wfirm final presentation

Institute for Regenerative Medicine

Optimization of Bioprinting

Parameters for Tissue Engineering

Summer Scholar: Sarthak “Ankit” Patnaik

Mentor/PI: Prafulla Chandra, Christina Ross,

Yuanyuan Zhang, Benjamin S. Harrison

Wake Forest Institute for Regenerative Medicine

Introduction

• More than 1 million civilian burn injuries in the US

every year require medical attention

• Trying to regenerate skin is very difficult because it has a

very sophisticated architecture

• Creating the tools needed to regrow and maintain

skin effectively is an important problem in

regenerative medicine

• Bioprinting is a powerful tool that can solve these

problems by using cells and biomaterials to print

new skin

3Institute for Regenerative Medicine

The Bioprinter - TissueJet

Computer interface

Valvejet pressure controllers (2)

Magnetic stirrer controllers (4)

Printing platform

Enclosed chamber with HEPA filtration


Printing platform

Substrate holder- with heater

Piezoelectric inkjet devices (4)

Inkjet print cartridge attached to heater & magnetic stirrer (4)

Valvejetdevices (2)

Valvejetprint cartridge(2)

X, Y & Z motion stages

Pressure-controlled sample inlet tube


Print cartridge- Isolated Print cartridge - In Bioprinter

Piezoelectric inkjet printhead

Sample tube- containing cell suspension

Pressure-controlled sample delivery tube

Magnetic stir bar (Red) Heater and Magnetic stirrer block


My Goal – Questions to Answer

• Optimizing the bioprinter: Does stir speed

and cartridge temperature affect the

printing process?

- How do these parameters affect average cell

distribution?

- How do these parameters affect cell viability

over time? What is our effective working time?

- Are these parameters optimal for multiple cell

types (mouse macrophages and fibroblasts)?


Results (macrophages)

0

20

40

60

80

100

120

0 60 120 180 240 300 360

Viability (%)

Time (min)

Viability vs. Time (Stir Speed)

No Stirring

Med Stirring

Max Stirring

* *

*Marksthelasttimepointwherep>0.05;showstheeffectiveworkingtimeforeachsetting• Medium Stirring has the longest effective working time of 2 hours; No Stirring has a working

timeof 60minutes,andMaximumStirringhasaworkingtimeof30minutes

*



No Stirring Medium Stirring

Maximum Stirring

Percentage of Drops with Cells

(n = 90) 27% 58%* 37%

Average cells per drop 1.2 ± 0.7 1.3 ± 0.7 1.4 ± 0.6

*p < 0.05 when t test is compared with other two settings• Stirring speed does not affect average cells per drop• Medium Stirring increases percentage of drops with cells in them by a

statistically significant amount



0

20

40

60

80

100

120

0 60 120 180 240 300 360

Viability (%)

Time (min)

Viability vs. Time (Temperature)

23° C

30° C

37° C

*Marksthelasttimepointwherep>0.05;showstheeffectiveworkingtimeforeachsetting• 30° C cartridges have the longest effective working time of 2 hours; 23° C cartridges have a

working timeof 90minutes,and37°Ccartridgeshaveaworkingtimeof60minutes

***



23 ° C 30 ° C 37 ° C


(n = 90) 27% 38%* 26%


*p < 0.05 when t test is compared with other two settings• Cartridge temperature does not affect average cells per drop• Setting the cartridge temperature to 30° C increases percentage of drops with

cells in them by a statistically significant amount


Results (Fibroblasts)

0

20

40

60

80

100

120

0 60 120 180 240 300 360

Viability (%)

Time (min)

Viability vs. Time (Stir Speeds)

No Stirring

Medium Stirring

Maximum Stirring

**

*Marksthelasttimepointwherep>0.05;showstheeffectiveworkingtimeforeachsetting• Medium Stirring has the longest effective working time of 2 hours; No Stirring and Maximum

Stirringhaveworkingtimesof90minutes

*



No Stirring Medium Stirring

Maximum Stirring


(n = 90) 26% 48%* 36%

Average cells per drop 1.2± 0.5 1.6 ± 0.7 1.7 ± 0.8

*p < 0.05 when t test is compared with other two settings• Stirring speed does not affect average cells per drop• Medium Stirring increases percentage of drops with cells in them by a

statistically significant amount



0

20

40

60

80

100

120

0 60 120 180 240 300 360

Viability (%)

Time (Min)

Viability vs. Time (Temperature)

23° C

30° C

37° C

*

*Marksthelasttimepointwherep>0.05;showstheeffectiveworkingtimeforeachsetting• 30° C cartridges has the longest effective working time of 2 hours; 23° C cartridges have a

working timeof 90minutes,and37°Ccartridgeshave aworkingtimeof60minutes

**



23 ° C 30 ° C 37 ° C


(n = 90) 22% 41%* 29%


*p < 0.05 when t test is compared with other two settings• Cartridge temperature does not affect average cells per drop• Setting the cartridge temperature to 30° C increases percentage of drops with

cells in them by a statistically significant amount


Conclusion• We have found that neither stir speed nor

temperature significantly affects average

number of cells per drop

• We have determined an optimal stir speed;

Medium Stirring both improves cell distribution

and lengthens effective working time

• We have also determined an optimal cartridge

temperature; 30° C cartridges have the same

effects as does medium stir speed

• We have shown that optimal parameters are

the same for both macrophages and fibroblasts


Future Directions

• We would like to keep repeating these

experiments with keratinocytes, another cell

type involved in skin printing

• There are other parameters on our bioprinter

that could possibly affect printing:

- Back Pressure

- Nozzle Diameter

• Use multiple optimized jets simultaneously to

print skin


Acknowledgements

• We would like to thank Eben Adarkwa and Dr.

Salil Desai for their training and assistance with

the printing process.

• This work was supported in part by USAMRMC

through contract #W81XWH-11-1-0658.

wfirm final presentation

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