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Ann Thorac Surg 1998;66:1653-1657
© 1998 The Society of Thoracic Surgeons

Fluorescence activated cell sorting: A reliable method in tissue engineering of a bioprosthetic heart valve

Simon P. Hoerstrup, MDa, Gregor Zünd, MDa, Andreina Schoeberlein, PhDa, Qing Ye, MDa, Paul R. Vogt, MDa, Marko I. Turina, MDa

a Department of Cardiovascular Surgery, University Hospital Zürich, Zürich, Switzerland

Accepted for publication May 19, 1998.

Address reprint requests to Dr Zünd, Department of Cardiovascular Surgery, University Hospital Zürich, Raemistrasse 100, CH 8091 Zürich, Switzerland
e-mail: (gregor.zund{at}chi.usz.ch)


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Techniques of tissue engineering are used to seed human autologous cells in vitro on degradable mesh to create new functional tissue like a bioprosthetic heart valve. A precondition is subsequent seeding of native-valve–analogous pure endothelial and myofibroblast cell lines. The aim of this study is to find a safe method of isolating viable cell lines out of tissues from the operating room.

Methods. Mixed cells from ascending aorta obtained from the operating room were incubated with an endothelial-specific fluorescent marker. The labeled cells were activated and sorted by flow cytometry. Isolated cell lines were cultured and thereafter square sheets of polymeric scaffold were seeded with myofibroblasts, followed by endothelial cells. The created tissue was stained with hematoxylin and eosin, van Gieson stain, and stains for factor VIII and CD34.

Results. Control culture samples (n = 25) revealed vital uncontaminated endothelial and myofibroblast cell lines. Microscopy of the seeded meshes (n = 16) demonstrated a tissue-like structure. Van Gieson stain showed production of collagen. Endothelial cells formed a superficial monolayer, demonstrated by factor VIII and CD34; no invasive formation of capillaries was detectable.

Conclusions. These results demonstrate that fluorescence activated cell sorting is a reliable and safe method to gain pure vital autologous cell lines out of human mixed cells for subsequent seeding on degradable mesh and that those cells are active to form new tissue.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Annually thousands of patients die as a direct result of heart valve dysfunction, and even more require heart valve replacement [1]. For treatment of heart valve disease, mechanical or biological valves are currently in use. The drawbacks of mechanical valves include the need for life-long anticoagulation associated with possible anticoagulation-related hemorrhage [2], the risk of thromboembolic events, prosthesis failure, and the inability of the device to grow [3, 4]. Although biological valves (homograft, xenograft) are considered to be hemodynamically excellent and relatively resistant to infections and they do not require anticoagulation treatment, these grafts have a limited durability resulting from their immunogenic potential [5].

Therefore, the optimal characteristics of a new prosthetic heart valve would include construction from viable autologous tissue, providing the ability to grow, repair, and remodel, thereby increasing durability without risk of rejection. Moreover, the need for long-term anticoagulation would be avoided.

In tissue engineering the material properties of synthetic compounds are chosen to enable delivery of dissociated cells onto a scaffold that will result in in vitro formation of new functional tissue. The new techniques of tissue engineering like in vitro seeding of myofibroblasts and endothelial cells derived from the potential recipients on biodegradable scaffolds to create a bioprosthetic heart valve are among the latest developments in this field [6–8].

Based on previous experience with endothelial cell seeding [9], our laboratory has focused on the creation of heart-valve–analogous functional human tissue with the techniques of tissue engineering. A precondition for the successful formation of autologous heart valve tissue is the subsequent seeding of the native valve analogous cell types, myofibroblasts and endothelial cells. Therefore, the aim of this study is the development and description of a reliable, safe, and efficient method to gain isolated pure cell lines (endothelial cells, myofibroblasts) out of human aortic mixed cell populations that are still viable and active for the formation of new functional tissue.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
The concept of our approach to formation of a tissue engineered heart valve includes isolation of cell lines out of mixed aortic cell population by flow cytometry followed by subsequent seeding of the pure cells (myofibroblasts, endothelial cells) to create a native valve analogous tissue (Fig 1).



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  		Fig 1. Concept of tissue engineering of a bioprosthetic heart valve. (FACS = fluorescence activated cell sorting.)

 
Mixed cell populations were obtained from the ascending aorta of explanted hearts of heart transplant patients. The harvested pieces of aortic tissue were immediately transferred to the laboratory and under sterile conditions were serially washed with phosphate-buffered saline solution (Gibco BRL-Life Technologies, Grand Island, NY) supplemented with 1x antibiotic-antimycotic solution (Gibco BRL-Life Technologies). Under a laminar flow hood (Forma Scientific, Marietta, OH) the pieces of tissue were fragmented into small compartments of about 1 mm in diameter. Thereafter these compartments were transferred into 75 cm2 culture dishes (Becton Dickinson, Lincoln Park, NJ) with Dulbecco’s modified Eagle’s medium (Gibco BRL-Life Technologies) supplemented with 10% fetal bovine serum and 1x antibiotic-antimycotic (Gibco BRL-Life Technologies). After 14 days’ culture time in a humidified incubator at 37°C with 5% CO2, the cells that had spread out of the tissue pieces and formed a confluent cell layer at the bottom of the culture dish were serially passaged to obtain freedom from residual tissue material.

