EXPERIMENTAL CELL RESEARCH 240, 1–6 (1998)ARTICLE NO. EX983936

A Simple Immunomagnetic Protocol for the Selective Isolationand Long-Term Culture of Human Dermal

Microvascular Endothelial Cells

Lisa Richard, Paula Velasco, and Michael Detmar1

Cutaneous Biology Research Center, Massachusetts General Hospitaland Harvard Medical School, Boston, Massachusetts 02129

uted to the rapid development of modern vascular biol-Endothelial cells involved in tumor angiogenesis, ogy. However, endothelial cells involved in the pathogen-

wound healing, and inflammation are predominantly of esis of tumor angiogenesis, wound healing, and inflam-microvascular origin and are functionally distinct from mation are predominantly of microvascular origin andlarge vessel-derived endothelial cells which have been display significant functional differences when comparedlargely used for in vitro vascular research. To overcome to large vessel-derived endothelial cells [3], in particularthe problems commonly involved in the culture of micro- with regard to their growth factor response [4, 5], prosta-vascular endothelial cells, including unreliable isolation glandin production [6], and their regulation of adhesiontechniques and low cell yields, we developed a simplified molecule expression [7–9]. Although techniques haveprotocol for the selective cultivation of human dermal been developed to isolate endothelial cells from small ves-microvascular endothelial cells (HDMEC) obtained from sels, most frequently from the skin [5, 10–14], the cultureneonatal foreskins, based on the transient, endothelial of human dermal microvascular endothelial cellscell-specific induction of E-selectin by tumor necrosis (HDMEC) has remained problematic because of difficult-factor-a (TNF-a). Subconfluent primary cultures, con- ies in cell isolation, low cell yields, and short life spanssisting of a mixture of endothelial cells, fibroblasts, and

of isolated cells. In particular, potential contamination ofkeratinocytes, were treated with TNF-a for 6 h, andHDMEC cultures with fibroblasts required time-consum-HDMEC were isolated by their selective binding to mag-ing density-gradient centrifugations [5, 13] or mechanicalnetic beads coupled with anti-E-selectin monoclonal an-removal of fibroblasts [11] and frequently prevented thetibody. After two immunomagnetic purification steps, ause of cultures in higher passage numbers. While dyna-homogenous population of HDMEC was obtained whichbeads coated with Ulex europaeus agglutinin, UEA-1showed typical cobblestone morphology, expressed[15], or with antibodies against thrombomodulin [16] orCD31 and von Willebrand factor, proliferated in re-platelet–endothelial cell adhesion molecule PECAM-1sponse to vascular endothelial growth factor, upregu-(CD31) [17] have been previously used to isolate endothe-lated the expression of intercellular adhesion molecule-

1 and vascular adhesion molecule-1 in response to TNF- lial cells, these markers may cross-react with other skina, and formed capillary-like tubes in a three-dimensional cell populations and/or may prevent detachment of dyna-collagen type I matrix. This simple technique may facili- beads due to persistent cell surface antigen expression.tate a more widespread use of microvascular endothelial We aimed to establish a simplified protocol for the isola-cell cultures obtained from different human or animal tion of HDMEC from neonatal human foreskins, takingorgans for functional in vitro studies. q 1998 Academic Press advantage of the transient, endothelial cell-specific induc-

tion of E-selectin by tumor necrosis factor-a (TNF-a) [18,19], predominantly in postcapillary venule endothelial

INTRODUCTION cells [20]. E-selectin-expressing cells can then be selec-tively isolated by magnetic dynabeads coupled to an anti-

The isolation and cultivation of endothelial cells from E-selectin monoclonal antibody. We report here the phe-large vessels, predominantly from human umbilical veins notypical and functional characterization of HDMEC ob-[1, 2], has become a routine procedure and has contrib- tained by this novel technique.

MATERIALS AND METHODS1 To whom reprint requests should be addressed at CBRC/Dept. ofDermatology, Massachusetts General Hospital East, Harvard Medi-cal School, Building 149, 13th St., Charlestown, MA 02129. Fax: Preparation of anti-E-selectin-coated magnetic beads. Five-hun-

dred microliters (15 mg) of dynabeads M-450 sheep anti-mouse IgG(617) 726-4453. E-mail: mdetmar@cbrc.mgh.harvard.edu.

