specific point (x, y) inside the collagen, to whereas time increases the normalized concentration never exceeds the previous point at the same time (dt), indicating no reversal of the chemical. This is an important characteristic of a chemotaxis chamber to allow direct flow of a chemoattractant without reverse flow.
By examining the last ten images at each x location, one is able to generate a plot of the steady-state concentration of rhodamine at each of the locations. By extrapolating across the entire sample length (Figure 5), one can to look at the whole sample. This indicates that the linear diffusion gradient is consistent across the entire sample length, which can be utilized for cell experiments (Figure 6).
Figure 1: A schematic of planar gradient diffusion system mold assembly in live cell imaging chamber. (1) A computer rendition of the complete system consisting of (from bottom to top) a bottom housing (c) a hydrophilic 22 mm coverslip, mold, top housing, and a glass cover. (2) Dimensions of mold where (a) cut inserts are shown. (3) Mold with collagen matrix with (b) hydrophobic coverslips on each side slid into the cut inserts to create the 3-well system. Please click here to view a larger version of this figure.
Figure 2: Example of rhodamine microscopy images in collagen. Representative images taken during rhodamine diffusion experiments, at same z-axis location, in the planar gradient diffusion system with a 1.0 mg/ml collagen concentration. Scale bar = 500 µm where (a) is 0 min (b) 3 min (c) 7 min, and (d) 15 min after rhodamine was added to chamber. Arrow indicates direction of diffusion of rhodamine through the collagen matrix. Please click here to view a larger version of this figure.
Figure 3: Example of analysis and summary of diffusion calculations generated by computational software code with a 2.5 mg/ml collagen concentration. (A) Black and white rendering of initial (dt = 0) image uploaded into computational software for analysis. (B) Specific points chosen by the user for intensity comparison and for diffusion calculations (x, y-axis are in pixels). (C) Diffusion profile of rhodamine throughout the collagen matrix where each line correlates to a red point in image B. (D) Concentration profile of rhodamine throughout the collagen matrix at steady-state. (E) Comparison between the smoothed data (blue dot) and true data from diffusion experiment (red dot) with difference between smoothed and true (green dot). (F) Normalized concentration of true (red dot) and fitted (green dot) data from computational software code throughout the experiment. Please click here to view a larger version of this figure.
Figure 4: Example of diffusion profile through collagen matrix using the planar gradient diffusion system at different points. Each line indicates a different selected data points chosen from the computational software code by the user. Collagen matrix at a density of (A) 1.0 mg/ml (B) 1.5 mg/ml (C) 2.0 mg/ml (D) 2.2 mg/ml and (E) 2.5 mg/ml. Please click here to view a larger version of this figure.
Figure 5: Example of a concentration (normalized) profile of rhodamine throughout the collagen matrix using the planar gradient diffusion system at steady-state. Circles are specific data points chosen though the computational software code by the user. Red line constructed using data points and extrapolating best line-fit (with R2 value shown) to show rhodamine concentration throughout the whole collagen matrix. Collagen matrix at a density of (A) 1.0 mg/ml (B) 1.5 mg/ml (C) 2.0 mg/ml (D) 2.2 mg/ml and (E) 2.5 mg/ml. Please click here to view a larger version of this figure.
Figure 6: Example of cell migration experiments. A Z-slice microscope image of a neutrophil migrating in the 3D planar gradient system. Neutrophil migrates towards the chemoattractant gradient (white arrow depicts direction of gradient and the triangle shows its concentration profile), where the star indicates the initial position of the cell at the start of the experiment. Subplot shows cell in 3D space, where arrow indicates next time-point direction. Scale bar = 10 µm. dt = 5 min between images (i.e., A-B). Please click here to view a larger version of this figure.
