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Serial Cultivation of Single Keratinocytes from the Outer Root Sheath of Human Scalp Hair Follicles

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Serial Cultivation of Single Keratinocytes from the Outer Root Sheath of Human Scalp Hair Follicles Serial Cultivation of Single Keratinocytes from the Outer Root Sheath of Human Scalp Hair Follicles A lain Limat, Ph.D. and Friedrich K. N aser, Ph.D. Cosmital SA, Marl y, Switze rl and (Resea rch Compa ny of We ll a AG, Darmstadt, F. R.G.) A m e tho d fo r the isolatio n of o uter root sheath k eratino­ h air follicle (yielding approximately 1. 5 X 104 cells) con­ cytes from pluck ed human hair follicles and for their sub­ sistentl y gave rise to a confluent 35-mm culture dish (with sequent cultivatio n h as been develop ed. T he selective tryp­ approximately 1.5 X 106 cells) w ithin abo ut 2 weeks. The sinizatio n of o uter root sheath k eratinocytes provided a o u ter root sh eath k eratinocytes can be serially p assaged for sin gle cell su spension of d efined o rigin within the h air fol­ up to 3 times and also cryopreserved . J In vest Derm ato! licle. T h e 3T3 feede r layer technique suppo rts su stained 87:485- 488, 1986 growth of these cells in that as little as o ne single pluck ed lucked hair folli cles were fi rst used as a convenient bi­ the fo llicles were transferred in to a 35-mm Petri dish. The re­ opsy material fo r the study of geneti c disorders and for maining liquid was sucked off and 10 fo llicles covered with 200 diagnosti c purposes. T hereafter, it beca me evident that ILl 0.1 % trypsin (1: 250) and 0.02% EDTA in phosphate-buffered hair fo llicles provide an interesting model system of saline (PBS) without Ca + + and M g + + (pH 7.2) and incubated epithelial ce ll s for more fundamenta l biomedi ca l re­ for 10 min at 3rc. T hereafter, a sin gle cell suspension was ob­ Psearch [1] . tained by vigorously pipetting the fo llicles in D M EM supple­ In recent years several meth ods have been devel oped for es­ mented with 10% newborn calf serum. For SO I11.e experiments, tablishing primary cul tures of human hair follicle cell s. Weterings the remaining keratinocytes still ad hering to the follicles were et al [2] were first able to grow human hair follicle keratinocytes sequentiall y removed by 2 additional trypsinization steps of 10 by expl anting plucked scalp hair follicles on bovine eye lens ca p­ min and 30 min, res pecti vel y. T he dissociated keratinocytes were sules. Si milarl y, Wells succeeded in explanting plucked human centrifuged fo r 10 min at 200 g and cells from one fo llicle resus­ hair follicles di rectl y on tiss ue cul ture plastic [3]. Recently, we pended in 2 ml of complete culture medium each. T he culture became interested in the study of epi theli al- mesenchymal inter­ medium consisted of 3 parts of D M EM (Seromed) , containing actions, especiall y concerning the hair fo llicle biology. For this sodium pyru vate and 1 g/liter glucose, and 1 part of Ham 's F-1 2 pu rpose, we aimed to initiate primary cultures of disaggregated (Seromed), supplemented w ith 10% fetal calf serum (Gibco), 5 hair follicle cells in order to in crease the proli fera ti ve ca pacity of ILg/ml in sulin (S igma), 0.4 ILg/ml hydrocortisone (S igma), 0.135 the primary hair keratinocytes and to get a more defined and mM adenine, 2 nM trii odothyronine, 0. 1 nM choleratoxin (S igma), standardized cul ture system . 10 ng/llll epidermal growth factor (Seragen) , and antibiotics (50 In this report, we describe a method for the growth of prim ary U / ml penicillin and 50 IL g/ml streptomycin) modified after Gilfix cultures starting from dissociated human hair follicles and for and Green [4]. T he keratinocytes were seeded at a density of2-3 2 their serial cul tivati on. T he method consists essentiall y in dis­ X 103 cells/clll2 on a 3T3 fee der layer (2 X 10" cells/cm ) (ATCC aggregating the outer root sheath keratinocytes with tryp­ CCL 92) prepared according to Rheinwald and Green [5]. T he sin/EDT A and by plating them on a pre form.ed 3T3 fee der layer. 