Vital Fluorescent Staining of Human Endothelial Cells, Fibroblasts, and Monocytes: Assessment of Surface Morphology James M. Crawford, MD, PhD, Edgar Milford, MD, Kay Case, BA, and Nina S. Braunwald, MD Division of Cardiac Surgew and Departments of Medicine and Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massacvhusetts

Vital fluorescent staining of human endothelial cells, fibroblasts, and monocytes seeded on a variety of sur- faces has been carried out to develop a method for studying cell growth and interactions on viable cells. Using 'special fluorochrome markers and monoclonal

n studies carried out at the National Heart Institute in I the 1960s, growth of nonthrombogenic autologous tissue layers was observed on the fabric lattices used to cover the support frames of homograft valves and rigid prostheses with moving poppets (1, 21. Moreover, flexible polyurethane leaflets of varying porosities that were nonthrombogenic when implanted in the valve annulus of host animals could be made [3]. This work was ex- tended to patients by covering ball valve prostheses with a similar fabric before implantation. Over the following decade, these fabric lattices were studied histologically after retrieval of the valves from patients coming to reoperation or autopsy [4, 51. These studies confirmed that use of a favorable lattice, in the absence of undue abrasion by a moving poppet, encourages growth of a living tissue layer which shows minimal calcification, even after a decade.

Recently, we have begun a series of experiments to examine the relationship between cell matrix materials and artificial surfaces, using vital fluorescent staining [6]. We hypothesized that the requirements for optimal cell growth were best studied in vitro in living cells actively growing on a polymer surface. Toward that end, the behavior of endothelial cells, blood monocytes, and fibro- blasts seeded on synthetic surfaces was monitored by fluorescence microscopy after cellular uptake of selected fluorescent markers.

Material and Methods Human umbilical vein endothelial cells were harvested with collagenase and subcultured in medium 199 (M.A. Bioproducts) with 20% fetal calf serum (Gibco), 50 pg/mL ECGS (Biomedical Technologies, Inc) and 100 pg/mL

Presented at the World Congress on Heart Valve Replacement, San Diego, CA, Jan 15-18, 1989.

Address reprint requests to Dr Braunwald, Division of Cardiac Surgery, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115.

antibodies, it was possible to differentiate different cell types as they grew on polymer surfaces similar to ones that are presently used on artificial heart valves and vascular grafts.

(Ann Thorac Surg 1989;48:S100-2)

heparin. Human monocytes were obtained from heparin- ized whole blood using a Ficoll gradient, and cultured in RPMI-1640 medium (Whittaker). Human fetal fibroblasts (MRCB) were obtained commercially, and cultured in Dulbecco's minimal essential medium. After 24 to 72 hours in culture, nonadherent cells were removed by gentle rinsing before the addition of solutions containing fluorescent probes in 0.5 mL of medium. Cells were grown on 12-mm coverslips coated with either 1% gelatin, 0.5% Biomer (a segmented polyether polyurethane; Ethi- con, Inc) plus human fibronectin, or Cell-Tak (an adhesive protein derived from the mussel Myelitis edulis; Biopoly- mers, Inc).

Endothelial cells were labeled with fluorescein isothio- cyanate (F1TC)-labeled or rhodamine-labeled Ulex euro- paeus I lectin (Vector Laboratories) after a one-hour incu- bation (50 pL). Monocytes were labeled with MOZ-FITC (one hour incubation; 50 pL) or M02-biotin (0.5 hour; 50 pL) followed by avidin-phycoerythrin (0.5 hour; 25 pL; Phycoprobes; Biomedia Corp). Fibroblasts were labeled with rhodamine 123 (one-hour incubation, 50 pL; Molec- ular Probes) or rhodamine-labeled phalloidin (two-hour or 24-hour incubation, 50 pL; Molecular Probes). Uptake of fluorescein diacetate over one minute (stock solution of 5 mg/mL in dimethyl-sulfoxide, diluted 1:1,000) was used to monitor viability of endothelial cells and fibroblasts previously labeled with the above fluorochromes; viability of monocytes was monitored by exclusion of 0.4% trypan blue. All fluorochromes were incubated with living cells; control experiments demonstrated fluorescent staining patterns similar to those seen in fixed cells.

Results Human umbilical vein endothelial cells were plated at low density (8 x lo4 cells per coverslip). In Figures 1A-C are shown the phase image (A) and staining of 24-hour- cultured endothelial cells with Ulex europueus-rhodamine lectin (B) and FITC-diacetate (C), demonstrating charac-

0 1989 by The Society of Thoracic Surgeons

Ann Thorac Surg 1989;48:SlOC-2

CRAWFORD ET AL slol VITAL FLUORESCENT STAINING OF HUMAN CELLS

Fig I . Vital fluorescent staining of endothelial cells, monocytes, and fibroblasts. Endothelial cells grown 24 hours on I % gelatin are shown by phase microscopy (A; x400), after staining one hour with Ulex europaeus-rhodamine lectin ( B ; x400), and after one minute uptake of FITC- diacetate, showing preservation of cell viability (C; ~ 4 0 0 ) . After being cultured for 48 hours, cell density is greater, as shown for a different plate ID: Ulex europaeus-rhodamine lectin; E: FITC-diacetate; ~ 4 0 0 ) . Monocytes stained with MO2-biotin-avidin are shown in ( F ) (x400). Fibro- blasts are shown under phase microscopy (G, 1) and stained one hour with rhodamine 123 ( H ; x1,OOO) or 24 hours with rhodamine-phalloidin (1; x1,OOO). Even after 24 hours of coincubation with rhodamine 123 f K ) , fibroblasts retain viability and their characteristic morphology (L; FlTC- diacetate; x400). (All magnifications are before 37% reduction.)

