We want to give thanks to older members of the lab that help to improve and refine these techniques to its actual state: Dr. Celia Martinez-Mora; Dr. Anna Mrowiec, Dr. Catalina Ruiz-Ca&#241;ada and Dr. Antonia Alcaraz-Garc&#237;a. We are indebted to the Hospital Cl&#237;nico Universitario Virgen de la Arrixaca for strongly supporting the development of these techniques. Also the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias. Plan Estatal I+D+I and Instituto de Salud Carlos III-Subdirecci&#243;n General de Evaluaci&#243;n y Fomento de la Investigaci&#243;n (Grant no.: PI13/00794); www.isciii.es. Fondos FEDER &#8220;Una manera de hacer Europa&#8221;. We also thank Universidad de Murcia, IMIB-Arrixaca and FFIS for administrative support and assistance. Finally, we want to give a special thanks to Dr. Isabel Mart&#237;nez-Argudo and the Facultad de Ciencias Ambientales y Bioqu&#237;mica, Campus Tecnol&#243;gico de la F&#225;brica de Armas, Universidad Castilla la Mancha, Toledo for their kind support in willingly ceding the Biomedicine and Biotechnology Laboratory to make possible the filmed part of this paper.

1. Artificial Wound Scratch Assay for Quantitative Studies
<ol>
	<li>Cell monolayer preparation
	<ol>
		<li>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,...