1 0014-4827/98 $25.00Copyright q 1998 by Academic Press

All rights of reproduction in any form reserved.

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2 RICHARD, VELASCO, AND DETMAR

ST (Dynal, Lake Success, NY) were added to 10 mg of mouse anti- without recombinant human VPF/VEGF165 (0.01–100 ng/ml; kindlyprovided by Dr. Stephen Ledbetter, Upjohn Laboratories, Kalama-human E-selectin monoclonal antibody (Genzyme, Cambridge, MA,

No. 2138-01) and were mixed overnight at 47C by end-over-end rota- zoo, MI) for 96 h. Medium and VPF/VEGF were replaced after 48 h.Cell counts were performed in triplicate cultures as described [22].tion. Magnetic beads, coated with anti-E-selectin antibody, were

washed four times with phosphate-buffered saline (PBS; Gibco BRL, For determination of [3H]thymidine incorporation, quadruplicate cul-tures were incubated for 6 h before harvesting with 1 mCi/ml [3H]-Grand Island, NY) containing 1% fetal calf serum (FCS; Gibco BRL),

using a magnetic particle concentrator, and were stored at 47C for thymidine (DuPont NEN, Boston, MA), and incorporated radioactiv-ity was determined as described [23].up to 1 month.

Isolation of cells from neonatal foreskins. Neonatal foreskins FACS analysis of adhesion molecule expression. Confluentwere obtained after routine circumcisions and were incubated in HDMEC on collagen type-I-coated 100-mm dishes were treated orHank’s balanced salt solution (HBSS) supplemented with penicillin not with 100 ng/ml TNF-a or with 50 ng/ml recombinant human(400 U/ml), streptomycin (400 mg/ml), and fungizone (1.0 mg/ml) (all VPF/VEGF165 for 20 h. After trypsinization, cells were resuspendedfrom Gibco BRL) at 377C for 2 h. Foreskins were then cut into approx- at a density of 11 106 cells/100 ml in cold Dulbecco’s modified Eagle’simately 2 1 2-mm squares and were incubated in 0.25% trypsin/PBS medium containing 3% FCS and were incubated with 10 ng/ml of(Sigma, St. Louis, MO), dermal side down, at 47C overnight. After two mouse monoclonal antibodies against intercellular adhesion mole-PBS washes, complete endothelial cell growth medium was added, cule ICAM-1 (CD54; clone HA58) or vascular cell adhesion moleculeconsisting of endothelial cell basal medium (EBM; Clonetics, Walk- VCAM-1 (CD106; clone 51-10C9) or with an equivalent amount ofersville, MD) supplemented with 1 mg/ml hydrocortisone acetate, 5 mouse IgG1 (isotype control; clone MOPG21; all from PharMingen)1 1005 M N-6,2 *-O-dibutyryl-adenosine 3 *,5*-cyclic monophosphate for 40 min at 47C. Cells were washed twice with PBS and incubated(Sigma), 20% heat-inactivated FCS, 100 U/ml penicillin, and 100 with 5 mg/ml FITC-conjugated anti-mouse IgG1 antibody (GI-6.5) atmg/ml streptomycin. This medium potently supports proliferation of 47C for 20 min. Cells were washed with PBS, fixed in 1% paraformal-endothelial cells, although it is also permissive for fibroblast growth. dehyde in PBS, and one-way analyzed using a Becton DickinsonAfter removal of the epidermal sheet, endothelial cells and other FACSort (Becton Dickinson, San Jose, CA).dermal cells were released into the medium by scraping the upperside of the dermis with a blunt scalpel blade [4] and were transferredthrough a 100-mm nylon mesh into a sterile centrifuge tube. The cells RESULTSwere counted, centrifuged at 300g for 5 min, resuspended in completeendothelial growth medium, and seeded at a density of 2 1 104 cells/

Primary cultures, established by plating dermal cellscm2 on fibronectin-coated dishes (50 mg/ml) at 377C/5% CO2 in ahumidified atmosphere. Two hours later, nonadherent cells were re- released from neonatal human foreskins, consisted ofmoved by two PBS washes. The culture medium was changed every a mixture of dermal microvascular endothelial cells,2–3 days. dermal fibroblasts, and some epidermal keratinocytes.