Collagen Cell Density | PBS | Fluorescent beads | NaOH | Collagen I Rat Tail | Media |
2.5 mg/ml | 10% | 6% | 2% | 70.20% | 11.80% |
2.2 mg/ml | 10% | 6% | 2% | 61.70% | 20.30% |
2.0 mg/ml | 10% | 6% | 2% | 56.10% | 25.90% |
1.5 mg/ml | 10% | 6% | 2% | 42.10% | 39.90% |
1.0 mg/ml | 10% | 6% | 2% | 28.10% | 53.90% |
Table 1: Custom collagen mixture adapted for the addition of fluorescent beads and cell viability.
Concentration of collagen Gel | Coefficient of Diffusion (cm2/sec) |
2.5 mg/ml | 1.03 x 10-6 ± 2.54 x 10-7 |
2.2 mg/ml | 1.28 x 10-6 ± 3.53 x 10-7 |
2.0 mg/ml | 6.48 x 10-6 ± 1.66 x 10-7 |
1.5 mg/ml | 1.05 x 10-5 ± 2.04 x 10-7 |
1.0 mg/ml | 1.39 x 10-5 ± 1.06 x 10-7 |
Water | 1.50 x 10-5 |
Table 2: Coefficients of diffusion.
The most critical steps for successful diffusion experiments with or without cells are: correctly setting up the mold assembly; developing the necessary manual dexterity to prevent damage during extraction of hydrophobic coverslips; ensuring to find a very good linear starting line to correctly calculate the diffusion coefficient; correct experimental calculations of both collagen and chemoattractant; correctly use of live cell imaging system to ensure matrix does not dry out; and maintaining a sterile, healthy culture.
When conducting diffusion experiments, it is imperative to make sure the diffusion plots match the traditional shapes of a linear diffusion model (Figure 4).
Running the analysis at several different x-locations on each sample, it is possible to calculate the average coefficients of diffusion for rhodamine in the various concentrations of collagens, as reported in Table 2. These results are consistent with previous research—coefficient of diffusion through the collagen gel decreases as the concentration of collagen decreased due to an increase of porosity in the matrix.
According to the diffusion model equation at steady state, one expects to have a linear concentration profile across the x values. By fitting a linear function to the points, one can see that they are all, in fact, linear. The R2 value of fitting a linear trend line for each of the best fit lines for the three collagen concentrations were at or above 0.92. The norms of the residuals in all extrapolated plots were .0227, indicating a good fit. This linear relationship is in agreement with the analytical solution. The concentration does not begin at 100%, as would be expected, because the imaging does not begin directly at the edge of the collagen sample. It is also impossible to perfectly calibrate the imaging.
Additionally, we were able to reference other papers that looked at diffusion in gels to compare results. Shenoy and Rosenblatt found the diffusion coefficient of BSA (radius = 4 nm) in a 30 mg/ml succinylated collagen solution to be 2.2 x 10-7 cm2/sec32. The radius of rhodamine is 0.78 nm, which explains its faster diffusion time through collagen24. In diffusion studies, it is common to utilize the radius to compare molecules. As the radius increases, the molecular weight also increases. Ramanujan et al. found the diffusion coefficient of dextran (radius = 6 nm) in polymerized 20% collagen gels (equivalent of 2.0 mg/ml concentration gels) to be 4.72 x 10-8 cm2/sec24. Again, this is comparable to the diffusion coefficient we observed for the 2.0 mg/ml concentration gel (D = 1.28 x 10-6 cm2/sec), if we take into account the molecule size difference. Leddy and Guilak studied the diffusion of dextrans through articular cartilage33. It is possible to compare diffusion through collagen matrices and through articular cartilage because approximately two-thirds of the mass of articular cartilage is polymeric collagen (predominately type II). Leddy and Guilak found the diffusion coefficient of 3 kDa dextran through articular cartilage to be between 6-10 x 10-7 cm2/sec33. Knowing that the system works properly we were then able to calculate the specific concentration anywhere within the system, using sawtooth and transient system heat transfer models. This additional metric can be used during cell experiments to track cell-concentration understanding and gradient slope.