At passage 2 or 3 the mixed cell populations were incubated with a specific fluorescent cell marker for human endothelial cells (Dil-Ac-LDL; Biomedical Technologies Inc, Stoughton MA). After 4 hours’ incubation (10 µg/mL medium) at 37°C, the medium containing Dil-Ac-LDL was removed and the cells were washed with probe-free medium several times. To produce a single cell suspension, the labeled mixed cultures (endothelial cells = LDL+, myofibroblasts = LDL-) were trypsinized by EDTA trypsin (1x; Gibco BRL-Life Technologies) and transferred in medium for cell sorting.

The sorting of the labeled cell suspension was done by flow cytometry with a fluorescence activated cell sorter (FACS; FACStar Plus; Becton Dickinson, Institute of Biomedical Engineering, Swiss Federal Institute of Technology and University of Zürich, Zürich, Switzerland). Activation of the labeled cells was provided by an argon laser ({lambda}ex = 514 nm), sorting of the activated cells by their emitted wavelength of 550 nm (Dil-Ac-LDL fluorescence was measured through a bandpass filter centered at 550 nm and amplified logarithmically). After sorting, there were two isolated cell lines: LDL-positive endothelial cells and LDL-negative myofibroblasts. These isolated cell lines were cultured for 3 weeks again and thereby passaged three times to obtain sufficient numbers of cells for the subsequent cell seeding. Before the seeding procedure was started, control samples of each cell culture were obtained and marked by Dil-Ac-LDL again for fluorescence microscopy.

Polymeric nonwoven scaffolds (n = 16) composed of polyglycolic acid with a fiber diameter of 12 to 15 µm and a polymer density of 70 mg/mL (Albany International Research, Mansfield, MA; a kind gift from Dr J. Vacanti, Children’s Hospital, Harvard Medical School, Boston, MA) were used for seeding as square sheets of 0.3 x 1 x 1 cm. The sorted human myofibroblasts were first seeded in eight to ten subsequent seeding procedures every 90 minutes. Each seeding procedure comprised 3.4 x 106 human myofibroblasts. The seeded cell-polymer constructions were cultured over a 21-day period. The medium was changed every 4 days. Thereafter the tissuelike structures were seeded with the initially sorted pure human aortic endothelial cells (eight seedings, 90-minute intervals, 2.8 x 106 cells per seeding). After another 7 days the constructs were fixed with 0.4% paraformaldehyde and stained with hematoxylin and eosin and van Gieson stains, and stains for factor VIII and CD34.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Flow cytometry
Before sorting by fluorescence activation, the human mixed cell populations (previously labeled with Dil-Ac-LDL) were submitted to analysis by flow cytometry (Fig 2). In the light of a negative control (see Fig 2A, without fluorescence labeling), discrimination between fluorescent negative (myofibroblasts) and positive cells (endothelial cells) (see Fig 2B) could be established. Cells were considered unambiguously fluorescent from greater than 102 upward (M2) and clearly nonfluorescent at less than 101 (M3). For sorting, the cell population in between 101 and 102 (M1) was excluded in order not to risk the inclusion of ambiguous cells in the sorted cell lines (Fig 2C).



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  		Fig 2. Histogram flow cytometry. (A) Negative control, unlabeled mixed cell population of human aortic tissue. (B) Positive assay, Dil-Ac-LDL–labeled mixed cell population of human aortic tissue (endothelial cells = LDL+ [fluorescent], myofibroblasts = LDL-). (C) "Gating" of significant areas for fluorescence activated cell sorting. (M3 = nonfluorescent [myofibroblasts]; M2 = fluorescent [endothelial cells]; M1 = with regard to fluorescence ambiguous cell population [safety margin for sorting procedure].)

 
Fluorescence microscopy
After cell sorting by FACS, the sorted cells were cultured and passaged for 3 weeks. To demonstrate the purity of these cell lines (endothelial cells, myofibroblasts) we obtained control samples of each cell culture before starting the seeding procedure. These samples were marked by Dil-Ac-LDL again and thereafter examined by fluorescence microscopy. Fluorescence microscopy demonstrated full fluorescence of the endothelial cell population (LDL+) and no fluorescence (apart from the natural fluorescent emission) of the myofibroblasts (LDL-) (Fig 3).



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  		Fig 3. Fluorescence microscopy: full fluorescence of the endothelial cell line (LDL+). (x100 before 35% reduction.)