Immunomagnetic purification of HDMEC. Recombinant human Subconfluent cultures were then treated with TNF-aTNF-a (100 ng/ml) (kindly provided by BASF-Knoll, Mannheim, Ger-for 6 h to selectively induce the expression of E-selectinmany) was added to subconfluent primary cultures, consisting of a

majority of endothelial cells, as well as some fibroblasts and keratino- in dermal microvascular endothelial cells, and endothe-cytes, to selectively induce the expression of E-selectin on HDMEC. lial cells were purified using magnetic beads coupledAfter 6 h, E-selectin-coupled magnetic beads were added (5 ml/ml to an anti-E-selectin monoclonal antibody. Dynabeadsmedium), and cultures were incubated at room temperature with

efficiently bound to the vast majority of cells with endo-gentle shaking for 5 min. After four thorough washes with PBS,thelial cell morphology, whereas in cultures not stimu-cultures were trypsinized, and cells with attached dynabeads were

isolated, using a magnetic particle concentrator. After additional lated by TNF-a, only a minority of cells bound anti-E-PBS washes, E-selectin-positive HDMEC were plated at 51 103 cells/ selectin-coupled dynabeads, predominantly endothelialcm2 on dishes coated with 50 mg/ml collagen type I (Vitrogen 100; cells undergoing mitosis [24]. After one immunomag-Collagen Corp., Palo Alto, CA). At 70–80% confluence, a second im-

netic purification step, first-passage cultures usuallymunomagnetic purification step was performed. For subcultures,consisted ofú99% HDMEC. Most magnetic beads weresubconfluent HDMEC were split at a ratio of 1:5.shed from the cell surface within 2 h after subculture,Immunocytochemistry. HDMEC were cultured on collagen-

coated LabTek chamber slides until they reached confluence. Slides due to downregulation of E-selectin without furtherwere washed with PBS, air dried, and stored at 0807C for up to 2 TNF-a stimulation, and could be removed by washesweeks. After thawing, slides were immersed in ice-cold acetone for with growth medium. Some beads were incorporated15 min and were subjected to immunocytochemistry as described

by HDMEC; however, the beads did not inhibit the[4], using a mouse monoclonal antibody against CD31 (PECAM-1;PharMingen), a rabbit antibody against von Willebrand factor growth or survival of HDMEC, since an appropriate(DAKO Corp.), or a mouse monoclonal pan cytokeratin antibody rec- proportion of bead-containing cells was observed overognizing human cytokeratins 4,5,6,8,10,13, and 18 (Sigma; clone C- several population doublings until the beads were fi-11). The squamous cell carcinoma cell line SCC-13 [21] was used

nally diluted out.as a positive control for cytokeratin immunocytochemistry and as aAfter a second immunomagnetic purification step,negative control for CD31 and von Willebrand factor staining. For

additional negative controls, primary antibodies were replaced with cultures displayed a characteristic cobblestone mor-an equivalent concentration of normal rabbit or mouse IgG (Phar- phology for up to eight further passages (Fig. 1A) andMingen). consisted of a homogenous population of HDMEC, as

Proliferation assays after VPF/VEGF stimulation. HDMEC were evaluated by their staining with an anti-CD31 antibodyseeded at 5 1 103 cells/cm2 into 24-well tissue culture clusters. Thewhich predominantly decorated HDMEC cell mem-following day, cultures were incubated in endothelial cell basal me-

dium with 2% fetal calf serum or in complete growth medium, with or branes in subconfluent and confluent cultures (Figs. 2A

