GB2442271A - In vitro human skin test assay protocol - Google Patents
In vitro human skin test assay protocol Download PDFInfo
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- GB2442271A GB2442271A GB0619005A GB0619005A GB2442271A GB 2442271 A GB2442271 A GB 2442271A GB 0619005 A GB0619005 A GB 0619005A GB 0619005 A GB0619005 A GB 0619005A GB 2442271 A GB2442271 A GB 2442271A
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- 238000003556 assay Methods 0.000 title claims abstract 3
- 238000000338 in vitro Methods 0.000 title abstract description 7
- 210000003491 skin Anatomy 0.000 claims abstract description 34
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0629—Keratinocytes; Whole skin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
- C12N5/0698—Skin equivalents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
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Abstract
An in vitro human skin test assay protocol is described wherein a colony of epidermal cells forming an island of such cells is produced from an explant of human skin tissue which has been treated with the enzyme Dispase to enable the removal of almost all the epidermal layer. Said island of cells develops from remaining epidermal cells and "autoengineers" itself to form a round multilayer structure. Laboratory tissue culture techniques using the natural cellular interactions between human epidermal and dermal cells of the skin is described. Said protocol can be used to determine whether a radioisotope, element, molecule or compound or any mixture thereof, applied to the skin topically, transdermally, or subcutaneously in solid, gaseous, liquid form or as a cream causes any effect on skin cellular architecture.
Description
An in vitro human skin assay test protocol which directly uses the
natural cellular interactions between un-manipulated autologous human epidermal and dermal cells.
Abstract An in vitro means of testing for indications of irritation or damage to human skin is described which employs the natural cellular interactions between epidermal and dermal cells and precludes any in vivo animal use. Said protocol has wide commercial applications besides the study of the structure of human skin.
Background. Scientific assessments of the skin irritation/damage potential of chemicals, cosmetics, gases, radiation, topical vehicles, electrical impulses are presently conducted by using laboratory reconstructed human epidennal' and dermal models (Epiderm)2. Such epidermal and dermal models are manufactured and sold e.g. by SkinEthic and Episkin, both subsidiary companies owned by L'Oreal, Paris, France. The common skin irritation protocol provides guidelines for these tests.
Commonly, these assessments can involve in vivo animal testing.
The skin is the largest organ in the human body and consists of the epidermis containing keratinocytes, melanocytes, Langerhans cells. The underlying dermis separated from the epidermis by a basement membrane area, is subdivided into two zones, the papillary dermis and the reticular dennis. Papillary fibroblasts are tissue cultured under laboratory conditions from skin dermatomed to a depth of 0.3 mm and reticular fibroblasts at a depth below 0.7 mm (see Fig 1 in Sorrell & Caplan 2004).
Tissue culture of cells is done in specially equipped sterile laboratories. Tissue culture flasks, dishes or multi-well plates and a specific nutrient solution (a culture medium) are employed. However, in the initial isolation and passaging (sub-culturing) of both epidermal keratinocytes and dermal fibroblasts from pre-screened skin tissue, researchers have employed enzymes, trypsin for keratinocytes, collagenase A for dermal fibroblasts. Trypsin continues to be used in spite of Barton & Marks (1981) report of changes (invagination of desmosomes, vacuolation, and redistribution of tonoflbnls) in suspensions of human keratinocytes directly attributed to this proteolytic enzyme. Furthermore, Krejci et al. (l99l) reported that human papillary dermis that has been treated by trypsin lacked laminin and collagen type IV in the basement membrane zone and supports keratinocyte attachment and differentiation less well. Trypsin or collagenase may not isolate a representative population of constitutive dermal fibroblasts from both the papillary and reticular dermis of the skin tissue even if the dermis were finely minced.
Dispase, on the other hand, is a neutral1rotease which is both a fibronectinase and type IV collagenase (Stenn et al. 1989) , dissolving the attachments between the basal keratinocytes and the basement membrane, without disturbing the desmosomal intercellular junctions between adjacent cells (Green 1991)7 This invention will describe a test protocol using an in vitro model comprised of normal human skin cells which will visibly show the effects of among other things, skin irritants and do so in a manner which will preclude (a) any doubts about the test results (b) Will not involve animal tests. There is no test described in the scientific literature which employs the cellular interaction between un-manipulated epidermal and dermal cells under tissue culture conditions as described in this invention.