 
Histomorphology
Microscopic examination of the seeded meshes demonstrated a well-grown homogeneous basic structure attached to the polymeric fibers, and van Gieson stain showed production of collagen matrices. The seeded human endothelial cells formed a superficial monolayer without signs of invasive capillary ingrowths into the basic tissue-like structure, as demonstrated by factor VIII and CD34 stains (Fig 4).



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  		Fig 4. Formation of endothelial monolayer. CD34 stain demonstrates a monolayer of endothelial cells (a) on the surface of a fibroblast core (b) with polymer fibers (c).

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
Tissue engineering is a new, expanding field in medical research, which potentially will lead to relevant clinical applications in the upcoming years [6]. In tissue engineering, human autologous cells are seeded in vitro on degradable polymeric scaffolds to create new functional tissue. The advantage of such tissue-engineered products is the ability to grow, repair, and remodel, without requirement for anticoagulation. Furthermore, because of the use of autologous cell lines, an increasing durability will possibly be achieved by absence of immunogenicity of the construction.

Tissue engineering is emerging experimentally, and tissue substitutes for liver, cartilage, bone, trachea, intestine, and urologic tissue are created [10–13]. With regard to cardiovascular surgery, Vacanti and Mayer at Children’s Hospital in Boston have become a leading research group in this field, especially concerning the creation of an autologous heart valve in the animal model [6, 7, 14].

Our laboratory has also focused on the construction of a tissue-engineered heart valve, and we tried to apply the experiences from the animal model to human cells [8]. For this reason we started with the construction of a living compound of human myofibroblasts followed by seeding of human endothelial cells.

A precondition for the formation of autologous heart valve tissue is the subsequent seeding of native valve-analogous cell types, namely, human myofibroblasts and human endothelial cells. For the subsequent seeding procedure it is very important to rely on a sufficient amount of pure human cells of each cell population. From our operating room we obtained human mixed cell populations of ascending aorta of transplant patients. As a consequence we had to look for a reliable, safe, and efficient method to isolate the myofibroblasts on the one hand and the endothelial cells on the other hand out of the mixed population to get absolutely pure cell lines of each type.

However, few efficient methods are available for isolation and purification of viable cell lines, the main problem being the presence and rapid overgrowth in culture of contaminating cells [15–17]. Among automated cell separation methods, immunomagnetic isolation has been tested, but it is less reliable concerning purity in comparison with fluorescence separation [18]. The results of this study demonstrate that FACS is a safe and efficient method to fulfill the above-mentioned criteria for cell separation for tissue engineering of cardiovascular tissue.

Fluorescence activated cell sorting has been already proved as a reliable method for cell separation with regard to several cell types. Schweitzer and associates [18] have described successful isolation of endothelial cells from human bone marrow. Moreover, isolation of microvascular endothelium derived from human lung has been presented by Carley and colleagues [19], and some research has been done in animal models [20].

To our mind, the reliability and safety of FACS results from the methodology of the procedure itself. The immunofluorescent labeling (Dil-Ac-LDL) of endothelial LDL receptors achieves a complete marking of the living endothelial cells, the precondition of precise cell sorting [21]. Moreover, in the light of the negative control (without fluorescent labeling), analysis by flow cytometry of the labeled mixed cell populations allows exact discrimination between fluorescent-negative myofibroblasts and positive endothelial cells. For the definite sorting procedure, we established an additional "safety margin." Cells were considered unambiguously fluorescent at greater than 102 and nonfluorescent at less than 101; that is, cell populations in between were excluded to realize pure endothelial and myofibroblast cell lines for the subsequent seeding procedure. Moreover, preceding the definite seeding procedure, newly relabeled (Dil-Ac-LDL) control samples of the cultured isolated cells revealed full fluorescence of the endothelial cells and, except for slight natural fluorescence, no emission of the myofibroblasts. This demonstrates that even after three cell passages and 3 weeks’ culture time, the populations remained pure and uncontaminated. Postsorting viability of the cell lines was proved by prompt trypsin reaction and excellent growth potential of the cells on the biodegradable meshes. The histomorphologic analysis of the seeded meshes showed a well-grown basic homogeneous structure of myofibroblasts and, most important, a superficial monolayer of pure endothelial cells without signs of invasive capillary ingrowth into the tissue. This fact is additional proof of purity of the seeded cell lines and moreover a good demonstration of the growth potential and viability of the postsorted cells, which should give them excellent applicability for subsequent seeding procedures of cardiovascular tissue engineering.

