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Ann Thorac Surg 1995;60:1627-1632
© 1995 The Society of Thoracic Surgeons
Department of Hematology-Oncology, Cornell University Medical Center, and Divisions of Cardiothoracic Surgery and Circulatory Physiology, College of Physicians & Surgeons, Columbia University, New York, New York
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Abstract |
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Methods. The blood-contacting surface of seven ThermoCardiosystems left ventricular assist devices was removed, washed with phosphate-buffered saline solution, and digested with 0.1% collagenase for 15 to 20 minutes. The hematopoietic cells released from the explants were isolated and analyzed by flow cytometry and immunohistochemical staining.
Results. More than 80% ± 6% of hematopoietic cells isolated in this fashion are of myelomonocytic origin and express CD14, CD15, and CD33 surface molecules. Four percent of cells express the CD34 surface marker, which suggests that the neointima is colonized by pluripotent hematopoietic stem cells. Continuous culture of these hematopoietic cells in the presence of the cytokines interleukin-3, c-kit ligand, granulocyte colony-stimulating factor, and granulocyte/macrophage colony-stimulating factor resulted in tenfold expansion by day 7 and 25-fold expansion by day 14.
Conclusions. Pluripotent hematopoietic cells with a high proliferative capacity colonize textured surfaces of left ventricular assist devices and may contribute to the development of a biologically nonthrombogenic neointima.
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Introduction |
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For editorial comment, see 1561.
The development of an LVAD with textured surfaces has decreased the number of thromboembolic events and eliminated the requirement of routine anticoagulation [8]. The reduced thrombogenicity of these textured surfaces is achieved by the adherence of a homogeneous cellular neointima to the blood-contacting surfaces [9, 10]. The neointimal surface of these LVADs is colonized by hematopoietic as well as nonhematopoietic cells [11–13]. Investigators have used immunohistochemical and electron microscopic techniques to characterize the morphology of the cellular components. Fibroblasts, myofibroblasts, monocytes, and multinucleated cells are the predominant cellular elements that are embedded within a collagenous fibrin mesh [11, 12]. This study was designed to elucidate the exact identity of hematopoietic cells formed on the surface and within the neointimal surfaces of LVADs.
In this study, we report a reproducible and simple technique for the isolation and cultivation of cellular elements within the LVAD neointimal surfaces. Characterization of hematopoietic cells with flow cytometry indicates that the majority of the hematopoietic cells present are myeloid or monocytic in origin. In addition, hematopoietic progenitor cells expressing CD34+ antigen are present as a small percentage of the total number of cells. Stimulation of these progenitor cells with cytokines results in their proliferation and differentiation into mature hematopoietic cells. These data suggest that textured biomaterials encourage recruitment and colonization of highly specialized hematopoietic and nonhematopoietic cells that have the capability to proliferate and produce an extracellular matrix that results in the formation of a nonthrombogenic neointima.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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). The neointima covering the textured surface was removed with a scalpel and placed in Dulbecco's modified Eagle's medium that had been supplemented with 10% fetal calf serum, penicillin (80 U/mL), and streptomycin (80 µg/mL). Prior to analysis, the explants were washed again with PBS.
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The remaining neointimal explants were washed with PBS and placed in six-well Costar (Cambridge, MA) tissue culture dishes. Subsequently, the explants were cut into small 2- to 3-mm fragments and passed through a 40-µm nylon filter (Falcon, Lincoln Park, NJ) to remove any adherent extracellular material. The retained explants were digested with 5 mL of 0.1% collagenase (Worthington Biochemical Corp, Freehold, NJ) at 37°C. After 15 to 20 minutes of collagenase digestion, the explants were disintegrated into smaller fragments by passage through a 22-gauge needle.
At this stage, the digested material, which consisted of single cells as well as undigested fibrin and collagen strands, was refiltered through a Falcon 70-µm nylon filter. The fragments retained on the filter, which were mostly undigested fibrin strands, were resuspended in PBS for further characterization. The mononuclear cells that flowed through the filter were washed twice with 1% bovine serum albumin in PBS.
After quantification of mononuclear cells, 2 x 104 cells were transferred to gelatin-coated plastic slides using a cytospin centrifuge for Wright-Giemsa staining or immunohistochemical analysis. Mononuclear cells, 5 x 104, were resuspended in PBS supplemented with 1% bovine serum albumin and 0.02% sodium azide (buffer A) for flow cytometric analysis. The remaining population of cells was cultured at 37°C in six-well Costar dishes for 2 hours. The nonadherent cells were removed and plated for proliferation assay or resuspended in buffer A for flow cytometric analysis. The adherent population was cultured with special stromal cell growth medium containing M199 medium (MA, Bioproducts), 20% fetal bovine serum with or without 2 ng/mL of basic fibroblast growth factor (Organon Teknika Corp) or 10 ng/mL of vascular endothelial cell growth factor (PeproTech, Rocky Hill, NJ), 2 mmol/L L-glutamine (Sigma, St Louis, MO), penicillin (80 U/mL), and streptomycin (80 µg/mL).
