Am J Physiol Gastrointest Liver Physiol 291: G1187-G1189, 2006.
First published June 15, 2006; doi:10.1152/ajpgi.00229.2006
0193-1857/06 $8.00
INNOVATIVE METHODOLOGY
Rat liver endothelial cells isolated by anti-CD31 immunomagnetic separation lack fenestrae and sieve plates
Laurie D. DeLeve,1
Xiangdong Wang,1
Margaret K. McCuskey,2 and
Robert S. McCuskey2
1Research Center for Liver Diseases and the Division of Gastrointestinal and Liver Diseases, Keck School of Medicine at the University of Southern California, Los Angeles, California; and 2Department of Cell Biology and Anatomy, University of Arizona, Tucson, Arizona
Submitted 24 May 2006
; accepted in final form 14 June 2006
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ABSTRACT
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The gold standard for the identification of sinusoidal endothelial cells (SEC) is the presence of fenestrae organized in sieve plates, which is characteristic of SEC in vivo. One of the methods currently in use to isolate SEC is immunomagnetic sorting for CD31. However, there is evidence to suggest that CD31 is not present on the surface of differentiated SEC. The present study used scanning electron microscopy to image rat hepatic endothelial cells isolated by anti-CD31 and immunomagnetic sorting and cells isolated by gradient centrifugation and centrifugal elutriation. Cells isolated by elutriation had well-developed fenestrae and sieve plates, whereas cells isolated by anti-CD31 and immunomagnetic sorting had significantly fewer fenestrae organized in sieve plates. In conclusion, cells isolated by anti-CD31 and immunomagnetic sorting lacked the hallmark features of SEC.
antigens; CD31; endothelial cells; cell separation; liver
HEPATIC SINUSOIDAL ENDOTHELIAL cells (SEC) are a unique cell type with nondiaphragmed fenestrae organized in sieve plates and without an underlying organized basement membrane. Fenestration is determined by specific cytoskeletal organization, membrane fusion, and contractile components and is affected by numerous factors (1, 2, 5). A method for isolating fenestrated SEC with good purity, viability, and yield was first described 20 years ago (6, 7, 10). The ability to isolate pure populations of cells with good yield has greatly enhanced our ability to study these cells. Unfortunately, isolation of SEC by gradient centrifugation and centrifugal elutriation is time consuming and requires special equipment. A much simpler method has been described that isolates SEC from a liver digest using anti-CD31 and immunomagnetic beads (8, 9). CD31 is present on the cell surface of many endothelial cells, but studies of SEC with flow cytometry of SEC isolated by Percoll density gradient centrifugation (12) or with confocal microscopy or scanning electron microscopy plus immunogold labeling for CD31 of SEC isolated by metrizamide density gradient centrifugation plus elutriation (4) have not found CD31 on the surface of SEC. The goal of the present study was to determine whether the population of cells isolated from a liver digest by anti-CD31 immunomagnetic sorting has the hallmark features of differentiated SEC, namely fenestrae organized in sieve plates as imaged by scanning electron microscopy.
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MATERIALS AND METHODS
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Preparation of liver digest.
SEC were isolated from male Sprague-Dawley rats (body wt 250–280 g). Experiments were performed in adherence with the guidelines outlined in the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (Revised 1985) prepared by the National Academy of Sciences. The experiments followed protocols approved by the Animal Care and Use Committee of the University of Southern California. Rats were sedated with pentobarbital and then treated with 200 µl porcine intestine heparin (1,000 units/ml). The liver was perfused for 10 min with calcium-free Gey's buffered saline (GBS) at 37°C at 10 ml/min, followed by perfusion in a recirculating fashion for 20 min with GBS containing 0.05% type 1a collagenase (Sigma Chemical, St. Louis, MO). The livers were mechanically dissociated, pressed through polypropylene mesh, and centrifuged, and the digest was resuspended in 50 ml GBS. One-fifth of the preparation was used to isolate cells by anti-CD31 immunomagnetic sorting and the remainder of the digest was used to isolate SEC by gradient centrifugation and centrifugal elutriation.
Isolation of cells by anti-CD31 antibody.
The hepatic digest was mixed with 0.2 ml (1:50) of rabbit anti-CD31 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA), incubated for 1 h at 37°C, washed, and then incubated for an additional 30 min with 4 beads/cell of magnetic beads coated with sheep anti-rabbit IgG (Dynal Biotech, Oslo, Norway). The suspension was placed on a magnetic particle concentrator for 5 min and magnetic beads were then harvested.
Isolation of cells by gradient centrifugation and centrifugal elutriation.
SEC from the remaining 80% of the liver digest were isolated by density gradient centrifugation and centrifugal elutriation as previously described (3, 11) with one modification. Instead of metrizamide gradient centrifugation, the initial separation was achieved with a 17% iodixanol (Accurate Chemical and Scientific, Westbury, NY) density gradient centrifugation. Elutriation was performed in a J2-21 Beckman centrifuge using the JE-6B rotor with standard chambers. The first elutriation was at 2,500 rpm and a pump speed of 27.5 ml/min. The first 100 ml of eluate was collected and centrifuged. The pellet was subjected to a second elutriation at 3,400 rpm. The first 100 ml were collected at a flow rate of 32 ml/min and discarded. Flow rate was increased to 61 ml/min, and 100 ml of eluate were collected and centrifuged. Purity was >98% as determined by positive staining for fluorescent acetylated low-density lipoprotein and a peroxidase stain to reveal contaminating Kupffer cells. Viability was >95% and yield averaged >100 million cells from the 80% of the liver digest.
