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 on cell function^1,5. Due to these limitations, research in cellular microarray technologies is burgeoning for molecular biological characterization, tissue engineering, and drug screening^1,6. The advantages of cellular microarrays include smaller sample use, minimal effects of cellular phenotype heterogeneity masking information, and most importantly the ability to automate assays for more high-throughput applications^1,7,8.

The pharmaceutical industry currently utilizes high-throughput cell-based screening assays with 2D cell monolayer cultures for drug screening in microtiter well plates⁹. Multiplexing cells in wells of microtiter plates offers the potential for higher throughput with unique experimentation options. Further, the current technologies for cellular microarrays allow the cells to dry which could dramatically alter the phenotype of the cells from in vivo^10,11. In order to overcome these problems, the MFCA was engineered and is shown in Figure 1. The design of the MFCA 3D fluidics enables the printhead tip in Figure 1 to be lowered into a bath and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and forms a reversible seal. The MFCA technology is uniquely suited to interface with submerged surfaces, which is required for both cell cultures and tissue slice systems, and is difficult or impossible with most other approaches. Pins or ink-jet printing will not work, and 2D microfluidic devices are not suited for deposition or interfacing with large arrays of discrete spots. Further, by miniaturizing and localizing the experiment - the cellular microarray - the MFCA overcomes the major problems associated with high-throughput cell-based screening assays.

The CFM uses 3D channel networks to cycle small volume fluid samples over microscopic spot locations on a surface^12,13. By printing with flow, biomolecules, cells, and other reagents are maintained in a liquid environment throughout the printing process, enabling the printing of sensitive biomolecules and cells without exposure to air, which hinders the current cell printing techniques. It is also possible to print directly from crude material such as hybridoma or supernatants provided there is a capture mechanism on the array surface. The objective of this manuscript is to explain in detail the submerged printing of two cell types onto a surface.

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 μl of the cell suspension and place in a 0.5 ml microcentrifuge tube.
   2. Add 10 μl of Trypan Blue to the cell suspension and stir with the tip of the pipette.
   3. Pipette 10 μl of the stained cells into a hemocytometer chamber.
   4. Count cells in hemocytometer at 10x magnification.
      1. Count the live cells (small, white balls) for 4 of the 9 large squares. Only count the live cells that do not appear dark blue from the Trypan Blue stain.
      2. Calculate the average number of cells per large square, resulting in the number of cells x 10⁴ cells/ml in the original cells suspension.
      3. Seed cells at a density of 1 x10⁵ cells/ml with a total of 5 ml in each T25 flask.
   5. Culture cells to between 70% and 80% confluency before beginning experiments. Refresh media every 2-3 days or as needed.

2. Printing Surface Preparation

1. Mark a rectangle with marker on the bottom of a tissue-culture-treated polystyrene (TCTPS) 12 well plate the size of the print head (7 mm x 19 mm).
2. Pipette 50 μl of fetal bovine serum into the rectangular area and spread it over the entire region with the tip.
3. Leave the Petri dish in a sterile biosafety hood O/N to allow the serum spot to dry completely. These plates should be prepared no more than 2 days prior to use.

3. Submerged Printing

1. Rinse the print head out with distilled water.
   1. Fill a 60 mm Petri dish with distilled water and dock the print head onto the surface.
   2. Flow distilled water through each of the lines for 2 min at 150 μl/min using a pneumatic pump.
   3. Discard the water from the Petri dish and fill it with clean water.
   4. Move the print head up and down in the water 3 times.
2. Dock the print head center over the serum-coated spot in the pre-prepared 12 well plate.
3. Prime the print head with pre-warmed complete media, 300 μl per channel.
4. Print suspended cells at a concentration of 50,000 cells/ml onto the surface and 60 μl/min, 100 μl per channel.
5. Place the print head, left docked against the surface, and the manifold in a cell culture incubator set to 5% CO₂ and 37 °C for 2 hr.
6. After the 2 hr incubation, remove the print head and place the lid on the culture dish. The time at which the print head removed is considered the 0 hr time point.

4. Standard Cell Culture

1. Add 1 ml of pre-warmed media to one of the serum-spotted TCTPS 12-well plate prepared in step 2.
2. Take 1 ml of the remaining cell suspension from step 2 and pipette into a single well of a 12-well TCTPS plate. This will produce a culture containing 50,000 cells in the dish.
3. Place the cell culture plate in an incubator at 37 °C for 2 hr.
4. After the 2 hr incubation begin timing; for consistency between printed and seeding samples, this is considered the 0 hr time point.

5. Culture Visualization

1. After 0, 2, 24, and 48 hr take cells from each of the two methods described above and image using a standard inverted microscope.
   1. Stain the cells using propidium iodide, a red fluorescent stain that is only incorporated into the nucleus of dead cells.
      1. Gently rinse the cells 3 times with 1 ml of phosphate buffered saline with calcium and magnesium (PBS++) to remove any debris.
      2. Add 1 ml of pre-warmed PBS++ to each culture vessel.
      3. Add 1 μl of propidium iodide stock solution (1 mg/ml) to each culture vessel.
      4. Incubate the cells stained with propidium iodide at 37 °C for 10 min.
   2. Image the cells using an inverted microscope at 10x and 40x magnification.
2. Discard the cells in an appropriate biohazard container.

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