Proliferation Assay
Mononuclear nonadherent cells, 4 x 104, were resuspended in 2 mL of a hematopoietic growth medium consisting of Iscove modified Dulbecco's medium (Bioproducts) supplemented with 20% fetal bovine serum, 2 mmol/L L-glutamine, penicillin (80 U/mL), and streptomycin (80 µg/mL) in the presence of four cytokines: human c-kit ligand, 20 ng/mL (kindly provided by Immunex, Seattle, WA); human interleukin-3, 50 ng/mL (Immunex); human granulocyte colony-stimulating factor, 100 ng/mL (Amgen, Thousand Oaks, CA); and granulocyte/macrophage colony-stimulating factor, 10 ng/mL (Immunex). Cultures were incubated at 37°C in 100% humidified 5% carbon dioxide in air. Each week, 1 mL of the proliferating cells was removed from the liquid culture for cell count and flow cytometric and immunohistochemical analyses. The cultures were supplemented with 1 mL of fresh hematopoietic growth medium, and reincubated for further analysis.
Characterization of Mononuclear Cells
METABOLIC LABELING WITH DIL-AC-LDL.
Nearly confluent monolayers of adherent cells were incubated with 10 µg/mL of Dil-Ac-LDL (acetylated low-density lipoprotein labeled with dioctadecyl 1,3,3,3,-tetramethyl-indocarbocyanine perchlorate) (Biomedical Technologies Inc, Stoughton, MA) for 4 hours at 37°C in PBS with 5% fetal bovine serum. The cells were washed with medium for 10 minutes and examined with a Nikon epifluorescence microscope equipped with phase-contrast optics. The Dil-Ac-LDL was visualized using standard rhodamine excitation/emission filter combinations.
IMMUNOFLUORESCENT FLOW CYTOMETRY.
Nonadherent cells, 5 x 104, obtained from collagenase digestion of neointimal explants were suspended in buffer A. These cells were stained with saturating amounts of fluorescein isothiocyanate (FITC)- or rhodamine- or phycoerythrin (PE)-conjugated antibodies to CD34 (Becton Dickenson, Immunotech), CD33 (Becton Dickenson, Immunotech, Coulter), CD15 (Becton Dickenson, Coulter), CD14 (Mo2; Coulter), CD45 (KC56; Coulter), and CD117 (Coulter). A complete list of the monoclonal antibodies used in this flow cytometric analysis is provided in Table 2. Controls were isotype-matched nonimmune immunoglobulins conjugated with FITC, rhodamine, or PE. Cell-associated immunofluorescence was assayed by quantitative flow cytometry using a Coulter Elite flow cytometer (Coulter, Hialeah, FL), available at Cornell University Medical College.
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For fluorescent immunohistochemical study, adherent monolayers were grown on polylysine-coated plastic slides, air-dried, and fixed with acetone or alcohol. Primary mouse monoclonal antibodies to factor VIII/von Willebrand factor (Dako), alpha-actin (AMAC), and vimentin (AMAC) were incubated on the slide for 1 hour or at 4°C overnight, washed three times with PBS, and counterstained with goat anti-mouse IgG1-FITC/PE. Photographs were taken with a Nikon fluorescence microscope on Kodak Ektachrome 160T ASA color film (Kodak Ltd, Liverpool, UK). The FITC or PE was visualized using standard FITC/PE excitation/emission filter combinations.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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shows the hematoxylin and eosin-stained sections of intact neointima from a 65-day old LVAD, demonstrating the colonization of the neointima with spindle-shaped nonhematopoietic cells as well as hematopoietic cells such as monocytes and neutrophils. Collagenase digestion of the LVAD neointima resulted in complete dissolution of these explants into a single mononuclear population of hematopoietic and nonhematopoietic cells. In most cases, neointimal explants were completely digested within 15 minutes in 0.1% collagenase at 37°C. However, thicker explants may require a longer period. Passage of the digested material through a needle enhanced the release of the cells embedded within the partially digested explants. At this stage, the digested explants formed a thick cellular liquid comprising mostly mononuclear cells and collagen and fibrin strands. Passage of this material through a 70-µm mesh resulted in isolation of mononuclear cells and entrapment of undigested granulation tissue. On average, 5 x 106 mononuclear cells were isolated from 0.5 cm2 of LVAD neointima.
