VERO Bioreactor Cell Culture and Vaccine Production4TR1: Capstone Project
Yang Liu - 0846882
Professor: Dr. Fei Geng
Introduction:
Background information:
Current technology:
Use of expensive bioreactor or roller bottles to culture vero cell for vaccine production
Vero cells used as they have defective immune systems and are easily infected with viruses
Optimum to develop a new method of cell culture for vaccine production on a large scale
Bioreactor Limitations:
Requires continuous batch bioreactor system
CO2 required for cell media buffering
Needs agitation but too much will shear the cells
Project Objectives:
Design an inexpensive method or adaption to the bioreactor system which will allow mammalian cells to grow.
Project Plan:Cell culturing and adaption
Culture adhering cells ( MCF-7), Vero recommended but difficult for conceptual testing
Roller incubation to adhere cells to Cytodex-1 and/or CultiSpher-S
MTT assay to test cell growth and adhering capabilities
Bioreactor design
Utilize Solidworks and AutoCAD to 3D print prototypes
Prototype development requirements:
Allows flow of media and agitation of bead
Can introduce new bare beads into the culture
Self-contained to limit contamination and bead breaching
Random flow within chamber to allow unpredictable bead movement allowing for cell exchange between beads
Testing bioreactor system with cells to determine efficiency of design
Results: Cytodex 1 Microcarriers
Metrics adhering:
Figure 1: MDCK cell adherence on Cytodex I microcarrier. Some individual cells can be seen adhered on the beads on March 13,2016.
Figure 2: A549 cell adherence on Cytodex 1 microcarriers on March 18, 2016
Results: CultiSpher-S Microcarriers
Figure 3: MCF 7 Cells adherence on CultiSpher-S on November 22, 2016 Figure 4: MCF 7 Cells adherence on CultiSpher-S
on November 24, 2016
MCF7 Cell culture growth condition:
Cell viability verification: MTT ASSAY
Beginning Cell Culture Cell Culture After Two Days
Absorbance(570nm)
Absorbance
Duplication Results
Average Absorbance
Absorbance
Duplication Results
Average Absorbance
Average Absorbance Total
Blank sample(bear beads)
0.184 0.205 0.1945 0.181 0.198 0.1895 0.192
Falcon Tube sample 1
0.397 0.326 0.3615 0.459 0.446 0.4525 0.407
Falcon Tube sample 2
0.442 0.269 0.3555 0.399 0.521 0.46 0.40775
Table 1: MCF7 Cell culture Viability Test With MTT Assay.
Figure 4: MCF7 Cell growth MTT assay result graph for tube 1.
Figure 5: MCF7 Cell growth MTT assay result graph for tube 2.
Mini-bioreactor design:
Bioreactor prototype 1 Bioreactor prototype 2
Conclusion:
Summary of the experiment:Successfully cultured MCF-7 cells to adhere on CultiSpher-S beadsDeveloped multiple prototypes of mini-bioreactor printable chambers
Established parameters used for conceptual testing of selected mini-bioreactor prototype
Tested most promising prototype and troubleshot conceptual testing errors
Next steps:Refine prototype design to eliminate leakage (Use better resolution setting on 3D Printer)Modify design to increase reusability and durability Test CultiSpher-S culture within bioreactor to establish growth feasibility Once growth feasibility has been proven, the final step will be adapting the culture to serum free
media to optimize growth and limit downstream processing
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