The authors have no acknowledgements.

An exempt approval (3002-13) to carry out research using de-identified tissue samples pursuant to the Department of Health and Human Services regulations, 45 CFR 46.101(b)(4) was granted. Full thickness skin is received from elective surgery. In this article, the tissue source is 66-year-old Caucasian female breast.
1. Preparation of Elastomer Coated Coverslips
<ol>
	<li>In a 20 mL glass vial, mix 0.107 g of Sylgard 184 curing agent with 5.893 g base. The total mixture mass is 6 g with a base to curing agent ratio of 55:1.</li>
	<li>After mixing with a glass rod to ensure homogeneity, place the glass vial in a vacuum chamber and degas to remove all bubbles.</li>
	<li>Place a glass cover-slip (55 mm x 25 mm) in the center of a spin coater. Add ~1 mL of the mixture onto the center of the cover-slip. Use a 5,000 &#181;L pipette with the end cut off with scissors. Spin coat the cover-slip at 2,000 rpm for 60 s.
	<ol>
		<li>Repeat this process to create 5-6 substrates.</li>
	</ol>
	</li>
	<li>Cure the cover-slips in an oven for 12 h at 60 &#176;C.</li>
	<li>Use a razor blade to partially remove the elastomer film from a sacrificial substrate. Use an indelible marker to mark the topside of the elastomer film and the exposed glass.</li>
	<li>Mount the sample on an inverted microscope and use a remote focus accessory to record the difference in z-height between the focal planes of the two marks. This corresponds to the elastomer substrate thickness, h.</li>
</ol>
2. Preparation of the Stratum Corneum
<ol>
	<li>Use a water bath or heated stir plate to heat a glass beaker half filled with deionized water (DW) to 60 &#176;C. Inside a biological safety cabinet, immerse the full thickness human skin in the water for 4 min.</li>
	<li>Immediately transfer the skin sample into a beaker containing DW cooled to &#60;10 &#176;C for 4 min. Half filling the beaker minimizes splashing of biohazardous material.</li>
	<li>Remove the skin from the beaker, place in a Petri dish, and gently isolate the epidermis using a pair of bent nosed tissue tweezers.</li>
	<li>Place the isolated epidermis basal side down in a Petri dish lined with gauze. Ensure the basal layer is fully in contact with the gauze.
	<ol>
		<li>Soak the gauze in a 0.25% (wt/vol) type IX-S porcine pancreas trypsin solution dissolved in 0.1 M phosphate buffered saline for 6-8 h at room temperature. Add only enough trypsin in the container to wet the gauze.</li>
	</ol>
	</li>
	<li>Lift the gauze with tissue tweezers and float it in a container partially filled with DW. Gently pull the SC to separate it from the gauze.</li>
	<li>Wash the stratum corneum 3-4 times in DW to remove residual epidermal tissue that remains attached to the SC.</li>
	<li>Float the isolated SC in a solution of 0.4% glycine max(soybean) trypsin inhibitor in DW. Use a plate shaker to agitate the tissue for 10 min.</li>
	<li>Float SC in a Petri dish partially filled with DW. Use a plate shaker to agitate the tissue for 10 min.</li>
	<li>Dry the isolated SC sheet on an ultra-fine plastic mesh for 48 h at room temperature (25 &#176;C, 40% relative humidity (R.H.)).</li>
	<li>Separate the SC from the mesh and cut out individual circular R=3 mm radius samples using a circular hole punch. Mark the center of the outmost face with a small spiral mark using an indelible marker. This provides a visual cue for recognizing the topside of the SC. 
	NOTE: The indelible mark should be applied in the center of the sample, where the drying deformations will be smallest. This will minimize the impact of the marker on recorded drying displacement profiles.</li>
</ol>
3. Sample Treatment and Deposition
<ol>
	<li>Agitate SC samples for 30 min in 15 mL DW containing 90 &#181;L fluorescent marker beads (505/515 nm, 1 &#181;m diameter, carboxylate-modified). This deposits beads onto the SC surface 
	NOTE: While deposition of large numbers of beads on the SC may marginally slow drying relative to samples without beads present12, it will maximize the spatial resolution of the in-plane deformation fields that can be subsequently obtained. The choice of bead volume added should therefore be made ad hoc.</li>
	<li>Remove SC samples and place them in a Petri dish partially filled with DW.</li>
	<li>Partially immerse a substrate in the DW at a shallow angle of 15-30&#176;.</li>
	<li>Pin an edge of the floating SC sample at the contact line between the substrate and water interface. Vertically withdrawing the substrate from the water will smoothly laminate the SC sample to the substrate without wrinkles or entrapped air bubbles.</li>
	<li>Repeat step 3.4 to place up to 6 SC samples onto each substrate. Leave at least a 2-3 mm gap between samples and avoid sample lamination close to the substrate edge. This prevents drying of one sample influencing drying displacements in another.</li>
	<li>Dry the mounted SC samples in laboratory conditions for 60 min. This allows residual water between the SC and substrate to evaporate and ensures complete tissue adhesion. 
	NOTE: At this juncture, SC samples can be treated with a chemical or cosmetic formulations7,8 by placing the substrates upside down in a desired solution for a requisite period of time. Repeat step 3.6 once the treatment step is performed. After drying, incomplete adhesion of SC samples to the substrate can be verified using transmitted light microscopy. Trapped bubbles under the SC sample or delaminated edges will form clear contrast variations in the sample with well-defined edges.</li>
	<li>Create a humidity chamber by placing a Petri dish partially filled with water into a hermetically sealed container.</li>
	<li>Place substrates into the chamber for 24 h to equilibrate to a relative humidity of 99%. Do not place the substrates into the Petri dish.</li>
</ol>
4. Microscope Environmental Control
<ol>
	<li>Achieve control of environmental conditions through a humidity control system connected to a microscope mountable perfusion chamber. Details of the humidity control system are provided in German et al. (2013)7 and Liu and German (2015)8.</li>
	<li>Mount the substrate on the microscope, place the perfusion chamber over the substrate and seal edges of perfusion chamber to the elastomer using vacuum grease.</li>
	<li>Once mounted, equilibrate internal air to 99% R.H. prior to experimentation. This prevents evaporation of water prior to experimentation. Once imaging in sections 5 or 7 has begun, reduce internal air humidity to desired value. 
	NOTE: In this article, SC samples are dried to 25% R.H.</li>
</ol>
5. Imaging in Plane Drying Displacements
<ol>
	<li>Acquire images of SC samples using an inverted microscope with 1X objective lens. Excite fluorescent beads using a light engine with FITC filter (503-530 nm emission bandpass). Multiple samples can be imaged sequentially throughout drying using an automated x-y stage.</li>
	<li>Record fluorescent and transmitted light images using a digital CCD camera at a resolution of 1,392 x 1,040 pixels. The field of view of each image is 8.98 x 6.71 mm, allowing a single image to capture a full SC sample. Take images with a frequency of 10 min for 16 h.</li>
</ol>
6. Substrate Preparation for Thickness Measurement
<ol>
	<li>In a chemical fume hood, place 1 mL silane (3-aminopropyltriethoxysilane; &#8805;98%) in some small plastic cap. Place elastomer substrates from section 1 and the cap in a sealed container for 5 h. Do not allow substrates to come directly in contact with the silane.</li>
	<li>Add 5 mg of EDC (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; &#8805;99%) in a 1.5 mL tube. Add 500 &#181;L of DW to the EDC. Agitate the solution for 10 s with a vortex mixer.</li>
