Preparation of IsolatedSheets of HumanStratum Corneum

ALBERT M. KLIGMAN, MD

AND

ENNO CHRISTOPHERS, MD

PHILADELPHIA

SummaryMethods of preparing intact, undenatured

sheets of human stratum corneum are de-scribed. The epidermis of split or full-thicknessskin is first separated by exposure to ammoniafumes or heat at 60 C. The horny layer is thenisolated by incubating in dilute trypsin solu-tion. Cantharidin blisters provide a conven-

ient way of obtaining stratum corneum fromliving subjects with minimum discomfort andno scarring.

Protection is the prime purpose of skin.It seals off the organism from its environ¬ment. In furry animals, hair bears the bruntof the external impacts. Man, however, byforfeiting his hair, has exposed his epidermisto the elements. Essentially naked, some

protective adaptation has been required. It isour thesis, which we shall elaborate in fur¬ther papers, that a specialized type of stratumcorneum now substitutes for the protectiveeffect of hair. The horny layer is the waterand chemical proofing of the skin.1,2 All or

part of the stratum corneum is the muchpublicized "barrier," the object of much re¬

cent research in cutaneous permeability.3The shielding value of the horny layer is

largely dependent on its anatomical construc¬tion, especially the tight bonding of hornycells into a coherent fabric. To discern howthe structure of the horny layer accounts forits functions requires that it be studied in itsnative state, as a tissue. Grinding, scraping,

and stripping the stratum corneum do violentinjury to the tissue and destroy its essentialcharacter. Because of its convenience, callusis often used as a model of keratinized tissue.It is a pathologic horny layer, however, andthe applicability of the findings have to bevalidated in each instance.

It is our purpose to describe ways of iso¬lating human stratum corneum in its nativestate.

TechniquesMethods Using Split-Thickness or Fidl-

Thickness Skin.—The starting material maybe autopsy skin, surgical specimens (breasts,amputations, etc), or traditional skin excisionusing the dermatome or knife. Thin razorblade shavings are often suitable. The tis¬sues may be frozen indefinitely. The fat, ifany, is trimmed away. Next, the epidermismust be separated in as gentle a fashion as

possible. The two methods which we havefound least traumatic and which preserve thehorny layer best are: (1) exposure to am¬monia fumes, and (2) moderately elevatedtemperature. The skin is placed in a closedvessel over concentrated ammonium hydrox¬ide solution for about 30 minutes. Bam¬berger, et al,4 immersed the skin inammonium hydroxide solution to separatethe epidermis. We wished, among otherthings, to prevent loss of water-soluble sub¬stances and have mainly avoided methodsrequiring prolonged immersion. Separationby heat is an ancient method utilized by thematchless Marcello Malpighi who simply

From the Department of Dermatology, Universi-ty of Pennsylvania School of Medicine.

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cooked the skin. Our modification is to placeskin for two to three minutes between twothick metal plates brought to 60 C by im¬mersion in a water bath. For most purposes,it appears permissible to immerse the skin inwater at 60 C for two minutes. With eitherheat or ammonia, the intact epidermis can

readily be teased off with forceps.The epidermal sheet is then placed, dermal

side down, on several sheets of filter papersaturated with a solution of trypsin,*1X10-4 (0.0001%), containing 0.5% so¬

dium bicarbonate. The pH should be between8.0 and 8.6. Phosphate buffer is also suitable.It is important to use salt-free, crystalline(lyophilized) trypsin. Separation can beeffected by a variety of proteolytic enzymes,including papain, pepsin, ficin, elastase, etc;but these are somewhat less efficient, andhigher concentrations must be used. Thefilter paper is just covered with the prote¬olytic solution in a small Petri dish which isincubated overnight at 37 C. A simple al¬ternative is to float the membrane on thetrypsin solution, dermal side down. Evensolutions as strong as 1% do not appear todestroy the horny layer, and separation ismuch faster. This abbreviated procedurewith 0.01% to 0.1% trypsin, will probablyserve most purposes quite well. We havefound, however, that solutions stronger than0.0001% are capable of solubilizing a goodlyportion of the fibrous protein of horny layer.For meticulous work, it seems safest to use