Invention In a tissue culture laboratory, a segment of pre-screened human skin tissue (fresh, or cadaver within 24 hours of death) will be aseptically dissected of all subcutaneous fat and inverted (that is, epidermis side facing down) in a pool of an enzyme named Dispase6 contained in a plastic tissue culture dish. The dish will be incubated (5% 02/95%C02) at 37 degrees Centigrade for a period of 12-16 hours. The skin tissue is removed and subjected to intense rinsing by gently swirling in a buffered saline solution followed by further rinsing with culture medium. The epidermis is almost completely stripped off with a pair of tweezers, leaving behind a speck of undisturbed epidermis. The dermis is mechanically scraped with 5 scrubbing strokes with the tip of a surgical scalpel blade avoiding contact with the speck of undisturbed epidermis.
The use of a dissection microscope (or magnifying glass) is recommended. The skin tissue is now lifted with a pair of tweezers with protective plastic tips, inverted and gently swabbed' onto a small area of the culture dish's substrate surface just beyond the point where the skin tissue will be placed.
A classic explant tissue culture procedure will be set up in a culture dish with the speck of epidermis skin tissue facing down in a volume of culture (nutrient) medium.
The culture medium is not changed but topped up' every 2-3 days to replace that amount of medium lost by evaporation. A phase contrast microscope will be needed to make certain specific observations.
The swabbing of the dish substrate will cause dermal flbroblasts to grow out as well as dermal flbroblasts will migrate away from the explant skin tissue and become established on the culture dish substrate. These will be the only cells observed in culture after a week of incubation. There will be an outgrowth of two distinct morphological types of dermal fibroblasts. Dermal fibroblasts with cell-cell interlinking tubular connectors' will organize first. This morphologic type of fibroblast is observed only in explant cultures of dermal skin tissue. The other type of dermal flbroblast, the more common type observed in primary cultures, subcultures and after enzyme digestions, will consist of a master' fibroblast with a thin lengthy proboscis' to which other fibroblasts with much shorter proboscis connected almost at right angles. There will be a preponderance of reticular fibroblasts which can be morphologically distinguished from papillary fibroblasts (see Figure 6 in SchOnherr et al. 1993) Subsequently, a colony of epidermal cells will be observed to move off of the dermal surface on to the plastic plate substrate (Fig. 1). At the leading end of the colony, cell attachment will be observed as migration concomitantly occurs (Fig. 2). Within 2-3 days, the dermal fibroblasts will retreat completely to one side leaving an open area for this advancing colony. (Fig. 3). After another 2-3 days, the epidermal cell colony will autoengineer' a rounded multi-layered island growing away from the dermal tissue (Fig. 4). This multi-layered island Will then move in the direction of the retreating fibroblasts.
Test protocol Test material may be introduced at various stages of the above tissue culture procedure. After the initial dermal fibroblast outgrowth, test material may be introduced Keep the dish incubated to make observations as to whether the epidermal colony will migrate from the explant skin tissue. If this occurrence does not manifest, there is a deleterious effect caused by the test material.
Alternatively, after the epidermal colony has migrated, test material may be introduced. If the multilayered autoengineered' round colony does not subsequently form or it displays a structural deformed' configuration, it can be assumed that the test material is causing a deleterious effect. This autoengineered' epidermal colony may be raised to the air-liquid interface as a means of carrying out further testing.
Of course, any material, gas or radiation causing a disruption of the collective structural integrity of the epidermal and dermal cells on the dish substrate will be indicative of a deleterious effect.
Scientific References 1Kandarova H., Liebsch M., Sthmidt E., Genschow E., Traue D., Spielman I-I., Meyer K., Steinhoff C., Tornier C., Dc Wever B.& Rosdy M. (2006) Assessment of the skin irritation potential of chemicals by using the SkmEthic reconstructed human epidermal model and the common skin irritation protocol evaluated in the ECVAM skin irritation validation study. Altern. Lab. Anim. 34(4):393-406.
2Ponec M., Boelsma E., Gibbs S.& Mommaas M. (2002) Characterization of reconstructed skin models. Skin Phannacol. Appi. Skin Physiol. 15 Suppi 1: 4-17.
3Sorrell J.M. & Caplan A.!. (2004) Fibroblast heterogeneity: more than skin deep.
1 Cell Sci. 117(5): 667-675.
4Barton S.P. & Marks R. (1981) Changes in suspensions of human keratinocytes due to trypsin. Arch Dermatol Res. 271(3): 245-257.
5Krejci N.C., Cuono C.B., Langdon R.C. & McGuire J. (1991) In vitro reconstitution of skin: fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dennis. J. Invest. Dermatol. 97(5): 843-848.