	<li>Add 0.076 g sodium tetraborate and 0.1 g boric acid to 20 mL DW. Mix using a magnetic stirrer at 70 &#176;C (1 h). Add boric acid until the pH is 7.4.</li>
	<li>Add 20 mL of borate buffer to a 50 mL centrifuge tube. Add 60 &#181;L of 1 &#181;m beads (535/575 nm, carboxylate-modified) to the borate buffer. Finally, add 200 &#181;L of EDC solution to bottle. Shake the tube to mix bead solution and then pour into a 10 cm diameter Petri dish.</li>
	<li>Remove the silanated substrates from the container and place them elastomer film-side down into the bead solution. Do so slowly to prevent bubbles from becoming trapped. Two substrates will fit in each Petri dish.</li>
	<li>Leave substrates to float in the bead solution for 45 min.</li>
	<li>Use tweezers to remove the substrates from the bead solution, then rinse in DW to remove unbound beads.</li>
	<li>Air dry the substrates. Blowing compressed air over the elastomer film surface reduces the formation of water spots.</li>
	<li>Seal the substrates in an opaque box to prevent photo-bleaching of the beads until SC sample deposition.</li>
</ol>
7. Imaging Thickness of SC
<ol>
	<li>Deposit SC samples on to a substrate using section 3. However, perform step 3.1 without adding fluorescent beads to the DW. Additionally, apply a 5 &#181;L drop of undiluted fluorescent marker bead solution (505/515 nm, 0.1 &#181;m diameter) to the surface of each deposited SC sample with a pipette before completing step 3.6.</li>
	<li>Establish measurements of SC thickness using the microscope with 40X objective lens. Measure the thickness of SC samples over time using a remote focus accessory to record the difference in z-height between the two bead layer focal planes located at the SC-substrate interface and the topside of the SC.</li>
	<li>Measure the thickness of 3 regions of each SC sample over a 3 h drying period. The thickness of SC samples reaches a steady state value within this time frame8.</li>
</ol>
8. Quantifying and Modeling Tissue Deformation
<ol>
	<li>Use particle image velocimetry13 to obtain spatially resolved in-plane drying displacements from the fluorescent images at each recorded time step.</li>
	<li>Use MATLAB to obtain azimuthally averaged radial and azimuthal displacement profiles from the displacement field of each radially symmetric SC sample. 
	NOTE: An example dataset (entitled &#39;d.mat&#39;) and MATLAB code (entitled &#39;PIV-processing.m&#39;) that performs both this step and step 8.3 has been provided in the supplemental information.</li>
	<li>Fit radial displacement profiles to a model7,8,14,15,16 describing drying SC as a shrinking linear elastic circular disk of time varying thickness, hSC, radius, R, and elastic modulus, ESC, adhered to a deformable elastic substrate with elastic modulus, E. Assume SC has a well-defined and constant Poisson&#39;s ratio, &#957;SC = 0.47,8. Obtain best fits using a minimum least squares approach. 
	NOTE: The model used for fitting describes radial displacements in terms of modified Bessel functions are: 
	<img alt="Equation" src="/files/ftp_upload/55336/55336eq2.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (1) 
	with <img alt="Equation" src="/files/ftp_upload/55336/55336eq3.jpg" />,&#160;<img alt="Equation" src="/files/ftp_upload/55336/55336eq4.jpg" /> and <img alt="Equation" src="/files/ftp_upload/55336/55336eq13.jpg" /> 
	The term <img alt="Equation" src="/files/ftp_upload/55336/55336eq6.jpg" /> corresponds to a penetration depth given by, 
	<img alt="Equation" src="/files/ftp_upload/55336/55336eq7.jpg" />. 
	<img alt="Equation" src="/files/ftp_upload/55336/55336eq8.jpg" /> denotes a substrate rigidity parameter; valid when sample sizes are much greater than the substrate thickness. Here, the parameters, <img alt="Equation" src="/files/ftp_upload/55336/55336eq9.jpg" /> and <img alt="Equation" src="/files/ftp_upload/55336/55336eq10.jpg" /> respectively denote the substrate thickness Poisson&#39;s ratio. The Poisson&#39;s ratio of the silicone elastomer substrate17 is &#957; = 0.5.</li>
	<li>Obtain model parameters &#945; and &#946; at each time step from the least squares fit of Equation (1) to the radial displacement profile.
	<ol>
		<li>Employ the fitting parameter &#946; to obtain SC elastic modulus, ESC, using the expression, 
		<img alt="Equation" src="/files/ftp_upload/55336/55336eq11.jpg" /></li>
		<li>Use fitting parameter &#945; to obtain the time varying contractile drying stress, PSC, using the expression, 
		<img alt="Equation" src="/files/ftp_upload/55336/55336eq12.jpg" /></li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In this article, we describe a technique that can be used to measure the dynamic drying behavior and mechanical properties of human SC. Previous studies have demonstrated that this technique can be used to quantify the effects of environmental conditions and chemical products commonly used in cosmetic cleansers and moisturizers on the dynamic drying behavior of SC7,8. There are a number of key steps in the protocol. Firstly, SC swells notably with water content; therefore, measurements of SC thickness as well as in-plane displacements are essential for accurately predicting the elastic modulus and drying stress magnitude. Secondly, samples need to be fully adhered to the substrate. Incomplete adhesion, non-radially symmetric samples or samples with small tears or holes should be avoided because they will significantly impact the distribution of drying deformations and the radial displacement profiles used for model fitting.
The technique can be used if a humidity control system is unavailable. Without environmental control, tissue samples will dry in laboratory conditions12. As such, the laboratory environment should be continuously monitored and maintained, as drying behavior and the repeatability of results will be impacted by both diurnal and seasonal variations in temperature and humidity.
Currently, the technique is limited only to samples that can adhere to the substrate and induce deformations within the elastomer film. While the technique can be readily adapted to test samples that undergo smaller in-plane displacements, by reducing the substrate elastic modulus12, results from samples that simply slip over the substrate will lack meaning.
Numerous in-vivo and ex-vivo techniques that can assess the drying behavior and mechanical properties of SC have been reported3,8,9,10,18,19,20,21. However, in-vivo techniques cannot fully distinguish mechanical changes in SC from the underlying epidermal and dermal layers. Moreover, ex-vivo techniques can typically only assess one sample per experiment. The method we report in this article allows up to 6 SC samples to be assessed per experiment. The size of the substrate and environmental chamber however could be scaled up to allow more samples to be assessed simultaneously. We estimate for n=6 SC samples, a timescale of ~13 h is required for preparation and testing, excluding substrate curing and tissue equilibration. In comparison, we estimate uniaxial tensometry testing would require more than twice this period. Significantly less SC tissue is also required per individual sample (0.28 cm2) in comparison with those required for tensometry9 (2.5 cm2). This technique further enables more physiologically relevant drying by preventing evaporation from the underside of the SC tissue. In addition to assessing drying behavior and mechanics in SC, we believe this technique could also be applied to studies of polymeric or colloidal systems that form a cohesive film upon drying.

