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Received Date : 25-Feb-2013 Revised Date : 27-Dec-2013 Accepted Date : 30-Jan-2014 Article type : Original Research
Isolation, purification, and cultivation of
primary retinal microvascular pericytes: a novel model using rats
Guanghui Liua, b, c, Chun Mengd, Mingdong Panc, Meng chene, Ruzhi Denga, b, Ling Lind, Li Zhaof, Xiaoling Liua, b, *
a
School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
b
State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health P. R. China, Wenzhou,
Zhejiang, China
c
Department of Ophthalmology, Affiliated People's Hospital (People's Hospital of Fujian Province), Fujian University of
Traditional Chinese Medicine, Fuzhou, Fujian, China
d
Department of Bioengineering, College of Biological Science and Biotechnology, Fuzhou University, Fuzhou, Fujian, China.
e
Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, US.
f
Department of Cardiology, Affiliated People's Hospital (People's Hospital of Fujian Province), Fujian University of Traditional
Chinese Medicine, Fuzhou, Fujian, China.
running title：cultivation of rat primary retinal pericytes
grant numbers and source(s) of support： Natural Science Foundation of Fujian Province, China (No. 2011J01197) National Natural Science Foundation of China (No. 81102619)
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/micc.12121 This article is protected by copyright. All rights reserved.
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corresponding author: School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, 270 Xueyuan Road, Wenzhou, Zhejiang, 325003, China; [email protected]
Abstract Purpose: To isolate, purify, and cultivate primary retinal microvascular pericytes (RMPs) from rats to
facilitate the study of their properties in vitro. Methods: Primary RMPs were isolated from weanling rats by mechanical morcel and collagenase
digestion, and purified by a step-wise combination of selective medium with different glucose concentrations, medium exchange, and partial enzymatic digestion. Morphology of RMPs was assessed by phase contrast microscopy. Further characterization was analyzed by immunofluorescence. Functional assay was evaluated by the pericytes- endothelial cells (ECs) coculture system. Results: RMPs migrated out of microvascular fragments after 24-48 hours of plating and reached
subconfluence on days 14-16. The cells showed typical pericyte morphology with large irregular triangular cell bodies and multiple long processes, and uniformly expressed the cellular markers α-SMA, PDGFR-β, NG2 and desmin, but were negative for vWF, GS , GFAP and SMMHC. Ninety-nine percent of the cell population had double positive staining for α-SMA and PDGFR-β. In the coculture system, RMPs can directly contact ECs
and move together to form the capillary-like cords.
Conclusions: RMPs can be readily obtained by our method. We report the first cultivation of primary
RMPs from rats and establish a simple method for their isolation and purification.
Keyword: Retina; Microvascular; Pericyte; Isolation; Purification
Abbreviations: α-SMA
α-smooth muscle actin
CLSM
confocal laser scanning microscopy
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DAPI
4', 6-diamidino-2-phenylindole
DM
diabetes mellitus
DMEM
Dulbecco's modified Eagle's medium
DR
diabetic retinopathy
ECs
endothelial cells
FBS
fetal bovine serum
GFAP
glial fibrillary acidic protein
GS
glutamine synthetase
H-DMEM-20
high-glucose (20mmol/L) DMEM supplemented with 20% FBS
L-DMEM-20
low-glucose (5mmol/L) DMEM supplemented with 20% FBS
MTT
methyl thiazolyl tetrazolium
NG2
nerve/glial antigen 2
PBS
phosphate-buffered saline
PDGFR-β
platelet-derived growth factor receptor-β
RMPs
retinal microvascular pericytes
SMMHC
smooth muscle myosin heavy chain
vWF
von Willebrand factor
Introduction Pericytes are important components of the microvasculature. They are strategically situated on the outer wall of
small blood vessels and are embedded within the basement membrane around endothelial cells (ECs) [1]. Pericytes play critical roles in microvascular biology, including: (1) the regulation ECs proliferation and differentiation [2, 3], (2) the stabilization and control of microvascular barrier [4, 5], (3) the regulation of vascular tone and perfusion pressure [6, 7], (4) and involvement in vasculogenesis and angiogenesis [8, 9]. Recent reports have also shown that pericytes are intimately involved in the pathogenesis of various retinal angiogenic diseases, particularly diabetic retinopathy (DR) [9, 10]. Compared with microvascular ECs though, the role of pericytes is much less understood
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[11]. This is partly due to difficulties in studying these cells in vivo, in obtaining the correct tissue, and in cultivating homogeneous pericyte populations in vitro [12]. Pericytes are morphologically, biochemically, and physiologically heterogeneous [13]. Thus far, in vitro
cultures of primary retinal microvascular pericytes (RMPs) have been mainly established from cells isolated from monkeys [14], humans [15-18], and bovine origin [19-25]. Although human retinas are useful sources for in vitro study, legal and ethical issues prevent researchers from utilizing them [18]. They are also scarce and not practical for routine isolation. Therefore, bovine retinas have been mostly used for in vitro assays. However, this source is limited as well and is inconvenient to obtain since bovine eyes are provided by slaughterhouses. Moreover, bovine animal models have not been used to study diabetes mellitus (DM), and some data derived from bovine RMPs are not in accord with those derived from human RMPs under the conditions that mimic DM [15, 26, 27]. In contrast, rat eyes are an abundantly available tissue source from which RMPs can be isolated. Rat-based in
vitro models are useful for investigating DR since there are several in vivo rat models of DM [28, 29]. Therefore, it is suitable to consider rat eyes as the main tissue source for routine cultivation of primary RMPs. Yet, to our knowledge, the culture of primary RMPs from rats has not been reported. Regardless of the starting tissue source, the major problems encountered in cultivating primary RMPs are the
high costs and the lengthy procedures required for purification. RMPs isolation protocols rely on enzymatic tissue digestion followed by either positive immunoselection [22] or microvascular outgrowth [30]. The former approach is complex, expensive, and results in relatively low yields of RMPs, while the latter often leads to heterogeneous populations with cellular contamination. Therefore, it appears beneficial to develop an economical and convenient protocol for the purification of primary RMPs. Here, we present a simple protocol for isolating, purifying and cultivating primary RMPs, using weanling rats
as the starting tissue source and using selective medium with different glucose concentrations as the key purification method. We demonstrated that the homogeneous RMPs populations produced by our method can readily proliferate in culture and uniformly express characteristic RMP cellular markers in vitro.
Materials and Methods Isolation of RMPs (a) Collection of retina samples.
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All experimental procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Ethics Committee for Animal Use in Research and Education at Wenzhou Medical College. All procedures were performed under aseptic conditions. The retinas were isolated from 20 male weanling rats (Sprague Dawley rat; SLAC, Shanghai, China), aged 3 weeks, by using a modified version of the method previously described [22]. After euthanasia of rats by cervical dislocation, the eyes were enucleated and immediately placed into ice-cold phosphate-buffered saline (PBS) containing 100 U/mL penicillin and 100 µg/mL streptomycin. They were then transferred into 75% ethanol for 1 minute and washed twice with PBS. Using ophthalmic microscopic surgical instruments, incisions were subsequently made in each eye posterior to the ciliary body and extended circumferentially while the eyes were in 60-mm-diameter tissue culture dish (Corning, NY, US) with PBS. Connective tissue was discarded. Retinas were pulled gently away from the posterior eyecups by microscopic iris repository. All retinas were washed twice in PBS to prevent contamination before use in further steps. (b) Enzymatic digestion. Retinas were mixed with a small amount of PBS and then finely minced into homogeneous fragments, which
were made as small as possible by crosscutting the tissue using two ophthalmic microscopic scalpels. The retinal fragments were suspended and incubated in 3 mL of 0.2% collagenase typeⅠfor 20 minutes at 37oC. After incubation, 3 mL of low glucose (5mmol/L) Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, US) supplemented with 20% fetal bovine serum (FBS; Hyclone, Logan, US) was added and mixed gently to suspend digestion. The solution was repeatedly pipetted up and down to break up clumps of cells and retinal fragments, and then filtered through 100 μm filtrate over a 55 μm nylon sieve (Falcon, Oxnard, US). The 55-100 μm filtrate was collected in 1.5 mL centrifuge tubes, and centrifuged at 2000 rpm (500 g) for 4 minutes at 4oC. The supernatant was
discarded, and the precipitated pellet containing retinal capillary-rich fragments was collected. The pellet was then re-suspended in 1.5 mL high glucose (20mmol/L) DMEM with 20% FBS (H-DMEM-20), and seeded into 6-well plates (Costar; Corning, NY, US) maintained with H-DMEM-20.
