Smooth muscle cells (SMC) are the p redominant cell type involved in the pathogenesis of atherosclerosis and restenosis after angioplasty [1]; however, they are also important in the formation and development of de novo blood vessels (vasculogenesis) through differentiation of mesenchymal cells under the influence of mediators secreted by the endothelial cells comprising newly formed vessels [2]. In angiogenesis, vascular SMC are formed by proliferation of existing SMC or maturation of pericytes [2, 3]. Experimental findings suggest a potential role of putative smooth muscle progenitor cells in the circulation or within adult tissues and the perivascular adventitia in the development of atherosclerotic plaques and biology of angiogenesis [4]. Modulation of vascular smooth muscle phenotype, SMC migration and hypertrophy are now recognized as key events in the development of arterial lesions in vascular diseases [1]. This has led to an increase in experimental research on SMC function in response to growth factors, extracellular matrix, modified lipoproteins, and other pro-atherogenic and pro-angiogenic mediators under controlled in vitro conditions to address the cellular mechanisms involved. Most of the methodologies used for vascular SMC isolation and culture have been developed to accomplish such studies [5].
In vivo, vascular SMC retain the characteristic of phenotypic modulation, ranging between the “contractile” and “synthetic” states. This plasticity allows smooth muscle cells to adapt to local environmental cues within the vessel wall, for example growth factors (platelet-derived growth factor, transforming growth factor-β1), contractile agonists (angiotensin II, endothelins), reactive oxygen species, inflammatory mediators, and biomechanical shear and stretch forces [5, 6, 7]. The healthy adult vasculature predominantly consists of the contractile-state SMC, whose main function is in the maintenance of vascular tone. They exhibit a characteristic “muscle-like” appearance, with up to 75 % of their cytoplasm containing contractile filaments. However, in culture, these cells are able to revert to a synthetic and proliferative phenotype, which is normally found in embryonic and young developing blood vessels [8]. These proliferative cells synthesize extracellular matrix components such as elastin and collagen, and consequently contain large amounts of rough endoplasmic reticulum and Golgi apparatus but few myofilaments in their cytoplasm [6, 7, 8]. The modulation of vascular SMC from a contractile to a synthetic phenotype is an important event in atherogenesis and restenosis, resulting in myointimal thickening and arterial occlusion. This may arise from damage to the endothelium and exposure of SMC to circulating blood components, such as oxidized lipoproteins, and pro-inflammatory cytokines and stimuli such as reactive oxygen species and growth factors released from endothelial cells, neutrophils, macrophages and platelets [1]. In addition, conditions such as hyperglycemia and hyperlipidemia have been shown to modulate SMC contractile phenotype to a synthetic phenotype, which plays a key role in the development of vascular diseases [9, 10].
The methodology chosen to isolate and culture vascular SMC can determine the initial phenotype of cells obtained in culture [5, 8]. The two main techniques commonly employed in SMC isolation from arterial and venous tissues are enzymatic dissociation, which readily yields a small number of SMC initially retaining a contractile phenotype, and explantation, which yields larger numbers of SMC after 2–3 weeks, in the synthetic and proliferative phenotypes. The lower yield of SMC following enzymatic dissociation is more suited for studies on single dispersed cells while tissue explantation of vascular tissues provides a better potential for obtaining longer-term cultures of confluent SMC monolayers. In addition to the source of SMC, alterations in the methods employed to culture SMC in vitro (e.g., serum, glucose, growth factor concentrations, biomechanical forces and oxygen tensions) can also influence their phenotype. These modifications, which may facilitate the culture of a contractile phenotype, include changes in culture medium source and composition, as well as surface coating of culture plasticware with matrix proteins [11, 12, 13]. This chapter describes two alternative techniques for vascular isolation and culture of SMC from arterial tissues, subculture, maintenance, and characterization of SMC phenot ype. In addition, a brief description of the isolation and identification of pericytes from human placental microvessels is also provided since these “muscle-like” perivascular cells are recognized to play a key role during angiogenesis for scaffolding, maturation, remodeling, and contraction of microvessels [3].
