Proposed Design for Murine Embryonic Stem Cell …beweb.ucsd.edu/courses/senior-design/projects/2011...Proposed Design for Murine Embryonic Stem Cell Bioreactor for Cardiomyocyte Cultivation

Proposed Design for Murine Embryonic Stem Cell

Bioreactor for Cardiomyocyte Cultivation

Constance Ardila,

Christopher Meeks,

Aaron Mitchell,

Edward Yragui

BENG 187B: Project Design

Professor: Dr. Melissa Micou

University of California, San Diego

December 1, 2011

Team #8 “A Stem Cell Bioreactor” Constance Ardila, Christopher Meeks, Aaron Mitchell, Edward Yragui

Advisor: Dr. Andrew McCulloch

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Table of Contents

Part I: Introduction ........................................................................................................................................ 3

The Need and How It Arose ..................................................................................................................... 3

Who will use the bioreactor, and where and how will the end users benefit from the solution ................ 3

The current state of the technology and how it will be improved with a new design ............................... 3

Needs Summary ........................................................................................................................................ 4

Design Goals ............................................................................................................................................. 4

Goals for Ethical Considerations .............................................................................................................. 5

Goals for Public Acceptance, Laws and Regulations ............................................................................... 5

Functional Requirements .......................................................................................................................... 6

Part II: Design Alternatives and Analysis ..................................................................................................... 7

Patenting Issues ......................................................................................................................................... 7

Specific Comparisons to current patents with the two design alternatives ........................................... 9

General Patent Requirements and FDA regulations for Tissue Culture Bioreactors .......................... 10

Ranking of Design Goals and Ranking Matrix ....................................................................................... 10

Ranking Matrix ................................................................................................................................... 10

Rationale for the Ranking Matrix ....................................................................................................... 10

Rating of Designs and Decision Matrix .................................................................................................. 11

Decision Matrix .................................................................................................................................. 11

Decision Analysis ............................................................................................................................... 11

Part III: Design Solution ............................................................................................................................. 12

Rationale for Final Design Choice .......................................................................................................... 12

Design description and resources required ............................................................................................. 13

Machined Items ....................................................................................................................................... 14

Parts List ................................................................................................................................................. 14

Additional Resources .............................................................................................................................. 14

Gantt Chart .............................................................................................................................................. 15

Summary of Strength and Weakness ...................................................................................................... 14

References ................................................................................................................................................... 17

Work Breakdown ........................................................................................................................................ 18



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Part I: Introduction The Need and How It Arose

A bioreactor is an apparatus used for growing and maintaining cells in culture for research applications. In order to study potential causes and cures for disease using stem cells, an effective bioreactor is needed in order to yield high-quality embryoid bodies (EBs). The current bioreactor model used by the client, Dr. Zambon, was developed by a mechanical engineering team. This bioreactor was closely based on an existing design.

1 The template design produced 12.5 x 10

6 cells/ml from an initial 2 x

105 cells/ml; at the time of reporting, the previous group did not test their design with cells.

1,2

The client expressed that the bioreactor's cell culture yield was low and the quality of the EBs was poor. Dr. Zambon also observed that after four days of culture in the bioreactor more than half of the cells were necrotic. Likewise, when the impeller was set at 60 rpm, many of the cells died. However, when set at a gentle speed (as low as 5 rpm), the cells began to aggregate underneath the impeller. The impeller blades were later rounded, lowering cell death, but this was still insufficient. The initial design was for a large-scale bioreactor capable of producing stem cells in great quantity.

1 Yet since the bioreactor is to be used

in a laboratory setting, the need arose to place emphasis on the quality rather than the quantity of EBs. The need is for the bioreactor to sustain and culture cells for at least a week with a stem cell yield

of 70%-80% following the developmental period. After this period, the EBs must be of acceptable size and quality, and the process must be as automated as possible to save costs.

More specific aspects of these needs were deduced. It was hypothesized that the high rate of cell death was caused in part by shear stresses formed by the impeller and the poor diffusion of gases, particularly oxygen, in the bioreactor. Automation via LabView is desired, and it should be able to regulate the exchange of media as well, with little human intervention. The budget is estimated at $1,500 and completion should be in Spring quarter.

The current state of technology addresses the previously defined needs. An STLV type bioreactor with a rotary orbital shaker, appears to be far superior to the group’s starting model, reducing shear stress to less than 2.5 dyn/cm

2.1,2,3,4,5

If an impeller model is a requirement, bulb shaped impellers would be a better choice than the existing blade construct.

3 Another alternative to reducing shear stress

would be to adulterate the media with additives like carboxymethylcellulose (10g/L) to increase the medium viscosity.

5 The use of a rotary orbital shaker would also address the hypothesized problem of

inadequate aeration. The cells may also be encapsulated in alginate beads or microcarriers may be used, but this may be expensive.

