EUROPEAN TISSUE REPAIR SOCIETY

TISSUE ENGINEERING II

PRIMARY CULTURES OF MURINE DERMAL MICROVASCULAR ENDOTHELIAL CELLS(mDMEC).
A USEFUL ANIMAL MODEL SYSTEM FOR IN VITRO WOUND ANGIOGENESIS STUDIES AND TISSUE ENGINEERING.

THE dermal microvascular endothelium with its strategic location plays a central role in physiological and pathological processes in the skin, including anti-thrombogenicity, blood vessel permeability and pressure control, metabolism of lipoproteins, tissue ageing, antigen presentation, tumour metastasis, and angiogenesis during wound healing.

Studies involving murine dermal microvascular endothelial cells have been generally impeded by the inherent difficulties in developing primary cultures of these cells. The in vitro cultures of macrovascular endothelial cells have proved to be useful in investigating some aspects of vascular physiology. However, studies of cultured macrovascular endothelial cells may not be valid to investigate specific microvascular behaviour and function. Human microvascular endothelial cell cultures have been established using a variety of isolation- and culture-methods for cells isolated from neonatal foreskin, adult human skin or tissue derived from vascular tumours.1 Methods based on continuous gradient centrifugation of cells obtained from dermis after enzymatic and mechanical disaggregation of tissue fragments have been described in the last decade.^2,3 Models for murine microvascular cell isolation are still limited and there is no current description of microvascular segment isolation of dermal origin.^4-7

Method of endothelial cell isolation

In the method described here, murine dermal microvascular endothelial cells (mDMEC) were derived from the skin of 3-day-old mice. After decapitation, the entire skin flap was dissected and placed immediately in Hank’s balanced salt solution (HBSS), containing concentrated antibiotic and antifungal compounds, (10,000 IU Penicillin, 10,000 µg ml^-1 Streptomycin, (GIBCO BRL, Carlsbad CA)), Fungizone (205 μg ml^-1 Sodium Deoxycholate and 250 μg ml^-1 Amphotericin B, (Omega Scientific, Tarzana, CA)) and Gentamycin (10 μg ml^-1 (Omega Scientific)). The tissue was washed in three sequential 5-minute incubations in this solution. The tissue flap was then incubated for 45 min at 37 °C in 0.05 mg ml-1 Dispase solution (0.94 units mg^-1, GIBCO BRL, Carlsbad, CA) with continuous agitation. The epidermis was then easily removed from the dermis by mechanical dissociation. The residual dermal fragments were incubated overnight at 4°C in a 4% type 1A Collagenase solution (342 units mg^-1, Worthington, NJ) with 4% bovine serum albumin in phosphate buffered saline (PBS). The dermal tissue fragments were passed through a 100 μm nylon mesh cell strainer (BD Labware, Franklin Lakes, NJ). The filtered cell suspension was diluted with HBSS and centrifuged for 5 min at 400g. The resulting cell pellet was resuspended in PBS and placed on a sterile Percoll gradient prepared by centrifuging a 35% Percoll solution at 30.000g for 15 min at 4ºC (Amersham Biosciences, Uppsala, Sweden). The gradient was then centrifuged for 10 min at 400g. Endothelial cells lay in the gradient between density 1.033 and 1.047, whereas nonendothelial cells have a density greater than 1.065 g/ml. The endothelial cells were collected, washed in HBSS, counted and plated at an average density of 7.500 cells cm^-2 on type IV collagen-coated plates (BD Biosciences, Bedford, MA). The growth medium consisted of complete Dulbecco’s Modified Eagle’s Medium (DMEM) plus 10 mM HEPES buffer, 5 μM Mercaptoethanol and Penicillin- Streptomycin (GIBCO BRL, Carlsbad CA). The medium was supplemented with 20% foetal bovine serum, 1% Endothelial Cell Growth Supplement (ECGS) (Sigma-Aldrich Chemical Co., St Louis, Mo) and recombinant Vascular Endothelial Growth Factor rh-VEGF (1.5 ng ml^-1) (Genentech, South San Francisco, CA).

Results

mDMEC Isolation and Culture
Purified cells were obtained, either from the skin of a single newborn mouse, or pooled skin from multiple mice. The gradient separation procedure following overnight collagenase digestion separates microvascular cells from nonendothelial cells such as dendritic cells, epithelial cells, melanocytes, fibroblasts and pericytes. The final purified cell yield averages 0.5 million cells per mouse. The collagen type IV-coated culture surfaces allowed adequate endothelial cell attachment within two hours. The plated cells typically reach confluence by day 5 and are then subcultured. At confluence the average cell density is approximately 40.000 cells cm^-2. The culture conditions described here inhibit fibroblast and keratinocyte growth and selectively stimulate the proliferation of endothelial cells.