<ol>
	<li>Singer, A. J., Clark, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+wound+healing.">Cutaneous wound healing.</a> N Engl J Med. 341 (10), 738-746 (1999).</li><li>Davidson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+wound+repair.">Animal models for wound repair.</a> Arch Dermatol Res. 290, S1-S11 (1998).</li><li>Alcaraz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+Membrane+Modifies+the+Genetic+Program+Induced+by+TGFss,+Stimulating+Keratinocyte+Proliferation+and+Migration+in+Chronic+Wounds.">Amniotic Membrane Modifies the Genetic Program Induced by TGFss, Stimulating Keratinocyte Proliferation and Migration in Chronic Wounds.</a> PLoS One. 10 (8), e0135324 (2015).</li><li>Ruiz-Canada, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+stimulates+cell+migration+by+modulating+Transforming+Growth+Factor-beta+signaling.">Amniotic membrane stimulates cell migration by modulating Transforming Growth Factor-beta signaling.</a> J Tissue Eng Regen Med. , (2017).</li><li>Schmierer, B., Hill, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta-SMAD+signal+transduction:+molecular+specificity+and+functional+flexibility.">TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility.</a> Nat Rev Mol Cell Biol. 8 (12), 970-982 (2007).</li><li>Hu, Y. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAK+and+paxillin+dynamics+at+focal+adhesions+in+the+protrusions+of+migrating+cells.">FAK and paxillin dynamics at focal adhesions in the protrusions of migrating cells.</a> Sci Rep. 4, 6024 (2014).</li><li>Martinez-Mora, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroin+and+sericin+from+Bombyx+mori+silk+stimulate+cell+migration+through+upregulation+and+phosphorylation+of+c-Jun.">Fibroin and sericin from Bombyx mori silk stimulate cell migration through upregulation and phosphorylation of c-Jun.</a> PloS one. 7 (7), e42271 (2012).</li><li>Bernabe-Garcia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oleanolic+acid+induces+migration+in+Mv1Lu+and+MDA-MB-231+epithelial+cells+involving+EGF+receptor+and+MAP+kinases+activation.">Oleanolic acid induces migration in Mv1Lu and MDA-MB-231 epithelial cells involving EGF receptor and MAP kinases activation.</a> PLoS One. 12 (2), e0172574 (2017).</li><li>Huang, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin-induced+proteome+alteration+during+cell+subculture+in+mammalian+cells.">Trypsin-induced proteome alteration during cell subculture in mammalian cells.</a> J Biomed Sci. 17, 36 (2010).</li><li>Liberio, M. S., Sadowski, M. C., Soekmadji, C., Davis, R. A., Nelson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+tissue+culture+coating+substrates+on+prostate+cancer+cell+adherence,+morphology+and+behavior.">Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior.</a> PLoS One. 9 (11), e112122 (2014).</li><li>Pierreux, C. E., Nicolas, F. J., Hill, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor+beta-independent+shuttling+of+Smad4+between+the+cytoplasm+and+nucleus.">Transforming growth factor beta-independent shuttling of Smad4 between the cytoplasm and nucleus.</a> Mol Cell Biol. 20 (23), 9041-9054 (2000).</li><li>Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H., Tomic-Canic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+and+cytokines+in+wound+healing.">Growth factors and cytokines in wound healing.</a> Wound repair and regeneration: official publication of the Wound Healing Society [and] the European Tissue Repair Society. 16 (5), 585-601 (2008).</li><li>Ansell, D. M., Holden, K. A., Hardman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+wound+repair:+Are+they+cutting+it?.">Animal models of wound repair: Are they cutting it?.</a> Exp Dermatol. 21 (8), 581-585 (2012).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453 (7193), 314-321 (2008).</li><li>Insausti, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+induces+epithelialization+in+massive+posttraumatic+wounds.">Amniotic membrane induces epithelialization in massive posttraumatic wounds.</a> Wound Repair Regen. 18 (4), 368-377 (2010).</li><li>Wu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+arrest+in+G0/G1+phase+by+contact+inhibition+and+TGF-beta+1+in+mink+Mv1Lu+lung+epithelial+cells.">Cell cycle arrest in G0/G1 phase by contact inhibition and TGF-beta 1 in mink Mv1Lu lung epithelial cells.</a> Am J Physiol. 270 (5 Pt 1), L879-L888 (1996).</li><li>Boukamp, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+keratinization+in+a+spontaneously+immortalized+aneuploid+human+keratinocyte+cell+line.">Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.</a> J Cell Biol. 106 (3), 761-771 (1988).</li><li>Marshall, J., Wells, C. M., Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Migration: Developmental Methods and Protocols. , 97-110 (2011).</li><li>Nyegaard, S., Christensen, B., Rasmussen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+accurate+quantification+of+cell+migration+using+human+small+intestine+cells.">An optimized method for accurate quantification of cell migration using human small intestine cells.</a> Metabolic Engineering Communications. 3, 76-83 (2016).</li><li>Szybalski, W., Iyer, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinking+of+DNA+by+Enzymatically+or+Chemically+Activated+Mitomycins+and+Porfiromycins,+Bifunctionally+"Alkylating"+Antibiotics.">Crosslinking of DNA by Enzymatically or Chemically Activated Mitomycins and Porfiromycins, Bifunctionally "Alkylating" Antibiotics.</a> Fed Proc. 23, 946-957 (1964).</li><li>Tomasz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitomycin+C:+small,+fast+and+deadly+(but+very+selective).">Mitomycin C: small, fast and deadly (but very selective).</a> Chem Biol. 2 (9), 575-579 (1995).</li><li>Sherry, D. M., Parks, E. E., Bullen, E. C., Updike, D. L., Howard, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+using+silicone+elastomer+masks+for+quantitative+analysis+of+cell+migration+without+cellular+damage+or+substrate+disruption.">A simple method for using silicone elastomer masks for quantitative analysis of cell migration without cellular damage or substrate disruption.</a> Cell Adh Migr. 7 (6), 469-475 (2013).</li><li>Dennis, E. A., Rhee, S. G., Billah, M. M., Hannun, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+phospholipase+in+generating+lipid+second+messengers+in+signal+transduction.">Role of phospholipase in generating lipid second messengers in signal transduction.</a> FASEB J. 5 (7), 2068-2077 (1991).</li><li>Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li></ol>

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 gap1,12 Thus...

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 cultures3,4,5,6; 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 healing3,4,7,8. 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.