The most commonly used growth medium in SMC culture is Dulbecco’s modified Eagle’s medium (DMEM) containing either 1000 or 4500 mg/L glucose; however, Medium 199 is also suitable (seeNote 1 ). The following additions to the basal medium are necessary prior to use: (final concentrations) 2 mM l-glutamine, 40 mM bicarbonate, 100 U/ml−1 penicillin, 100 μg/ml streptomycin, and 10 % (v/v) fetal calf serum (FCS). Sterile stocks of these components are usually prepared and stored as frozen aliquots as described below. The complete medium can be stored at 4 °C for up to 1 month and is prewarmed to 37 °C prior to use in routine cell culture. Culture medium without the FCS component is used during the isolation procedures.
Hanks’ balanced salt solution (HBSS) is used as a tissue specimen collection medium. The following additions, from sterile stock solutions, are necessary prior to use (final concentrations): 100 μg ml−1 Gentamycin, 0.025 M HEPES and 20 mM bicarbonate. The HBSS can be stored in aliquots at 4 °C for up to 2 weeks.
l-Glutamine (200 mM) stock solution: Dissolve 5.84 g l-glutamine in 200 ml tissue culture grade deionized water and sterilize by passing through a 0.22 μm filter. Aliquots of 5 ml are stored at −20 °C and 4 ml used in 400 ml of medium.
Bicarbonate (4.4 %, 0.52 M) solution: Dissolve 44 g NaHCO3 in 1000 ml tissue culture grade deionized water, and sterilize by auto claving for 10 min at 115 °C. Aliquots of 15 ml are stored at 4 °C for up to 6 months and 2 aliquots used in 400 ml of medium.
Penicillin and streptomycin stock solution (80× concentrate): Dissolve 480 mg penicillin (G sodium salt) and 1.5 g streptomycin sulfate in 200 ml tissue culture grade deionized water and sterilize by passing through a 0.5 μm pre-filter and a 0.22 μm filter. Aliquots of 5 ml are stored at −20 °C and 1 aliquot used in 400 ml of medium.
Gentamycin solution (80× concentrate): Dissolve 750 mg gentamycin sulfate in 100 ml tissue culture grade deionized water and sterilize by passing through a 0.22 μm filter. Aliquots of 5 ml are stored at −20 °C and 1 aliquot used in 400 ml of HBSS.
HEPES solution (1 M): Dissolve 47.6 g of HEPES in 200 ml tissue culture grade deionized water and sterilize by passing through a 0.22 μm filter. Aliquots of 5 ml can be stored at −20 °C and 2 aliquots used in 400 ml of HBSS.
Trypsin solution (2.5 %): Trypsin from porcine pancreas is dissolved (2.5 g 100 ml−1) in PBS-A and sterilized by passing through a 0.22 μm filter. Aliquots of 10 ml are stored at −20 °C.
EDTA solution (1 %): EDTA disodium salt is dissolved (500 mg/50 ml−1) in tissue culture grade deionized water and sterilized through a 0.22 μm filter. Aliquots of 5 ml are stored at 4 °C.
Trypsin (0.1 %)–EDTA (0.02 %, 0.5 mM) solution is prepared by adding 10 ml trypsin (2.5 %) and 5 ml EDTA (1 %) to 250 ml sterile PBS-A. This solution is prewarmed to 37 °C before use to detach cells from culture flasks and stored at 4 °C for up to 2 months.
Collagenase, Type II. Dissolve collagenase in serum free medium (3 mg ml−1) on ice. Particulate material is removed by filtering the solution through a 0.5 μm pre-filter and then sterilize by passing through a 0.22 μm filter. This enzyme solution can be stored long term as 5–10 ml aliquots at –20 °C until use.
Elastase, type IV from porcine pancreas. Immediately before use, elastase is dissolved in serum free medium (1 mg/ml) and the pH of solution adjusted to 6.8 with 1 M HCl. This solution is then sterilized by passing through a 0.22 μm filter and kept on ice.
Antibody to specific SMC antigen, e.g., monoclonal mouse α-smooth muscle actin.
Normal rabbit serum.
Fluorescein isothiocyanate conjugated rabbit anti-mouse secondary antibody.
Methanol (100 %).