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3IMMUNOMAGNETIC ISOLATION OF MICROVASCULAR ENDOTHELIAL CELLS

specific feature of endothelial cells. Accordingly,HDMEC (studied between passage 2 and 12) reactedto VPF/VEGF treatment with a highly increased rateof proliferation, with a minimal effective concentrationof 0.3 ng/ml (Figs. 4A and 4B). The extent of stimula-tion of cell proliferation was higher when cells werecultured in endothelial cell basal medium with additionof 2% FCS, compared to cells grown in complete growthmedium (Fig. 4). Treatment of HDMEC with TNF-a,in passages 2 to 8, potently induced the expression ofintercellular adhesion molecule-1 (ICAM-1; CD54; Fig.5A) and, to a lesser extent, of vascular cell adhesionmolecule-1 (VCAM-1; CD106; Fig. 5B).

DISCUSSION

We have established a simple immunomagnetic pro-tocol for the selective isolation of HDMEC from neona-tal foreskins. Taking advantage of the transient, endo-thelial cell-specific induction of E-selectin by TNF-a[18, 19], we used magnetic beads coupled to an anti-E-selectin monoclonal antibody to selectively detect andisolate E-selectin expressing endothelial cells in mixedprimary cultures stimulated with TNF-a. Fibroblastsand epidermal keratinocytes, frequent contaminants inprimary HDMEC cultures, were not inducible for E-

FIG. 1. (A) Monolayer culture of HDMEC (passage 6) with char- selectin expression and, therefore, were easily removedacteristic cobblestone morphology. (B) HDMEC (passage 6) main- during the immunomagnetic purification process. Aftertained for 6 months without subculture: Monolayer culture with few a second purification step, a homogenous populationtube-like structures and no evidence of contaminating fibroblasts or

of HDMEC was obtained. These cells showed typicalother cell populations; 175.cobblestone morphology, expressed the endothelial cellmarkers CD 31 and von Willebrand factor, did not reactwith an anti-cytokeratin antibody, showed uptake ofand 2B). Staining for von Willebrand factor revealed

the intracytoplasmic granular staining pattern charac- acetylated low density lipoproteins, spontaneouslyformed tubes in a three-dimensional collagen type Iteristic for endothelial cells (Figs. 2C and 2D). HDMEC

also showed a rapid uptake of acetylated LDL, leading matrix, responded to the mitogenic effect of VPF/VEGF[27], and upregulated the cell adhesion moleculesto a characteristic intracytoplasmic staining pattern

(not shown). In contrast, staining for cytokeratins was ICAM-1 and VCAM-1 in response to TNF-a. Therefore,the cells isolated using this immunomagnetic purifica-completely absent in HDMEC cultures (Fig. 2F),

whereas the squamous cell carcinoma cell line SCC-13 tion protocol showed the typical morphology and func-tional behavior of HDMEC in culture.used as a positive control showed strong cytokeratin

reactivity (Fig. 2E), but complete absence of staining The major advantages of the described immunomag-netic protocol are: (I) Its simplicity and avoidance offor CD31 or von Willebrand factor (not shown). The

purity of the endothelial cell preparations was further time-consuming steps such as density-gradient centrif-ugations [5, 13] or mechanical removal of fibroblastsconfirmed by the persistence of typical monolayers with

some degree of capillary-like tube formation even after [11]. Moreover, neonatal foreskins are easily obtainedfrom routine circumcisions. We have also applied the6 months without passaging, without any evidence for

fibroblast overgrowth (Fig. 1B). When confluent immunomagnetic protocol to successfully isolate andculture HDMEC from adult skin, although with a lowerHDMEC cultures were overlaid with a collagen type

I gel (500 mg/ml Vitrogen 100 in endothelial growth cell yield (data not shown). (II) The use of a simplifiedgrowth medium without addition of human serum ormedium), they formed capillary-like tubes within 4-6

h (Fig. 3), a typical, although not specific, behavior of recombinant growth factors. Addition of up to 30% hu-man serum to the growth medium has been reportedcultured endothelial cells.