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Biowest, Nuaill&#233;, France</td>
			<td>L0102-500</td>
			<td>Optional 10 % FBS supplement</td>
		</tr>
		<tr>
			<td>Eagles&#8217;s Minimum Essential Medium (EMEM)</td>
			<td>Lonza</td>
			<td>BE12 -662F</td>
			<td>Optional 10 % FBS supplement</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605E</td>
			<td>Use at 2 mM</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Thermo Fisher Scientific, Waltham, MA USA</td>
			<td>DE17603A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>T4049</td>
			<td>Dilute as appropriate</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>P9155</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS) (10x)</td>
			<td>Gibco by Life Technologies</td>
			<td>14200-067</td>
			<td>Dilute to 1x</td>
		</tr>
		<tr>
			<td>24-well culture plates</td>
			<td>BD FALCON//SARSTED</td>
			<td>734-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>SARSTEDT</td>
			<td>83-3920</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>E9644</td>
			<td>Used 10 ng/mL</td>
		</tr>
		<tr>
			<td>Round cover glass</td>
			<td>MENZEL-GL&#196;SER</td>
			<td>MENZCB00120RA020</td>
			<td>SHORT DEPTH OF FIELD</td>
		</tr>
		<tr>
			<td>Reinforced razor blade no. 743</td>
			<td>Martor (through VWR)</td>
			<td>MARO743.50</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;l sterile aerosol pipet tips</td>
			<td>VWR</td>
			<td>732-0541</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;l sterile aerosol pipet tips</td>
			<td>VWR</td>
			<td>732-0528</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera coupled phase contrast microscope</td>
			<td>Motic Spain</td>
			<td>Moticam camera 2300 3.0 M Pixel USB 2.0; Motic Optic AE31</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>ZEISS Microimaging, Germany</td>
			<td>LSM 510 META</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Culture dish</td>
			<td>BD FALCON</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti c-Jun antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-1694</td>
			<td>Used 1:100</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG (polyclonal goat ) AF 488</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Used 1:400</td>
		</tr>
		<tr>
			<td>Hoechst-33258</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>14530</td>
			<td>Used 1:1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 phalloidin (in methanol) (red)</td>
			<td>Invitrogen</td>
			<td>A12381</td>
			<td>Used 1:100</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2323</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>BD DIFCO</td>
			<td>232100</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health, USA</td>
			<td>Release 1.50i</td>
			<td></td>
		</tr>
		<tr>
			<td>Zen LSM 510 image processing software</td>
			<td>ZEISS Microimaging, Germany</td>
			<td>Release 5.0 SP 1.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

microscopy based methods for the assessment of epithelial cell migration during in vitro wound healing

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.

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...

Watch this Scientific Journal Video about Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing at JoVE.com

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

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.

