Localized Induction of Gene Expression in Embryonic Stem Cell Aggregates Using Holographic Optical Tweezers to Create Biochemical Gradients

Glen R Kirkham,Thomas Upton,Stephanie Allen,Kevin M Shakesheff,James Ware,Lee Dk Buttery

doi:10.1007/s40883-019-00114-5

Abstract

Three-dimensional (3D) cell models that mimic the structure and function of native tissues are enabling more detailed study of physiological and pathological mechanisms in vitro. We have previously demonstrated the ability to build and manipulate 3D multicellular microscopic structures using holographic optical tweezers (HOTs). Here, we show the construction of a precisely patterned 3D microenvironment and biochemical gradient model consisting of mouse embryoid bodies (mEBs) and polymer microparticles loaded with retinoic acid (RA), embedded in a hydrogel. We demonstrate discrete, zonal expression of the RA-inducible protein Stra8 within mEBs in response to release of RA from polymer microparticles, corresponding directly to the defined 3D positioning of the microparticles using HOTs. These results demonstrate the ability of this technology to create chemical microgradients at definable length scales and to elicit, with fidelity and precision, specific biological responses. This technique can be used in the study of in vitro microenvironments to enable new insights on 3D cell models, their cellular assembly, and the delivery of drug or biochemical molecules for engineering and interrogation of functional and morphogenic responses.Graphical abstract

Highlights

Our ability to understand the complex biology and physiology of cells and tissues is being advanced through innovative approaches to reproduce their multicellular three-dimensional (3D) interactions, structure, and function in vitro
Using an instrument called holographic optical tweezers, we have shown how a nondamaging light source can literally work as a pair of microscopic tweezers
The embryonic stem (ES) cells were serum starved for 24 h, and this data indicates that there were no deleterious effects on embryoid bodies (EBs) formation or stimulated by RA gene 8 (Stra8) gene expression and background Stra8 expression remains negligible

Summary

Introduction

Our ability to understand the complex biology and physiology of cells and tissues is being advanced through innovative approaches to reproduce their multicellular three-dimensional (3D) interactions, structure, and function in vitro. The level of precision and accuracy of control of cell interactions and delivery of bio-instructive signals that can be achieved is varied among these technologies, and the ability to work interactively and precisely at the scale of individual cells and their microenvironment is a particular challenge [9]. Meeting this challenge, we have established the use of holographic optical tweezers to enable simultaneous imaging and micromanipulation of multiple cells, as well polymer microparticles within 3D culture environments [10]. These can be precisely positioned and assembled, within a matter of minutes, into predetermined 3D microtissue structures, with accuracy of control from micron- to millimeterlength scales (10)

Results

Discussion

Conclusion