25 cm2 and 75 cm2 tissue culture flasks and 90 mm petri dishes.
Sterile Pasteur, 5 and 10 ml pipettes.
Sterile 30 ml universal containers and 10 ml centrifuge tubes.
Scalpel handles and blades, small scissors, watchmaker’s forceps, and hypodermic needles.
Cork board for dissection covered with aluminum foil, both sterilized by thorough spraying with 70 % ethanol.
Sterile conical flasks of various sizes.
Lab-Tek chamber slides (Nunc).
In this laboratory, cells are routinely isolated from human umbilical arteries, a readily available source of hu man vascular SMC. As soon as possible after delivery, the whole umbilical cord, obtained with prior ethical approval and consent of mothers, is placed in the HBSS collection medium and stored at 4 °C. Cords collected and stored in this way can be used for SMC isolation up to 48 h after delivery. SMC can also be isolated from arteries following harvesting of endothelial cells from the corresponding umbilical vein [14]. Immediately prior to proceeding with SMC isolation, 5 cm lengths of the umbilical artery should be carefully dissected out from the cord, ensuring minimal surrounding connective tissue remains, and stored in new collection medium. Other common sources of arterial tissue are from human, mouse, rat, or porcine aortae, which should be carefully dissected out from the body, cleaned of extraneous tissues and stored in the collection medium at 4 °C as soon as possible (seeNote 2 ). Isolation of SMC/pericytes from microvessels can be performed using human placental or bovine retinal tissue [15].
As much surrounding connective tissue as possible should be dissected away from around the artery and the tissue washed with new HBSS collection medium. The artery is then placed on the sterile dissection board and covered with HBSS to keep it moist.
The artery is fixed to the dissection board at one end using a hypodermic needle and then cut open longitudinally, using small scissors, with the luminal surface upward.
The endothelium is removed along the whole length of the artery by scraping the cell layer off with a sterile scalpel blade and the tissue re-moistened with HBSS.
The thickness and nature of the arterial wall will vary depending on the source of tissue; however, in general the arterial intima and media are peeled into 1–2 mm width transverse strips using watchmaker’s forceps and a scalpel. Muscle strips are transferred in to a 90 mm petri dish containing HBSS. This procedure is repeated for the whole surface of the vessel.
Most of the HBSS medium is then aspirated off and the muscle strips cut into 1–2 mm cubes using scissors or a scalpel blade. The cub es are then washed in new HBSS and transferred into a sterile conical flask of known weight and the mass of tissue measured to determine volume of enzyme solution needed for digestion.
Collagenase solution in serum free culture medium is next added to the tissue to give a ratio of tissue (g) to enzyme solution (ml) of 1:5 (w/v). The flask is then covered with sterile aluminum foil and shaken in a water bath at 37 °C for 30 min.
Elastase solution is then prepared and directly added to the solution containing the tissue and collagenase. The flask is returned to the shaking water bath at 37 °C and every 30 min during the following 2–5 h the suspension mixed by pipetting in the sterile safety cabinet.
At each 30 min interval, a 10 μl sample of suspension is transferred to a hemocytometer to check for the appearance of single cells. This is repeated until the tissue is digested and there are no large cell aggregates visible. Digestion should not proceed for longer than 5 h to avoid loss of cell viability (seeNote 3 ).
The cell suspension is finally divided into 10 ml centrifuge tubes and centrifuged at 50–100 × g for 5 min. The supernatants are carefully aspirated and 2–5 ml of prewarmed complete culture medium added to resuspend the cells using a sterile pipette. Cells are then seeded into 25 cm2 culture flasks at a density of about 8 × 105 cells ml−1 and placed into a 37 °C, 5 % CO2 incubator with the flask cap loose.
Viable SMC should adhere to the flask wall within 24 h and all the medium is then replaced with fresh prewarmed complete culture medium. Half of the culture medium is replaced every 2–3 days until a confluent SMC monolayer is obtained.
The artery is cut into 2 mm cubes with a scalpel blade and placed on to the surface of a 25 cm2 culture flask using a sterile Pasteur pipette, ensuring that the luminal surface is in contact with the flask wall. The artery should be kept continually moist with the HBSS and a small drop of serum containing medium should be placed on each cube when placed in the flask.