the weakest effective concentration and toavoid immersion. After incubation, the tis¬sue is smoothed out on a flat surface andthe mushy epidermis removed by firm rub¬bing with a moistened cotton-tipped applica-

tor. This cleaning process can be carried outwith surprising vigor without injury to thehorny layer. The product is a smooth, trans¬parent, tough, resilient sheet (Fig 1). Todry for storage, it is briefly floated on waterand lifted out on a steel wire mesh of coarse

grade. In this form it can be kept indefinitelyat room temperature.

The Cantharidin Blister Technique.—Thismethod was devised in order to secure good-sized sheets of stratum corneum from livingsubjects. Circular sheets of horny layer as

large as 4 cm in diameter, suitable for perme¬ability studies, can be prepared with minordiscomfort to the subject and with the fur¬ther great advantage of not leaving a scar.

Many samples can be obtained from differ¬ent regions of the same person with verylittle trouble.

Use is made of the capacity of cantharidinto produce an intraepidermal blister ; hence,no scarring. For most body areas a 0.2%solution of cantharidin in acetone is satis¬factory. For sites where the stratum corneum

is thick or dense, as the hands and feet, fore¬head, scalp, etc, the concentration is increasedto 0.5%. To obtain a good-sized disk, 0.3ml is placed into a glass or metal cylinder, 4cm in diameter, and the acetone evenly evap¬orated to dryness under a stream of air. (Anordinary hair dryer will do.) A layer ofwater-soaked Webril,f (a nonwoven cloth ofhigh water-holding capacity) or other wetcloth, is placed over and beyond the applica¬tion site and fastened occlusively to the skinby overlapping strips of impervious plastictape.f Eight to ten hours later, the turgidclear blister is cut away with a scissors (Fig

THE EPIDERMIS

The epidermis k a thin, totalU cellular niehrbrarie ¿¿void of lymphatics.blood vessels and connective tissue. It is parasitic on the corium, derivingits nourishment from rich capillarv plexuses therein. IiHknurtatofy,processesin the dermis will, therefore, almost inevitably alter the epidermis patholog¬ically. Being nearest the eye, these epidermal chartgrcs are conspicuous. Itis no wonder that the changes of blistering, scaling, thickening and dis¬coloration figure heavily in the description of sMrt dweasc'.Funcrionally,the epidermis cannot be considered apart from the substrate on which itrests.

Fig 1.—Sheets of strat¬um corneum from a whitesubject (left) and a

Negro (right) have beenplaced over a printed textto show transparency, aswell as the greater mela¬nin content of Negrohorny layer. Such sheetsare very thin but toughand flexible.

* Worthington, Inc., Freehold, NJ.t Curity.\s=dd\Blenderm, Minnesota Mining Co.

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Fig 2.—An eight-hour cantharidin blister. Blisters4 cm in diameter like this one can be prepared frompractically any body area. Obviously, the hornylayer has been stretched by the fluid.

2). Remnants of epidermis cling to theundersurface of the horny layer (Fig 3).Debridement of the loosely adherent epithe¬lium is accomplished by firm rubbing with a

cotton-tipped applicator as before. Enzy¬matic treatment is not required. Stratumcorneum sheets prepared both by the blistermethod and by split-thickness excisions plusheat and trypsin from the same subject havebeen compared with respect to a number ofparameters. The essential qualities, such as

water diffusion, stress-strain characteristics,etc, appear to be equally well preserved byeither technique.

The follicles and eccrine sweat ducts are,of course, ruptured during isolation of thehorny layer. The "holes" so created do notimpair the barrier function. Actually thestratum corneum is not a flat sheet pierced byappendageal outlets. The horny layer con¬

tinues well down into the follicular orificesand into the "beakers" where sweat ductsemerge. The horny, cone-shaped infundibulaof the follicles hang from the undersurfaceof stratum corneum sheets (Fig 4). Finally,one should be aware that separated stratumcorneum is in a somewhat "relaxed" stateand occupies a larger area than in situ, owingto flattening of the rete ridges. If one cutsout a disk of split thickness skin and freesthe epidermis by heat or ammonia, the area

of the separated sheet will be found to haveincreased by about 20%. No further changein area occurs when the stratum corneum issubsequently separated.

