Optical reprogramming of human cells in an ultrashort femtosecond laser microfluidic transfection platform

Abstract

Induced pluripotent stem cell (iPS cell) technology can be used to produce unlimited numbers of functional cells for both research and therapeutic purposes without ethical controversy. Typically, viruses are applied for efficient intracellular delivery of genes/transcription factors to generate iPS cells. However, the viral genomic integration may cause a risk of mutation as well as tumor formation therefore limits its clinical application. Here we demonstrate that spatially shaped extreme ultrashort laser pulses of sub-20 femtoseconds induce transient membrane permeabilisation which enables contamination-free transfection of cells in a microfluidic tube with multiple genes at the individual cell level in order to achieve optical reprogramming of large cell populations. We found that the ultrashort femtosecond laser-microfluidic cell transfection platform enhanced the efficacy of iPS-like colony-forming following merely a single transfection. Illustration of the spatially shaped femtosecond laser-assisted microfluidic cell transfection platform for production of iPS cell colonies.

Materials for Improving Immune Cell Transfection

“…Microfluidics refers to the manipulation of fluids at micro‐scale and such technology has been used extensively in cell processing and diagnostics. [ 63 ] A large number of microfluidic platforms have been created for transfection with kinds of mechanisms involving microinjection, [ 64 ] cell squeezing, [ 65 ] shear stresses, [ 66 ] vortex shedding, [ 67 ] deterministic mechanoporation, [ 68 ] compressive forces, [ 69 ] laser, [ 70 ] and electrical fields. [ 71 ] Microfluidic technology has also been combined with biochemical polymers to enhance transfection efficiency.…”

Recent Advances in Microfluidic Platforms for Programming Cell‐Based Living Materials

“…Various physical strategies have been reported to generate transient nanopores on the cell membranes. [ 151–158 ] For example, Chung and co‐workers developed a hydroporation strategy to induce rapid cell deformation using fluid inertia in microfluidic platforms with cross‐ or T‐shaped channels. [ 151–153 ] Figure 5a shows the most recent design of microfluidic hydroporation.…”

Xeno- and transgene-free reprogramming of mesenchymal stem cells toward the cells expressing neural markers using exosome treatments

“…However, the use of c-Myc and viral transfection prevent the use of iPS cells in the clinic. Another approach used a plasmid with the Epstein-Barr virus (EBV) promotor to induce transcription factors Oct4, Sox2, Klf4, c-Myc, and Lin28 [7], which did not require the use of a virus, but the use of tumorigenic genes still pose problems. Evidence suggests that the different origins of the material cells, as well as the various combinations of transcription factors, affect the efficiency of the reprogramming and cell conversion [31].…”

“…However, this technology is still worth using for modeling diseases and drug screenings in vitro. To overcome the issues, many researchers attempted to create iPS cells in a safer and faster manner through various methods [5][6][7][8][9].…”