Cultivation of RMPs The suspended fragments were incubated at 37oC with 5% CO2 atmosphere. Cell growth was monitored under
phase contrast microscopy (Olympus, Tokyo, Japan). The medium was changed daily (days 3, 4, and 5) until day 5, after which the medium was changed every 3 days. H-DMEM-20 was replaced with low glucose DMEM with 20%
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FBS (L-DMEM-20) on day 3. When primary RMPs cultures were near confluence (above 80%), they were further propagated by treatment with 1 mL of 0.25% trypsin (Santa Cruz Biotech, Santa Cruz, US) and 0.02% EDTA for 3 minutes and then were either split into a 1:3 ratio to passage to additional 6-well plates or harvested for cryopreservation. For proliferation assays, cells at passages 3 were counted and seeded into 96-well plates (1×104
cells per well). Medium was added at a volume of 100 μL and was replaced every 3 days with fresh media. Plates were incubated for a total of 12 days. Methyl thiazolyl tetrazolium (MTT) assay was then performed according to the literature [31].
Weeding contaminating cells. The following techniques were used to obtain pure populations of RMPs. After 48 hours of incubation, any
unattached fragments, cells or debris were discarded and the media was removed without agitation and replaced with L-DMEM-20. On days 4 and 5, the plates were rinsed twice to remove any loosely adherent cellular contamination. Old medium was removed and replaced with fresh L-DMEM-20. This weeding procedure was performed during medium exchange. When RMPs reached 80% confluence, they were incubated with trypsin. Trypsinization was observed under
phase contrast microscopy. It was observed that cells showed different detachment times. Contaminating cells began to detach first after 1-2 minutes of trypsinization, followed by RMPs after 3-5 minutes. After determination and optimization of detachment times, the effect of trypsin was neutralized right after the time period in which only contaminating cells detached and before RMPs detached. The detached contaminating cells were then gently mixed into the culture medium by gently swirling the culture plate back and forth, after which the medium containing detached contaminating cells was discarded. RMPs remained attached. New trypsin was added to the RMPs, which were then incubated for passage.
Morphological and immunofluorescence characterization of RMPs Morphological characterization was performed using phase contrast microscopy, and immuno-fluorescence
characterization was analyzed using the confocal laser scanning microscope (CLSM) LSM 710 (Zeiss, Jena, Germany). The cells were characterized with the cellular characterization kit according to the manufacturer’s protocol. Briefly, the cells were grown in glass bottom cell culture dishes (NEST, Wuxi, China). The dishes were washed for 5 minutes three times with PBS, and cells were fixed in 4% paraformaldehyde at room temperature for
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20 minutes. Subsequently, the cells were washed extensively with ice-cold PBS and incubated for 2 hours at room temperature with blocking buffer containing 5% FBS and 0.3% Triton X-100 in PBS. The primary antibodies were diluted with working concentrations of the blocking buffer. The cells were incubated overnight at 4oC with primary antibodies against the following markers: α-smooth muscle actin (α-SMA; Sigma, St. Louis, US), desmin (Santa
Cruz Biotech, Santa Cruz, US), platelet-derived growth factor receptor-β (PDGFR-β; Santa Cruz Biotech, Santa Cruz, US), nerve/glial antigen 2 (NG2; Chemicon, Temecula, US), von Willebrand factor (vWF; Santa Cruz Biotech, Santa Cruz, US), glutamine synthetase (GS; Chemicon, Temecula, US), glial fibrillary acidic protein (GFAP; Santa Cruz Biotech, Santa Cruz, US), and smooth muscle myosin heavy chain (SMMHC; Bioss, Beijing, China). Afterwards, the cells were washed once with PBS and three times with blocking solution. The cells were then incubated for 2 hours at room temperature with fluorescent secondary antibodies of goat anti-rabbit IgG TRITC and anti-mouse IgG FITC (Santa Cruz Biotech, Santa Cruz, US). For double staining, this procedure was repeated with different primary antibodies and appropriate secondary antibodies. Finally, the labelled cells were incubated with a solution containing 4', 6-diamidino-2-phenylindole (DAPI; Invitrogen, Karlsruhe, Germany) at a dilution of 1:1000. Parallel control cultures that were either stained only for primary antibodies or only for secondary antibodies alone were maintained as well.
RMPs and ECs coculture The rat retinal ECs were obtained as previously described [32, 33]. According to the previous literatures [34,
35], RMPs and ECs were plated at a 1:10 ratio, direct-contact assay was evaluated by 2-dimensional (2D) RMPsECs coculture system under routine culture conditions, and capillary-tube formation assay was evaluated by 3dimensional (3D) system in Matrigel (BD Bioscience, La Jolla, US). ECs and RMPs were labelled by cell tracker (Molecular Probes, Eugene, US) before 3D coculture in Matrigel. After 48 hours of coculture the cells were analyzed by fluorescent microscopy (Nikon, Tokyo, Japan).
Results Cultivation of RMPs Heterogeneous cells and microvascular fragments were observed immediately after plating cultures (Fig 1. A).
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The fragments started attaching to culture dishes after 4-5 hours of incubation, and cells were adherent to culture dishes within 12-24 hours. RMPs migrated out of the retinal fragments 24-48 hours after plating (Fig 1. B). Culture media was changed at 48 hours, when unattached contaminating cells were discarded and heterogeneous cells remained in culture. Most of these remaining cells were RMPs with irregular triangular morphology; a few cells appeared to have smaller and rounder cell bodies (Fig 1. C). We did not observe any cobble-stone patterns typical of ECs cultures [36] or any characteristic bipolar morphology typical of Müller cells cultures [37]. Contaminating cells were gradually eliminated by the method described above by rinsing plates when changing medium at 48 hours and on days 4 and 5. This resulted in only very few contaminating cells left in culture (Fig 1. D). On day 8, RMPs appeared in primary cultures as either large isolated cells containing typical stress fibres (Fig 1.
E) or as loose colonies of polygonal and elongated cells with ruffled edges (Fig 1. F). The large isolated cell may grow from a single cell digested by collagenase, and the clone grow from a central tissue fragments. On day 14, either overlapping layers containing quantities of cells (Fig 1. G) or cells sparsely spreading on the bottom of dish (Fig 1. H) were observed. Growth of the primary RMPs were even slower. The cells approximated 80% confluence on days 14-16 and reached a total number of about 1.3×105 primary cells per well as determined by hemocytometry.
The subcultures grew faster than the primary cultures and reached confluence on days 12-14（Fig 2）. RMPs can
be successfully cryopreserved and recultured without loss of typical features; they can be repeatedly passaged 9 times without obvious loss of characteristic phenotype.
