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-cell variability across the population), or required reagents are difficult to obtain or prohibitively expensive (e.g. purified cell-secreted factors). Such issues can be successfully addressed by scaling down culture size, which has the added benefit of reducing consumption of all reagents required for in vitro analysis^1,2. Unfortunately, conventional equipment and methods do not support precise and reproducible manipulation of micro-scale cultures and the microfluidics-based automated systems currently available^3-11 are too complex and specialized for routine use by most laboratories.

In this report, we describe assembly and use of 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 architecture (Figure 1) is modular in design: a set of fibronectin-coated microcapillaries ("cell perfusion chambers" module) serves as the site for establishment, maintenance, and stimulation of micro-scale cultures; and a digital microfluidics (DMF)^12,13 device outfitted with "transfer" microcapillaries ("central hub" module)^14,15 routes cells and reagents to and from the perfusion chambers. DMF enables the user to individually address multiple droplets simultaneously and to change or re-order the manipulations (i.e. reconfigure sample processing trains) without altering the device hardware. Its tremendous flexibility is evident in its recent emergence as a key technology in a wide range of applications, including cell culture^16,17, enzyme assays^18,19, immunoassays^20,21, DNA analysis^22,23, protein processing,^24,25 and clinical specimen processing.^26,27 Our central hub takes advantage of the flexibility inherent to DMF devices, and further enhances it through the addition of microcapillary interfaces, which provides opportunity to carry out a subset of manipulations (e.g. cell culture) in specialized peripheral modules, rather than on the DMF device itself. Compartmentalization of processing trains in this way also simplifies design of the platform architecture (no need to build a DMF device that can carry out all processing steps) and facilitates its evolution as new functions are required (simply integrate new peripheral modules as necessary). Transport of cells and reagents within the central hub is driven by electrowetting forces generated by sequential activation of electrodes within the DMF device^13,28; transport to, from, and within the perfusion chambers is powered by pressure changes generated by a high-precision syringe pump. All of these fluid movements are controlled via a simple electronic interface and automated through use of pre-determined scripts.

As a representative example, we demonstrate the use of the platform for the study of transcriptional responses elicited in immune cells upon challenge with bacteria (Figure 2). Carrying out these experiments on the platform enabled us to work with small numbers of cells (~1,000 per experimental condition), minimize experiment-to-experiment variability, conserve reagents, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, this 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.

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 Populate the Cell Perfusion Chambers

1. Coat the interior surfaces of sterilized (autoclaved) microcapillaries (fused-silica; 679 μm o.d., 534 μm i.d; 15 cm long) with fibronectin, by instructing the syringe pump to draw a sterile fibronectin solution (30 μl of 50 μg/ml) into each microcapillary, incubating (37 °C for 2 hr), instructing the syringe pump to withdraw the solution, and letting the microcapillaries dry (room temperature (RT) for 6 hr).
2. Connect each fibronectin-coated microcapillary (perfusion chamber) to polycarbonate tubing (500 μm o.d., 100 μm i.d.) and the tubing to the multi-port syringe pump, using CapTite fittings (6 perfusion chambers, 8-port syringe pump).
3. Using tape, secure the body of each perfusion chamber to a heat block set to desired temperature (37 °C).
4. Immerse the open ends of the perfusion chambers in a reservoir (microcentifuge tube or microtiter plate well) containing cells (P388D.1 murine macrophages) suspended in fresh growth medium (RPMI-1640, 10% FBS, 500 units/ml penicillin, 500 μg/ml streptomycin) at desired concentration (10⁶ cells/ml).
5. Instruct the syringe pump to draw cells (10 μl, 10⁴ cells) into each perfusion chamber followed by enough air to move the liquid plug (~4.5 cm) to the section secured to the heat block.
6. Allow the cells to adhere to the fibronectin-coated interior surfaces of the perfusion chambers (37 °C for 1 - 2 hr). In the meantime, transfer the open ends of the perfusion chambers from the cells reservoir to a new reservoir containing fresh growth medium.
7. After the adherence period, instruct the syringe pump to send the liquid plugs (conditioned media and unattached cells) to waste, withdraw fresh medium (10 μl), and follow with enough air to position the new liquid plugs (fresh medium) over the adhered cells.
8. Continue to carry out medium exchanges (i.e. repeat Step 1.7) at regular intervals (every 2 hr) until the cell populations are sufficiently equilibrated and expanded (16 - 24 hr of micro-scale culture generated populations of ~1,000 cells/chamber).

