Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Podsumowanie

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

Streszczenie

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Wprowadzenie

Cell migration is required for wound healing, as it is responsible for the final closure of the epithelial gap and restoration of the disrupted surface¹. Performing artificial wounds in animal models allows for the replication of this complex process in near physiological conditions². However, this approach often results in costly and complicated experimental procedures, that potentially lack precision for the study of distinct processes, due to the intricate nature of the wound-healing process.

In vitro culture of epithelial cell lines provides a helpful alternative to animal models for researching the role that these cells play in wound healing and the effects of treatment on cell migratory behavior. The physiology of epithelial cells is often studied by molecular techniques using non-confluent cultures^3,4,5,6; however, the disruption of the epithelium integrity is usually achieved by fine mechanical incisions. In cell culture, this implies that negligible number of cells may be exposed to the wound gap, and they represent a too small sample for molecular biology techniques. However, these lesions can be studied at the microscopic scale, taking advantage of the innate migratory properties of some epithelial cell lines, such as the Mink Lung Epithelial cell (Mv1Lu) or the spontaneously immortalized human keratinocyte (HaCaT) cell lines.

Here we described a method for microscopy that is suitable to obtain quantitative data on the migration of epithelial cells in the context of wound healing^3,4,7,8. Moreover, we present additional methods that are helpful to study qualitatively molecular and morphological changes occurring on epithelial monolayers during migration. Overall, these methods provide a framework to study both the dynamics and morphological changes involved with epithelial cell behavior and response to treatments during wound healing.