CommentWith native sheets of stratum corneum

one may systematically examine how thisdelicate membrane can fulfill its biologicalobjectives. Measurements are underway ofits thickness, tensile strength, hygroscopicity,permeability, elasticity, light transmission,etc. One cannot work with this tissue, too

frequently thought of as a porous, disinte-

Fig 3.—Microscop¬ic view of the edge of an

intraepidermal cantharidinblister. The remnants ofepidermis which remainloosely attached to thestratum corneum can bewiped away by rubbingwith a moistened cottonpledget. Hematoxylin andeosin; reduced 32% frommag X 90.

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Fig 4.—Undersurfaceof the separated stratumcorneum of the scalpshowing horny stalks ofruptured hair follicles.The small, round holesbetween the follicles aresweat gland orifices.

grating, mass of dead cells, without comingto admire its marvelous construction andtoughness. The stratum corneum must notbe visualized as a kind of unorganized kera¬tin dump. It is a fabric, a tissue whose im¬portant qualities stem as much from the cellmembranes and the forces which hold thesetogether, as the fibrous protein they contain.Isolation of the stratum corneum opens new

pathways for investigating disorders inwhich the horny layer figures prominently,viz, the puzzling "dry skin" problem, agedskin, chapping, chemical irritability, etc.

To our knowledge, Matoltsy was the firstto notice in passing that the horny layerwould separate as a sheet when newborn rat

epidermis was incubated in 2 M urea and 1%trypsin.5 Large sheets of "stratum corneum"were observed by Rollins to form underplaster of paris casts worn for a long time byorthopedic patients, but this is certainly an

undefined material.6Recently, Gilbert et al described a rack

whereby skin could be stretched in order toobtain an epidermal sheet a la Van Scott.7Evidently, a pure sheet of stratum corneum

is obtainable by reducing the stretching forcebelow that which shears off the whole epi¬dermis. Also recently Onken and Moyershowed that immersion of human skin in 3%trypsin solution would release the stratum

corneum.8 This concentration is much higherthan needed and doubtlessly alters the chemi¬cal composition.

We are indebted to the inmates of HolmesburPrison for serving as volunteers and to the ad¬ministration (Edward Hendrick, Superintendent)for use of the facilities.

Albert M. Kligman, MD, Department of Derma¬tology, University of Pennsylvania School ofMedicine, Philadelphia 4, Pa.

REFERENCES1. Rothman, S.: Physiology and Biochemistry of

Skin, Chicago : University of Chicago Press, 1954.2. Blank, I. H., and Gould, E.: Penetration of

Anionic Surfactants Into Skin, J Invest Derm 33 :

327, 1959.3. Szakall, A.: Experimentelle Daten zur

Klarung der Funktion der Wasserbarriere in derEpidermis des lebenden Menschen, Berufsderma-tosen 6:171, 1958.

4. Bamberger, J. P.; Suntzoff, V.; and Coudry,V. F.: Methods for Separation of Epidermis FromDermis, J Nat Cancer Inst 2:413, 1942.

5. Matoltsy, A. G.: "The Chemistry ofKeratinization," in Biology of Hair Growth, NewYork: Academic Press, Inc., 1958, p 135.

6. Rollins, T. G.: Means of Obtaining StratumCorneum, Correspondence, Arch Derm 85 :116, 1962.

7. Gilbert, D.; Mier, P. D.; and Jones, T. E.:An Improved Technique for the Isolation ofEpidermis From Human Skin, J Invest Derm 40 :

165, 1963.8. Onken, H. D., and Moyer, C. A.: The Water

Barrier in Human Epidermis, Arch Derm 87:584,1963.

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