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shows the results of the flow cytometric analysis of the mononuclear cells obtained directly from collagenase digestion of LVAD neointima. Of the mononuclear cells, 88% ± 6% expressed CD45 antigen, which is present on all hematopoietic cells but absent in nonhematopoietic cells. Approximately, 4% ± 2% of CD45+ cells also expressed the CD34 and CD117 surface markers. CD34 is expressed on the primitive hematopoietic progenitor cells, which can differentiate into myeloid, erythroid, and megakaryocytic progenitor cells [14, 15]. CD117 is the cluster designation of c-kit receptor, which is also expressed simultaneously on CD34+ progenitor cells.
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). Of the nonadherent CD45-positive cells, 5% ± 2% stained with CD34 and CD117.
Wright-Giemsa staining of the mononuclear cells isolated from digestion of LVAD neointimal explants demonstrated a preponderance of monocytes, neutrophils, and multinucleated giant cells (Fig 3). In addition, a small number of lymphocytelike cells with prominent nucleoli were detected (see Fig 3A). These latter cells resemble primitive hematopoietic progenitor cells that reside in bone marrow but also circulate in the peripheral blood at a very low frequency. Like lymphocytes, these cells have large nuclei and prominent nucleoli, which are characteristic of hematopoietic stem cells.
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, a large number of adherent cells took up Dil-Ac-LDL, which is a feature of monocytic cells. Phase contrast microscopy revealed the morphology of the adherent spindle-shaped cells, which appeared to be fibroblasts or myofibroblasts (Fig 5). The majority of the adherent cells were positive for alpha-actin and vimentin but negative for factor VIII/von Willebrand factor (data not shown).
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). By day 7 of culture, 50% ± 4% of the proliferating hematopoietic cells were monocytic expressing CD14 antigen. The majority of cells on day 14 or 21 of culture were myelomonocytic expressing CD15 and CD33 antigens. On day 21, 55% ± 7% of cells were monocytic expressing CD14 antigen. In parallel studies, freshly isolated mononuclear cells and hematopoietic cells proliferating in liquid culture were also stained with monoclonal antibody to lymphoid and erythroid progenitors. No staining was detected with monoclonal antibody to CD3, CD4, CD8, CD19, CD20, or glycophorin A. |
Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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To characterize the hematopoietic and nonhematopoietic cells within the neointima of explanted LVADs, we developed a technique to isolate and cultivate these cells. Collagenase digestion of LVAD explants results in release of mononuclear cells embedded in the neointima. Plating these cells on a plastic dish results in attachment of the majority of spindlelike cells and multinucleated cells. The majority of the nonadherent cells are hematopoietic cells expressing CD45 antigen. A very small percentage of these hematopoietic cells have the morphologic and phenotypic characteristics of CD34-positive pluripotent hematopoietic cells [15, 16]. Even though CD34-positive progenitor cells comprise only 0.03% to 0.09% of the circulating hematopoietic cells, they comprise 4% ± 2% of mononuclear cells in LVAD neointima [17]. Incubation of these cells in the presence of cytokines results in proliferation of a large number of myeloid cells, particularly CD14-positive monocytic cells.
Initial colonization of the LVAD with circulating pluripotent hematopoietic cells and spindle cells may produce a specialized nonthrombogenic matrix. The differentiation of hematopoietic progenitor cells to more mature hematopoietic cells is likely regulated by the production of cytokines within the neointima by monocytes or fibroblasts. Several studies have reported that the extracellular matrix produced by stromal cells are a major reservoir for various cytokines, including granulocyte/macrophage colony-stimulating factor, granulocyte colony-stimulating factor, fibroblast growth factor, and vascular endothelial cell growth factor [17–19]. Thus, soluble cytokines that are present in the circulating blood or produced locally by neointima cells may accumulate in the neointima. This highly enriched microenvironment is conducive to the proliferation and differentiation of a wide variety of cells. Pluripotent CD34-positive cells that circulate may attach to the neointima, proliferate, and undergo differentiation within this microenvironment [20].
Whether the hematopoietic cells within the LVAD neointima originate only from the circulating blood or are also a product of progenitor cells within the LVAD surface is critical to understanding the complex nature of the neointima. The absence of a complex cellular matrix on the more thrombogenic smooth-surface biomaterials suggests that cellular constituents of the neointima formed on the surface of textured biomers may mediate thrombogenicity. An understanding of the specialized cells that contribute to LVAD neointimal formation may allow creation of artificial biomaterials that will decrease thrombogenic complications in cardiovascular procedures.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Address reprint requests to Dr Oz, Department of Cardiothoracic Surgery, Columbia-Presbyterian Medical Center, MHB 7GN-435, 177 Fort Washington Ave, New York, NY 10032.
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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