The outer most layer of the epidermis, or stratum corneum (SC) consists of cohesive corneocyte cells surrounded by a lipid rich matrix1,2. The composition and structural integrity of SC is essential for maintaining correct barrier functionality3, which prevents invasion from microorganisms and resists both mechanical forces and excessive water loss4. The capacity of personal care products to maintain or degrade skin barrier function is of great interest to skin healthcare and the cosmetic industry5. The daily application of personal care products is known to alter the mechanical properties of the SC6,7,8. For example, surfactants contained in cosmetic cleansers can cause significant increases in the elastic modulus and a build-up of drying stresses in SC, increasing the tissue&#39;s propensity to crack7,9. Glycerol contained in nearly all cosmetic moisturizers can soften SC and decrease the build-up of drying stresses8,10,11, reducing the likelihood of tissue rupture.
The method detailed in this article is capable of quantifying the dynamic drying behavior and mechanical properties of SC drying in controlled environments7,8. Previously, this technique has been demonstrated to be capable of elucidating the effect of different cosmetic products on changes in the dynamic drying behavior and mechanical properties of SC tissue. This is achieved by quantifying drying-induced shrinkage of human SC tissue adhered to a soft elastomer substrate, fitting drying displacements with a simple contractility model, and then extracting the elastic modulus and drying stress from the fitted profile. When testing of multiple SC samples is required, this method offers a more rapid alternative to uniaxial tensometry, utilizes significantly less tissue and provides more physiologically relevant drying by preventing evaporation from the sample underside.