The cubes are distributed evenly on the surface with a minimum of 12–16 cubes per 25 cm2 flask. The flask is then placed upright and 5 ml serum containing medium added directly to the bottom of the flask before transferring into a 37 °C, 5 % CO2 incubator with its cap loose. To facilitate adherence of the explanted tissue to the culture plastic substrate, the flask is kept upright for 2–4 h in the incubator before the flask is carefully placed horizontally such that the medium completely covers the attached muscle cubes.
The explants should be left undisturbed for 4 days, inspecting daily for infections (seeNote 4 ). Every 4 days any unattached explant cubes should be removed and half the medium replaced with fresh prewarmed serum containing medium. Cells will initially migrate out from the explants within 1–2 weeks.
After 3–4 weeks there should be sufficient density of SMC around the explants for removal of the tissue. Using a sterile Pasteur pipette, the cubes are gently dislodged from the plastic flask surface and aspirated off with the culture medium. The culture medium is replaced and cells are then left for a further 2–4 days to proliferate and form a confluent SMC monolayer (seeNote 5 ).
Dissect a central section of the placenta and wash thoroughly in serum-free medium. Ensure that the section chosen is distant from any large blood vessels and the outer membrane.
Manually dissect and cut the tissue into small 5 mm2 pieces and incubate in serum-free media containing 3 mg/ml collagenase for 3 h at 37 °C in a shaking water bath.
Separate the microvessels by passing the suspension through a 70 μM mesh filter (Falcon) and wash through two times with serum-free media.
Remove the microvessels from the mesh filter and place into a 25 cm2 culture flask containing medium supplemented with 20 % serum.
After 24 h the medium is removed and attached cells washed once with warmed sterile PBS to remove floating debris. Fresh growth medium is added and following 5–6 days, pericytes and endothelial cells proliferate out from the microvessel fragments.
As the culture medium does not contain endothelial cell growth supplements, any endothelial cells present initially no longer survive after two rounds of trypsinization leaving a pure culture of pericytes which can be characterized by their morphology and antigen expression detected by immunofluorescence.
Once a confluent monolayer has been attained in a 25 cm2 flask by either isolation method, the SMC can be subcultured (passaged) into further 25 cm2 flasks or a 75 cm2 flask. The culture medium is removed and cells are washed twice with prewarmed sterile PBS-A to remove traces of serum.
Prewarmed trypsin–EDTA solution (0.5 ml for 25 cm2 flask or 1 ml for a 75 cm2 flask) is added to cover the cells and the flask incubated at 37 °C for 2–4 min. The flask is then examined under the microscope to ensure cells have fully detached. This can also be facilitated by vigorous tapping of the side of the flask three to six times to break up cell aggregates (seeNote 6 ).
Serum containing medium (5 ml) is added to stop the action of the trypsin which can reduce SMC viability through prolonged exposure. The cell suspension is then drawn up and down a sterile Pasteur pipette four to six times to further break up any cell clumps.
The cells are then transferr ed into new culture flasks at a split ratio of 1:3 and sufficient serum containing medium added to the new flasks (5 ml in a 25 cm2 and 10 ml in a 75 cm2 flask). The flasks are returned to the 37 °C, 5 % CO2 incubator and the culture medium changed as described in Subheading 3.2, step 10.
Smooth muscle cells can be passaged between 10 and 20 times, depending on species, before their proliferation rate significantly decreases. Phenotypic changes of enzyme dispersed SMC to the “proliferative” state occurs following passaging, the extent of which depends on the vessel type and species from which the SMC are derived, the culture medium and their seeding density (further discussed below) [5, 6, 8, 16].
