0022-202X/ 78/ 7005-0280$02.00/ 0 THE J OURNAL OF lNVESTIGATlVE DERMAT OLOGY, 70:280-284, 1978 Vol. 70, No. 5 Copyright © 1978 by The Williams & Wilkins Co. Printed in U.S.A. COUNTING AND SIZING OF EPIDERMAL CELLS IN NORMAL HUMAN SKIN PAUL R. BERGSTRESSER, M.D., ROBERT J. PARISER, M.D., AND J. RICHARD TAYLOR, M.D. Department of M edicine (PRB), University of Texas Health Science Center at Dallas, Dallas, Texas and Department of Dermatology (RJP, JRT) University of Miami School of Medicine and Miami Veterans Administration Hospital, Miami, Florida, U.S.A. During keratinocyte maturation, individual cells un­ MATERIALS AND METHODS dergo an orderly succession of biochemical and struc­ Subjects tural changes. In certain skin disorders alterations in keratinocyte numbers, volumes, and epidermal skin Six male patients on the Dermatology Service at the Miami Veterans Administration thickness occur. To assess such alterations Hospital were selected on the basis of skin diseases and to pro­ limited to the face or feet. Each gave informed consent vide base line and was biopsied values for normal human epidermis, a at 3 normal sites: (a) volar forearm, (b) upper back, and (c) upper, computer assisted histologic technique was developed. lateral thigh. The 6 subjects ages were 50, 72, 46, 61, 67 and 60 Skin biopsies were taken from normal skin on the respectively. forearm, back and thigh of 6 adult men. Whole specimens of epidermis were separated from the dermis with col­ Epidermal Whole Mounts lagenase, fixed, stained, and mounted for microscopic For human skin, thin free-hand 4 X 4 mm scapel biopsies were taken examination. From the three dimensional coordinates of under local 1% Xylocaine anesthesia and were placed in RPMI-1640 epidermal nuclei, epidermal cell volumes, surface den­ (Roswell Park Memorial Institute-Gibco Co.) medium. For pig skin, sity of epidermal cells, and epidermal thickness were 0.3 mm thick keratome strips measuring 3 X 4 em were taken with a determined. Measurement of cell volumes in this way motor driven Castrovievjo keratome from the backs of domestic pigs compared favorably with electronic cell sizing of disag­ and were also placed in RPMI-1640 medium. After 15 min the biopsies gregated epidermal cells in matched samples. The mean were incubated in the same medium containing 1 mg/ ml crude bacterial densities of nucleated cells per 104 p. 2 surface area were collagenase (Sigma) for 30-60 min at 37 °C. After incubation, light 452, 483, and 487 for the forearm, tension with 2 pairs of forceps caused a complete separation at the back and thigh respec­ epidermal-dermal interface. tively. This If the incubation was continued more than technique will be used to make similar as­ 1 hr, individual keratinocytes appeared in the incubation medium. sessments in disorders of abnormal keratinocyte matu­ Dermal-epidermal separation using 2 N NaBr and other ionic solu tions ration. was attempted but discarded because nuclear morphology became distorted and the entire epidermal sheet appeared shrunken. Separated epidermal specimens were placed under a cover glass to prevent curling Keratinocyte volumes, keratinocyte populations and three and were fixed in acidic Bouins solution for 2 hr [16]. They were t hen dimensional epidermal structure play an important role in rinsed for 2 days in 4 c hanges of 70% ethyl alcohol. Specimens were dermatologic investigation. Although biochemical studies are stained with aqueous iron hematoxylin, then were cleared slowly reported on the basis of epidermal weight, DNA, and protein through absolute alcohol to xylene, and were mounted, basal layer up, content, epidermal matabolism occurs at the level 9f single on microscope slides. Epidermis prepared in this manner could be keratinocytes. To determine epidermal cell turnover times by studied at all powers with transmission light microscopy. The stratum corneum became transparent with adequate xylene clearing, and compartmental analysis, quantitation of the populations in each epi­ dermal cell boundaries became imperceptable. The nuclei stained well compartment is required [1-3]. The processes which control and were seen easily. epidermal proliferation and maturation are reflected in epider­ mal compartment populations, epidermal thickness, and indi­ Keratinocyte Suspensions vidual cell volumes [ 4- 7]. Pig keratinocyte preparations were made from 0.3 mm thick Castro­ Previous studies of cell populations and epidermal dimen­ vievjo keratome strips of skin from the back. These strips were cut to sions have been based on thin histologic sections [8-11]. This 3 x 3 em squares and were incubated at 37 °C for 30-45 min in technique requires numerical coiTections [12] and considerable Dulbecco's phosphate-buffered saline (PBS) (Gibco), to which crude labor, particularly when it is extended to serial sections. Precise bacterial collagenase (Sigma, 1 mg/ml), ethylenediaminetetraacetate