<table><tbody><tr><td>Silicone elastomer base</td><td>Dow-Corning</td><td>1064291</td><td></td></tr><tr><td>Silicone elastomer Curing Agent</td><td>Dow-Corning</td><td>1015311</td><td></td></tr><tr><td>FluoSpheres Carboxylate 0.1 &amp;micro;m yellow green fluorescent 505/515&amp;nbsp;</td><td>Thermo Fisher</td><td>F8803</td><td></td></tr><tr><td>FluoSpheres Carboxylate 1 &amp;micro;m yellow green fluorescent 505/515&amp;nbsp;</td><td>Thermo Fisher</td><td>F8823</td><td></td></tr><tr><td>FluoSpheres Carboxylate 1 &amp;micro;m nile red fluorescent 535/575&amp;nbsp;</td><td>Thermo Fisher</td><td>F8819</td><td></td></tr><tr><td>Trypsin from porcine pancreas</td><td>Sigma-Aldrich</td><td>T6567</td><td></td></tr><tr><td>Trypsin inhibitor type II-s</td><td>Sigma-Aldrich</td><td>T9128</td><td></td></tr><tr><td>(3-aminopropyl)triethoxysilane</td><td>Sigma-Aldrich</td><td>440140</td><td></td></tr><tr><td>Sodium tetraborate</td><td>Sigma-Aldrich</td><td>221732</td><td></td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B0294</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>P7059</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(3-Dimethylaminopropyl)-&lt;em&gt;N&lt;/em&gt;-ethylcarbodiimide hydrochloride</td><td>Sigma-Aldrich</td><td>E7750</td><td></td></tr><tr><td>Vortexer mixer</td><td>VWR</td><td>58816-123</td><td></td></tr><tr><td>6 mm diameter hole punch</td><td>Sigma-Aldrich</td><td>Z708860</td><td></td></tr><tr><td>SOLA 6-LCR-SB&amp;nbsp;</td><td>Lummencor light engine</td><td>No.3526</td><td></td></tr><tr><td>Cfi Plan Achro Uw 1X Objective</td><td>Nikon Plan UW</td><td>MRL00012</td><td></td></tr><tr><td>CFI Plan Fluor 40X Oil Objective 1.3 na - 0.20&amp;nbsp;mm wd</td><td>Nikon Plan Fluor</td><td>MRH01401</td><td></td></tr><tr><td>Nikon Eclipse Ti-U inverted microscope&amp;nbsp;</td><td>Nikon</td><td>MEA53200</td><td></td></tr><tr><td>Clara-E Camera</td><td>Andor</td><td>DR-328G-C02-SIL</td><td></td></tr><tr><td>Remote Focus Attachment E-RFA Ergo Design</td><td>Nikon</td><td>99888</td><td></td></tr><tr><td>Ti-S-E Motorized Stage</td><td>Nikon</td><td>MEC56110</td><td></td></tr></tbody></table>