Morphological and phenotypic characterization of mDMEC
Cultured mDMEC form contact-inhibited monolayers with morphological characteristics that closely resemble endothelial cell cultures from other species. The typical ‘cobblestone’ appearance is shown in Figure 1 (a and b). The cells maintain their uniform appearance through eight serial passages, suggesting a homogeneous cell population.

The cellular identity of mDMEC was confirmed by assessment of structural and functional parameters: The isolated mDMEC cultures have positive immmunoreactivity to anti-CD 31/PECAM (platelet endothelial cell adhesion molecule) (Figure 2a). In addition, mDMEC also displayed the characteristic immunoreactive endothelial Nitric Oxide Synthetase (eNOS) in the Golgi-vesicular compartment (Figure 2b). The cells also reacted to antibodies directed against von Willebrand Factor (vWF), which is commonly used for endothelial cell identification (Figure 2c). A functional assay performed on sub confluent mDMEC cultures demonstrated the uptake of Di-acetylated LDL (Molecular Probes, Eugene, OR) mediated through the endothelial cell Scavenger Receptor (Figure 2d). The primary mDMEC cultures retain these phenotypic characteristics up to subculture 5.

Gene expression studies
Quantitative real-time RT-PCR studies were used to evaluate the gene expression of endothelial phenotypic markers including CD 31 (PECAM), eNOS, Flk-1 and Tie-2 (Figure 3).

Cell Sorting
To address the homogeneity of the mDMEC cultures fluorescence- activated cell sorting analysis was done using anti- CD31 antibody (Figure 4). As shown, primary cultures of mDMEC are homogeneous.

Figure 1. (1a) mDMEC 3 days after seeding. (1b) mDMEC after reaching confluence.

Figure 2. mDMEC Immunofluorescence Staining. Cells were cultured on collagen-coated microscope slides and stained with the indicated antibodies. Nuclei were stained using DAPI for contrast. (2a) Anti-murine CD31 (PECAM); (2b) Anti- eNOS-stain; (2c) Polyclonal rabbit anti-vWF; (2d) DI-acetylated LDL-uptake.

Figure 3. Gene Expression. Total RNA from mDMEC cultures was used for reverse transcriptase followed by real-time PCR reaction using murine TaqMan probe-primer sets. (3a) CD31; (3b) eNOS; (3c) Flk-1 – VEGF-tyrosine kinase receptor; and (3d) Tie-2 – Angiopoetin-Receptor.

Figure 4. Cell Sorting Profile.
Anti-CD-31 labeled cells (solid line) were analyzed by FACS. Control cells received non-specific IgG.

Tube Formation in 3D Collagen Gels
The ability of mDMEC to form tubes in a 3D matrix gel was assessed using an in vitro model for angiogenesis as previously described.^8,9 Tube formation was induced by stimulating the cultures with various concentrations of murine recombinant Leptin (Calbiochem, San Diego CA) (Figure 5).

Conclusions

Murine microvascular endothelial cells have been difficult to culture for several reasons. Primary cultures are difficult to prepare and are often contaminated by fibroblast and other stromal cells. Furthermore, the components of certain culture media do not fully satisfy the growth requirements. ^1,3,10,11 The most useful techniques have involved either perfusion of target organs with digestive enzymes or the digestion or homogenization of an entire organ. ^5,7 Techniques of EC isolation of dermal origin often utilize neonatal foreskin, adult human skin or skin fragments from large mammals.1 This is the first report on the isolation and culture of dermal microvascular endothelial cells from a murine source. The strategy for the isolation of mDMEC involves enzyme digestion and a Percoll gradient purification. The media formulation provides culture conditions that favour endothelial cell proliferation and help prevent overgrowth by contaminating non-endothelial cells. The advantage of generating primary endothelial cultures using the skin from individual mice is of particular interest to investigators using transgenic and null murine models where the availability of animals is limited and costly. Cultured mDMEC may be used to generate models systems to analyze basic aspects of microvascular biology and to investigate possible applications for tissue engineering.

Figure 5. 3D- fibronectincollagen Gel Assay.
mDMEC cells were incubated in Type I collagen-fibronectin gels. Complete medium was used as control. Tube formation induced by 15nM and 25 nM murine recombinant Leptin.

References

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8. Sierra-Honigmann MR, Nath AK, Murakami C, Garcia-Cardena G, Papapetropoulos A et al. Biological Action of Leptin as an Angiogenic Factor. Science 1998; 281: 1683–1686.
9. Schechner JS, Nath AK, Zheng L, Kluger MS, Hughes CWC et al. In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse. Proc Natl Acad Sci 2000; 97: 9191–9196.
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Corresponding authors:
Erhan Demir and M. Rocio Sierra-Honigmann
Department of Surgery,
Division of Plastic and Reconstructive Surgery,
Cedars Sinai Medical Center,
Los Angeles, CA, USA
Erhan.Demir@cshs.org
or Rocio.Honigmann@cshs.org