from Small and Large Vessels

times in order to a successful primary culture. Concentrations and incubation times suggested here were optimized for murine aortas. 6. After the cells are attached and growing, one should proceed with the endothelial cell characterization to ensure that the cells obtained are positive for endothelial cell markers, such as platelet endothelial cell adhesion molecule (PECAM) and von

Willebrand’s factor (vWF). 7. To ensure that the phenotype of cells is maintained with passaging, it is important to characterize cells at all passages. At some point in passaging (usually around passage 6–7 in our experience), the phenotype changes and expression of receptors and ion channels change. We suggest that cells beyond this point of passage are not used for further experiments.

References

1. Mensah GA (2007) Healthy endothelium: the scientific basis for cardiovascular health promotion and chronic disease prevention. Vascul Pharmacol 46(5):310–314 2. Yetik-Anacak G, Catravas JD (2006) Nitric oxide and the endothelium: history and impact on cardiovascular disease. Vascul Pharmacol 45(5):268–276 3. Manconi F, Markham R, Fraser IS (2000)

Culturing endothelial cells of microvascular origin. Methods Cell Sci 22:89–99 4. Michiels C (2003) Endothelial cell functions.

J Cell Physiol 196:430–443 5. Sumpio BE, Riley JT, Dardik A (2002) Cells in focus: endothelial cell. Int J Biochem Cell Biol 34(12):1508–1512 6. Vita JA, Keaney JF (2002) Endothelial function: a barometer for cardiovascular risk?

Circulation 106:640–642 7. Furchgott RF, Zawadzki JV (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine.

Nature 288:373–376 8. Forstermann U, Munzel T (2006) Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 113:1708–1714 9. Busse R, Edwards G, Feletou M, Fleming I,

Vanhoutte PM, Weston AH (2002) EDHF: bringing the concepts together. Trends

Pharmacol Sci 3:374–380 10. Garlanda C, Dejana E (1997) Heterogeneity of endothelial cells specific markers. Arterioscler

Thromb Vasc Biol 17:1193–1202 11. Suh SH, Vennekens K, Manolopoulos VG,

Freichel M, Schweig U, Prenen J, Flockerzi V,

Droogmans G, Nilius B (1999) Characterisation of explanted endothelial cells from mouse aorta: electrophysiology and Ca2+ signalling.

Eur J Physiol 438:612–620

Chapter 27

Isolation and Culture of Vascular Smooth Muscle Cells from Small and Large Vessels

Augusto C. Montezano, Rheure A.M. Lopes, Karla B. Neves, Francisco Rios, and Rhian M. Touyz

Abstract

Primary culture of vascular smooth muscle cells is an important in vitro model for the dissection of molecular mechanisms related to a specific physiological or pathological response at the cellular level. Cultured cells also provide an excellent model to study cell biology. This chapter describes a user-friendly and practical protocol for isolation of vascular smooth muscle cells from small and large vessels by enzymatic dissociation, which can be applied to vessels from different species, including rodents and humans.

Key words Vascular smooth muscle cells, Primary culture, Small vessels, Large vessels

1 Introduction

Smooth muscle cells (SMC) can be cultured from several human tissues such as placenta, bladder, umbilical cord and vessels (VSMCs). VSMCs cells are the major component of arteries, veins, and microvessels and are major regulators of vascular function. Primary culture of VSMCs represents an in vitro model, which retain a high degree of plasticity and can be used to study responses that range from contraction to proliferative or secretory responses. Interestingly, the phenotype of VSMC is heterogeneous and varies according to the vessel size, vessels type (conduit versus resistance), age, species and vascular bed. The layer from where the cells derive can also impact on phenotype. For example, intimal VSMC presents lower levels of contractile proteins and more organelles such as mitochondria and sarcoplasmic reticulum and gene expression may vary in different cell layers [1–4].

VSMC can be easily isolated from aorta by the explant technique and are mainly useful to study responses related to atherosclerosis. However, this isolation protocol may be selective to a specific type of VSMCs, and may not represent the whole content

Rhian M. Touyz and Ernesto L. Schiffrin (eds.), Hypertension: Methods and Protocols, Methods in Molecular Biology, vol. 1527, DOI 10.1007/978-1-4939-6625-7_27, © Springer Science+Business Media LLC 2017 349

of a vessel or part of it. On the other hand, cells isolated from small-resistance arteries, such as mesenteric arteries isolated by enzymatic digestion, are suitable to verify events that regulate arterial blood pressure, since those vessels contribute to control of total peripheral vascular resistance [5].