The expression of the VPF/VEGF receptors, KDR as necessary to sustain HDMEC growth [28]. In ourexperience, pure HDMEC cultures could be efficiently[25] and Flt-1 [26], has been widely considered as a

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4 RICHARD, VELASCO, AND DETMAR

FIG. 2. HDMEC, passage 5, cultured on chamber slides: (A, B) Membrane-bound staining of subconfluent HDMEC with anti-CD31antibody. A, 168; B, 1136. (C, D) Characteristic granular cytoplasmic staining pattern after immunohistochemical labeling of HDMEC forvon Willebrand factor. A, 168; B, 1136. (E) Pronounced staining for cytokeratins in SCC-13 squamous cell carcinoma cells used as positivecontrol; 1136. (F) Complete absence of staining for cytokeratins in HDMEC; 1136.

propagated with the addition of 20% FCS only, growth factors and cytokines. HDMEC growth mediahave been routinely supplemented with epidermalavoiding the high costs and the potential contamina-

tion risks associated with human serum. Previously, growth factor (EGF). However, we have been unable todetect mitogenic effects of EGF on HDMEC in vitro,the use of prepartum maternal serum has been sug-

gested to enhance the growth of HDMEC in vitro [28]. and HDMEC did not show any detectable expressionof EGF receptors on Northern or Western blots or tyro-This has not been necessary in our experience; more-

over, prepartal maternal serum contains a number of sine phosphorylation after EGF stimulation (M. Det-mar et al., manuscript in preparation). (III) The tre-additional growth factors, including VPF/VEGF [29],

complicating investigations into the in vitro effects of mendous expansion potential of the cells under the

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5IMMUNOMAGNETIC ISOLATION OF MICROVASCULAR ENDOTHELIAL CELLS

FIG. 3. Spontaneous formation of tube-like structures 16 h afteroverlay of a collagen type I gel onto confluent HDMEC, passage 5; 175.

chosen culture conditions. Using the growth mediumdescribed above and splitting subconfluent cultures ata 1:5 ratio onto collagen type I-coated dishes, HDMEC

FIG. 5. Potent induction of ICAM-1 and moderate induction ofVCAM-1 after 24 h treatment of HDMEC with TNF-a (100 ng/ml).No effect was detected after treatment with VPF/VEGF (50 ng/ml).FACS analysis, mean fluorescence. Data represent one experimentof three with comparable results.

proliferated for more than 35 population doublings be-fore reaching proliferative senescence (data notshown). Even when HDMEC are only used before com-pleting 30 population doublings, one foreskin prepara-tion potentially yields as many as 1 1 1012 HDMEC,making frequent endothelial cell isolation proceduresunnecessary.

In summary, we have established a reliable protocolto isolate and propagate microvascular endothelialcells obtained from neonatal human foreskins. Thetime- and cost-efficient technique avoids major disad-vantages of previously described culture protocols andmay enable a more widespread use of skin microvascu-lar endothelial cell cultures that are functionally dis-tinct from endothelial cell cultures derived from largevessels. Moreover, we have preliminary evidence that

FIG. 4. VPF/VEGF is a potent mitogen for HDMEC. HDMEC the technique is also useful to isolate microvascularwere incubated with or without recombinant human VPF/VEGF for endothelial cells from other normal human organs and4 days. (A) [3H]thymidine incorporation (CPM; mean { SD, n Å 4) in from malignant tumors, as well as from a variety ofendothelial cell basal medium/ 2% FCS (open circles) or in complete

tissues in different species, facilitating organ- or tu-endothelial cell growth medium containing 20% FCS (filled circles).(B) Cell numbers (mean { SD, n Å 3). mor-specific vascular research at the in vitro level.

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6 RICHARD, VELASCO, AND DETMAR

This work was supported by Deutsche Forschungsgemeinschaft 15. Jackson, C. J., Garbett, P. K., Nissen, B., and Schrieber, L.(1990). Binding of human endothelium to Ulex europaeus-1Grant De483/3-2 and by NIH/NCI Grant CA69184.coated dynabeads: Application to the isolation of microvascularendothelium. J. Cell Sci. 96, 257–262.

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Received August 26, 1997Revised version received December 5, 1997

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