Scanning electron microscopy.
Cells isolated by either method were plated onto coverslips and cultured overnight. Then, the cells were fixed with 2% glutaraldehyde for 2 h at room temperature after which they were dehydrated in a graded ethanol series, critical point-dried, fractured, sputter-coated with 10 nm gold, and examined using a FEI XL35 scanning electron microscope. The number of fenestrae per cell was counted in 10 randomly selected cells from each group.
Statistical analysis.
Numerical data represent means ± SE. Results with P < 0.05 by two-sided Student's t-test were considered significant.
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RESULTS
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Figure 1 demonstrates scanning electron microscopy of cells isolated by immunomagnetic sorting for anti-CD31 and of cells that were isolated by density gradient centrifugation and centrifugal elutriation. Cells isolated by centrifugal elutriation had 540.3 ± 20.0 fenestrae/cell (range 440–620) in contrast to 118.7 ± 12.9 fenestrae/cell (range 59–181) in cells isolated by immunomagnetic sorting (P < 0.0001). Fenestrae in the former group were organized in well-developed sieve plates.
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Fig. 1. Scanning electron microscopy. Endothelial cells were isolated from the liver by gradient centrifugation and centrifugal elutriation (A) and immunomagnetic sorting for anti-CD31 (B). Note that most of the cells in A have fenestrae organized in sieve plates (indicated by arrowheads) characteristic of sinusoidal endothelial cells in vivo whereas few of the cells in B demonstrate this phenotype.
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The yield of cells isolated from 20% of the liver digest by anti-CD31 immunomagnetic sorting was 0.17 x 106 ± 0.01 x 106 (n = 3). The yield of cells isolated from the remaining 80% of digest by gradient centrifugation and elutriation was 100.67 x 106 ± 5.07 x 106 (n = 3).
To examine whether anti-CD31 immunomagnetic separation promotes loss of fenestrae, cells were isolated after elutriation by anti-CD31 immunosorting. There was no difference in the morphology on scanning electron microscopy between cells isolated by elutriation alone vs. elutriation followed by immunomagnetic separation.
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DISCUSSION
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In this study, cells isolated from a liver digest by anti-CD31 immunomagnetic sorting lacked the hallmark features of SEC, i.e., fenestrae organized in sieve plates, and were therefore not differentiated SEC. This is not entirely surprising given that a previous study using scanning electron microscopy combined with immunogold labeling for CD31 did not demonstrate CD31 on the surface of fenestrated SEC (4). The yield of cells isolated by anti-CD31 in the present study was less than 1% of that isolated by elutriation. The very low cell yield either may reflect the relative inefficiency of the method itself or may indicate that the population of cells isolated by this method is present in the liver in small numbers. On the other hand, surface expression of CD31 is a good marker for the nonfenestrated endothelial cells seen in capillarization (4), and anti-CD31 immunomagnetic separation could be a simple method for isolating capillarized cells in liver disease models or in the pseudocapillarization of aged animals.
Of note, studies in the literature have used and a commercial vendor of human hepatic endothelial cells is currently utilizing anti-CD31 immunomagnetic sorting to isolate SEC. The validity of studies using these cells may now need to be reexamined.
At present, the single best phenotypic feature defining a SEC is the presence of fenestrae in sieve plates. Uptake of fluorescent acetylated low-density lipoprotein is a tool that is frequently used to determine purity of an SEC isolation; a peroxidase stain is often used in combination with this to reveal contaminating Kupffer cells, which also take up fluorescent acetylated low-density lipoprotein. However, uptake of fluorescent acetylated low-density lipoprotein merely reveals the presence of endothelial cells and does not confirm that the cell in question is indeed an SEC. Furthermore, SEC that are allowed to dedifferentiate in culture maintain the ability to take up fluorescent acetylated low-density lipoprotein (L. D. DeLeve, unpublished observation), so uptake of fluorescent acetylated low-density lipoprotein also does not indicate that the cell in question has sustained the phenotype in culture.
For our understanding of SEC biology and pathology to progress, it is important for investigators to study a cell type that is as close as possible to that present in vivo. Several methods are currently in use to isolate SEC. We suggest that before a method for isolation of SEC is accepted for general use the method should be validated by electron microscopy studies that demonstrate that the majority of cells obtained contain fenestrae with a diameter of 
100–150 nm that are organized in sieve plates.
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GRANTS
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This paper was supported by National Institutes of Health Grants DK-66423 and AA-12436.
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FOOTNOTES
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Address for reprint requests and other correspondence: L. DeLeve, USC Keck School of Medicine, 2011 Zonal Ave.-HMR 603, Los Angeles, CA 90033 (e-mail: deleve{at}usc.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Copyright © 2006 by the American Physiological Society.
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ABSTRACT
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