Considering the different species and preparation, to isolate pulmonary artery smooth muscle cells from rat, I was using a different mixture of enzymes, consisting of:

In 2ml of HEPES (no Ca2+, no EGTA and 1g/L glucose):

·         4mg of colagenase Type XI     (0.2% w/v)

·         2mg of papaine                       (0.1% w/v)

·         2mg of trypsin inhibitor         (0.1% w/v)

·         20µl of DTT 100mM stock      (1 mM)

The numbers in brackets are final concentration in the solution.

Maybe this combination, adjusting the volumes because I was departing from a much lower amount of tissue, would help you to get rid of the endothelial cells.

Good luck

I obtained abundant and pure VSMCs following our protocol!

can follow our publications,

A Novel Smooth Muscle-specific Enhancer Regulates Transcription of the Smooth Muscle Myosin Heavy Chain Gene in Vascular Smooth Muscle Cells, JBC.

http://www.jbc.org/content/270/52/30949.long

Somasundaram C et al.

http://www.bio.unipd.it/bam/PDF/6-1/art.31-36.pdf

Primary VSMC cultures were begun from the medial smooth muscle cell layers of adult male rabbit (2 kg) aorta. Isolation of the thoracic aorta was followed by direct mechanical stripping of transverse layers of medial smooth muscle including endothelium. Strips (Graphic1 mm) of tissue were then incubated in 4 ml per aorta of 0.25% Trypsin for 10 min at 37°C. Endothelial cells were removed by incubation of strips with 5 ml of collagenase (2 mg/ml) in M199 medium for 60 min at 37°C in a CO2 incubator. Smooth muscle cells were dispersed by incubation with 2 ml of elastase (0.25 mg/ml) for 60 min at 37°C, followed by addition of 5 ml of collagenase and DNaseI to a final concentration of 2.5 μg/ml for a further 1-2 h. Smooth muscle cells were resuspended in M199 medium containing 10% fetal calf serum (FCS), 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml fungizone. Cells were counted by trypan blue exclusion and routinely showed >95% viability. Cells were seeded at 1 × 104/cm2 and allowed to attach, and the medium was changed after 48 h. Cells were occasionally checked for endothelial cells and were routinely negative when stained for von Willebrand antigen. Cells were split 1:3 during passaging and were used at passage number 2 unless otherwise stated.

Thank you Jesus and Chandra. I will follow your instructions and let you know.

You are welcome, Neha!

Hi Neha could please share the picture of endothelial cells you are getting. 

Anjali: I don't have the pictures. If you have the protocol to obtain purified smooth muscle cells or to purify the smooth muscle cells from a mixture of smooth muscle and endothelial, kindly share it with me.