Characterization of RMPs RMPs were primarily analyzed and characterized based upon morphology under phase contrast microscopy.
Cells were confirmed as RMPs by observing a typical pericyte morphology with irregular shape, long processes, and large and flat cell bodies [9, 38-41]. In the cultures, almost all of the cells showed typical pericyte morphology (Fig 1. I). Further immunofluorescence confirmation was performed by double immunostaining. The expression of
pericyte markers α-SMA, PDGFR-β, NG2 and desmin was detected by CLSM. Double staining for α-SMA and
PDGFR-β revealed that 99% of the cells in culture showed positive immunofluorescence for both markers (Fig 3. AD), confirming the purity of RMPs in culture. Cells in culture were also positive for NG2 (Fig 3. E-H) and, to a lesser extent, desmin (Fig 3. I-L). In the population positive for α-SMA, >99% of cells were positive for desmin while >85% of cells were positive for NG2. The relative heterogeneity of expression of NG2 is consistent with the
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previous report [34]. The expression of these 4 markers did not significantly change in RMPs from passages 3 to 7. Additionly, the expression of α-SMA and PDGFR-β were analyzed in the cells at 48 hours after incubating. The result showed 100% double-positive staining for these two markers in RMPs. (Fig 4). None of the cells in culture expressed positive staining for vWF, GS, GFAP, and SMMHC, which are specific
markers, respectively, for ECs, Müller cells, glial cells, and vascular smooth muscle cells (vSMCs). Thus, there was no contamination of cultures by ECs, Müller cells, glial cells, or vSMCs (Fig 3. M-X). The negative control cultures that were stained either only for primary antibodies or only for secondary antibodies all showed negative staining (Fig 5).
Functional assay Pericytes-ECs coculture was used as a means to assess pericyte function in vitro. In our 2D coculture system,
RMPs was recruited by ECs and extended their processes to contact ECs directly in the dish bottom after 48 hours of plating (Fig 6). In 3D system, the cells moved toward one another as they do in blood vessels in vivo and formed cords liking capillary-tube in the Matrigel after 48 hours of plating. Some cords condensed to form large aggregates (Fig 7).
Discussion In this report, we describe a simple method for cultivation of primary RMPs from rats without the need for
positive cell selection. Our method achieved a nearly homogenous population of RMPs from which readily expanding subpopulations of RMPs can be generated. In our protocol, primary RMPs were isolated from the weanling rats and largely purified by the selective
medium, a prerequisite for successful cultivation. Key features of our method that differ from previously published reports include: (1) a starting tissue source of RMPs originating from a readily available rat model, weanling rats, (2) collagenase digestion of retinal fragments limited to only 20 minutes without agitation, (3) significantly less requirement of expensive materials and sophisticated instrumentation, (4) usage of high glucose and low glucose media changes to selectively stimulate pericytes, and (5) partial trypsinization to selectively remove contaminating cells. The technical aspects and tools required for our protocol are simple, thereby making the isolation, purification and cultivation of RMPs feasible for any cell biology laboratory. Traditionally, a major difficulty in the cultivation of primary RMPs in the past has been limited tissue sources
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[11, 42]. Though some reported model systems of primary RMPs have been developed from various species, including monkey [14], human [15, 18], and bovine [19-25], the study of RMPs has lagged far behind that of ECs for the difficulties of in vivo study, tissue source and in vitro cutivation mentioned above. Looking at the last four decades, the annual number of publications regarding pericytes is far less compared with the numbers of publications regarding ECs. The key words “endothelial cell (s)” produce 10,217 (8,783) citations in PubMed for the year 2010, while the key word “pericyte (s)” resulted in only 256 (221) [11, 42]. Furthermore, only a small number of these publications are about RMPs. Rats are a common study model and are easily obtainable. Yet, they have been scarcely used in the culture of primary RMPs. This is primarily attributable to the smaller size of the rat eye, making it harder to dissect, thus resulting insufficient cell yield from rat eyes in comparison with those of bovine eyes [42]. There has been only one previous report in the literature regarding an immortalized pericyte cell line from tsA58 Tg rats, which are uncommon model source harboring the temperature-sensitive simian virus 40 large Tantigen gene and dealt with unconventional protocol [42]. In the current study, the weanling rats were adopted for retinal source. The rat at the age of 3 weeks had not only developed retinal vessels [43] but also the cells with good proliferative ability [44, 45]. Due to the complex components of retina and unique structures of eyeball wall, the protocol for RMPs isolation is somewhat different with those for other tissues, such as brain, coronary, and skeletal muscle [11, 22, 34, 46, 47]. Additionally, the conventional instruments used for adult rats and bovine are not suitable for the weanling rats because of their small eyeball. In present report, retinas of the weanling rats can be successfully isolated by using ophthalmic microscopic instruments without the need of a dissecting microscope. One crucial aspect in isolating a whole rat retina was in utilizing microscopic iris repositories to gently pull the retinas away from the posterior eyecups. Additionally, since our RMPs can recover from cryopreservation without losing their characteristic phenotype, a sufficient amount of accumulated cells can be achieved by cryopreservation for large demand although the cells from a single rat may not enough [42]. A major difficulty of obtaining homogeneous RMPs populations is in the elimination of contaminating cells
such as gliacytes. A number of purification techniques are available in pericytes cultivation that utilize sequential sieving [30], gradient centrifugation [48], weeding non-pericytes [20], fluorescent-activated cell sorting [22], and magnetic beads [22]. However, these techniques are limited by high monetary costs, lengthy time requirements, or viable cell-consumption. They all generally require expensive and sophisticated instrumentation and complex procedures. In Kondo’s report [42], RMPs are cloned from a single cell and keep 100% homogeneity, since the SV
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40 large T-antigen gene is activated by low temperature (33oC) and the culture become immortalized in the early stage. But monoclone isn’t appropriate for primary RMPs due to their limited proliferation. The method described in our study for eliminating contamination is a step-wise combination of selective medium, medium exchange, and partial enzymatic digestion that does result in a purified, homogeneous population of RMPs. It is a relatively simpler and less costly method for eliminating gliacytes than the above previous methods. Contaminating ECs are also reported in pericyte culture [34, 49]. Owing to uncoated plate and inhibition of 20% FBS [20], migration and adherence of ECs did not appear in our study. During this previous year, we repeated our culture method more than 50 times, showing good repeatability and reproducibility. Cultured cells, as well as those recovered from cryopreservation storage, uniformly express characteristic pericyte surface and intracellular proteins and lack ECs, vSMCs, and glial cell markers. As reported previously [50], although a number of pericyte markers has been identified in the past, the lack of
reliable markers has still hindered pericyte identification. The expression of pericyte markers are dynamic [51] and varies depending on the species and tissue studied. No single marker is able to identify all pericytes [52, 53]. Therefore, we decided that the use of multiple markers and high-resolution confocal microscopy was a better approach to identify and study pericytes in our cultures. Four of the best-described markers used to identify
pericytes are α-SMA, desmin, PDGFR-β and NG2 [54]. Since 1985, α-SMA has been used as a pericyte marker [55] and has been considered to be a general marker for
pericytes [50, 56]. In primary cultures, nearly 100% of pericytes express α-SMA by day 7 [57]. Many investigators
have used α-SMA to identify RMPs in vitro[18, 24, 33]. Desmin is an intermediate filament proteins and is an