6Stenn K.S., Link R., Moellmann G., Madri J. & Kuklinska E. (1989) Dispase, a neutral protease from Bacillus polymxa, is a powerful fibronectinase and type IV collagenase. J. Invest. Dermatol. 93(2): 287-290.
7Green H. (1991) Cultured cells for the treatment of disease. Sci. Amer. (Nov.) p. 96-102.
8SchOnherr E., Beavan L.A., Hausser H., Kresse H.& Cuip L.A. (1993) Differences in decorin expression by papillary and reticular fibroblasts in vivo and in vitro. Biochem. J. 290(3): 893-899.
9Normand J. & Karasek M.A. (1995) A method for the isolation and serial propagation of keratinocytes, endothelial cells, and fibroblasts from a single punch biopsy of human skin. In Vitro Cell. Dcv. BioI.-Animal 31: 447-455.
Mack J.A., Anand S.& Maytin E.V. (2005) Proliferation and comification during development of the mammalian epidermis. Birth Defects Res. C Embryo Today 75(4): 3 14-329.
Claims (16)
1. The said test protocol described herein will allow the straight forward testing of materials which need to be assessed for human topical use e.g. sunscreens. An ultraviolet radiation (UV) index can be firmly standardised.
2. Excessive exposure to the sun leading to skin cancer can be firmly evaluated by the introduction of known cancer cells to the ongoing tissue culture of the epidermal and dermal cells described by said method.
3. No in vavo animal use is required.
4. Skin irritants can be fully evaluated since the migrating epidermal island contains a full complement of epidermal cells.
5. Cosmetics and topical vehicles can be tested with said method.
6. Gases, viruses, bacteria, fungi, vaccines can be tested with said method.
7. Microbiological agents including nanoparticles can be tested for possible endocytosis by the epidermal and dermal cells described herein.
8. The said method will facilitate medical research into sicin conditions e.g. psoriasis, eczema, epidermolysis bullosa, just to name three of these clinical manifestations.
9. The said method will lead to an improved method for the manufacture of cultured epithelial autografts (CEAs) used in the clinical management of burns, wounds and for cosmetic applications. Autologous serum could be used for cell growth.
10. Radiation levels which can be tolerated by the skin can be solidly established by said method.
Ii. Wound care and skin care products can be re-evaluated.
12. The full complement and type of cells emanating from a dermal explant tissue in addition to dermal fibroblasts has not been described in the scientific literature (see Norinand & Karasek 1995). This can now be achieved.
13. Transdermal delivery to aid anti-aging procedures as well as the study of aging are facilitated, since the epidermal development program first seen in the foetus is recapitulated for the entire life of the human being (see Mack et al. 2005).
14. Said method employs no growth factor supplement. Said method will provide a means of testing the effects of growth factors on epidermal-mesenchymal cell layers' cross-talk.
15. From a patient's skin biopsy, said method will allow the isolation of autologous dermal cells and collagen which having been frozen, may be used later in cosmetic surgery.
16. Said method allows the introduction of stem cells (eznbiyonic stem cells, autologous mesenchymal stem cells) to study cellular interactions which may influence phenotypic expression by natural means. Presently, a veiy expensive cocktail (consisting of various supplements) is used to force' alteration of cellular phenotypic expression in a particular direction.
Claim 17: Said methodology, involving a series of steps described as autoengincering' in this patent application, may be used, as is as a commercial skin test/assay protocol for an ktyjlro model of human skin and its reorganization after injury'.
Claim 1$: Human monoclonal antibodies may be raised from both the epidermal (full complement of unmanipulated cells) and dermal cefl&
16. Said method allows the introduction of stem cells (embryonic stem cells, autologous mesenchymal stem cells) to study cellular interactions which may influence phenotypic expression by natural means. Presently, a very expensive cocktail (consisting of various supplements) is used to force' alteration of cellular phenotypic expression in a particular direction.
Amendments to the claims have been filed as follows
I. The said test protocol described herein Will allow the straight forward testing of materials which need to be assessed for human topical use e.g. sunscreens. An ultraviolet radiation (UV) index can be firmly standardised.
2. Excessive exposure to the sun leading to skin cancer can be firmly evaluated by the introduction of known cancer cells to the ongoing tissue culture of the epidermal and dermal cells described by said method.
3. No in vivo animal use is required.
4. Skin irritants can be folly evaluated since the migrating epidermal island contains a full complement of epidermal cells.