measuring and modeling contractile drying in human stratum corneum

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both cleansing agents and daily variations in ambient moisture; both of which can alter the water content of the tissue. Reductions in water content from severe barrier dysfunction or low humidity environments can alter SC stiffness and cause a build-up of drying stresses. In extreme conditions, these factors can cause mechanical rupture of the tissue. We have established a high throughput method of quantifying dynamic changes in the mechanical properties of SC upon drying. This technique can be employed to quantify changes in the drying behavior and mechanical properties of SC with cosmetic cleanser and moisturizer treatments. This is achieved by measuring dynamic variations in spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. In-plane radial displacements acquired during drying are azimuthally averaged and fitted with a profile based on a linear elastic contractility model. Dynamic changes in drying stress and SC elastic modulus can then be extracted from the fitted model profiles.

Figure 1(a) shows a representative fluorescent image of an SC sample coated with fluorescent beads (section 3). The corresponding transmitted light image of the sample is shown in Figure 1(b) overlaid with a quiver plot of spatially resolved drying displacements that form after 16 h drying at 25% R.H. Due to the circular symmetry of the samples, these displacements can be azimuthally averaged. Figure 1(c) shows radial (ur, solid red line) and azimuthal (u&#952;, dashed blue line) displacement profiles plotted against the dimensionless radial position, r/R. Here, R denotes the mean SC sample radius, r/R = 0 denotes the sample center and r/R = 1 denotes the edge. Standard deviations at each radial position are denoted by the shaded regions around the mean. These variations are primarily caused by the structural heterogeneity of the SC3,7,12. Throughout drying, azimuthal displacements remain small. Radial displacement profiles however increase monotonically from center to edge and grow in magnitude until an equilibrium is reached.
<img alt="Figure 1" src="/files/ftp_upload/55336/55336fig1.jpg" /> 
Figure 1: Circular SC sample (6.2 mm diameter) adhered to an elastomer substrate with elastic modulus E = 16 &#177; 1 kPa after drying for 15 h in a 25 &#177; 1% R.H. environment. (a) Fluorescent image of the SC sample highlighting the deposited fluorescent marker beads used for tracking spatially resolved in-plane drying displacements. (b) Quiver plot of spatially resolved in-plane drying displacements overlaid on a transmitted light image of the SC sample. (c) Azimuthally averaged radial (ur, solid red line) and azimuthal (u&#952;, blue dashed line) displacements of the sample plotted against dimensionless radial position, r/R. Positive values of ur correspond to contractile displacements. Shaded regions surrounding the lines indicate the standard deviation about the mean at each radial position. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55336/55336fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Profiles recorded at 30 min intervals are plotted in Figure 2(a) and show the time evolution of in-plane displacements. The average SC thickness, hSC, is plotted in Figure 2(b). Decreases of SC during drying primarily occur over the first 2 h.
<img alt="Figure 2" src="/files/ftp_upload/55336/55336fig2.jpg" /> 
Figure 2: (a) Overlay of radial displacement profiles (ur, solid red lines) at 30 min intervals over a 15 h drying period in 25% R.H. conditions plotted against dimensionless radial position r/R for a typical SC sample. Positive values of ur correspond to contractile displacements. (b) Average SC sample thickness, (hSC, n=3), plotted against drying time. (c) Radial displacement profiles from (a) overlaid with minimum least squares fits (blue dashed line) of Equation (1) for the first and last recorded radial displacement profile. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55336/55336fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Fitting displacement profiles with the linear elastic contractility model described by Equation (1) provides further insight into the mechanical properties of drying SC. Displacement profiles at each time step are fitted with the model using a minimum least squares approach, as shown in Figure 2(c). The contractile drying stress, PSC, and elastic modulus, ESC, are subsequently extracted from the model at each time step. Average changes in these parameters (based on 3 individual SC samples) are shown respectively in Figures 3(a) and 3(b). Both parameters increase rapidly over the first 2 h drying period and reach a plateau within 5 h.
<img alt="Figure3" src="/files/ftp_upload/55336/55336fig3.jpg" /> 
Figure 3: (a) Averaged SC elastic modulus, ESC, plotted against drying time over a 15 h period. (b) Average contractile drying stress, PSC, plotted against drying time over a 15 h period. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55336/55336fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measuring and Modeling Contractile Drying in Human Stratum Corneum at JoVE.com