intracellular marker [54] expressed by immature and mature pericytes [58, 59]. In vivo studies have shown that desmin is a more sensitive marker for pericytes than other pericyte markers in adult rat retina [60] and can label more pericytes in mouse retina [61]. In vitro study, desmin is used as a routine marker for pericytes cultivation [3, 27, 62, 63]. Desmin and α-SMA are both intracellular markers that are viewed as the first line of evidence of the presence of pericyte contractile proteins [64]. The double positive immunostaining of α-SMA and, to a lesser extent, desmin in our culture cells suggested that rat RMPs maintain contractile functions in vitro. It was consistent with what found in Berrone et al [18] studies. But compared with low positive expression (5%-30%) of α-SMA in freshly isolated brain pericytes reported by Dore-Duffy [50, 65], our result demonstrated a little difference in that 100% of RMPs at 48 hours expressed the smooth muscle phenotype. We inferred the difference may be owed to the source
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heterogeneity [13], theirs from brain and ours from retina. In addition to involvement in hemodynamic processes, pericytes also play a crucial role in vasculogenesis and
angiogenesis. For this reason, PDGFR-ß is one of the most widely used markers for pericytes [33, 52]. PDGFR-ß plays a significant role in pericyte recruitment and in the initiation and maturation of the microvasculature [54]. Knockout of the genes encoding PDGFR-β in mice results in a reduced number of pericytes and subsequent vessel malformation and dysfunction [66, 67]. NG2 is another established, specific marker for pericyte identification [68] that is expressed on the surface of activated pericytes during vessel formation [69]. The previous studies on both normal [70] and pathological [71] angiogenesis of retina suggest that NG2 can act as a reliable marker for microvascular pericytes. Our cultures showed a strong positive immunofluorescence for PDGFR-ß staining. Positive expression of NG-2 was also observed. This demonstrated that rat RMPs can also be used to study retinal angiogenesis in vitro. Aside from the phenotypic analysis outlined above, the pericytes-ECs coculture, as a means to verify the
function of pericytes, further evidenced that the cells derived from our culture can act as pericytes in microvessle in
vivo. In the coculture system, not only RMPs can directly contact ECs by multi processes but also the cocultured
cells can move together to form the capillary-like cords, which were in accordance with Bryan’s report [35]. The results demonstrated that rat RMPs acquired by our method can retain the ability to closely associate with ECs. In the foregoing, both phenotypic analysis and functional assay strongly supported the claim that our method
obtains highly pure RMPs and suggested that the cells could be useful as a model to investigate DR. RMPs loss is a histopathological hallmark of early DR and is a key event in the pathogenesis of DR [53, 72]. It
has been well demonstrated in vitro that pericyte contractility and survival is reduced in high glucose environments
in a dose-dependent manner [73, 74]. Previous reports indicate that long time exposure of pericytes to a high glucose concentration of 28 to 30 mmol/L induces significant mitochondria fragmentation, increased membrane potential heterogeneity, and increased numbers of apoptotic RMPs [27, 74]. But RMPs are less affected by a short exposure time of less than 48 hour to high glucose concentrations (＜25mmol/l) [75, 76]. It is suggest that exposure to glucose at 20 mmol/L leads to insignificant cell death when compared to 5 mmol/L glucose [77]. This is confirmed by the successful use of high glucose/low glucose media changes to selectively stimulate pericytes in our study. Neuron apoptosis is another important component of DR. It has been shown both clinically [51] and in the
laboratory [52] that degenerative changes occur in nerve cells prior to vascular cells of the retina. The results from
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our study suggest that gliacytes are more sensitive to high glucose concentrations than pericytes, and thus may be more susceptible to injury from hyperglycemia. Müller cells and other glial cells showed decreased adherence to the culture plate surface, while RMPs cultured in 20 mmol/L glucose migrated out of the retinal fragment within 24-48 hours as previously reported [78] without any obvious delay. This suggests that different cell types of the retina have varying tolerance levels to high glucose concentrations. This variant glucose sensitivity is an interesting finding needing further investigation.
Perspectives The present study is the first report of the cultivation of primary RMPs from rats and demonstrates a simple and
reproducible method to successfully isolate and purify primary RMPs. This method would be useful for further investigations on RMPs properties and facilitate subsequent studies to elucidate the roles of RMPs in retinal angiogenic diseases.
Acknowledgements Confocal laser scanning microscopy was performed at Biomedical Research Center of Fujian Academy of
Integrative Medicine. The authors thank Research Intern Li Zuanfang for his valuable assistance with confocal microscopy. This research was supported by a grant from Natural Science Foundation of Fujian Province, China (No. 2011J01197) and National Natural Science Foundation of China (No. 81102619)
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Fig 1. Phase contrast photomicrographs of primary rat RMPs in culture. A: Heterogeneous cells and
microvascular fragments were observed 20 minutes after seeding. B: RMPs (broad black arrow) migrated out of retinal fragments (slim black arrow) at 48 hours. C: A small loose colony of RMPs (broad white arrow) mixed with contaminating cells (slim white arrow) at 72 hours. D: RMPs predominating in culture and a few contaminating cells (slim black arrow) left. RMPs appeared in primary cultures as large isolated cells (E) or loose colonies on day 8 (F). Pro-confluent primary RMPs showed both multilayered (G) or sparsely spread (H) organization on day 14. I: Characteristic feature of RMPs with irregular triangular morphology stained with DAPI by CLSM with 200 ms exposure time. A-I: Scale bar = 50 μ m.
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Fig 2. Growth curve of RMPs. The population of RMPs entered log phase by day 4, transited to stationary
phase by day 10.
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Fig 3. Immunofluorescence of RMPs at passage 3 by CLSM. A-D: Positive double immunostaining of α-SMA
and PDGFR-β. Positive staining of α-SMA in RMPs cytoplasm and clear fiber bundles (B). E-H: Positive double
immunostaining of α-SMA and NG2. I-L: Positive double immunostaining of α-SMA and Desmin. M-P: Double
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staining of α-SMA and SMMHC showed positive α-SMA expression and negative SMMHC expression. Q-T: Double staining of α-SMA and GS showed positive α-SMA expression and negative GS expression. U-X: Double
staining for vWF and GFAP with negative immunostaining for both markers. A-X: Scale bar = 50 μ m.
Fig 4. Immunofluorescence of RMPs at 48 hours by CLSM. A: Nuclear DAPI staining. B: Positive staining of
α-SMA in RMPs cytoplasm and clear fiber bundles. C: Cell contours. D: Positive staining of PDGFR-β. E: Overlay. A-E: Scale bar = 25 μ m.
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Fig 5. Negative control of immunofluorescence. RMPs were either stained for primary antibodies or
secondary antibodies alone. The nucleuses were positively labelled with DAPI, and the expression of relevant antibody was negative (overlay). A: DAPI + α-SMA + PDGFR-β; B: DAPI + α-SMA + NG2; C: DAPI + α-SMA + Desmin; D: DAPI + α-SMA + GS; E: DAPI + vWF + GFAP; F: DAPI + α-SMA + SMMHC; G: DAPI + goat anti-
rabbit IgG TRITC; H: DAPI + goat anti-mouse IgG FITC. A-H: Scale bar = 50 μm.
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Fig 6. RMPs interactions with ECs in 2D coculture system. RMPs and ECs plated at a 1:10 ratio. The cocultured cells were labelled with red cell tracker. A centrally located pericyte extending many processes (white arrows) that contact multiple surrounding ECs. Scale bar = 10um
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Fig 7. Cords formation in 3D coculture system. RMPs and ECs plated at a 1:10 ratio. RMPs were labelled with
green cell tracker and ECs with red one. A: RMPs align with ECs capillary-like cord in Matrigel (slim black arrow). Some cords condensed to form large aggregate (broad black arrow). B-D: The green-labelled RMPs (B, slim white arrow) were closely invested with the red-labelled ECs (C) to form vessel-like structures (D, merge). Scale bar = 10um.
