Hello.
I am interested in performing co-culture in 100 mm dish with transwells (75 mm) with endothelial cells and AoSMC. any advice how to perform it. after the treatment i would like also to scrape the cells in the bottom of the petri and onto the transwell membrane for collecting protein; especially for the second case. is possible? also what do you suggest? putting EC on the transwell and SMC on the petri dish or the contrary.
thank you in advance
Why do you want to co-culture? A cleaner method to modulate activity is to use conditioned medium from the alternate cell type and then look at function. There are always problems associated with co-culturing cell types, unless you use cell types from different species, so you know which species is doing what. You would stain cells with species-specific antibodies, but before hand you need to make sure the antibodies do not cross react and stain the cells from both species.
Check out this paper. Different type of vascular smooth muscle cells but the method should work for you as well.
Am J Physiol Lung Cell Mol Physiol. 2011 Oct;301(4):L527-35. doi: 10.1152/ajplung.00091.2011. Epub 2011 Jul 29.
Myoendothelial gap junctional signaling induces differentiation of pulmonary arterial smooth muscle cells.
Gairhe S1, Bauer NN, Gebb SA, McMurtry IF.
Author information
Abstract
Myoendothelial gap junctions are involved in regulating systemic arterial smooth muscle cell phenotype and function, but their role in the regulation of pulmonary arterial smooth muscle cell (PASMC) phenotype is unknown. We therefore investigated in cocultured pulmonary arterial endothelial cells (PAECs) and PASMCs whether myoendothelial gap junctional signaling played a role in PAEC-dependent regulation of PASMC phenotype. Rat PAECs and PASMCs were cocultured on opposite sides of a porous Transwell membrane that permitted formation of heterotypic cell-cell contacts. Immunostaining showed expression of the gap junctional protein connexin 43 (Cx43) on projections extending into the membrane from both cell types. Dye transfer exhibited functional gap junctional communication from PAECs to PASMCs. PASMCs cocultured with PAECs had a more contractile-like phenotype (spindle shape and increased expression of the contractile proteins myosin heavy chain, H1-calponin, and α-smooth muscle cell-actin) than PASMCs cocultured with PASMCs or cocultured without direct contact with PAECs. Transforming growth factor (TGF)-β1 signaling was activated in PASMCs cocultured with PAECs, and the PASMC differentiation was inhibited by TGF-β type I receptor blockade. Inhibition of gap junctional communication pharmacologically or by knock down of Cx43 in PAECs blocked TGF-β signaling and PASMC differentiation. These results implicate myoendothelial gap junctions as a gateway for PAEC-derived signals required for maintaining TGF-β-dependent PASMC differentiation. This study identifies an alternative pathway to paracrine signaling to convey regulatory signals from PAECs to PASMCs and raises the possibility that dysregulation of this direct interaction is involved in the pathogenesis of hypertensive pulmonary vascular remodeling.
You may want to check work from the Isakson lab at UVA, they have extensive experience in this area.
The method for coculture is here:
https://www.cvrc.virginia.edu/Isakson/methods.html
And the method for collecting proteins from each side of the transwell, and then from the transwell itself is published in this paper:
http://www.ncbi.nlm.nih.gov/pubmed/20133900
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