Single cell-laden protease-sensitive microniches for long-term culture in 3D.
Single cell-laden three-dimensional (3D) microgels that can serve to mimic stem cell niches in vitro, and are therefore termed microniches, can be efficiently fabricated by droplet-based microfluidics. In this technique an aqueous polymer solution along with a highly diluted cell solution is injected into a microfluidic device to create monodisperse pre-microgel droplets that are then solidified by a polymer crosslinking reaction to obtain monodisperse single cell-laden microniches. However, problems limiting this approach studying the fate of single cells include Poisson encapsulation statistics that result in mostly empty microniches, and cells egressing from the microniches during subsequent cell culture. Here, we present a strategy to bypass Poisson encapsulation statistics in synthetic microniches by selective crosslinking of only cell-laden pre-microgel droplets. Furthermore, we show that we can position cells in the center of the microniches, and that even in protease-sensitive microniches this greatly reduces cell egress. Collectively, we present the development of a versatile protocol that allows for unprecedented efficiency in creation of synthetic protease-sensitive microniches for probing single stem cell fate in 3D.
John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA. mooneyd@seas.harvard.edu and Wyss Institute for Biologically Inspired Engineering, Cambridge, MA 02138, USA.
Philipp S
0000-0002-0272-3988
PS
Lienemann
John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA. mooneyd@seas.harvard.edu and Wyss Institute for Biologically Inspired Engineering, Cambridge, MA 02138, USA.
Torsten
T
Rossow
John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA. mooneyd@seas.harvard.edu and Wyss Institute for Biologically Inspired Engineering, Cambridge, MA 02138, USA.
Angelo S
AS
Mao
Department of Obstetrics, University Hospital Zurich, University of Zurich, Schmelzbergstr. 12, 8091 Zurich, Switzerland.
Queralt
Q
Vallmajo-Martin
Department of Obstetrics, University Hospital Zurich, University of Zurich, Schmelzbergstr. 12, 8091 Zurich, Switzerland.
Martin
M
Ehrbar
John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA. mooneyd@seas.harvard.edu and Wyss Institute for Biologically Inspired Engineering, Cambridge, MA 02138, USA.
David J
DJ
Mooney
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D060833:Cellular Microenvironment / Q000502:physiology*; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D010447:Peptide Hydrolases; D059010:Single-Cell Analysis / Q000379:methods*
D010447:Peptide Hydrolases