Adhesive strength-based, label free isolation of stem cells in regenerative medicine

1. Low adhering derivatives Undifferentiated stem cells Heterogeneous population Adhesion strength Stem cells Adhesive strength-based, label free isolation of stem cells in regenerative medicine High adhering derivatives Efraín A. Cermeño1,2, Ankur Singh4, Todd McDevitt2,3, Andrés J. García1,2 1Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA; 2Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta ,GA; 3Wallace H. Coulter Dept. of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA; 4Sibley School of Mechanical & Aerospace Engineering, Cornell University, Ithaca, NY • Background Adhesion Strength Measurements Results • A • STEM CELLS • Have the ability to self renew and differentiate • Potential uses intissue repair, drug discovery, and disease modeling • Hard to culture and maintain pure populations • Fig. 1: Adhesion of human induced pluripotent stem cells (hiPSCs) undergoing reprogramming and differentiation.(A, B) Adhesion strength of the indicated cell types on fibronectin (FN) and laminin (LM) (A) and of cells during reprogramming (B). Adhesion strength for undifferentiated (UD) and spontaneously differentiating (SD) cultures of hiPSCs and hESCs on FN or LM. • C • A • B • B • C • E • D • REGENERATIVE MEDICINE • Aims to re-establish normal function of tissues • Utilizes stem cells to regenerate damaged tissues • Eliminates problems such as immune rejection associated with current approaches • Fig. 2: Adhesion strength of different cell populations. Cells were grown in on matrigel coated coverslips. After 24hrs, spinning disk experiments were performed and the adhesion strength measured • Fig. 4: Adhesion strength–based enrichment of hiPSCs from differentiating cultures. (A,B)Flow cytometry histograms (Alexa Fluor 488–TRA-1-60) showing purification (A) and survival efficiencies (B) of hiPSCs processed as indicated. (C) Enrichment efficiency of hiPSCs upon repeated passaging with the indicated methods. P0 cells for all plots were from the same batch with 90% TRA-1-60+ cells; recovered cultures were propagated for 5–6 d. (D,E)Cell survival (D) and growth curves (E) of cells on Matrigel after passaging as indicated. D, day. • HYPOTHESIS • Different cell types will have unique adhesion signatures that can be exploited for cell purification • OBJECTIVE • Utilize a microfluidic system to purify stem cell populations based on their adhesion strengths Results • A • Fig. 3: Adhesion strength–based isolation of pluripotent stem cells in microfluidic devices. (A,B) Selective isolation of hiPSCs at a shear stress of 85–125 dynes cm−2 when cocultured with IMR90 cells at low (A) and high (B) density. (C) Heterogeneous reprogramming culture seeded into a μSHEAR device and subjected to a shear stress of 100 dynes cm−2 for 5 min. The white arrowheads indicate a hiPSC colony that is detached by flow. The red arrowheads indicate IMR90 fibroblasts. Scale bars, 200 μm. (D,E) Enrichment of hiPSCs and hESCs isolated at 85–125 dynes cm−2 from a coculture with IMR90 and mouse embryonic fibroblast cells, respectively. Graphs show mean ± s.d. (*P < 0.05, n = 3). Conclusions • B • Different cell populations have distinct adhesive signatures • The μSHEAR microfluidic system is capable of purifying cell populations based on their adhesion strength signatures • μSHEAR based purification of hiPSCs and hESCs is fast and resulted in high yields, purity, and survival μSHEAR Microfluidic Platform • C Differentiation Acknowledgements Adhesion strength change • E • D This work has been supported by: NSF Stem Cell Biomanufacturing IGERT NIH grant 5R43NS080407 Ti:GER Program • Undifferentiated stem cells have a unique adhesive signature that changes when they undergo differentiation. Devices were sterilized, coated with a matrix and heterogeneous populations of cells were then introduced. After 24 hours, cells were exposed to fluid flow at predetermined PBS flow rates. Recovered cell/colonies were either stained and quantified by flow cytometry or plated on Matrigel-coated plates containing media and evaluated after 24hrs References Singh, Ankur. Nature Methods (2013)