5. Cosmetics and topical vehicles can be tested with said method.
6. Gases, viruses, bacteria, fungi, vaccines can be tested with said method.
7. Microbiological agents including nanoparticles can be tested for possible endocytosis by the epidermal and dermal cells described herein.
8. The said method will facilitate medical research into skin conditions e.g. psoriasis, eczema, epidennolysis bullosa, just to name three of these clinical manifestations.
9. The said method Will lead to an improved method for the manufacture of cultured epithelial autografis (CEAs) used in the clinical management of bums, wounds and for cosmetic applications. Autologous serum could be used for cell 10. Radiation levels which can be tolerated by the skin can be solidly established by said method.
11. Wound care and skin care products can be re-evaluated.
12. The full complement and type of cells emanating from a dermal explant tissue in addition to dennal fibroblasts has not been described in the scientific literature (see Norinand & Karasek 1995). This can now be achieved.
13. Transderinal deliveiy to aid anti-aging procedures as well as the study of aging are facilitated, since the epidermal development program first seen in the foetus is recapitulated for the entire life of the human being (see Mack et al. 2005).
14. Said method employs no growth factor supplement. Said method will provide a means of testing the effects of growth factors on epidennal.
mesenchymal cell layers' cross-talk IS. From a patient's skin biopsy, said method will allow the isolation of autologous dennal cells and collagen which having been frozen, may be used later in cosmetic stwgeiy.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0619005A GB2442271B (en) | 2006-09-27 | 2006-09-27 | An in vitro human skin assay protocol which directly uses the natural cellular interactions between un-manipulated autologous human epidermal and dermal cells |
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| GB0619005A GB2442271B (en) | 2006-09-27 | 2006-09-27 | An in vitro human skin assay protocol which directly uses the natural cellular interactions between un-manipulated autologous human epidermal and dermal cells |
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| GB0619005D0 GB0619005D0 (en) | 2006-11-08 |
| GB2442271A true GB2442271A (en) | 2008-04-02 |
| GB2442271B GB2442271B (en) | 2011-06-15 |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1103601A1 (en) * | 1999-11-26 | 2001-05-30 | Menicon Co., Ltd. | A method for culturing cell and a culture vessel |
| US20020028192A1 (en) * | 1998-03-24 | 2002-03-07 | S. Dan Dimitrijevich | Non-contracting tissue equivalent |
| WO2002046375A1 (en) * | 2000-12-08 | 2002-06-13 | Xgene Corporation | In vitro synthesis of a layered cell sorted tissue |
| EP1312669A1 (en) * | 2000-07-21 | 2003-05-21 | Cellseed Inc. | Cultured epidermal cell sheet, laminated cultured skin sheet and process for producing the same |
| EP1314440A2 (en) * | 2001-11-22 | 2003-05-28 | Nipro Corporation | Cultured skin and method of maufacturing the same |
| WO2003051419A1 (en) * | 2001-12-19 | 2003-06-26 | Henkel Kommanditgesellschaft Auf Aktien | Skin/hair equivalent with reconstructed papillae |
-
2006
- 2006-09-27 GB GB0619005A patent/GB2442271B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020028192A1 (en) * | 1998-03-24 | 2002-03-07 | S. Dan Dimitrijevich | Non-contracting tissue equivalent |
| EP1103601A1 (en) * | 1999-11-26 | 2001-05-30 | Menicon Co., Ltd. | A method for culturing cell and a culture vessel |
| EP1312669A1 (en) * | 2000-07-21 | 2003-05-21 | Cellseed Inc. | Cultured epidermal cell sheet, laminated cultured skin sheet and process for producing the same |
| WO2002046375A1 (en) * | 2000-12-08 | 2002-06-13 | Xgene Corporation | In vitro synthesis of a layered cell sorted tissue |
| EP1314440A2 (en) * | 2001-11-22 | 2003-05-28 | Nipro Corporation | Cultured skin and method of maufacturing the same |
| WO2003051419A1 (en) * | 2001-12-19 | 2003-06-26 | Henkel Kommanditgesellschaft Auf Aktien | Skin/hair equivalent with reconstructed papillae |
Non-Patent Citations (1)
| Title |
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| Int J Exp Path, Vol 83, 2002, DE Solomon, "An in vitro examination of an extracellular matrix scaffold for use in wound healing", 209-216 * |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2442271B (en) | 2011-06-15 |
| GB0619005D0 (en) | 2006-11-08 |
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| PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 20150927 |