Protokół

1. Artificial Wound Scratch Assay for Quantitative Studies

1. Cell monolayer preparation
   1. Working under sterile conditions, seed and grow Mv1Lu or HaCaT epithelial cells in culture flasks using serum-supplemented medium. Refresh medium once every 24-48 h. After cells reach 80% confluence, detach cells using an appropriate method, i.e., trypsinization⁹.
      NOTE: Mv1Lu and HaCaT epithelial cell lines are cultured using EMEM and DEMEM culture medium, respectively, supplemented with 2 mM L-Glutamine, and incubated at 37 °C with a controlled atmosphere of 5% CO₂. Every cell line must be assayed thoroughly to determine cell concentration and timing required to reach full confluency. Typically for Mv1Lu or HaCaT cells, 2-4 x 10⁴ or 4-6 x 10⁴ cells/well, respectively, are seeded in a 175 cm² culture flask, and at 80% confluence yields up to 35 x 10⁶ Mv1Lu or 1 x 10⁷ HaCaT cells.
   2. In either a 12-well or 24-well culture plate, seed in each well 2 mL of cells. Refresh medium once every 24-48 h. Ensure cell numbers are high enough to reach 100% confluence after 2 or 3 days.
   3. After the cells reach confluence, remove the serum-supplemented medium and wash twice with fresh serum-free medium. Keep cells in serum-free medium for 24 h before the scratching.
      NOTE: Mv1Lu or HaCaT cells do not have special substrate requirements; however, for cell lines susceptible to detaching spontaneously in serum-free conditions, pre-coating of the culture plate surface using a poly-L-lysine solution should be considered¹⁰.
2. Monolayer scratching
   1. Working in a sterile environment, scratch the culture monolayer by firmly dragging the narrow end of a 20 µL or 200 µL sterile micropipette tip, depending on the desired scratch width. Keep the tip perpendicular to the culture surface to maximize gap homogeneity.
      1. Place the culture plates over a dark surface to clearly monitor the cell monolayer. If needed, perform two perpendicular scratches (cross-shaped) on each well to study up to 4 migration areas.
   2. Wash detached cells by gently removing culture medium and gently adding fresh serum-free medium. Repeat two times, or until most unattached cells have been removed.
3. Experimental procedure and data collection
   1. Take up to 4 pre-treatment reference images centered on the scratch gap from each well using an inverted phase-contrast microscope incorporating a CCD camera. Select interest areas by aligning the edge of microscope field to the adjacent intersection of scratches.
      NOTE: Typically, images are acquired using a 10x magnification and a 1,280 x 1,024 pixel image size. Selecting interest areas as described above helps for easy localization and validation of up to 4 measurements per well.
   2. Once all the images are acquired, designate treatment wells; exchange the medium in the wells with fresh medium that contains selected treatments.
      NOTE: Alternatively, for chemicals or compounds that do not disturb culture medium homogeneity, treatments can be inoculated directly into the culture medium.
   3. Incubate the scratched plate (37 °C with a controlled atmosphere containing 5% CO₂) until the conditions under observation reach 90% scratch gap closure. Avoid full closure.
      NOTE: Total gap closure nullifies post-treatment picture collection as reference areas are not detectable and the time reference for gap closing cannot be established. In the case of Mv1Lu or HaCaT cells, 16-19 h are required to reach 90% confluence.
   4. Stop cell migration by fixing the cells; gently replace the experimental culture medium with 1 mL of 4% formalin in PBS. Incubate for 15 min at RT.
   5. Wash the cells to remove excess formalin; gently replace the solution in the wells with fresh PBS. Repeat at least two times.
      NOTE: At this stage, after sealing, plates can be stored at 4 °C indefinitely.
   6. Take post-treatment reference images of each well from the original reference areas captured after scratching. Use the same equipment (inverted optical phase-contrast microscope incorporating a CCD camera) and matching picture settings (magnification and digital resolution/density).
      NOTE: For cells that migrate individually and fill the gap independently of the formation of a coherent front (e.g., MDA-MB-231 breast cancer human cells), the time-course images may allow the calculation of individual cell migration speeds.
4. Image analysis and quantification of migration
   1. Using image processing software (e.g., ImageJ), define scratch gap limits and determine the gap surface for up to four measurements in pre-treatment pictures. Record the data as "pre-treatment gap surface" (PREGAP). Repeat the same procedure for post-treatment pictures. Record the data as "post-treatment gap surface" (POSTGAP).
      1. Open the recorded image using ImageJ. In the "image" menu, set image type to 8 bit. In the "process" menu, go to "filters" submenu and apply "variance" filtering.
      2. In the "image" menu, enter "adjust" submenu and set threshold to black and white (B&W), make sure "Dark background" is not selected. In the "process" menu, go to "binary" submenu,  and select "fill holes".
      3. In the "analyze" menu, select "set measurements" and activate "area". Then draw the measurable area following the migrating edge contour thus delimiting the gap.
      4. In the "analyze" menu, select "analyze particles" and record "total area" values for the drawn area surface.
   2. Introduce PREGAP and POSTGAP total area values in a spreadsheet to quantify absolute migration for each set of individual samples as the difference of gap surface measurements: PREGAP − POSTGAP [Arbitrary units]. Additionally, normalize absolute migration data of each condition to control samples: (SAMPLE / CONTROL) * 100 [%].
      NOTE: For cells that migrate individually and fill the gap independently of the formation of a coherent front, (e.g., MDA-MB-231 breast cancer human cells), the absolute migration can be calculated by counting cells invading a central strip either in the control or the treated samples.
   3. Plot quantification outputs (see Figure 1). Perform statistical analysis if necessary. Consider Student's t-test when comparing two conditions or Analysis of Variance test for greater number of conditions.