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Received Date : 25-Feb-2013 Revised Date : 27-Dec-2013 Accepted Date : 30-Jan-2014 Article type : Original Research
Isolation, purification, and cultivation of
primary retinal microvascular pericytes: a novel model using rats
Guanghui Liua, b, c, Chun Mengd, Mingdong Panc, Meng chene, Ruzhi Denga, b, Ling Lind, Li Zhaof, Xiaoling Liua, b, *
a
School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
b
State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health P. R. China, Wenzhou,
Zhejiang, China
c
Department of Ophthalmology, Affiliated People's Hospital (People's Hospital of Fujian Province), Fujian University of
Traditional Chinese Medicine, Fuzhou, Fujian, China
d
Department of Bioengineering, College of Biological Science and Biotechnology, Fuzhou University, Fuzhou, Fujian, China.
e
Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, US.
f
Department of Cardiology, Affiliated People's Hospital (People's Hospital of Fujian Province), Fujian University of Traditional
Chinese Medicine, Fuzhou, Fujian, China.
running title：cultivation of rat primary retinal pericytes
grant numbers and source(s) of support： Natural Science Foundation of Fujian Province, China (No. 2011J01197) National Natural Science Foundation of China (No. 81102619)
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/micc.12121 This article is protected by copyright. All rights reserved.
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corresponding author: School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, 270 Xueyuan Road, Wenzhou, Zhejiang, 325003, China; [email protected]
Abstract Purpose: To isolate, purify, and cultivate primary retinal microvascular pericytes (RMPs) from rats to
facilitate the study of their properties in vitro. Methods: Primary RMPs were isolated from weanling rats by mechanical morcel and collagenase
digestion, and purified by a step-wise combination of selective medium with different glucose concentrations, medium exchange, and partial enzymatic digestion. Morphology of RMPs was assessed by phase contrast microscopy. Further characterization was analyzed by immunofluorescence. Functional assay was evaluated by the pericytes- endothelial cells (ECs) coculture system. Results: RMPs migrated out of microvascular fragments after 24-48 hours of plating and reached
subconfluence on days 14-16. The cells showed typical pericyte morphology with large irregular triangular cell bodies and multiple long processes, and uniformly expressed the cellular markers α-SMA, PDGFR-β, NG2 and desmin, but were negative for vWF, GS , GFAP and SMMHC. Ninety-nine percent of the cell population had double positive staining for α-SMA and PDGFR-β. In the coculture system, RMPs can directly contact ECs
and move together to form the capillary-like cords.
Conclusions: RMPs can be readily obtained by our method. We report the first cultivation of primary
RMPs from rats and establish a simple method for their isolation and purification.
Keyword: Retina; Microvascular; Pericyte; Isolation; Purification
Abbreviations: α-SMA
α-smooth muscle actin
CLSM
confocal laser scanning microscopy
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DAPI
4', 6-diamidino-2-phenylindole
DM
diabetes mellitus
DMEM
Dulbecco's modified Eagle's medium
DR
diabetic retinopathy
ECs
endothelial cells
FBS
fetal bovine serum
GFAP
glial fibrillary acidic protein
GS
glutamine synthetase
H-DMEM-20
high-glucose (20mmol/L) DMEM supplemented with 20% FBS
L-DMEM-20
low-glucose (5mmol/L) DMEM supplemented with 20% FBS
MTT
methyl thiazolyl tetrazolium
NG2
nerve/glial antigen 2
PBS
phosphate-buffered saline
PDGFR-β
platelet-derived growth factor receptor-β
RMPs
retinal microvascular pericytes
SMMHC
smooth muscle myosin heavy chain
vWF
von Willebrand factor
Introduction Pericytes are important components of the microvasculature. They are strategically situated on the outer wall of
small blood vessels and are embedded within the basement membrane around endothelial cells (ECs) [1]. Pericytes play critical roles in microvascular biology, including: (1) the regulation ECs proliferation and differentiation [2, 3], (2) the stabilization and control of microvascular barrier [4, 5], (3) the regulation of vascular tone and perfusion pressure [6, 7], (4) and involvement in vasculogenesis and angiogenesis [8, 9]. Recent reports have also shown that pericytes are intimately involved in the pathogenesis of various retinal angiogenic diseases, particularly diabetic retinopathy (DR) [9, 10]. Compared with microvascular ECs though, the role of pericytes is much less understood
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[11]. This is partly due to difficulties in studying these cells in vivo, in obtaining the correct tissue, and in cultivating homogeneous pericyte populations in vitro [12]. Pericytes are morphologically, biochemically, and physiologically heterogeneous [13]. Thus far, in vitro
cultures of primary retinal microvascular pericytes (RMPs) have been mainly established from cells isolated from monkeys [14], humans [15-18], and bovine origin [19-25]. Although human retinas are useful sources for in vitro study, legal and ethical issues prevent researchers from utilizing them [18]. They are also scarce and not practical for routine isolation. Therefore, bovine retinas have been mostly used for in vitro assays. However, this source is limited as well and is inconvenient to obtain since bovine eyes are provided by slaughterhouses. Moreover, bovine animal models have not been used to study diabetes mellitus (DM), and some data derived from bovine RMPs are not in accord with those derived from human RMPs under the conditions that mimic DM [15, 26, 27]. In contrast, rat eyes are an abundantly available tissue source from which RMPs can be isolated. Rat-based in
vitro models are useful for investigating DR since there are several in vivo rat models of DM [28, 29]. Therefore, it is suitable to consider rat eyes as the main tissue source for routine cultivation of primary RMPs. Yet, to our knowledge, the culture of primary RMPs from rats has not been reported. Regardless of the starting tissue source, the major problems encountered in cultivating primary RMPs are the
high costs and the lengthy procedures required for purification. RMPs isolation protocols rely on enzymatic tissue digestion followed by either positive immunoselection [22] or microvascular outgrowth [30]. The former approach is complex, expensive, and results in relatively low yields of RMPs, while the latter often leads to heterogeneous populations with cellular contamination. Therefore, it appears beneficial to develop an economical and convenient protocol for the purification of primary RMPs. Here, we present a simple protocol for isolating, purifying and cultivating primary RMPs, using weanling rats
as the starting tissue source and using selective medium with different glucose concentrations as the key purification method. We demonstrated that the homogeneous RMPs populations produced by our method can readily proliferate in culture and uniformly express characteristic RMP cellular markers in vitro.
Materials and Methods Isolation of RMPs (a) Collection of retina samples.
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All experimental procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Ethics Committee for Animal Use in Research and Education at Wenzhou Medical College. All procedures were performed under aseptic conditions. The retinas were isolated from 20 male weanling rats (Sprague Dawley rat; SLAC, Shanghai, China), aged 3 weeks, by using a modified version of the method previously described [22]. After euthanasia of rats by cervical dislocation, the eyes were enucleated and immediately placed into ice-cold phosphate-buffered saline (PBS) containing 100 U/mL penicillin and 100 µg/mL streptomycin. They were then transferred into 75% ethanol for 1 minute and washed twice with PBS. Using ophthalmic microscopic surgical instruments, incisions were subsequently made in each eye posterior to the ciliary body and extended circumferentially while the eyes were in 60-mm-diameter tissue culture dish (Corning, NY, US) with PBS. Connective tissue was discarded. Retinas were pulled gently away from the posterior eyecups by microscopic iris repository. All retinas were washed twice in PBS to prevent contamination before use in further steps. (b) Enzymatic digestion. Retinas were mixed with a small amount of PBS and then finely minced into homogeneous fragments, which
were made as small as possible by crosscutting the tissue using two ophthalmic microscopic scalpels. The retinal fragments were suspended and incubated in 3 mL of 0.2% collagenase typeⅠfor 20 minutes at 37oC. After incubation, 3 mL of low glucose (5mmol/L) Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, US) supplemented with 20% fetal bovine serum (FBS; Hyclone, Logan, US) was added and mixed gently to suspend digestion. The solution was repeatedly pipetted up and down to break up clumps of cells and retinal fragments, and then filtered through 100 μm filtrate over a 55 μm nylon sieve (Falcon, Oxnard, US). The 55-100 μm filtrate was collected in 1.5 mL centrifuge tubes, and centrifuged at 2000 rpm (500 g) for 4 minutes at 4oC. The supernatant was
discarded, and the precipitated pellet containing retinal capillary-rich fragments was collected. The pellet was then re-suspended in 1.5 mL high glucose (20mmol/L) DMEM with 20% FBS (H-DMEM-20), and seeded into 6-well plates (Costar; Corning, NY, US) maintained with H-DMEM-20.
