Passive Mechanical Properties of Polymer-Coated Cells for High Speed Sorting Using Antigen Specific Lysis (ASL)

ASL is a new cell sorting technique that sorts cells by coating the target cells with a temporary cross-linked polymer, and then exposing the entire cell population to harsh conditions to lyse the uncoated cells. Purity of ASL with surfactant based lysis exceeds 95% while the batch process takes 1h. We seek to eliminate damaging surfactant residues by using osmotic lysis. The objective of this study is to relate the mechanical properties of polymer coatings to the prevention of osmotic lysis.

Methods and Materials

Micropipette aspiration technique was used to test the cortical tension of both uncoated Jurkat cells and polymer-coated Jurkat cells. The data was analyzed using Laplaceâ??s Law and the calculated cortical tension is compared. The hypothesis is that by providing a greater tensile reinforcement to the cells, high modulus coating yield more live cells than low modulus coating during a hypotonic treatment.

Cells were prepared at a concentration of ~500,000 cells/mL, they were washed twice with PBS and then suspended in 1mM EDTA and 0.5% FBS. Then the cell solution was placed in a chamber on the microscope stage, a glass micropipette prefilled with PBS is used to aspirate cells. The pressure is created by two water reservoirs connected to the pipette and is measured by a pressure transducer.

Jurkat cells were polymer coated by first labeling the cells with photoinitiator (eosin) through standard immunolabeling against CD45 with eosin probes. Cells were then suspended in a pH 7.4 phosphate buffered solution of PEG diacrylate (Mn 575, 3500, or 8000), triethanol amine, and vinyl pyrollidinone. Polymerization commenced with irradiation (20 mW/cm2, 10 minutes, 530 nm LED lamp). Upon rinsing, the polymer coated cells were suspended in the same solution as the uncoated cells and were then tested.

Tension values were calibrated using the neutrophilsâ?? cortical tension is ~15±3 pN/µm, which falls in the lower end of literature values.

Results/Discussion

The cortical tension of Jurkat cells was tested and was found to be ~20±4 pN/µm. Upon coating with a PEG (Mn 575) diacrylate coating, the cortical tension increased to 700±49 pN/µm; upon coating with a PEG (Mn 3500) diacrylate coating, the cortical tension increased to 2917±274 pN/µm. Cells protected with both PEG diacrylate coatings resisted swelling in pure deionized water and retained viability.

Conclusion

The polymer coating prevents cells from osmotic lysis by providing tensile reinforcement. By finding the correlation between tensile reinforcement and polymer type and thickness, we can potentially control the mechanical properties of the cells by changing the coating and thus potentially be able to sort cells more effectively, which has great potential for future clinical applications.