(136e) Characterization of Microfluidic Devices for Cell Separation Via Adhesion to Peptide-Functionalized Surfaces

INTRODUCTION AND BACKGROUND

Over 80 million people suffer from cardiovascular disease, with over 36 percent of all deaths in
North America today citing CVD as a cause ¹. Tissue engineering and cell injection strategies are attractive methods of regenerating or repairing ailing hearts but both research fields suffer from the limitation of selecting an ideal cell source. Recent research has identified the presence of resident cardiac progenitors in the adult heart. These rare cells represent a potential autologous, expandable source of cardiac cells if they can be isolated in high enough concentrations. To that end we propose the use of alginate-peptide hydrogels to render glass surfaces selectively adherent to various cell types in the neonatal rat heart model. In this study we modify alginate with the tetra-peptides, REDV (arg-glu-asp-val), VAPG (val-ala-pro-gly) and RGDS (arg-gly-asp-ser), which have been found to capture endothelial cells, smooth muscle cells and fibroblasts respectively ^2,3. Using this method we aim to sequentially deplete a heterogeneous heart isolate via surface adherence of cells, and enrich the cell suspension in non-adherent cardiac progenitor cells. Our goal is to characterize these novel surfaces for use in the clinical setting in the form of low-cost, easily fabricated microdevices to isolate cells from patient biopsies for further expansion.

MATERIALS AND METHODS

Alginate modification:

The peptides RGDS (American Peptides), REDV (American Peptides) and VAPG (Sigma) were attached to a low viscosity sodium salt of alginate (Sigma A2158) using EDC chemistry (EDC, S-NHS, Pierce Biotechnology). Alginate at 18.75mg/ml in MES buffer was activated by EDC for 2 hours and then reacted with the peptides at various concentrations for 20 hours. Following the 20-hour reaction with the peptides, the alginate was then dialysed for two days (3500 MWCO) and then lyophilized.

Surface modification:

12mm diameter circular glass coverslips were plasma treated and then modified alginate was immediately pipetted onto the cleaned surfaces. Coverslips were then placed into a spin coater to create a thin, uniform layer of alginate on the surfaces. The coverslips were removed from the spin coater and placed into 1mM CaCl₂ to gel. Gels were incubated at 4°C overnight before use.

Cell seeding:

Cells were isolated from 1-2 day old Sprague-Dawley rats as described previously ⁴ and a 60-minute preplating step was used to remove most of the non-adherent cardiomyocytes. The adhered preplated cells were expanded for a week and then trypsinized (a step which also kills cardiomyocytes). The alginate-modified surfaces were washed in sterile PBS containing calcium ions and preplate cells were resuspended in serum-free culture medium and carefully pipetted onto the coverslips. Unmodified alginate-coated surfaces as well as bare glass surfaces were used as controls. The seeded coverslips were incubated at 37°C for 45 minutes and then non-adherent cells were gently removed and culture medium added. Adherent cells were imaged at random regions of the surfaces and counted using image analysis software.

RESULTS AND DISCUSSION

One area of concern with this strategy is prevention of non-specific cell adhesion on the surfaces. The presence of the alginate between the exposed peptides on the hydrogel surface should prove non-adhesive to cells, allowing us to evaluate the efficacy of the peptide groups at cell capture. The total number of cells was counted and averaged over several surfaces, and the modified alginate surfaces compared to the unmodified alginate surfaces at the low end, and to the bare glass surfaces at the high end of cell adhesion.

Figure 1: Brightfield images of representative regions of coated surfaces after cell seeding and incubation

Figure 2: Cell counts of total adhered cells averaged over surface type. Peptide concentration on modified alginate surfaces is 0.6mg/ml.

At a peptide concentration of 0.6mg/ml, the RGDS surfaces showed an increased level of cell adhesion, almost comparable to bare glass levels. REDV surfaces did not show significantly greater adhesion than unmodified alginate surfaces at this concentration while VAPG surfaces showed only a slight improvement. It is to be noted that the preplate cell suspension being seeded on the surfaces is homogeneous, consisting of mainly fibroblasts, with a small percentage of endothelial and smooth muscle cells. It is to be expected then, that lower cell numbers may be seen on the surfaces specific to capturing ECs and SMCs.

A major advantage of the alginate system is that the hydrogel can be easily dissoluted using a chelating agent such as EDTA, allowing for release of any captured cells. These cells can then be cytospun onto slides and immunostained for specific endothelial, fibroblast and smooth muscle markers, such as CD31, prolyl-4-hydroxylase and alpha-smooth muscle actin respectively, to identify the captured cell types. In our ongoing studies we use these techniques to assess the effectiveness and specificity of each hydrogel at various peptide concentrations. Finally, we hope to use each surface type to serially deplete a cell suspension of ECs, SMCs and lastly FBs and characterize the cells present in the final output.

REFERENCES

1.       The American Heart Association Heart Disease and Stroke Statistics -- 2008 Update

2.       Plouffe, B. D., et al., Peptide mediated selective adhesion of smooth muscle and endothelial cells in microfluidic shear flow, Langmuir. 2007 Apr 24;23(9):5050-5.

3.       Plouffe, B. D., et al., Microfluidic depletion of endothelial cells, smooth muscle cells, and fibroblasts from heterogeneous suspensions, Lab Chip. 2008 Mar;8(3):462-72.

4.       Radisic, M. et al., Medium perfusion enables engineering of compact and contractile cardiac tissue, Am J Physiol Heart Circ Physiol. 2004 Feb;286(2):H507-16

ACKNOWLEDGEMENTS

This work was supported by grants from NSERC (DG), CFI (LOF), ORDCF (ARTEC) and
University of
Toronto Open Fellowships to HTHA and MFC.