The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

Summary

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

Abstract

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

Introduction

Recent advances in the pharmaceutical industry have led to increased interest in using cellular microarrays in the drug discovery process for drug screening and cytotoxicological analysis^1,2,3. The development of in vitro high-throughput assays and screening methods using cell microarrays would facilitate the rapid and cost-effective development of drug candidates as well as advance the fundamental understanding of the cell^1,4. The traditional approach to screening with cells uses conventional well-plate platforms; however this approach is limited due to the high cost, limited throughput, and limited ability for quantitative information ....

Protocol

1. Cell Culture Preparation

1. Store NIH/3T3 cell stocks in liquid nitrogen until ready for use.
2. Prepare complete media for NIH/3T3 cells using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 10 mM HEPES buffer, 50 units/ml penicillin, and 50 μg/ml streptomycin.
3. Thaw cells for 2-3 min in a shaking water bath at 37 °C.
4. Resuspend cells in 5 ml of complete media and centrifuge at 1,500 x g for 3 min.
5. Remove the cell supernatant without disturbing the cell pellet.
6. Resuspend the cells in 5 ml of media and count cells using a hemocytometer.
   1. Remove 10 _....

Results

The fibroblast cell line NIH/3T3 cells were printed or seeded onto a submerged surface. The cells were grown to a density of 5 X 10⁴ cells per ml. Cells were seeded using traditional cell culture techniques. Cells were printed using a large-format, twelve-flow cell printhead where the channels are larger (~500 µm) than the CFM used for proteins and other biomolecules. The cells were printed or seeded onto a serum coated surface. The printing process is shown in Figure 1.

Discussion

The 3D microfluidic printing technology described here is uniquely capable of microfluidically printing arrays of cells into a liquid filled well, i.e. a submerged surface. By printing onto submerged surfaces, cell microarrays can be produced that maintain the physiologically relevant cellular phenotype of cells as well as the ability to multiplex cells in the bottom of a single well Figure 4. The results of this study show that microfluidically printing cells results in cell attachment with com.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Continuous flow microspotter	Wasatch Microfluidics
NIH/3T3 cells	ATCC	CRL-1658
Dubbleco's Modified Eagle Medium	Invitrogen	11965-092	Base media for cells
HEPES buffer	Invitrogen	15630-080	Cell media additive (control pH)
Sodium pyruvate	Invitrogen	11360-070	Cell media additive
Penicillin-streptomycin	Invitrogen		Cell media additive
Trypan blue	Invitrogen	15250-061	Stain cell sfor counting
Hemocytometer	Fisher	267110	Cell chamber to count cells
Nikon Eclipse TS100	Nikon		Used to check on cells
Nikon Eclipse TE2000-U	Nikon		Used for collecting images
Phosphate buffered saline (with calcium and magnesium)	Invitrogen	14040-133	Rinsing cells before passaging and before staining with PI
TrypLE Express	Invitrogen	A12177-01	Used to remove cells from surface