In conclusion, FACS proved to be a reliable, safe, and efficient method to gain vital uncontaminated human autologous cell lines (myofibroblasts, endothelial cells) out of human aortic mixed cell populations. Therefore, FACS is a promising technique fulfilling the needs of tissue engineering cardiovascular tissues such as a bioprosthetic heart valves, especially with regard to cell viability and activity for new tissue formation. In the near future, new high-speed cell sorters will enable sorting procedures of even higher cell numbers and thereby may provide an almost unlimited supply of pure human cell lines for tissue engineering.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
We thank Mrs Eva Niederer (Institute of Biomedical Engineering, Swiss Federal Institut of Technology and University of Zürich) for her kind help and technical assistance.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Heart and stroke facts. 1994 statistical supplement. Dallas, TX: American Heart Association, 1994.
  2. Cannegieter S.C., Rosendaal F.R., Briet E. Thromboembolic and bleeding complications in patients with mechanical heart valve prostheses. Circulation 1994;89:635-641.[Abstract/Free Full Text]
  3. Renzulli A., DeLuca L., Caruso A., Verde R., Galzerano D., Cotrufo M. Acute thrombosis of prosthetic valves: a multivariate analysis of the risk factors for a lifethreatening event. Eur J Cardiothorac Surg 1992;6:412-420.[Abstract]
  4. Hammermeister K.E., Sethi G.K., Henderson W.G., Oprian C., Kim T., Rahimtoola S. A comparison of outcomes in men 11 years after heart valve replacement with a mechanical valve or bioprosthesis. N Engl J Med 1993;18:1289-1296.
  5. Yacoub M., Rasmi N.R., Sundt T.M., et al. Fourteen years’ experience with homovital homografts for aortic valve replacement. J Thorac Cardiovasc Surg 1995;110:186-193.[Abstract/Free Full Text]
  6. Langer R., Vacanti J.P. Tissue engineering. Science 1993;260:920-925.[Abstract/Free Full Text]
  7. Shinoka T., Breuer C.K., Tanel R.E., et al. Tissue engineering heart valves. Ann Thorac Surg 1995;60:513-516.
  8. Zünd G., Breuer C.K., Shinoka T., et al. The in vitro construction of a tissue engineered bioprosthetic heart valve. Eur J Cardiothorac Surg 1997;11:493-497.[Abstract]
  9. Pasic M., Muller-Glauser W., von Segesser L., Odermatt B., Lachat M., Turina M. Endothelial cell seeding improves patency of synthetic vascular grafts: manual versus automatized method. Eur J Cardiothorac Surg 1996;10:372-379.[Abstract]
  10. Cao Y., Vacanti J.P., Ma X., et al. Generation of neo-tendon using synthetic polymers seeded with tendocytes. Transplant Proc 1994;26:3390-3391.[Medline]
  11. Sakata J., Vacanti C.A., Schloo B., Healy G.B., Langer R., Vacanti J.P. Tracheal composites tissue engineered from chondrocytes, tracheal epithelial cells and synthetic degradable scaffolding. Transplant Proc 1994;26:3309-3312.[Medline]
  12. Vacanti J.P., Morse M.A., Saltzman W.M., Domb A.J., Perez-Atayade A., Langer R. Selective cell transplantation using bioabsorbable artificial polymers as matrices. J Pediatr Surg 1988;23:3-9.
  13. Vacanti J.P. Beyond transplantation. Third Annual Samuel Jason Mixter Lecture. Arch Surg 1988;123:545-549.[Abstract/Free Full Text]
  14. Shinoka T., Ma X., Breuer C.K., et al. Tissue engineering heart valves: autologous valve leaflet replacement study in a lamb model. Circulation 1996;94(Suppl 2):164-168.
  15. Karasek M.A. Microvascular endothelial-cell culture. J Invest Dermatol 1989;93(Suppl 2):33S-38S.[Medline]
  16. Imcke E., Ruszak Z., Mayer da Silva A., Detmar M., Orfanos C.E. Cultivation of human dermal microvascular endothelial cells in vitro: immunocytochemical and ultrastructural char-acterization and effect of treatment with three synthetic retinoids. Arch Dermatol Res 1991;283:149-157.[Medline]
  17. Drake B.L., Loke Y.W. Isolation of endothelial cells from human first trimester decidua using immunomagnetic beads. Human Reprod 1991;6:1156-1159.[Abstract/Free Full Text]
  18. Schweitzer C.M., van der Schoot C.E., Dräger A.M., et al. Isolation and culture of human bone marrow endothelial cells. Exp Hematol 1995;23:41-48.[Medline]
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  20. Sahagun G., Moore S.A., Fabry Z., Schelper R.L., Hart M.N. Purification of murine endothelial cell cultures by flow cytometry using fluorescein-labeled Griffonia simplicifolia agglutinin. Am J Pathol 1989;134:1227-1232.[Abstract]
  21. Voyta J.C., Netland P.A., Via D.P., Zetter B.R. Specific labeling of endothelial cells using fluorescent acetylated-low density lipoprotein. J Cell Biol 1984;99:81A.



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