Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Summary

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Abstract

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Introduction

Delivery of macromolecules to the cell cytoplasm is a critical step in therapeutic and research applications. Nanoparticle mediated delivery, for example, has shown potential in gene therapy^1,2, while protein delivery is a promising means of affecting cellular function in both clinical³ and laboratory⁴ settings. Other materials, such as small molecule drugs, quantum dots, or gold nanoparticles, are of interest in applications ranging from cancer therapeutics^5,6 to intracellular labeling^7,8, and single molecule tracking⁹.

The cell membrane is largely impermeable to macromolecu....

Protocol

1. Storage

1. Store the reservoirs, holders, O-rings and microfluidic devices in 70% ethanol. Use a container (e.g. jar or beaker) that has a lid to prevent evaporation and contamination by dust or outside particles. Place the devices in one container (1), reservoirs and O-rings in a second container (2), and holders in the third (3).

Note: The use of 70% ethanol for storage is to maintain sterility. If the only components of the solution are ethanol and water (i.e. no denaturing agents), all system components should be fully compatible and will not degrade over time.

2. Change ethanol ....

Results

Figure 1 contains a descriptive schematic of the microfluidic delivery system. Figures 2a-b illustrate typical results from treating HeLa cells with different device designs in the presence of fluorescently conjugated 3 kDa dextran²⁰. If the procedure is followed correctly, system performance will be sensitive to device type and operating speed. Therefore, one should optimize these conditions for a given application before proceeding to more complex experiments. In the range o.......

Discussion

Certain aspects of the described experimental procedure (i.e. factors other than chip design and operating speed) may need to be optimized depending on the cell type and delivery material the system is applied to. The discussion that follows addresses some of the most common factors to consider when designing experiments.

To improve the delivery signal for fluorescently labeled compounds, one needs to address sources of background fluorescence. Surface binding and endocytosis, for exa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Device Holder & Plastic reservoir	SQZ Biotechnologies	Holder
LSRFortessa Analyzer	Becton Dickinson	N/A	Flow cytometry machine used at the Koch Institute Core Facilities
Microfluidic device	SQZ Biotechnologies	Cell Squeeze
O-Rings	McMaster	9452K311
Pressure system to operate device	SQZ Biotechnologies	Pressure System
Tweezers
Ultrasound bath