Measuring and Modeling Contractile Drying in Human Stratum Corneum

This article describes a method of quantifying the dynamic drying behavior and mechanical properties of stratum corneum by measuring spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. This technique can be used to measure how different chemical treatments alter drying and tissue mechanical properties.

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both ...

measuring-and-modeling-contractile-drying-in-human-stratum-corneum

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

6.6K Views.  Binghamton University. This article describes a method of quantifying the dynamic drying behavior and mechanical properties of stratum corneum by measuring spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. This technique can be used to measure how different chemical treatments alter drying and tissue mechanical properties.

Video: Measuring and Modeling Contractile Drying in Human Stratum Corneum

Resolving Water, Proteins, and Lipids from In Vivo Confocal Raman Spectra of Stratum Corneum through a Chemometric Approach

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in human subjects. This information is very important for skin-related diseases and characterizing skin care product performance. This protocol illustrates a method for confocal Raman spectra collection and the subsequent analysis of the spectral dataset leveraging chemometrics. The goal of this method is to establish a standard protocol for data collection and provide general guidance for data analysis. Preprocessing (e.g., removal of outlier spectra) is a critical step when processing large datasets from clinical studies. As an example, we provide guidance based on prior knowledge of a dataset to identify the types of outliers and develop specific strategies to remove them. A principal component analysis is performed, and the loading spectra are compared with spectra from reference materials to select the number of components used in the final multivariate curve resolution (MCR) analysis. This approach is successful for extracting meaningful information from a large spectral dataset.

Here, we present a protocol for collection of confocal Raman spectra from human subjects in clinical studies combined with chemometric approaches for spectral outlier removal and the subsequent extraction of key features.

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in ...

Chemistry

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

Efficient 3D Skin Models for Flexible Research Application

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

Author Spotlight: Enhancing Skin Model Diversity with Cost-Effective 3D Cellular Models

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

Medicine

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Neuroscience

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This paper describes a protocol for assessing changes in contractility in adult human primary ventricular cardiomyocytes utilizing the MyoBLAZER, a non-invasive optical method that preserves the normal physiology and pharmacology of the cells. This optical recording method continuously measures contractility transients from multiple cells in parallel, providing both medium-throughput and valuable information for each individual cell in the field of view, enabling the real-time tracking of drug effects. The cardiomyocyte contractions are induced by paced electrical field stimulation, and the acquired bright field images are fed to an image-processing software that measures the sarcomere shortening across multiple cardiomyocytes. This method rapidly generates different endpoints related to the kinetics of contraction and relaxation phases, and the resulting data can then be interpreted in relation to different concentrations of a test article. This method is also employed in the late stages of preclinical development to perform follow-up mechanistic studies to support ongoing clinical studies. Thus, the adult human primary cardiomyocyte-based model combined with the optical system for continuous contractility monitoring has the potential to contribute to a new era of in vitro cardiac data translatability in preclinical medical therapy development.

This protocol describes how to measure contractility in adult human primary cardiomyocytes from donor hearts with the MyoBLAZER system, a reliable platform for assessing drug-induced changes in contractility during preclinical development.

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This ...

Measuring and Modeling Contractile Drying in Human Stratum Corneum

Summary

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Chapters in this video

Resolving Water, Proteins, and Lipids from In Vivo Confocal Raman Spectra of Stratum Corneum through a Chemometric Approach

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes