1. Designing a Rotating Vessel For Production of 3-D Cardiac Tissue
[Roli Kumar]
[Advisors: Dr. Joey Barnett]
11/7/2018

2. Overview Importance of designing and building a rotating vessel
objectives
Protocol for Cell Dissociation
Design (Building + Testing)
Results
11/7/2018

3. Background: Importance of 3-D Tissue
Replacement of damaged heart tissue caused by
disease
myocardial infarction (heart attack)
surgical procedures
radiational procedures
Research
a new route to drug delivery
11/7/2018

4. Myocardial Infarction
Single leading cause of death
Reduced blood supply to the area
irreversible damage
limited regenerating potential
11/7/2018

5. Objectives
Place cardiomyocytes in a rotating vessel and the cells should adhere to the walls.
The cells should divide and become confluent.
With continuous rotation the cells should adhere one on top of another
Therefore formation of 3-D tissue and they will connect and beat.
11/7/2018

6. Tasks 1. Develop a protocol for dissociation of cardiomyocytes (CMC)
2. Design and test the rotating vessel
3. Display results with photographs
11/7/2018

7. Protocol for Dissociation of Cardiomyocytes
10 day old chick embryos are used
Dissect ventricular portion
Add trypsin
Isolate myocytes from the red blood cells
Add myocytes to fetal calf serum
spin it down
get pour off fetal calf serum
suspend myocytes in growth medium
dilute according to the no. of cells necessary
11/7/2018

8. How many cells are needed?
1.6X 10^5 cells/cm^2
surface area= SA of cylinder+SA half circle
SA=2(pi)rh +.5(4(pi)r^2)
No. of cells= SA sphere X (cell/cm^2)
No. of cells= 3,455,200 cardiac cells
11/7/2018

9. Apparatus Set-Up Plastic Vessel Stainless Steel Holder Plastic Rod*
07/16/96
Apparatus Set-Up
Plastic Vessel
Stainless Steel Holder
Plastic Rod
Rotating Platform
Magnetic Stirrer
11/7/2018 *

10. Design Specifications (Needs)
End-over end mixing
measurements at various speeds
problem: too fast, too slow
Solid body rotation
suspending cells in a liquid medium
proper cell to growth medium ratio
Aeration
medium usually contain bicarbonate
Problem: CO2 is produced in a closed container
11/7/2018

11. Design Specifications (Actual)
End over end mixing
Rotational speeds 100 rotations per minute
Solid Body Rotation
A ratio of 3 X 10^5 cell/ml
Container holds 10 ml
Aeration
A Hepes buffer was used
11/7/2018

12. Results
Visually see the difference between the monolayer growth in a plate and 3-D growth in a rotating vessel
Pictures were taken of the monolayer growth after day 1 and day 5
Pictures have been taken after day 1 to show that the myocytes adhered to the wall.
11/7/2018

13. Current & Future Work Finding how long cells grow in Hepes buffer?
Continuously rotate until multiple layers adhere to the wall and one and another
Take Pictures of 3-D tissue
11/7/2018