CELL PRINTER OPTIMIZATION FOR BIOMEMS APPLICATIONSMichael Lopez, Muhaimeen Hossain, Ashley Bui, Alec S.T. Smith, Catia Bernabini, Bonnie Berry, James J. Hickman

1NanoScience Technology Center, University of Central Florida, Orlando, FL, USA

Introduction

Methods

Results

References and

Acknowledgements

Future Plans

Conclusion

Drug development is an arduous process,

sometimes taking years to get a drug from research to the

market. Human clinical trials are expensive and often fail

despite positive outcomes obtained from animal models.

Moreover, the use of animals for drug testing carries

ethical concerns and coupled with difficulty in translating

results from animals to humans, highlights the need for

creating human-based platforms for reliable in vitro drug

testing. By creating a “body-on-a-chip” system, we can

efficiently test drugs on human systems while

simultaneously cutting the costs and the risks associated

with utilizing human subjects.

Currently, print parameters are being optimized for

use in printing a two-dimensional cell pattern on

BioMEMS (Biological Microelectromechanical Systems)

for electrophysiological testing. These tests will allow us

to monitor cell viability and revise our cell culturing

techniques. Combining various human cell types cultured

into one organized, in vitro system will produce a “human-

on-a-chip” product that has the capability to replace all

former methods of testing drug toxicity and physiological

response.

The reengineering and optimization of a cell printer

would pave the way for future experiments that seek to

change the way drugs are developed and tested.

Advantages of this printing include:

• Allowing the precise placement of cells on a

micrometer scale

• Capability to print multiple cell types within a single

device or substrate

• Ability to control the geometric placement of cells in

patterns determined by the user

Specific applications also include providing reliable

placement of muscle and neural cells for the formation of

neuromuscular junctions, allowing us to:

• Analyze the data retrieved from cells grown in vitro with

in vivo processes

• Follow the eventual path of printing of human cells

NanoScience Technology Center Hybrid Systems Laboratory

• RegenHU cell printer (Figure 2)

• C2C12 Mouse Myoblast Cell Lines

• Printed and maintained using DMEM with FBS (with

serum) or lab-developed serum free medium (M1)

• Once cells were confluent, differentiated using

DMEM with B27

• Testing of cell viability under various conditions:

• Humidity

• Temperature

• Cell Density

• Cell suspension settling time

• Print media such as serum, without serum,

hydrogels, PVP, and tissue culture mineral oil

Once conditions are optimized, printing will be done

on cantilevers to form neuromuscular junctions

accurate enough to be less than a millimeter apart.

Figure 3. C2C12 cultures printed in two types of media.

The above phase-contrast images are of printed C2C12

cells on glass cover slips coated with DETA. DMEM with

FBS (top) and with M1 (bottom) were the media used to

mature the cells. All of the cells were then differentiated in

DMEM with B27.

The ability to culture cells in vitro with comparable

properties to in vivo conditions is a progressive stride

within the medical field, specifically the pharmaceutical

industry. To maximize the effectiveness of drugs, it is

essential that proper testing models are available. Human

clinical trials are expensive and present numerous risks—

an in vitro “body-on-a-chip” model would significantly

reduce or eliminate the need for animal testing and would

significantly accelerate the drug development, testing, and

approval process.

Figure 4. Hygrometer display. Readout system of a

humidity sensor placed inside the sealed cell printer.

• The hygrometer has been programmed to measure

humidity once every two seconds, to ensure stability in

the system.

• Optimal humidity was determined to be 85 – 95%.

Figure 2. RegenHU Cell printer setup. The above setup of a cell printer (A) and computer (B) equipped with sophisticated

software capable of controlling various cell printing parameters. Specifically, the software is capable of controlling the X, Y, and

Z positions of the printhead while utilizing applications, such as BioCAD, to design print patterns.

Figure 5. Immunostained Neurons. The above confocal

images represent neurons stained for various segments of

their cell bodies. In the future, neurons will be printed at

optimal viability levels onto cantilevers and MEAs.

Figure 1. Cantilever Designs.

Neurons (red line) and Muscle

Cells (red dots) will be printed

onto cantilevers. This will provide

geometric placement of

appropriate cell types for the

formation of neuromuscular

junctions that are able to be

stimulated electronically.

RESULTS: Tests indicate optimal cell condition is printing C2C12s in serum containing medium

with a 5 minute cell adherence period in the printer under 85-95% humidity.

1. Huh, D. et al., (2012) Lab Chip. 12: 2156–2164

2. Song, H. et al., (2013) Lab Chip. 13: 1201

3. Smith, A. et al., (2014) Technology. 1: 37-48

This work was supported by NIH Grant Numbers R01 EB005459 and

UH2TR000516 .

GOAL: Optimize method of printing various cell

types onto chemically patterned substrates for

use in body-on-a-chip applications.