Cultivation of RMPs The suspended fragments were incubated at 37oC with 5% CO2 atmosphere. Cell growth was monitored under
phase contrast microscopy (Olympus, Tokyo, Japan). The medium was changed daily (days 3, 4, and 5) until day 5, after which the medium was changed every 3 days. H-DMEM-20 was replaced with low glucose DMEM with 20%
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FBS (L-DMEM-20) on day 3. When primary RMPs cultures were near confluence (above 80%), they were further propagated by treatment with 1 mL of 0.25% trypsin (Santa Cruz Biotech, Santa Cruz, US) and 0.02% EDTA for 3 minutes and then were either split into a 1:3 ratio to passage to additional 6-well plates or harvested for cryopreservation. For proliferation assays, cells at passages 3 were counted and seeded into 96-well plates (1×104
cells per well). Medium was added at a volume of 100 μL and was replaced every 3 days with fresh media. Plates were incubated for a total of 12 days. Methyl thiazolyl tetrazolium (MTT) assay was then performed according to the literature [31].
Weeding contaminating cells. The following techniques were used to obtain pure populations of RMPs. After 48 hours of incubation, any
unattached fragments, cells or debris were discarded and the media was removed without agitation and replaced with L-DMEM-20. On days 4 and 5, the plates were rinsed twice to remove any loosely adherent cellular contamination. Old medium was removed and replaced with fresh L-DMEM-20. This weeding procedure was performed during medium exchange. When RMPs reached 80% confluence, they were incubated with trypsin. Trypsinization was observed under
phase contrast microscopy. It was observed that cells showed different detachment times. Contaminating cells began to detach first after 1-2 minutes of trypsinization, followed by RMPs after 3-5 minutes. After determination and optimization of detachment times, the effect of trypsin was neutralized right after the time period in which only contaminating cells detached and before RMPs detached. The detached contaminating cells were then gently mixed into the culture medium by gently swirling the culture plate back and forth, after which the medium containing detached contaminating cells was discarded. RMPs remained attached. New trypsin was added to the RMPs, which were then incubated for passage.
Morphological and immunofluorescence characterization of RMPs Morphological characterization was performed using phase contrast microscopy, and immuno-fluorescence
characterization was analyzed using the confocal laser scanning microscope (CLSM) LSM 710 (Zeiss, Jena, Germany). The cells were characterized with the cellular characterization kit according to the manufacturer’s protocol. Briefly, the cells were grown in glass bottom cell culture dishes (NEST, Wuxi, China). The dishes were washed for 5 minutes three times with PBS, and cells were fixed in 4% paraformaldehyde at room temperature for
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20 minutes. Subsequently, the cells were washed extensively with ice-cold PBS and incubated for 2 hours at room temperature with blocking buffer containing 5% FBS and 0.3% Triton X-100 in PBS. The primary antibodies were diluted with working concentrations of the blocking buffer. The cells were incubated overnight at 4oC with primary antibodies against the following markers: α-smooth muscle actin (α-SMA; Sigma, St. Louis, US), desmin (Santa
Cruz Biotech, Santa Cruz, US), platelet-derived growth factor receptor-β (PDGFR-β; Santa Cruz Biotech, Santa Cruz, US), nerve/glial antigen 2 (NG2; Chemicon, Temecula, US), von Willebrand factor (vWF; Santa Cruz Biotech, Santa Cruz, US), glutamine synthetase (GS; Chemicon, Temecula, US), glial fibrillary acidic protein (GFAP; Santa Cruz Biotech, Santa Cruz, US), and smooth muscle myosin heavy chain (SMMHC; Bioss, Beijing, China). Afterwards, the cells were washed once with PBS and three times with blocking solution. The cells were then incubated for 2 hours at room temperature with fluorescent secondary antibodies of goat anti-rabbit IgG TRITC and anti-mouse IgG FITC (Santa Cruz Biotech, Santa Cruz, US). For double staining, this procedure was repeated with different primary antibodies and appropriate secondary antibodies. Finally, the labelled cells were incubated with a solution containing 4', 6-diamidino-2-phenylindole (DAPI; Invitrogen, Karlsruhe, Germany) at a dilution of 1:1000. Parallel control cultures that were either stained only for primary antibodies or only for secondary antibodies alone were maintained as well.
RMPs and ECs coculture The rat retinal ECs were obtained as previously described [32, 33]. According to the previous literatures [34,
35], RMPs and ECs were plated at a 1:10 ratio, direct-contact assay was evaluated by 2-dimensional (2D) RMPsECs coculture system under routine culture conditions, and capillary-tube formation assay was evaluated by 3dimensional (3D) system in Matrigel (BD Bioscience, La Jolla, US). ECs and RMPs were labelled by cell tracker (Molecular Probes, Eugene, US) before 3D coculture in Matrigel. After 48 hours of coculture the cells were analyzed by fluorescent microscopy (Nikon, Tokyo, Japan).
Results Cultivation of RMPs Heterogeneous cells and microvascular fragments were observed immediately after plating cultures (Fig 1. A).
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The fragments started attaching to culture dishes after 4-5 hours of incubation, and cells were adherent to culture dishes within 12-24 hours. RMPs migrated out of the retinal fragments 24-48 hours after plating (Fig 1. B). Culture media was changed at 48 hours, when unattached contaminating cells were discarded and heterogeneous cells remained in culture. Most of these remaining cells were RMPs with irregular triangular morphology; a few cells appeared to have smaller and rounder cell bodies (Fig 1. C). We did not observe any cobble-stone patterns typical of ECs cultures [36] or any characteristic bipolar morphology typical of Müller cells cultures [37]. Contaminating cells were gradually eliminated by the method described above by rinsing plates when changing medium at 48 hours and on days 4 and 5. This resulted in only very few contaminating cells left in culture (Fig 1. D). On day 8, RMPs appeared in primary cultures as either large isolated cells containing typical stress fibres (Fig 1.
E) or as loose colonies of polygonal and elongated cells with ruffled edges (Fig 1. F). The large isolated cell may grow from a single cell digested by collagenase, and the clone grow from a central tissue fragments. On day 14, either overlapping layers containing quantities of cells (Fig 1. G) or cells sparsely spreading on the bottom of dish (Fig 1. H) were observed. Growth of the primary RMPs were even slower. The cells approximated 80% confluence on days 14-16 and reached a total number of about 1.3×105 primary cells per well as determined by hemocytometry.
The subcultures grew faster than the primary cultures and reached confluence on days 12-14（Fig 2）. RMPs can
be successfully cryopreserved and recultured without loss of typical features; they can be repeatedly passaged 9 times without obvious loss of characteristic phenotype.