2. Artificial Migration Front Assay for Topographical Studies

1. Cell monolayer preparation
   1. Working in sterile conditions, place one layer of sterilized round coverslips in empty culture plates until the plate's surface is completely covered.
      NOTE: Up to 12 and 33 coverslips fit on 5 cm and 10 cm diameter plates, respectively.
   2. On the culture plate, gently seed an adequate volume of cells at a concentration that allows 100% confluence after 2 or 3 days. Make sure no coverslip overlaps neighboring coverslips and prevent coverslips from floating by applying gentle pressure with a sterile micropipette tip. Refresh the medium every 24-48 h.
      NOTE: Every cell line must be assayed thoroughly to determine cell concentration and timing required to reach full confluency. Typically for Mv1Lu or HaCaT cells, 2-3 x 10⁶ or 2.5-4 x 10⁶ cells/10 cm-diameter plate, respectively, are seeded. For smaller plates, cell numbers must be scaled down.
   3. After the cells reach confluence, remove the serum-supplemented medium and replace with fresh serum-free medium. Keep the cells in serum-free medium for 24 h before the artificial wound is performed.
      NOTE: Mv1Lu or HaCaT cells do not have special substrate requirements; however, for cell lines susceptible of detaching spontaneously in serum-free conditions, pre-coating of the culture surface using a poly-L-lysine solution should be considered¹⁰.
2. Monolayer wounding
   1. Using sterile tweezers, gently move a coverslip to a clean 10 cm plate containing fresh serum-free medium. Avoid damaging the monolayer by holding the coverslip at the periphery with tweezers. Avoid excessive pressure that could result in coverslip breakage.
   2. Create artificial wounds by dragging a sterilized razor blade in a transverse line over the center of the coverslips. Drag 3-4 mm back-and-forth, in order to completely remove the central monolayer strip. Make sure no cell debris remains attached to the wounded edges.
      NOTE: On a 12-mm diameter round coverslip, the incision is made at the mid of the coverslip. Make sure to leave a streak of cells opposite the main front large enough (at least 2 mm wide) to avoid tilting of the coverslip in the following steps.
   3. Transfer wounded coverslips with cells facing up, to a clean 6-well plate containing at least 2 mL fresh serum-free medium. Fit up to 4 wounded coverslips/well.
   4. Gently wash two times using fresh serum-free medium to remove detached cells.
3. Experimental procedure and sampling
   1. Gently exchange the medium in the wells with fresh medium that contains selected treatments.
      NOTE: Alternatively, for chemicals or compounds that do not disturb culture medium homogeneity, treatments can be inoculated directly into the culture medium. Proceed with caution to avoid any potential cell detachment.
   2. Keep the plate inside a cell incubator (37 °C, 5% CO₂) for the desired experimental timing.
   3. Stop cell migration by fixing the cells; gently replace the experimental culture medium with 1 mL of 4% formalin in PBS. Incubate for 15 min at RT.
      NOTE: For time course experiments, remove the samples from the culture plate and fix them in a separate plate to avoid formalin fumes affecting the other, ongoing experimental conditions.
   4. Wash the cells to remove excess formalin; gently replace the solution in the wells with fresh PBS. Repeat at least two times.
      NOTE: At this stage, after sealing, plates can be stored at 4 °C indefinitely.
4. Immunofluorescence (IF) and topological analysis
   1. Perform IF staining and imaging on individual coverslips as described elsewhere^3,4,11.
      1. Permeabilize for 10 min by immersing coverslips in a 0.3% Triton X-100 solution in PBS.
      2. Block for 30 min using the following blocking solution: 0.3% Bovine Serum Albumin; 10% Fetal Bovine Serum; 5% skim milk; 0.3% Triton X-100 solution in PBS.
         NOTE: Blocking solution without milk can be prepared in advance and stored at -20 °C.
      3. Incubate for 1 h at room temperature inside a moist chamber, with primary antibody diluted in milk-free blocking solution. Place the coverslips upside-down in the 15 µL antibody solution to ensure proper distribution.
         NOTE: The antibody working dilution is variable and will depend on the antibody in use. For example, the Rabbit anti-c-Jun antibody requires a 1/100 dilution.
      4. Wash three times by dipping the coverslips in a in 0.1% Triton X-100 solution in PBS.
      5. Incubate the coverslips in secondary antibody diluted in milk-free blocking buffer for 30 min.
         NOTE: The antibody working dilution is variable and will depend on the antibody in use. For example, the Goat-anti-Rabbit (488) requires a 1/400 dilution for optimal resolution. Other labeling reagents such as phalloidin and Hoechst-33258 can be added to the incubation buffer at this stage.
      6. Wash three times by dipping the coverslips in 0.1% Triton X-100 solution in PBS.
      7. Mount the coverslips upside-down in a 10 µL mounting media drop (e.g., Vectashield) placed on a microscope slide. Leave the slides in the dark at room temperature overnight for the mounting medium hardening. Afterwards, store at 4 °C in the dark indefinitely.
   2. Obtain either epifluorescence or confocal microscopy images of structural changes occurring at the migrating front edge.
      NOTE: Topological studies can be performed by tiling pictures from the migrating front to the inner monolayer. Semi-quantitative studies are possible by software based analysis on local fluorescence intensities.