analyses of epidermal cell dimensions in histologic sections have (EDTA) (10 - 5M), and glucose (1 mg/ml) were added. Dermal-epider­ been pe1formed by stereology, a difficult technique which relies mal separation was accomplished by light traction with forceps. Epi­ on thin sections, but no assessment of cell populations has been dermal sheets were placed onto glass slides (stratum corneum side up), made [13-15]. The stratum corneum was carefully blotted dry, and a 3 X 3 em square We have developed a computer assisted technique to deter­ of cellophane adhesive tape was applied to the stratum corneum. Human epidermal sheets were obtained from the roofs of suction mine cell populations, individual cell volumes, and epidermal blisters raised with an apparatus which applied a suction of 25 em Hg dimensions in fixed whole mounts of epidermis. Epidermal and maintained a suction chamber temperature at 40°C for 45-60 min. thickness and epidermal keratinocyte populations were deter­ Histologic sections of the blister roofs confirmed the s ubepidermal mined directly, and cell volumes were calculated from the three location of the cleavage. The epidermal sheets were next incubated at dilnensional location of all cell nuclei. Statistical assumptions 37°C for 30 min with constant stirring in PBS to which bovine pan­ and potential artifacts of this method are discussed. creatic trypsin (Calbiochem, A grade, 2.5 mg/ ml), EDTA (10 - oM) , and glucose (1 mg/ml) were added. Trypsinization released all nucleated epidermal cells into suspension, leaving a s heet of stratum corneum behind. The cell suspensions were then passed through a nylon mesh Manuscript received July 19, 1977; accepted for publication Novem­ with openings of 0.1 X 0.1 mm to remove debris and large cell clumps. ber 21 , 1977. Smaller clumps were broken up by syringing the suspensions gently Reprint requests to: Paul R. Bergstresser, M.D ., Division of Der­ through a 20-gauge needle several times. The cells were pelleted by matology, University of Texas Health Science Center at Dallas, 5323 centrifugation, resuspended in PBS with EDTA (10 - oM) and glucose Harry Hines Blvd., Dallas, Texas 75235. (1 mg/ml), and syringed again. The result was a nearly monodispersed Abbreviation: cell suspension. Five ml of this suspension was slowly added to 20 ml of PBS: phosphate buffered saline Bouin's fixative with constant stirring. After 1 hr of fixation, the cells 280 May1978 COUNTING AND SIZING OF EPIDERMAL CELLS 281 were pelleted by centrifugation and resuspended in PBS with EDTA 25 - 5 (10 - M) and glucose (1 mg/ml) for electronic s izing. - Derived from Epidermal Whole Mounts Cell Sizing: E lectronic c::J Derived from Electronic Sizi ng 20 I- A Coulter Counter model ZH with a 70 iJ. aperture t ube was used for e lectronic s izing of suspended cells. In this device particles passing t hrough the aperture produce electronic s ignals w hose amplitude is proportional to particle volume. These signals were recorded and !z 15 - displayed on a 100-channel Coulter Channelyzer. The apparatus was w a djusted so that a linear range of volumes (as determined by particles u a:: of known volume) with respect to channel number was obtained for w particles up to 1050 iJ.a in volume. 17,500 pig and 24,000 human keratin­ (L 10 1- ocytes were counted. Cell Sizing: Epidermal Matrix At oil immersion power (1000 X), an aJ"ea to be counted was examined 5 1-- with a 10 division by 10 division square grid (American Optical Co. # 1408A) in the eyepiece. Each division measured 9.8 iJ. by 9.8 iJ. · The 2 e ntire grid covered an area of 9600 iJ. At this power most nuclei I ll occupied 1/4 of the area in 1 small division and a portion of a repre­ I sentative field is illustrated in Fig 1. The center of each nucleus was 100 300 500 700 900 1100 estimated in two dimensions to an accuracy of 1/ lOth of 1 division Cell Volume ( fl-3) (approximately 1 iJ.). In this way, 2 coordinates were assigned for each nucleus. FIG 2. Comparison of volume histograms of human epidermal cells. The depth of foc us of the microscope used in this study at 1000 x Stippled bars represent the histogram derived from electronic sizing. magnification was approximately 3 iJ.· Nuclei outside of the 3 iJ. limit Solid bars represent the histogram derived from epidermal whole were imperceptable, and the absence of cytoplasmic stain permitted mounts. s ufficient transmitted light to see all epidermal nuclei as the plane of focus passed through the specimen. The vertical coordinate for the center of each nucleus was determined with a precision of 1 iJ. by reading the vertical dimension, 19.5 iJ. diameter spherical ragweed pollen was t he vertical scale on the objective control. Care was required to prevent soaked in xylene on a glass slide, covered with mounting medium and displacement o f the s pecimen or eyepiece grid after b eginning t he examined under oil immersion.