| Specific SMC protein markers | Function |
|---|---|
| Smooth muscle myosin heavy chain | SMC contractile protein |
| Calponin | Contractile regulator |
| SM22α | Cytoskeletal protein |
| Desmin | Intermediate filament |
| H-Caldesmon | Actin binding protein |
| Metavinculin | Actin binding protein |
| Sm oothelin | Cytoskeletal protein |
| α-Smooth muscle actin | Contractile protein |
Effect of cell culture conditions on SMC phenotype marker gene expression. Commercially sourced human aortic S MC isolated using enzymatic dissociation were cultured for 2 days in basal medium (DMEM supplemented with 10 % FCS), and compared to cells cultured in either proprietary medium, DMEM containing lower serum (5 % FCS), or cells cultured in basal DMEM on cultureware coated with either fibrillar or monomeric collagen. Expression of mRNA for smoothelin and caldesmon, determined by qPCR, was significantly upregulated in cells cultured on monomeric collagen, compared to basal medium (indicated by the dashed line). Expression of myosin heavy chain and smooth muscle alpha-actin 2 was also enhanced in monomeric collagen coated cultures. Cells cultured on fibrillar collagen also exhibited a similar pattern of mRNA expression to monomeric collagen, although to a lesser extent. SMC grown in proprietary medium exhibited an mRNA marker profile consistent with a synthetic phenotype, compared to the basal DMEM medium. (*p < 0.05, ***p < 0.001, n = 5)
In sterile conditions, dilute stock type I rat tail collagen (Corning®—354236) to a concentration of 50 μg/ml in 0.02 N (0.02 M) acetic acid. Make enough of this diluted solution to cover the culture wells sufficiently (~40 μl/well in a 96-well tray; ~1 ml/well in a 6-well tray).
Add diluted collagen solution to cultureware and incubate for 1 h at room temperature.
After 1 h, remove collagen solution and rinse twice in sterile PBS.
The plates can be used immediately, or dried and stored at 4 °C until required.
Smooth muscle cells are subcultured into Lab-Tek slide wells and characterized after 48 h.
The culture medium is removed from the wells and cells gently washed three times with serum free culture medium before being fixed with ice-cold methanol (100 %) for 45 s, and then further washed three times with ice-cold PBS-A.
Cells are then incubated with a mouse monoclonal anti-smooth muscle α-actin antibody at 1:50 dilution with PBS-A for 60 min at room temperature. As a negative control, some cells are incubated with PBS-A only at this stage.
The primary antibody or PBS-A is then removed and cells washed three times with PBS-A and incubated for 5 min with normal rabbit serum at 1:20 dilution.
After a single wash with PBS, cells are then further incubated for 30 min at room temperature with FITC-conjugated rabbit anti-mouse IgG (Santa Cruz) diluted 1:50 in PBS-A.
Finally cells are washed three times with PBS-A and viewed under a microscope equipped for epifluorescence with appropriate filters for FITC.
Visualization of positive staining with this technique should reveal cells with a three-dimensional network of long, straight, and uninterrupted α-actin filaments running in parallel to the longer axis of the cells and an underlying row of parallel filaments along the smaller cell axis, with no cytoplasmic staining between filaments.
Although pericytes are related to vascular smooth muscle cells, pericytes can to a degree be distinguished by marker expression [25]. There is as yet, no specific molecular marker for pericytes, although, a number of markers that are commonly present in pericytes, albeit not exclusively, can be used for detection [26]. Commonly used markers include: desmin and α-smooth muscle actin, both contractile filaments; regulator of G protein signaling 5 (RGS-5), a GTPase-activating protein; neuron-glial 2 (NG2), a chondroitin sulfate proteoglycan; and platelet-derived growth factor receptor beta (PDGFRβ), a tyrosine-kinase receptor [3, 25, 26, 27]. In a similar manner to SMC phenotype determination, assessment of multiple markers would be required to give a positive presence of pericytes. It must be stressed that the markers mentioned above in cases cannot identify all pericytes and that expression can vary between species, which may lead to greater difficulty in true identification of pericytes. One example of this is α-smooth muscle actin, which is not expressed to an extent in skin or CNS pericytes under normal circumstances but is upregulated durin g retinopathy and in subcutaneously transplanted tumors [25].
Confluent SMC cultures should be detached from one 75 cm2 flask as described in Subheading 3.5.
Following centrifugation of the cell suspension for 5 min at 1000 rpm, the supernatant is aspirated and the cell pellet resuspended well in serum containing culture medium with an additional 10 % (v/v) dimethyl sulfoxide (DMSO) and transferred to a suitable cryovial.