3T3 cell proli fera ti on had been halted by a treatment with 6 ILg/ml Cells fro m only one single hai r fo llicle give rise to a confluent mitom ycin C (Sigm a) fo r 8 h. 35-mm Petri dish within about 2 weeks with a multipli cation The cultures were incubated at 37°C in a humidified atm osphere 3 factor of 10 and 12 es timated cell generati ons. of 5% CO2 in air and the medium changed tw ice a week. As a rule, the fee der layer was renewed once a week after selective M ATERI ALS AN D M ETH ODS rem oval of the 3T3 cells using 0.02% EDT A [5]. C ell Isolation and Cultivation Hair follicles were plucked from the scalp and the bulk of th e hair shafts cut off. T he fo lli cles Subculture After 2-3 weeks in primary culture, the residual were immersed in D ulbecco's modified Eagle's medium (D MEM ) 3T 3 cells were removed with 0.02% EDTA and the keratinocytes buffered with 25 mM H EPES (N-2-hydroxyethylpiperazine-N '- detached and disaggregated to si ngle cells by incubating with 2-ethanesulfonic acid) and supplemented with 400 U /ml penicillin 0.1 % trypsin (1: 250) and 0.02% EDTA in PBS (pH 7.2) at 37°C and 400 ILg/ml streptomycin. T hose in anagen phase, indicated for 15-30 min. T he dispersed cells were replated in cul ture me­ by the visible bulb and intact outer root sheath, were ca refull y dium at a density of 50 cells to 104 cells/cm2 on a fres h 3T3 feeder selected under the dissecting microscope. After 2 additional rinses, layer. Cryopreservation For storage in liquid nitrogen the cells were released from culture dishes as described' above. After 1 wash Manuscrip t received October 16, 1985; accepted for publi ca tion March with culture medium, 106 cells were res uspended in 1 ml culture 28, 1986. medium containing 10% glycerol, transferred into a cryotube Reprint requ ests to: Alain Limat, Ph. D., Cos mital SA, Rtc de Chcsa li es (Nunc), and placed within a styrofoam container in a - 80°C 21, CH-1723 Marl y, Sw itze rl and . Abbrevi ations: freeze r. After 24 h the tubes were transferred to a liquid nitrogen DMEM: Dul becco's modi fied Eagle's med ium ta nk. To recover the cells fro m frozen storage, the tubes were PBS : phos phate-buffe red sa line warmed to 37°C and the suspension diluted in culture m edium . 0022-202X/86/lG03.50 Copyri ght © 1986 by The Society for In vestigative Derm atology, Inc. 485 486 L1MAT AND NOSER THE JOURNAL OF INVESTIG ATIVE DERMATOLOGY Figure 1. Plucked adult human scalp hair follicle. A , Whole hai r follicle with its usu­ all y almost intact outer root sheath in the portion between the upper part of the hair bulb and the presumptive level of the se­ baceous gland which is normally absent. Unstained, bar = 0.5 mm. B, Transversal secti on of the middle third of a hair follicle. The peripheral layer of the columnar basa l cells is often preserved over an extensive portion of the circumferen ce. H&E, bar = 50 /-Lm. A The cell s were washed and then inoculated at a density of 2 x final enzym atic digestion of 30 min removed the remainder of 104 cells/cm 2 onto a feeder layer. the outer root shea th cell s, leavin g behind the denuded inner root sheath (Fig 4) . Histologic Examination Confluent cultures of hair follicle ke­ ratinocytes grown in culture dishes were detached as epithelial Cell Culture Primary cultures of hair follicle keratinocytes could sheets according to Green et al. [6] and Watt [7]. Briefly, the be routinely es tablished by plating cells disaggregated from one cultures were incubated with Dispase II (Boehringer Mannheim) single hair follicle on a preformed 3T3 feeder layer in a 35-mm at 1. 2 units/ml in PBS. After about 15 min at 37°C, the epithelial culture dish or from 10 follicles on a 3T3 feeder layer in a 100- sheet started to separate from the plasti c surface. The detachment mm culture dish. The seedin g efficiency of the keratinocytes at could be accelerated by carefully lifting the sheet with tweezers. the initiation of the primary cultures was 2.6 ± 0.5% (n = 4) . Pieces of the sheet were punched out, stretched over a Whatman After 2-3 days in culture, groups of2-4 keratinocytes surrounded N o.1 fi lter paper,· and washed with PBS. They were th en fixed by 3T3 cell s were visible. These cells formed quickly expanding in Bouin's solu tion for 24 h and processed for wax embedding. colonies pushing away the 3T3 cells. The keratinocytes were of Seri al sections were prepared at 7 J.l.m and stained with hema­ similar small size and closely packed in a polygonal epitheloid toxylin and eosin . arrangement (Fi g 5) . As long as the colonies were of limited size, In order to facilitate their handling the plucked hair follicles the weekly renewal of the feed er layer proved to be beneficial.
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