Characterization of RMPs RMPs were primarily analyzed and characterized based upon morphology under phase contrast microscopy.
Cells were confirmed as RMPs by observing a typical pericyte morphology with irregular shape, long processes, and large and flat cell bodies [9, 38-41]. In the cultures, almost all of the cells showed typical pericyte morphology (Fig 1. I). Further immunofluorescence confirmation was performed by double immunostaining. The expression of
pericyte markers α-SMA, PDGFR-β, NG2 and desmin was detected by CLSM. Double staining for α-SMA and
PDGFR-β revealed that 99% of the cells in culture showed positive immunofluorescence for both markers (Fig 3. AD), confirming the purity of RMPs in culture. Cells in culture were also positive for NG2 (Fig 3. E-H) and, to a lesser extent, desmin (Fig 3. I-L). In the population positive for α-SMA, >99% of cells were positive for desmin while >85% of cells were positive for NG2. The relative heterogeneity of expression of NG2 is consistent with the
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previous report [34]. The expression of these 4 markers did not significantly change in RMPs from passages 3 to 7. Additionly, the expression of α-SMA and PDGFR-β were analyzed in the cells at 48 hours after incubating. The result showed 100% double-positive staining for these two markers in RMPs. (Fig 4). None of the cells in culture expressed positive staining for vWF, GS, GFAP, and SMMHC, which are specific
markers, respectively, for ECs, Müller cells, glial cells, and vascular smooth muscle cells (vSMCs). Thus, there was no contamination of cultures by ECs, Müller cells, glial cells, or vSMCs (Fig 3. M-X). The negative control cultures that were stained either only for primary antibodies or only for secondary antibodies all showed negative staining (Fig 5).
Functional assay Pericytes-ECs coculture was used as a means to assess pericyte function in vitro. In our 2D coculture system,
RMPs was recruited by ECs and extended their processes to contact ECs directly in the dish bottom after 48 hours of plating (Fig 6). In 3D system, the cells moved toward one another as they do in blood vessels in vivo and formed cords liking capillary-tube in the Matrigel after 48 hours of plating. Some cords condensed to form large aggregates (Fig 7).
Discussion In this report, we describe a simple method for cultivation of primary RMPs from rats without the need for
positive cell selection. Our method achieved a nearly homogenous population of RMPs from which readily expanding subpopulations of RMPs can be generated. In our protocol, primary RMPs were isolated from the weanling rats and largely purified by the selective
medium, a prerequisite for successful cultivation. Key features of our method that differ from previously published reports include: (1) a starting tissue source of RMPs originating from a readily available rat model, weanling rats, (2) collagenase digestion of retinal fragments limited to only 20 minutes without agitation, (3) significantly less requirement of expensive materials and sophisticated instrumentation, (4) usage of high glucose and low glucose media changes to selectively stimulate pericytes, and (5) partial trypsinization to selectively remove contaminating cells. The technical aspects and tools required for our protocol are simple, thereby making the isolation, purification and cultivation of RMPs feasible for any cell biology laboratory. Traditionally, a major difficulty in the cultivation of primary RMPs in the past has been limited tissue sources
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[11, 42]. Though some reported model systems of primary RMPs have been developed from various species, including monkey [14], human [15, 18], and bovine [19-25], the study of RMPs has lagged far behind that of ECs for the difficulties of in vivo study, tissue source and in vitro cutivation mentioned above. Looking at the last four decades, the annual number of publications regarding pericytes is far less compared with the numbers of publications regarding ECs. The key words “endothelial cell (s)” produce 10,217 (8,783) citations in PubMed for the year 2010, while the key word “pericyte (s)” resulted in only 256 (221) [11, 42]. Furthermore, only a small number of these publications are about RMPs. Rats are a common study model and are easily obtainable. Yet, they have been scarcely used in the culture of primary RMPs. This is primarily attributable to the smaller size of the rat eye, making it harder to dissect, thus resulting insufficient cell yield from rat eyes in comparison with those of bovine eyes [42]. There has been only one previous report in the literature regarding an immortalized pericyte cell line from tsA58 Tg rats, which are uncommon model source harboring the temperature-sensitive simian virus 40 large Tantigen gene and dealt with unconventional protocol [42]. In the current study, the weanling rats were adopted for retinal source. The rat at the age of 3 weeks had not only developed retinal vessels [43] but also the cells with good proliferative ability [44, 45]. Due to the complex components of retina and unique structures of eyeball wall, the protocol for RMPs isolation is somewhat different with those for other tissues, such as brain, coronary, and skeletal muscle [11, 22, 34, 46, 47]. Additionally, the conventional instruments used for adult rats and bovine are not suitable for the weanling rats because of their small eyeball. In present report, retinas of the weanling rats can be successfully isolated by using ophthalmic microscopic instruments without the need of a dissecting microscope. One crucial aspect in isolating a whole rat retina was in utilizing microscopic iris repositories to gently pull the retinas away from the posterior eyecups. Additionally, since our RMPs can recover from cryopreservation without losing their characteristic phenotype, a sufficient amount of accumulated cells can be achieved by cryopreservation for large demand although the cells from a single rat may not enough [42]. A major difficulty of obtaining homogeneous RMPs populations is in the elimination of contaminating cells
such as gliacytes. A number of purification techniques are available in pericytes cultivation that utilize sequential sieving [30], gradient centrifugation [48], weeding non-pericytes [20], fluorescent-activated cell sorting [22], and magnetic beads [22]. However, these techniques are limited by high monetary costs, lengthy time requirements, or viable cell-consumption. They all generally require expensive and sophisticated instrumentation and complex procedures. In Kondo’s report [42], RMPs are cloned from a single cell and keep 100% homogeneity, since the SV
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40 large T-antigen gene is activated by low temperature (33oC) and the culture become immortalized in the early stage. But monoclone isn’t appropriate for primary RMPs due to their limited proliferation. The method described in our study for eliminating contamination is a step-wise combination of selective medium, medium exchange, and partial enzymatic digestion that does result in a purified, homogeneous population of RMPs. It is a relatively simpler and less costly method for eliminating gliacytes than the above previous methods. Contaminating ECs are also reported in pericyte culture [34, 49]. Owing to uncoated plate and inhibition of 20% FBS [20], migration and adherence of ECs did not appear in our study. During this previous year, we repeated our culture method more than 50 times, showing good repeatability and reproducibility. Cultured cells, as well as those recovered from cryopreservation storage, uniformly express characteristic pericyte surface and intracellular proteins and lack ECs, vSMCs, and glial cell markers. As reported previously [50], although a number of pericyte markers has been identified in the past, the lack of
reliable markers has still hindered pericyte identification. The expression of pericyte markers are dynamic [51] and varies depending on the species and tissue studied. No single marker is able to identify all pericytes [52, 53]. Therefore, we decided that the use of multiple markers and high-resolution confocal microscopy was a better approach to identify and study pericytes in our cultures. Four of the best-described markers used to identify
pericytes are α-SMA, desmin, PDGFR-β and NG2 [54]. Since 1985, α-SMA has been used as a pericyte marker [55] and has been considered to be a general marker for
pericytes [50, 56]. In primary cultures, nearly 100% of pericytes express α-SMA by day 7 [57]. Many investigators
have used α-SMA to identify RMPs in vitro[18, 24, 33]. Desmin is an intermediate filament proteins and is an