teristic morphology with excellent viability. Confluence was achieved after 48 to 72 hours in culture: Figures 1D-E demonstrate the increased cellular density with retention of rhodamine-lectin staining and viability at 48 hours. Cell morphology and fluorochrome uptake were consistent and similar whether the cells were grown on 1% gelatin (Figs 1 A-E), Cell-Tak, or Biomer with fibronectin. Fibro-

blasts and monocytes were not stained by Ulex europaeus- fluorochrome.

Monocytes stained uniformly with M02-biotin/avidin/ phycoerythrin (Fig 1F) and MO2-FITC while retaining viability, as documented by uptake of FITC-diacetate. Endothelial cells and fibroblasts did not stain with the M02-conjugated probes.

s102 CRAWFORD ET AL VITAL FLUORESCENT STAINING OF HUMAN CELLS

Ann Thorac Surg 1989;48:S100-2

Optimal staining of MRC-5 fibroblasts with rhodamine 123 was achieved after one hour; overnight incubation of fibroblasts was optimal for staining with rhodamine- phalloidin. Fibroblasts were consistently labeled with these probes, but exhibited contrasting patterns of fluo- rescence. Rhodamine-phalloidin produced a limited pat- tern of punctate intracellular fluorescence (Fig lG, phase; Fig lH, fluorescence). In contrast, bright intracellular structures were visualized with rhodamine 123 (Fig 11, phase; Fig lJ, fluorescence). In both cases, cells retained viability, even after overnight incubation (Fig lK, over- night rhodamine 123; Fig lL, overnight rhodamine 123 followed by one-minute incubation with FITC-diacetate).

Comment In these studies we have demonstrated that a specific group of fluorescent markers can be applied to living cells, with no evidence of toxicity over many hours in culture. Endothelial cells, monocytes, and fibroblasts have been examined while growing on a variety of biomatrix mate- rials, using fluorochromes that enable identification of cellular types and their easy visualization by fluorescence microscopy. The lectin from Ulex europaeus has previously been shown to bind to surface glycoproteins and juxtanu- clear structures representing the Golgi apparatus in endo- thelial cells (71, whereas the monoclonal antibody M02 binds to surface antigens on monocytes (81. Although rhodamine 123 and rhodamine-phalloidin (which bind to intracellular mitochondria and F-actin, respectively) [9, 101 do not specifically label fibroblasts, they have proved to be particularly useful in visualizing growth of these cells on a biomatrix.

The experimental approach presented here will enable detailed studies of the cells that normally line cardiovas- cular channels and their interaction with biomatrix mate- rials. We can thus examine the requirements for, and modulation of, tissue layer growth on the biosynthetic components of artificial heart valves and cardiac assist devices, as well as the effects of biomatrix composition on

cellular morphology. Moreover, this in vitro approach will allow exploration of the effects of clinical treatment mo- dalities on tissue growth (eg, focal destruction of the vascular lining by laser treatment). The knowledge gained may be critically important for the future design of car- diovascular prosthetic devices and our understanding of the tissue response to these materials.

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Bull B, Fuchs JCA, Braunwald NS. Mechanisms of formation of tissue layers on the fabric lattice covering intravascular prosthetic devices. Surgery 1969;65 :W. Braunwald NS, Fuchs JCA, Boncheck LI. Simplified insertion of aortic homograft valves with nonthrombogenic prosthetic frames. Surgery 1968;63:3844. Braunwald NS, Reis RL, Pierce GE. Relation of pore size to tissue ingrowth in prosthetic heart valves: an experimental study. Surgery 1965;57:741-8. Bull BS, Braunwald NS. Human histopathologic response to a completely fabric-covered prosthetic heart valve. Ann Surg 1971;174:755-61. Schoen FJ, Goodenough SH, Ionescu MI, Braunwald NS. Implications of the late morphology of Braunwald-Cutter mitral heart valve prosthesis. J Thorac Cardiovasc Surg 1984;88:208-16. Braunwald NS, Viles A, Case K, Milford E. Vital fluorescent staining of endothelial cells and monocytes on a surface for the assessment of cellular morphology and interactions [Ab- stract]. ASAIO 1988:30. Holthofer H, Virtanen I, Kariniemi AL, et al. Ulex europaeus I lectin as a marker for vascular endothelium in human tissues. Lab Invest 1982;47:60-6. Todd RF 111, van Agthoven A, Schlossman SF, Terhorst C. Structural analysis of differentiation antigens Mol and Mo2 on human monocytes. Hybridoma 1982;1:329-37. Goldstein S, Korczack LB. Status of mitochondria in living human fibroblasts during growth and senescence in vitro: use of laser dye rhodamine 123. J Cell Biol 1981;91:392-8. Barak LS, Yocum RR, Webb WW. In vivo staining of cyto- skeletal actin by autointernalization of nontoxic concentra- tions of nitrobenzoxadiazole-phallacidin. J Cell Biol 1981; 8 ~ 6 ~ 2 .