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Human skin sample methods and models for validating hypotheses for mechanisms driving skin pigmentation

a skin pigmentation and human skin technology, applied in the field of human skin sample methods and models for validating hypotheses of mechanisms driving skin pigmentation, can solve the problems of inability to account for intrinsic intricate matrices of cells constantly interacting, easy to use single cell types, and difficult to generalize results to human skin

Inactive Publication Date: 2014-11-13
THE PROCTER & GAMBLE COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for developing an ex-vivo human skin model and screening methods to identify agents that can change skin tone, such as lightening agents. This involves culturing human skin tissue and contacting it with a test agent, then analyzing changes in gene expression to identify the effective agent. The invention also provides methods to prepare skin tissue for screening and validate the role of specific genes in driving skin pigmentation. The technical effects include improved precision and accuracy in identifying effective agents for skin toning and increased efficiency in developing new skin care products.

Problems solved by technology

However these relatively simple models often lack much of the intra and intercellular complexities of human skin.
For example, cell cultures of single cell types are easily utilized but overly simplistic and have severe limitations for generalizing results to human skin.
Second, such cultures cannot account for intrinsic intricate matrices of cells constantly interacting as a unit.
Multi-celled cultures are limited due to difficulties in creating the integrated mechanical structure of native tissue and in ensuring that the cells comprise the extracellular components necessary to maintain cellular genetic expression levels at a normal physiological level.
Models that include stem cells treated to mimic human skin are suitable for their intended purposes, but fall victim to similar concerns and limitations.
While having additional complexity, animal models suffer from limitations including the genetic variation with respect to human skin; in the analysis of obtained results there is always a concern that human tissues react differently from animal tissues and the ability to generalize results is compromised.
In addition, extrinsic factors can affect test results with animals, and stressors unrelated to the test agent could also affect results.
Skin-equivalent models are limited by lack of cellular interconnectivity, permeability concerns, and anatomical simplicity.
These models by their very nature are limited in that they can have reduced barrier function that can lead to aberrant sensitivities to tested agents.
In addition the organotypic skin equivalent models are also missing normal skin structures such as glands that can affect skin response.
Previous attempts to utilize ex-vivo human skin as an assay model had limitations such as brief life-spans with low vitality or viability.
Previous attempts at such models included small biopsies of skin floating directly in media, which is not analogous to the normal environment of the skin and resulted in the tissue having a limited lifespan.
However such models are still limited by transiency of the construct, delicacy, and even xenogeneic concerns.
As discussed more fully hereafter, the challenges and uncertainties associated with developing a practical screening method for tone agents are numerous.

Method used

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  • Human skin sample methods and models for validating hypotheses for mechanisms driving skin pigmentation
  • Human skin sample methods and models for validating hypotheses for mechanisms driving skin pigmentation
  • Human skin sample methods and models for validating hypotheses for mechanisms driving skin pigmentation

Examples

Experimental program
Comparison scheme
Effect test

example 1

Skin Preparation

[0106]Individual experiments generally comprise processing of human surgical waste skin used for related ex-vivo (explant) studies. An example embodiment is herein described below:

[0107]Materials for preparation included: Dulbecco's Modified Eagle Medium plus Glutamax, antibiotic and antimycotics, 1×phosphate buffer solution (PBS (-ca, -mg)), 150×15 mm sterile culture dishes, sterile gauze 4×4, disposable safety scalpels, 4 mm disposable biopsy punch, 6-well cell culture inserts, disinfected tweezers, cotton-Tipped Applicators, 6-well culture plates, ruled, disinfected cutting mat (12×12), scraper handle, tissue freezing medium, biopsy cassettes, frozen tissue freezing vessels, 10% formalin, shipping containers for fixed tissue, and biohazard / sharps containers.

[0108]Skin preparation involved preparation of media in advance of the arrival of skin (3×media including 500 ml DMEM plus 15 ml of antibiotic and antimycotics; The 3× referred to 3-fold levels of antibiotics / a...

example 2

Culturing

[0111]An example embodiment of culturing using ex-vivo skin is herein described below:

[0112]Culturing involved setting up culture plates: putting 2.5 ml of media or treatment into each well of a 6-well tissue plate, adding one Millicell culture insert to each well, and putting 100 μl of 1×DMEM on the center of each insert membrane. Squares were selected randomly placed into each of the inserts (one piece of skin per insert), on top of the media on the membrane. The plates were stored at either 33° C. (though an additional non-limiting example would be 37° C. depending on the experiment endpoint). As a control, baseline tissue measurements were collected from excess waste (cryogenics were used for snap freezing). Baseline biopsy collection involved two 4 mm punches for MTT; One 4 mm punch was snap frozen in OCT for fresh histology; One 4 mm punch was fixed in 10% formalin and sent out for paraffin embedding. PCR baseline was also be taken with six 4 mm punches snap frozen in...

example 3

mRNA Processing, Nanodrop-Quantification and Purity Assessment, RNA Quantification Gel Protocol, cDNA Synthesis. and PCR Setup mRNA Processing

[0119]mRNA was processed for the ex-vivo skin methods. An example embodiment is herein described below:

[0120]For cryopreparation and extraction the following equipment was obtained and prepared: 2 ml round bottom tubes were prepared with 1 ml of TRIZOL as was a 5 mm stainless steel bead for each sample; a dry ice bucket was obtained and liquid nitrogen was placed in the bucket. Biopsy punches were maintained on dry ice, and samples remained frozen. Cryobags and covaris cryoprep were used in freeze fracturing of samples. To freeze fracture the samples, biopsies were placed in a cryo bag (2 per sample) and dipped in liquid nitrogen, then placed in cryoprep (setting 4). The sample bag was taken out and dipped back into nitrogen. A flattened disk was removed and placed into corresponding 2 ml tubes with TRIZOL. The disk was kept frozen in the TRIZ...

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Abstract

Human skin tissue sample methods and models for validating hypotheses for mechanisms driving skin pigmentation as well as methods for driving skin pigment levels in ex-vivo skin tissue. The method includes providing a first cultured human skin tissue sample and testing the effectiveness of the test agent for driving skin pigmentation, and comparing the transcriptional profile generated from the treated human skin tissue sample to the transcriptional profile of a control to validate the hypothesis that the test agent drives skin pigmentation.

Description

TECHNICAL FIELD[0001]One aspect of the invention relates to novel ex-vivo skin models and screening methods for validating hypotheses for mechanisms driving skin pigmentation.BACKGROUND OF THE INVENTION[0002]Skin pigmentation levels and regulation are determined and controlled by a complex system of interrelated genes, proteins and a milieu of intracellular and extracellular compounds, some of which have yet to be elucidated. Skin models capable of mimicking aspects of cellular processes integral to the skin pigmentation pathway(s) are therefore desired in order, for example, to identify skin-active agents effective in modulating skin pigmentation.[0003]Modeling techniques to study skin physiology and skin responses to agents have historically included a variety of specific techniques, from the culturing of a single cell type or a small number of co-mingled cell types, to fabricating human tissue equivalents, to developing animal models. However these relatively simple models often ...

Claims

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Application Information

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IPC IPC(8): G01N33/50C12Q1/68
CPCG01N33/5023C12Q1/6876G01N33/5088G01N33/5044C12Q2600/136G01N33/5082G01N2500/10
Inventor FINLAY, DEBORAH RUTHHAKOZAKI, TOMOHIRO NMNBASCOM, CHARLES CARSONMATHENY, HEATHER EILEEN
Owner THE PROCTER & GAMBLE COMPANY
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