2. Fabricate the DMF Device and Assemble the Hub Architecture

1. As described previously^14,29, pattern the bottom plate of the DMF device with forty-six indium tin oxide (ITO) electrodes (drivers of droplet actuation) by photolithography and etching, and coat with 5 μm of Parylene-C and 50 nm of Teflon-AF. Form the top plate of the DMF device by coating an unpatterned ITO glass substrate with Teflon-AF, as above.
2. Using metal compression frames, fix the DMF plates into a polymer cast^14,30 with recesses that maintain a 400 μm spacing between the plates; this spacing, combined with the actuation electrode size (2.5 mm²), defines the droplet volume (2.5 μl per electrode). Note that DMF assembly can be accomplished by simpler means²⁴.
3. Insert Teflon-coated transfer microcapillaries (360 μm o.d., 100 μm i.d.; 3.5 - 4.0 cm long) into the space between the DMF plates, positioning each to extend to the edge of its cognate actuation electrode.
4. To the opposite end of each transfer microcapillary affix a CapTite fitting.
5. Engage the electrical connectors to supply voltage to the ITO electrodes. Electrode activation sequences are automated through use of a computer-controlled electronic interface running pre-determined scripts.
6. Optional: Move the assembled DMF hub onto the translational stage of a microscope (SZ-6145TR (Olympus, Japan)) equipped with a charge-coupled device (CCD) camera (DCR-HC96 (Sony, Japan)) to facilitate tracking of droplets.³¹

3. Connect the Perfusion Chambers to the DMF Hub and Stimulate the Cell Populations

1. Remove the open ends of the perfusion chambers from the medium reservoir (see Step 1.6) and insert them into the CapTite fittings affixed to the ends of the transfer microcapillaries of the DMF hub (see Step 2.4).
2. Instruct the syringe pump to send the liquid plugs (conditioned media) in the perfusion chambers to waste.
3. Instruct the DMF hub to draw stimulus (E. coli BioParticles at 100 μg/ml in fresh medium with 0.1% Pluronic F127 w/v) from an on-board reservoir and deliver a droplet of appropriate size (10 μl) to each transfer microcapillary. The on-board reservoirs are comprised of cylindrical-shaped compartments (1.5 x 1.5 cm each) and connected to the DMF Hub via tubing.
4. Instruct the syringe pump to draw the stimulus plugs into the transfer microcapillaries, and follow with enough air to position the plugs over the cell populations in the perfusion chambers.
5. Incubate the cells with stimulus (37 °C for 1 - 4 hr). Optional: Exchange for fresh stimulus (i.e. repeat Steps 3.2-3.4) at regular intervals.

4. Terminate Stimulation and Recover Cell Lysates for Analysis

1. Optional: If a post-stimulation period is required, exchange the stimulus-containing liquid plugs for fresh medium (i.e. repeat Steps 3.2 - 3.4, substituting fresh medium for stimulus), and continue to incubate and exchange as needed.
2. Exchange the liquid plugs in the perfusion chambers for wash buffer (Prelude Direct Lysis Module Buffer B) (i.e. repeat Steps 3.2 - 3.4, substituting wash buffer for stimulus), and incubate as needed (5 min).
3. Exchange the liquid plugs (wash buffer) for lysis solution (Prelude Direct Lysis Module Buffer A with 0.1% Pluronic F127 w/v) (i.e. repeat Steps 3.2 - 3.4, substituting lysis solution for stimulus), and incubate as needed (5 min).
4. Instruct the syringe pump to send the liquid plugs (cell lysates) to the DMF hub.
5. Disassemble the DMF hub, remove the top plate of the DMF device (using care not to tilt the plate sideways, as this can result in droplet mixing), and collect the lysates using a Pipetman or syringe for off-platform analysis (gene expression profiling via qPCR).

5. Clean Platform Hardware for Re-use

1. Immerse the transfer microcapillaries and CapTite fittings in 10% bleach (v/v in water) for 10 min at RT, rinse well with deionized water, and dry using nitrogen gas.
2. Immerse the DMF device plates in 10% bleach for 10 min at RT, rinse well with isopropanol followed by deionized water, and dry using nitrogen gas followed by baking at 160 °C for 10 min.
3. The syringe pump's polycarbonate tubing, and the DMF device's polymer cast and compression frame, can be re-used without cleaning. The perfusion chamber microcapillaries are discarded after each experiment.

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)