VSMCs, that are freshly isolated and in primary culture, can be used for electrophysiology experiments at early days (from 5 to 7 days). It is important take into consideration that VSMC have a finite number of divisions, after which they lose their phenotype, undergo growth arrest and become senescent [1, 6]. These observations raise an important aspect of working with cultured VSMCs, where one should always confirm that the process of culturing VSMCs does not affect the expression of receptors, channels, transporters, enzymes, or any other protein important to the study. As such characterizing cells at different passages is important to ensure that cells do not undergo dedifferentiation with loss of receptors, channels etc.

Once isolated by enzymatic digestion, primary culture VSMCs retain the original phenotype, providing an important in vitro model to study disease-associated signaling pathways. For this reason, this chapter only concentrates on the isolation of VSMCs by enzymatic dissociation. This protocol has been demonstrated to not alter the expression of smooth muscle specific marker α-actin, and has many advantages as described above. In addition, the protocol described in this chapter is relatively fast, reducing risks of cell contamination by other resident cells from the vasculature and microorganisms. VSMCs isolated by enzymatic dissociation have been successfully used in many experimental approaches such as RT-PCR, northern blot, immunoblotting, live cell microscopy and imaging protocols (i.e., immunofluorescence).

2 Materials

1. Ham’s F12 nutrient mix (pH 7.2) containing: penicillin/ streptomycin (1×), HEPES (15 mM), l-glutamine (1 mM), sodium bicarbonate (14 mM). 2. Pre-digestion mix: 30 mL of complete Ham’s F12 nutrient mix containing 90 mg collagenase type I (≥125 U/mg) (see

Note 1). 3. Digestion mix for small vessels: 12.5 mL of complete Ham’s

F12 nutrient mix containing 25 mg of bovine serum albumin (BSA, 2 %), 25 mg of collagenase type I (≥125 U/mg), 1.5 mg of elastase (≥4 U/mg), 4.5 mg of soybean trypsin inhibitor (see Note 2). 4. Digestion mix for large vessels: 25 mL of complete Ham’s F12 nutrient mix containing 50 mg of BSA, 50 mg of collagenase

type I (≥125 U/mg), 3 mg of elastase (≥4 U/mg), 9 mg of soybean trypsin inhibitor (see Note 2). 5. Dulbecco’s Modified Eagle Medium (DMEM) (pH 7.2) containing: low d-glucose (5.5 mM), penicillin/streptomycin (1×),

HEPES (25 mM), l-glutamine (4 mM), sodium bicarbonate (44 mM), sodium pyruvate (1 mM), fetal bovine serum (10%). 6. 100 mm petri dishes. 7. Syringes and needles (20 G, 18 G). 8. 100 μm nylon filter. 9. 50 mL or 15 mL centrifuge tubes. 10. Cell culture flasks (25 or 75 cm2) or sterile coverslips inserted in a 6-well plate (see Note 3). 11. Surgical material: toothed forceps, straight fine forceps, angled fine forceps, small sharp scissors, microdissecting scissors.

3 Methods

3.1 VSMC Isolation from Small Vessels (i.e., Mesenteric Arteries from Rodents)

1. Isolate the whole mesenteric bed and place it in cold complete

Ham’s F12 nutrient mix (see Note 4). 2. Clean the mesenteric bed by carefully removing the fat with two fine forceps in a 100 mm petri dish containing Ham’s F12 nutrient mix. First, identify the main mesenteric artery and with one of the fine forceps hold the mesenteric bed. Once the mesenteric bed is stably secured, with the help of the second fine forceps, remove the fat from the artery tree by gently pulling it (see Note 1). 3. Preheat the digestion mix at 37 °C for 5 min. 4. Once cleaned, incubate the mesenteric bed in a centrifuge tube containing the digestion mix for small vessels for 30 min, at 37 °C and under agitation. The digestion is complete when the vessels appearance is similar to cotton. Do not digest the vessels to a point where it is not possible to see the artery tree. 5. Syringe the vascular bed through a 20 G needle four times in order to obtain a homogenized solution. 6. Filter cells and tissue debris through a 100 μm nylon filter, collecting the cell solution in a centrifuge tube. 7. Centrifuge the cell solution for 3 min at 300 × g. 8. After the centrifugation it may be possible to see a cell pellet (it will depend on the amount of tissue available for the culture and the efficacy of the previous steps). Aspirate the supernatant and resuspend the cell pellet in 5 mL of DMEM (as described in Subheading 2, item 5).