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

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

The overall goal of these epithelial cell culture artificial wounding procedures is to study cell migration from both a quantitative and qualitive perspective, allowing assessment of the effects of specific treatments of interest on wound healing. This method can help answer key questions in the wound-healing field, as they allow the precise characterization of the roles of specific migration processes during epithelial disruption. The main advantage of this approach is that it allows the correlation of cell migratory measurements with changes in the behavior of relevant molecular markers.Implication of these techniques extend towards a therapy of clinical wounds, as it offers a convenient setup for early drug testing and for wound closure. Generally in to this method will start because of difficulties in obtaining consistent reproduceability in the wounds with an generation. For an artificial wound scratch assay, begin by seeding each well of a culture plate with two milliliters of epithelial cells of interest in complete medium.Enculture the cells at 37 degrees Celsius and five percent carbon dioxide for two to three days until 100%confluency. When the cells are ready, wash the cells two times with fresh serum-free medium followed by 24 hours of culture in fresh serum-free medium. The next day, place the plate in a sterile work area and drag the narrow end of a sterile micro-pipette tip across the bottom of each well two times to generate a cross-shaped wound.A consistent wound gap is achieved by applying constant, firm pressure while smoothly moving the tip along the epithelial surface. Excessive pressure will deform the tip, causing an irregular wound radius. Gently remove the supernatant to discard the detached cells and carefully add fresh serum-free medium to the wells.Using in inverted phase contrast microscope connected to a charge coupled device camera, align the edge of the microscope field with the adjacent intersection of the scratches to obtain up to four pre-treatment reference images of the scratch gap in each well. When all of the images have been acquired, replace the medium in the designated treatment wells with fresh medium, supplemented with selected treatments of interest and return the plates to the cell culture incubator. When at least one condition reaches about 90%scratch gap closure, gently replace the supernatant with one milliliter of four percent formalin in PBS for a 15 minute incubation at room temperature.Then gently wash the cells with PBS at least three times to remove any excess formalin and image the wells as just demonstrated, using the same imaging parameters in the original reference areas captured after scratching. To analyze the images using the appropriate image processing software, open the recorded, pre-treatment image of interest and under the Image menu, set the image type to 8-bit. Under the Process menu, select the Filters sub-menu and apply Variance filtering.Under the Image menu, open the Adjust sub-menu and set the Threshold to black and white. In the Process menu, select the Binary sub-menu and apply Fill Holes. Under the Analyze menu, select Set Measurements and activate Area.Then draw the measurable area, following the migrating edge contour to delimit the gap. Under the Analyze menu, select Analyze Particles and record the total area values for the drawn area surface. To quantify the absolute migration for each set of individual samples as the difference between the gap surface measurements, export the pre and post-treatment total gap surface area values to a spreadsheet and plot the quantification outputs.For an artificial migration front assay, under sterile conditions, add one layer of up to 33 sterilized round coverslips into empty 10 centimeter culture plates. When the plate surface is completely covered, gently seed the epithelial cells of interest at a concentration that allows for 100%confluence after two or three days of culture. If necessary, gently depress the coversips with a sterile micropipette tip to prevent floating.When the cells reach confluency, replace the supernatant with serum-free medium for another 24 hours of culture. Then, use sterile tweesers to carefully transfer one coverslip into a new 10 centimeter plate containing fresh serum-free medium, taking care to hold the coverslip along its periphery. To create the artificial wounds, drag a sterilized razor blade back and forth across each coverslip to make a three to four millimeter transverse line over the center of each cell culture to completely remove the central monolayer strip.Take care to place the razor blade edge heavily across the coverslip surface when making the wound to safeguard against an irregular edge shape and the generation of flopping epithelial portions. Transfer up to four wound coverslips, cell side up, into individual wells of a six well plate, containing at least two milliliters of serum-free medium and gently wash each well two times with fresh serum-free medium to remove any detached cells. When all of the detached cells have been discarded, gently replace the wash medium with fresh medium supplemented with the treatments of interest, and place the plate in the cell culture incubator.At the end of the appropriate experimental period, fix the cells with four percent formalin in PBS as just demonstrated, and stain the cells with the appropriate fluorescent conjugated antibodies of interest. Then image the structural changes occurring at the migrating front edge by fluorescence microscopy according to the experimental parameters of interest. In this experiment, the wound gaps were measured before and after treatment with epidermal growth factor, a well-known inducer of epithelial cell proliferation and migration.The absolute migration was then calculated as the difference between the pre and post-treatment gap surface areas. Treatment with epidermal growth factor potentiates cell cytoskeletal dynamics as observed in these laser scanning microscopy images of F-Actin staining of control and growth factor stimulated cells. Further, c-Jun over-expression is observed with an increased expression of the protein apparent in the cells adjacent to the migrating front.While attempting this procedure, it's important to remember to check the integrity of the wound edges for the presence of epithelial cell flaps that may disrupt the migration quantification. After watching this video, you should have a good understanding of how to both quantitatively and qualitatively assess the migratory behavior of the

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Medicine

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

In this study, we present a real-time imaging method using confocal microscopy to observe cells moving toward damaged tissue by ex vivo incubation with the cochlear epithelium containing the organ of Corti.

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...