intracellular marker [54] expressed by immature and mature pericytes [58, 59]. In vivo studies have shown that desmin is a more sensitive marker for pericytes than other pericyte markers in adult rat retina [60] and can label more pericytes in mouse retina [61]. In vitro study, desmin is used as a routine marker for pericytes cultivation [3, 27, 62, 63]. Desmin and α-SMA are both intracellular markers that are viewed as the first line of evidence of the presence of pericyte contractile proteins [64]. The double positive immunostaining of α-SMA and, to a lesser extent, desmin in our culture cells suggested that rat RMPs maintain contractile functions in vitro. It was consistent with what found in Berrone et al [18] studies. But compared with low positive expression (5%-30%) of α-SMA in freshly isolated brain pericytes reported by Dore-Duffy [50, 65], our result demonstrated a little difference in that 100% of RMPs at 48 hours expressed the smooth muscle phenotype. We inferred the difference may be owed to the source
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heterogeneity [13], theirs from brain and ours from retina. In addition to involvement in hemodynamic processes, pericytes also play a crucial role in vasculogenesis and
angiogenesis. For this reason, PDGFR-ß is one of the most widely used markers for pericytes [33, 52]. PDGFR-ß plays a significant role in pericyte recruitment and in the initiation and maturation of the microvasculature [54]. Knockout of the genes encoding PDGFR-β in mice results in a reduced number of pericytes and subsequent vessel malformation and dysfunction [66, 67]. NG2 is another established, specific marker for pericyte identification [68] that is expressed on the surface of activated pericytes during vessel formation [69]. The previous studies on both normal [70] and pathological [71] angiogenesis of retina suggest that NG2 can act as a reliable marker for microvascular pericytes. Our cultures showed a strong positive immunofluorescence for PDGFR-ß staining. Positive expression of NG-2 was also observed. This demonstrated that rat RMPs can also be used to study retinal angiogenesis in vitro. Aside from the phenotypic analysis outlined above, the pericytes-ECs coculture, as a means to verify the
function of pericytes, further evidenced that the cells derived from our culture can act as pericytes in microvessle in
vivo. In the coculture system, not only RMPs can directly contact ECs by multi processes but also the cocultured
cells can move together to form the capillary-like cords, which were in accordance with Bryan’s report [35]. The results demonstrated that rat RMPs acquired by our method can retain the ability to closely associate with ECs. In the foregoing, both phenotypic analysis and functional assay strongly supported the claim that our method
obtains highly pure RMPs and suggested that the cells could be useful as a model to investigate DR. RMPs loss is a histopathological hallmark of early DR and is a key event in the pathogenesis of DR [53, 72]. It
has been well demonstrated in vitro that pericyte contractility and survival is reduced in high glucose environments
in a dose-dependent manner [73, 74]. Previous reports indicate that long time exposure of pericytes to a high glucose concentration of 28 to 30 mmol/L induces significant mitochondria fragmentation, increased membrane potential heterogeneity, and increased numbers of apoptotic RMPs [27, 74]. But RMPs are less affected by a short exposure time of less than 48 hour to high glucose concentrations (＜25mmol/l) [75, 76]. It is suggest that exposure to glucose at 20 mmol/L leads to insignificant cell death when compared to 5 mmol/L glucose [77]. This is confirmed by the successful use of high glucose/low glucose media changes to selectively stimulate pericytes in our study. Neuron apoptosis is another important component of DR. It has been shown both clinically [51] and in the
laboratory [52] that degenerative changes occur in nerve cells prior to vascular cells of the retina. The results from
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our study suggest that gliacytes are more sensitive to high glucose concentrations than pericytes, and thus may be more susceptible to injury from hyperglycemia. Müller cells and other glial cells showed decreased adherence to the culture plate surface, while RMPs cultured in 20 mmol/L glucose migrated out of the retinal fragment within 24-48 hours as previously reported [78] without any obvious delay. This suggests that different cell types of the retina have varying tolerance levels to high glucose concentrations. This variant glucose sensitivity is an interesting finding needing further investigation.
Perspectives The present study is the first report of the cultivation of primary RMPs from rats and demonstrates a simple and
reproducible method to successfully isolate and purify primary RMPs. This method would be useful for further investigations on RMPs properties and facilitate subsequent studies to elucidate the roles of RMPs in retinal angiogenic diseases.
Acknowledgements Confocal laser scanning microscopy was performed at Biomedical Research Center of Fujian Academy of
Integrative Medicine. The authors thank Research Intern Li Zuanfang for his valuable assistance with confocal microscopy. This research was supported by a grant from Natural Science Foundation of Fujian Province, China (No. 2011J01197) and National Natural Science Foundation of China (No. 81102619)
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Fig 1. Phase contrast photomicrographs of primary rat RMPs in culture. A: Heterogeneous cells and
microvascular fragments were observed 20 minutes after seeding. B: RMPs (broad black arrow) migrated out of retinal fragments (slim black arrow) at 48 hours. C: A small loose colony of RMPs (broad white arrow) mixed with contaminating cells (slim white arrow) at 72 hours. D: RMPs predominating in culture and a few contaminating cells (slim black arrow) left. RMPs appeared in primary cultures as large isolated cells (E) or loose colonies on day 8 (F). Pro-confluent primary RMPs showed both multilayered (G) or sparsely spread (H) organization on day 14. I: Characteristic feature of RMPs with irregular triangular morphology stained with DAPI by CLSM with 200 ms exposure time. A-I: Scale bar = 50 μ m.
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Fig 2. Growth curve of RMPs. The population of RMPs entered log phase by day 4, transited to stationary
phase by day 10.
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Fig 3. Immunofluorescence of RMPs at passage 3 by CLSM. A-D: Positive double immunostaining of α-SMA
and PDGFR-β. Positive staining of α-SMA in RMPs cytoplasm and clear fiber bundles (B). E-H: Positive double
immunostaining of α-SMA and NG2. I-L: Positive double immunostaining of α-SMA and Desmin. M-P: Double
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staining of α-SMA and SMMHC showed positive α-SMA expression and negative SMMHC expression. Q-T: Double staining of α-SMA and GS showed positive α-SMA expression and negative GS expression. U-X: Double
staining for vWF and GFAP with negative immunostaining for both markers. A-X: Scale bar = 50 μ m.
Fig 4. Immunofluorescence of RMPs at 48 hours by CLSM. A: Nuclear DAPI staining. B: Positive staining of
α-SMA in RMPs cytoplasm and clear fiber bundles. C: Cell contours. D: Positive staining of PDGFR-β. E: Overlay. A-E: Scale bar = 25 μ m.
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Fig 5. Negative control of immunofluorescence. RMPs were either stained for primary antibodies or
secondary antibodies alone. The nucleuses were positively labelled with DAPI, and the expression of relevant antibody was negative (overlay). A: DAPI + α-SMA + PDGFR-β; B: DAPI + α-SMA + NG2; C: DAPI + α-SMA + Desmin; D: DAPI + α-SMA + GS; E: DAPI + vWF + GFAP; F: DAPI + α-SMA + SMMHC; G: DAPI + goat anti-
rabbit IgG TRITC; H: DAPI + goat anti-mouse IgG FITC. A-H: Scale bar = 50 μm.
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Fig 6. RMPs interactions with ECs in 2D coculture system. RMPs and ECs plated at a 1:10 ratio. The cocultured cells were labelled with red cell tracker. A centrally located pericyte extending many processes (white arrows) that contact multiple surrounding ECs. Scale bar = 10um
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Fig 7. Cords formation in 3D coculture system. RMPs and ECs plated at a 1:10 ratio. RMPs were labelled with
green cell tracker and ECs with red one. A: RMPs align with ECs capillary-like cord in Matrigel (slim black arrow). Some cords condensed to form large aggregate (broad black arrow). B-D: The green-labelled RMPs (B, slim white arrow) were closely invested with the red-labelled ECs (C) to form vessel-like structures (D, merge). Scale bar = 10um.
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