3.2 VSMC Isolation from Large Vessels (i.e., Aorta from Rodents or Humans)

9. Add the cell solution to a 25 cm2 flask and keep it in a humidified 37 °C/5 % CO2 incubator (see Note 5). 10. Replace the medium after 24 h to a fresh 5 ml DMEM (see

Note 6). 11. Keep changing the medium every 2 days until cells reach confluence and are ready for either further culture (passages) or the experimental protocol.

1. Isolate the whole vessel, remove the perivascular tissue and remaining blood, and place it in cold Ham’s F12 nutrient mix. 2. Incubate the large vessel in the pre-digestion mix for 15 min at 37 °C under agitation. This step will facilitate the removal of the adventia and intima layers. 3. In a 100 mm petri dish containing Ham’s F12 nutrient mix, cut the large vessel longitudinally and remove the endothelial cells (intima layer) by gently scrapping the inner surface of the vessel. 4. Under a dissection microscope, turn the vessel with the inner surface down. With a fine forceps remove the adventitia layer by gently peeling it off the vessel wall. This step is important to decrease the contamination of the primary culture with fibroblasts. 5. Preheat the digestion mix for large vessels at 37 °C for 5 min. 6. Cut the vessel in small pieces to facilitate digestion. Pieces should range in size between 2 and 5 mm (see Note 7). 7. Incubate in a centrifuge tube containing the digestion mix for large vessels for 60–90 min, at 37 °C and under agitation (see

Note 8). The digestion is complete when the vessel appearance is similar to cotton. Do not digest the vessel to a point where it is not possible to see it anymore (see Note 9). 8. Syringe the vascular bed through an 18 G needle (one time) and then through a 20 G needle (four times) in order to obtain a homogenized solution. 9. Filter cells and tissue debris through a 100 μm nylon filter, collecting the cell solution in a centrifuge tube. 10. Centrifuge the cell solution for 3 min at 300 × g. 11. After the centrifugation it may be possible to see a cell pellet (it will depend on the amount of tissue available for the culture and the efficacy of the previous steps). Aspirate the supernatant and resuspend the cell pellet in 5 mL of DMEM. 12. Add the cell solution to a 25 cm2 flask and keep it in a humidified 37 °C/5 % CO2 incubator (see Note 5). 13. Replace the medium after 24 h to a fresh 5 ml DMEM (see

Note 6).

14. Keep changing the medium every 2 days until cells reach confluence and are ready for either further culture (passages) or the experimental protocol.

4 Notes

1. The pre-digestion solution may be necessary to facilitate further cleaning of large vessels from rats and humans. Is also extremely useful for the cleaning of mesenteric beds from rats.

It is not recommended to perform the pre-digestion step in vessels from mice, due to these vessels being very fragile. If necessary, the concentration of collagenase type I suggested here must be decreased. 2. Concentrations of collagenase type I and elastase may vary accordingly to the material available. Large vessels from humans may require higher concentrations of both. Moreover, collagenase type II may be used as well [2, 3]. 3. The amount of material available for culture will determine if one will use a cell culture flask or a coverslip inserted in a 6-well plate, i.e., a pull of many vessels vs. a single vessel. If a coverslip is to be used, ensure that all instruments are properly sterile to avoid contamination. 4. Avoid damage of the intestine during the collection of the mesenteric bed, since this could be a major source of contamination.

This protocol can also be used for the extraction and culture of

VSMCs from the microvasculature of human material. 5. If one is culturing VSMCs from a single small vessel (usually the case for human material) or a single animal, the cell pellet should be resuspended in 500 μL of DMEM and transferred to the top of a sterile coverslip inserted in a 6-well plate. Cells will grow in the coverslip and migrate to the surface of the plate.

Once cells are found in the surface of the plate, one should transfer the coverslip to another well by using a sterile fine forceps, allowing the primary culture to expand. 6. Cells may take a couple of days to adapt to the culture and acquire the appropriate shape of a VSMC. One should be aware that VSMCs in culture can be heterogeneous and display differences in shape, size, and growth. 7. Pieces that are too small may lead to a poor cell population, as the same for bigger pieces of vascular tissue (increasing the digestion time and efficiency) 8. Digestion time may vary accordingly to the nature of the material.

Usually human large vessels require longer periods of digestion. 9. Cell viability will be affected by the digestion protocol.