Label-free cell sorting strategies via biophysical and biochemical gradients

Abstract

Isolating active mesenchymal stem cells from a heterogeneous population is an essential step that determines the efficacy of stem cell therapy such as for osteoarthritis. Nowadays, the gold standard of cell sorting, fluorescence-activated cell sorting, relies on labelling surface markers via antibody-antigen reaction. However, sorting stem cells with high stemness usually requires the labelling of multiple biomarkers. Moreover, the labelling process is costly, and the high operating pressure is harmful to cell functionality and viability. Although label-free cell sorting, based on physical characteristics, has gained increasing interest in the past decades, it has not shown the ability to eliminate stem cells with low stemness. Cell motility, as a novel sorting marker, is hence proposed for label-free sorting active stem cells. Accumulating evidence has demonstrated the feasibility in manipulating directional cell migration through patterning the biophysical, biochemical or both gradients of the extracellular matrix. However, applying those findings to label-free cell sorting has not been well discussed and studied. This review thus first provides a brief overview about the effect of biophysical and biochemical gradients of the extracellular matrix on cell migration. State-of-the-art fabrication techniques for generating such gradients of hydrogels are then introduced. Among current research, the authors suggest that hydrogels with dual-gradients of biochemistry and biophysics are potential tools for accurate label-free cell sorting with satisfactory selectivity and efficiency.

Translational potential of this article: The reviewed label-free cell sorting approaches enable us to isolate active cell for cytotherapy. The proposed system can be further modified for single-cell analysis and drug screening.

Keywords: Cell sorting; Gradients; Hydrogels; Stem cells.

Figure 1

(A) Gradients induced by automatic diffusion ; (B) Gradients induced by dynamic mixture ; (C) Gradients induced by stationary greyscale mask /sliding greyscale mask ; (D) Gradients controlled by current ; (E) Gradients controlled by temperature gradients ; (F) Gradients induced by shape ; (G) Gradients induced by compression-relaxation ; (H) Gradients induced by interstitial flow. P1≠P0 and the stress gradient was applied on the gel (yellow part) . (I) Gradients controlled by microfluidics ; (J) Gradients built by 3D printing . VGEF = vascular endothelial growth factor; PEG = polyethylene glycol; TMSPMA = 3-(Trimethoxysilyl)propyl methacrylate; GelMA = gelatin methacryloyl.

Figure 2

(A) Two Distinct stimulating light to generate dual gradients (B) Overlaying the second gradient on the first gradient. SMCC = sulfo-sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; DTT = dithiothreitol; NVP = N-vinylpyrrolidone.

Figure 3

Design of the dual gradients hydrogel scaffold.