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