Wyniki

Artificial Wound Scratch Assay for Quantitative Studies: Assessing Epidermal Growth Factor (EGF) Promotion of Migration:

EGF is a well-known inducer of epithelial cells proliferation and migration, and thus a positive control for quantifying migration promotion. Mv1Lu and HaCaT cell monolayers were used in the wound scratch assays and pre-treatment pictures were obtained. After inoculation with 10 ng/mL EGF, cells were incubated fo...

Dyskusje

Upon skin or mucous membrane disruption, barrier function is restored by the actions of numerous cell types, including fibroblasts or epithelial and immune cells. Conjointly, these cells undergo a complex process involving apoptosis, proliferation, differentiation, and importantly, fibroblast and epithelial cell migration, which is the ultimate mechanism responsible for restoration of the disrupted tissue and the closure of the superficial epithelial gap^1,12 Thus...

Materiały

Name	Company	Catalog Number	Comments
Dulbecco’s Modified Eagle Medium (DMEM)	Biowest, Nuaillé, France	L0102-500	Optional 10 % FBS supplement
Eagles’s Minimum Essential Medium (EMEM)	Lonza	BE12 -662F	Optional 10 % FBS supplement
L-Glutamine	Lonza	BE17-605E	Use at 2 mM
Fetal Bovine Serum (FBS)	Thermo Fisher Scientific, Waltham, MA USA	DE17603A
Trypsin-EDTA	Sigma-Aldrich, St Louis, MO, USA	T4049	Dilute as appropriate
Poly-L-Lysine	Sigma-Aldrich, St Louis, MO, USA	P9155
Dulbecco's Phosphate Buffered Saline (DPBS) (10x)	Gibco by Life Technologies	14200-067	Dilute to 1x
24-well culture plates	BD FALCON//SARSTED	734-0020
6-well culture plates	SARSTEDT	83-3920
Epidermal Growth Factor (EGF)	Sigma-Aldrich, St Louis, MO, USA	E9644	Used 10 ng/mL
Round cover glass	MENZEL-GLÄSER	MENZCB00120RA020	SHORT DEPTH OF FIELD
Reinforced razor blade no. 743	Martor (through VWR)	MARO743.50
200 µl sterile aerosol pipet tips	VWR	732-0541
20 µl sterile aerosol pipet tips	VWR	732-0528
Digital camera coupled phase contrast microscope	Motic Spain	Moticam camera 2300 3.0 M Pixel USB 2.0; Motic Optic AE31
Confocal microscope	ZEISS Microimaging, Germany	LSM 510 META
10 cm Culture dish	BD FALCON	353003
Rabbit polyclonal anti c-Jun antibody	Santa Cruz Biotechnology	sc-1694	Used 1:100
Anti-rabbit IgG (polyclonal goat ) AF 488	Invitrogen	A11008	Used 1:400
Hoechst-33258	Sigma-Aldrich, St Louis, MO, USA	14530	Used 1:1000
Alexa Fluor 594 phalloidin (in methanol) (red)	Invitrogen	A12381	Used 1:100
Bovine Serum Albumin	Santa Cruz Biotechnology	SC-2323
Triton X-100	Sigma-Aldrich, St Louis, MO, USA	T9284
Skim milk	BD DIFCO	232100
ImageJ	National Institutes of Health, USA	Release 1.50i
Zen LSM 510 image processing software	ZEISS Microimaging, Germany	Release 5.0 SP 1.1