Aggi-E Challenge The Perfusion Bioreactor Section Group 2a. Diego Garcia, Megan Maladecki, Nathanael Marshall, Ria Rao

Transcription

1 Aggi-E Challenge The Perfusion Bioreactor Section Group 2a Diego Garcia, Megan Maladecki, Nathanael Marshall, Ria Rao

2 Types of Bioreactors Spinner Flask Scaffolds are fully submerged in a media-filled flask with a magnetic stir bar at the bottom for media agitation. Rotating Wall Vessel Two concentric cylinders are rotated in a zero-gravity environment to create waves in the media. This makes a semi-static condition for the submerged scaffolds within the bioreactor. Perfusion The scaffold(s) is/are suspended in a media bath with a pump continuously pumping media from a media reservoir through the scaffold(s). [1] [1] [1]

3 Advantages of Perfusion Perfusion eliminates stagnation of cells on the outermost layer of the scaffold. All cells receive approximately equal amounts of media saturation. The flow rate can be adjusted to help control the pressure to which the cells are subjected. The media reservoir can be easily replenished with fresh media. Perfusion bioreactors have a relatively low material and fabrication costs. [2]

4 Materials Pump 4 Channels allow for independent control of up to 4 tubular flow rates. 12 Rollers minimize pulsatile flow through the tubes and scaffold and allow for a higher degree of continuous flow accuracy. Output flow of the pump ranges from ml/min. Tubing Masterflex platinum-cured silicone tubing is bioinert, autoclavable, and flexible. Valves Pinch valves are used to restrict flow in certain portions of the device. Pinch valves are easily adjustable and do not contact the fluid flowing through the restricted tubing. Media Reservoir A sterile container holds media for pump intake of cell media. The media reservoir contains excess media so that it takes longer for the cells to deplete the nutrients in the media.

5 Bioreactor Design Material: polysulfone Polysulfone is bioinert and cells do not adhere easily to it. The autoclave will not damage polysulphone. A combination of a lathe and drill can easily produce the design. Polysulfone has been used successfully. [2] Function Silicone tubing enters and exits through the holes in the lid. The inlet and outlet tubings are connected to the scaffold within the bioreactor. The bioreactor contains a media bath and media flows through tubing and scaffold lumen. The outlet flow of the bioreactor is restricted, causing transluminal flow through the scaffold radially. The offset slits in lid and body allow for free gas exchange.

6 Mimic Scaffolds While the Parker rotary jet spinner was being completed, mimic scaffolds were used to simulate porous scaffold transluminal flow. The scaffolds used were: 1. Straw Scaffold Materials: plastic straw and Parafilm This was cheaply and easily constructed, but did not mimic porous flow well. 2. Silicone Tubing Scaffold Materials: segment of silicone tubing Silicone tubing was punctured using a 27 gauge needle many times to approximate a porous structure. However, the elasticity of the silicone tended to press the pores closed under pressure. 3. Inflexible Tubing Scaffold Materials: segment of scrap tubing The advantages to this scaffold were that the tubing was less flexible than silicone, and the transluminal flow was approximately porous. 4. Paper filter Scaffold Materials: PTFE paper filters This scaffold was made from rolled filter paper. The advantages to this were that the porosity was known (2.5 microns) and the thickness could be controlled. However, when wet, it

7 Future Directions Seeded Scaffolds Ultimately, the goal of the bioreactor is to maintain cell life and growth within a tubular scaffold. We could try to seed a different, more biocompatible mimic scaffold or wait for the scaffolds of interest to be generated and seeded using the Parker rotary jet spinner. Cell-Staining Assays Assays are essential tools for the analysis of a cell culture s health. Live or Dead assay - Stains live cells green and dead cells red. This will be used to determine the survival rate of cells within a scaffold. MTT assay - Timed staining that stains cells by varying amounts based on cell viability, type, and environment. This assay will be used to determine the viability of surviving cells. Scaffolding Scaffolding can be added to the interior of the bioreactor to add structural support for the scaffold. This scaffolding could also help make scaffold access easier by having a section that could be pulled up through the convenient access lid, taking the scaffold with it. The scaffolding could be machined using the polysulfone left over from machining the bioreactor. Bioreactor Motion Most of the scaffold perfusion occurred through the bottom edge of the tubular scaffold. This effect could be reduced by gently rocking the bioreactor.

8 References 1. A. Yeatts, D. Choquette, & J. Fisher, (2013). Bioreactors to Influence Stem Cell Fate: Augmentation of Mesenchymal Stem Cell Signaling Pathways via Dynamic Culture Systems. Biochimica et Biophysica Acta, 1830(2), D. Butler, S. Goldstein, R. Guldberg, X. Guo, R. Kamm, C. Laurencin, R. Tranquillo, (2009). The Impact of Biomechanics in Tissue Engineering and Regenerative Medicine. Tissue Engineering. Part B, Reviews, 15(4), InBreath 3D Bioreactor for Hollow Organs, Bronchus, Trachea & Blood Vessels, [Online]. Available: [Accessed: 1-Oct Dec-2016] 4. Cole-Parmer US - Fluid Handling and Analysis Supplies from Cole Parmer. N.P., n.d. Web. 16 Dec. 2016