Time duration of experiments may be a limiting factor when using our approach. Therefore, it is important to use a cover for the live cell imaging system to ensure the matrix does not dry out. Using a cover during experiments, we were able to successfully monitor the diffusion of rhodamine up to 7 hr and 4 hr during cell experiments without the gel drying up. If one does not use a cover, the gel might dry up faster and significantly affect the diffusion and migration of cells.
The protocol described herein uses a well-controlled planar diffusion model/system to be able to measure cell forces in a 3D environment during chemotaxis. Here we present a newly developed simple system that can be utilized for TFM/DVC experiments. Using this system, the researchers will be able to: 1) successfully model diffusion through 3D collagen matrices and verify, by fitting the data to analytical expression, that the planar gradient diffusion system, when seeded with cells, should promote chemotaxis and not chemokinesis; 2) calculate the coefficient of diffusion for collagen of five different concentrations; and 3) successfully find the specific concentration of the chemoattractant within the diffusion chamber. With this approaches, one can further examine cell chemotaxis with specific metrics not easily and readily available without the laborious approaches of multifaceted systems and complex diffusion gradients.
The authors have nothing to disclose.
The authors would like to acknowledge Drs. Jonathan Reichner and Angle Byrd for cell experiment insight. The National Science Foundation Graduate Research Fellowship Program (GRFP) supported this work.
Silicone elastomer kit | Dow Corning Corp | 182 SIL ELAST KIT .5KG | a two-part misture with a 10:1 mix |
Live cell imaging chamber | Live Cell Instrument | CM-B18-1 | CMB for 18mm round coverslips |
22mm Glass Coverslip | Fisher Scientific | NC0180281 | Neuvitro Corp. cover slip 22mm 1.5 |
Machined aluminum metal cube | |||
Hobby utility knife | X-Acto | X3201 | |
3-(aminopropyl) trimethoxysilane | Sigma-Aldrich | 281778-5ML | |
Glutaraldehyde | Polysciences, Inc | 00216A-10 | Glutaraldehyde, EM Grade, 8% |
50 ml tube | Fisher Scientific | 14-432-22 | Standard floor model and tabletop centrifuges |
Glass petri dish | Fisher Scientific | 08-747A | Reusable Petri Dishes: Complete (60 x 15mm) |
Forceps | Fisher Scientific | 22-327-379 | Fine Point Forceps |
Cover glasses | Fisher Scientific | 12-518-105A | Rectangle; 30 x 22mm; Thickness No. 1 |
Tridecafluoro-1,1,2,2-tetrahydrooctyl | Gelest | SIT8174.0 | |
Acetic acid | Sigma-Aldrich | 320099 | Acetic acid ACS reagent, ≥99.7% |
Hexane | Sigma-Aldrich | 296090 | Hexane |
anhydrous, 95% | |||
Ethyl alcohol | Sigma-Aldrich | E7023 | 200 proof, for molecular biology |
High-precision diamond scribing tool | Lunzer | PV-081-3 | Straight extended tip scribe, .020" (.50mm) diameter by .200" (5.0mm) tip length |
Vacuum grease | Dow Corning | 14-635-5C | High-Vacuum Grease |
15 ml tube | Fisher Scientific | 14-959-49D | 15 ml conical centrifuge tubes with hydrophobic, biologically inert surface |
10X phosphate buffered solution | Fisher Scientific | BP399-500 | 1.37 M Sodium Chloride, 0.027 M Potassium Chloride, and 0.119 M Phosphate Buffer |
1N sodium hydroxide | Sigma-Aldrich | 38215 | Sodium hydroxide concentrate |
Collagen I, rat tail | BD Biosciences | 354236 | Rat tail |
Micro centrifuge tube | Fisher Scientific | 02-681-332 | Volume: 2.0 ml; O.D. x L: 13 x 40 mm; sterile; single-wrapped |
Carboxylate-modified microspheres | Invitrogen | F-8813 | Carboxylate-modified microspheres, 0.5 µm, yellow-green fluorescent (505/515), 2% solids |
Rhodamine | Sigma-Aldrich | 83689 | Rhodamine B for fluorescence |