A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Summary

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Abstract

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (≥10⁵) in milliliter-scale volumes (≥0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 μl). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

Introduction

The study of cells maintained in culture (in vitro analysis) has provided invaluable insight into the fundamental principles and molecular mechanisms governing complex biological systems and human health. The conventional methods for culture, stimulation, and collection of cells for analysis, which utilize Petri dishes and microtiter plates, were designed for study of large populations of cells (≥10⁵) in milliliter-scale culture volumes (≥0.1 ml). However, there are many instances in which only limited quantities of cells are available (e.g. primary cells), or small populations of cells are desirable (e.g. to reduce cell-to-....

Protocol

This general protocol is designed to support application of the platform to a wide variety of studies; aspects specific to the representative study described in this report are separated out in brackets. Figure 2 illustrates a representative study carried out using the protocol. Note that in the protocol, all "instruct" commands are automated through use of pre-determined scripts. Note too that Step 2 can be carried out in parallel with Step 1 (e.g. during Steps 1.7 and 1.8), and that Step 2.1 is often bypassed (because patterned/coated DMF device plates can be washed and re-used; see Step 5.2).

1. Assemble and Popul....

Results

As a demonstration of the automated platform, we used it to carry out a study in which small populations of immune cells were grown in micro-scale cultures, challenged with bacteria, and lysed for off-platform analysis of pro-inflammatory responses (Figure 2).

Each of six cell perfusion chambers was seeded with 10⁴ immune cells (P388D.1 murine macrophages) resuspended in 10 μl of growth medium. After an adherence period (37 °C for 2 hr) and a medium exchange, ~500 c.......

Discussion

We have developed a simple and versatile automated platform for micro-scale cell culture and stimulation experiments. The platform enables us to work with small culture volumes and cell populations (1 - 20 μl and 100 - 2,000 cells, per chamber); culture sizes could be further reduced through use of microcapillaries of smaller diameter. Working at these scales reduces the cost of routine studies and makes feasible studies that require use of precious reagents and/or cells. Platform-executed experiments also sh.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Prelude Direct Lysis Module	NuGEN	1400-24
Trypan Blue (0.4% w/v)	GIBCO	15250-061
Cell Stripper	Cellgro	25-056-C1
Ovation PicoSL WTA	NuGEN	3310-048
Agencourt RNAClean XP	Beckman Coulter Genomics	A63987
pHrodo BioParticles	Invitrogen	P35361
CCL4 TaqMan qRT-PCR assay	Applied Biosystems	Mm00443111_m1
CCL5 TaqMan qRT-PCR assay	Applied Biosystems	Mm01302428_m1
PTGS2 TaqMan qRT-PCR assay	Applied Biosystems	Mm00478374_m1
TNF TaqMan qRT-PCR assay	Applied Biosystems	Mm00443258_m1
GAPDH TaqMan qRT-PCR assay	Applied Biosystems	Mm99999915_g1
Pluronic F127	Sigma Chemical	2594628
Fluorinert FC-40	Sigma Chemical	51142-49-5
Parylene C dimer	Specialty Coating Systems	28804-46-8
Teflon-AF	DuPont	AF1600
Polyimide tape	ULINE	S-11928
Indium tin oxide (ITO) coated glass substrates	Delta Technologies	CB-40IN-1107
DMF hub Teflon-coated fused-silica microcapillaries	Polymicro Technologies	TSU100375
Perfusion chamber microcapillaries	Polymicro Technologies	TSP530700
Tubing and microcapillary fittings	Sandia National Laboratories
Polycarbonate tubing	Paradigm Optics	CTPC100-500-5
8-port precision syringe pump equipped with 30 mm (500 μl capacity) syringes	Hamilton Company	54848-01
Parylene-C vapor deposition instrument	Specialty Coating Systems	PDS 2010 Labcoter 2
High-voltage function generator	Trek	615A-1 615-3
MVX10 microscope	Olympus		Optional (facilitates tracking of droplets on DMF hub)
QIClick digital CCD camera	QImaging	QIClick-F-CLR-12	Optional (facilitates tracking of droplets on DMF hub)