To facilitate gradual freezing, the cryovial is then stored at 4 °C for 1 h, transferred to −20 °C for 1 h min and −70 °C for 1 h before being immersed into liquid nitrogen for long term storage. Alternatively, cryovials can be placed in a dedicated “freezing chamber” containing isopropanol that lowers their temperature by 1 °C per minute when placed directly in a −70 °C freezer.
To defrost cells, the cryovial should be rapidly warmed to room temperature by placing at 37 °C and the cell suspension transferred to a 25 cm2 culture flask. 20 ml of prewarmed serum containing culture medium is then added to the flask and the medium changed after 24 h.
Alternatively, on defrosting, the cells can be centrifuged at 50–100 × g for 5 min in serum containing medium. The supernatant is aspirated to remove the DMSO, the cell pellet resuspended well in prewarmed serum containing medium and then transferred into a 25 cm2 flask. Cells should be passaged once prior to use in experiments.
The three techniques for SMC isolation described yield cells with very different proliferative properties in culture. If larger quantities of SMC in culture are required, the explant isolation technique is recommended, although initially slower to yield cells. Enzymatic dispersion may provide more cells in the contractile phenotype, but the initial yield may be low and subcultures less readily proliferate through as many passages. Future studies are likely to address the isolation and characterization of stem cells which can differentiate into smooth muscle progenitor cells. It remains to be elucidated whether these progenitor cells participate in processes leading to angiogenesis and the pathogenesis of vascular diseases. A better understanding of vascular SMC and pericyte biology can be achieved when cells are cultured under physiological conditions that encompass biomechanical forces (e.g. fluid shear stress and stretch) and oxygen levels (e.g. 1−10 kPa) found in vivo of relevance to arterial and venous circulations and the microcirculation in metabolically active tissues and tumours (seeNote 8 ). Elucidation of the molecular mechanisms underlying their phenotypic modulation is required to identify therapeutic strategies to target angiogenesis and treat cardiovascular diseases.
Other “smooth muscle cell optimized” proprietary culture media are commercially available such as Smooth Muscle Basal and Growth Media (Lonza, or PromoCell), which are based on MCDB131 medium. These media may help to promote more rapid outgrowth of cells from tissue explants, but it should be noted that they may contain components which could alter SMC function, such as insulin, basic fibroblast growth factor (bFGF) and hydrocortisone.
For maximal SMC yield and viability, cells should be isolated from as soon as possible after harvesting the vessels. This also reduces the risk of infections since tissues are often handled and excised under nonsterile conditions.
If the SMC yield and viability is low following enzyme dispersion, soybean trypsin inhibitor, at a final concentration of 0.1 mg/ml−1, can be added to the enzyme solution to inhibit the action of nonspecific proteases which may contaminate commercial elastase.
Should infections frequently occur following explantation, additional antibiotics and fungicides can be supplemented to the culture medium for the initial 24 h following isolation and then the medium replaced. Gentamycin (25 μg ml−1) and Amphotericin B (2 μg/ml−1) are commonly used and 5 ml aliquots of these can be stored at −20 °C as 2.5 mg/ml−1 and 0.2 mg/ml−1 stocks respectively.
When the explanted tissue i s removed, SMC can also be detached by trypsinization and redistributed evenly in the same flask as described in Subheading 3.4. This is advisable if cells have grown in a very dense pattern around the explants and will facilitate obtaining the confluent monolayer of cells.
The trypsin–EDTA solution should be prewarmed to 37 °C only immediately prior to use and not left in a heated water bath for extended periods to prevent loss of activity. Cells should not need incubation with trypsin–EDTA at 37 °C for longer than 5–7 min to detach from the flask and this may indicate that a fresh solution should be prepared.
Coating of culture vessels with monomeric collagen may provide a physiologically relevant matrix for supporting SMC growth, as well as, maintenance of a cell phenotype of relevance to the experimental model.
Long term adaptation and culture of vascular smooth muscle cells and pericytes under physiological oxygen tensions of relevance to tissue conditions in vivo can be achieved using a workstation with temperature, humidity, oxygen and carbon dioxide control (e.g. SCI-tive, Baker Ruskinn, Bridgend, Wales, UK).
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