5

Who will use the bioreactor, and where and how will the end users benefit from the solution

The bioreactor is first and foremost designed to meet the needs of the client, Dr. Zambon. It will be used in a laboratory setting to conduct research on cardiomyocytes. However, this bioreactor has the potential of being used in any academic or industrial laboratory which needs high-quality EBs, produced in an efficient and cost effective manner. Globally, adult stem cell research is conducted in most developed countries, notably the United States, and a fair number of developing ones as well. Embryonic stem cell (ESC) research is conducted in a smaller, but still significant, number of countries.

6 Market size

varies, but the estimated size for stem cell research products in general was estimated at $872 million in 2009, with a prediction of continual growth in coming years.

6 A fraction of this would contribute to

current stem cell bioreactor development.7 The users will benefit from an increase in EB production,

homogeneity, lifespan, and viability within the bioreactor, along with the effective use of reagents. This will allow for a better lab-scale production of EBs in a controlled and monitored environment. This system will provide researchers the tools to recapitulate developmental processes that are relevant to their research goals involving stem cells. With a larger starting population, research on stem cells can be expanded and data can be acquired more readily with higher statistical significance.

By having an automated method for mass producing a homogeneous population of healthy EBs, the cost, time and manpower associated with maintaining EB formation will be reduced significantly, allowing for these resources to be saved for other aspects of research. This will increase the overall productivity of the lab and prove to be more cost and time effective.

The current state of the technology and how it will be improved with a new design

Currently, the Hanging Drop method and Static Suspension Culture (SSC) are used for homogeneous ESC production. Hanging Drop method allows the cells to aggregate and form highly uniform EBs that reduce variability in experiments. Without the presence of factors such as leukemia inhibitory factor (LIF), the EBs begin to differentiate to form the three initial germ layers of



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embryogenesis: the endoderm, ectoderm, and mesoderm.8 Once formed, the EBs can be chemically

treated to terminally differentiate into a specific cell type. This method has been shown to successfully yield cardiomyocytes.

9 However, this is a challenging method which is limited in the amount of

homogeneous EBs that can be produced.10

The other method, SSC, has also been used to successfully generate EBs in a media solution in greater numbers. However, the cardiomyogenic differentiation has also proved cumbersome and yields a low number of beating cardiac cells.

11

Stirred bioreactors have been shown to increase the amount of EB formation through controlled conditions and monitoring of the cell culture environment.

12 Since the entire ESC culture experiences

identical conditions while differentiating, this leads to a more homogeneous population of EBs similar to the Hanging Drop Method. In addition, the bioreactor’s larger size and nutrient mass allows for a greater population of EBs to develop and expand similar to SSCs. These factors combine to produce an efficient means of generating a large homogeneous population of EBs intended for cardiomyogenesis.

When comparing the two technologies, stirred bioreactors enhanced EB development by 15-fold, while the SSC method only improved development by 4-fold.

13 The obvious effects that the improved

technology has on EB development and homogeneity will prove beneficial to researchers in universities globally focused on investigating the differentiation process of cells on a large scale.

This is consistent since the health of the ESC depends on the following engineering factors: hydrodynamic shear stress by stirring of the medium; the thresholds of forces to be determined; and the mechanics of the cells. The fluid shear stress depends on the relative differences in fluid velocities that EBs experience while inside the bioreactor and it has been shown that there is an ideal amount of shear for different types of cells.

12 This shear stress is commonly achieved by stirring via an impeller, but can

also be achieved through circulating the media using pumps or machines to physically rotate the entire container the EBs are in. At low shear stresses the culture might become clustered, making stem cell extraction difficult, while at high shear stress the culture experiences a large amount of cell death.

12

Furthermore, stem cell mechanics must be considered when dealing with gas perfusion in the medium since some cells can attach to the gas bubbles and rupture when the bubble bursts.

12 Therefore it is

crucial to correlate mechanical limitations of stem cells and the macromechanics of the design, such as impeller length and diameter, in order to provide adequate cell health, production and differentiation. This will lead to a successful design to satisfy the proposed needs. Needs Summary

Human Embryonic Stem Cells (hESCs) and Mouse Embryonic Stem Cells (mESCs), have shown a high degree of regenerative potential along with an apparent unlimited ability to divide.

14 These cells

are only available from native embryonic tissue in very limited quantities, are cost prohibitive to procure in large amounts, and are very sensitive to environmental cues that may hamper cell growth. Thus, it is the need to obtain a high yield of cardiomyocytes from EBs grown in a reliable, inexpensive, and contamination free manner. Our client, Dr. Zambon, currently doing research with cardiomyocytes, is in need of a bioreactor that can support mouse ESC growth and division for 5-14 days while retaining the potential to differentiate into cardiomyocytes. An approximate 100-fold increase of embryoid bodies resulting in a 0.5% yield of cardiomyocytes after 12 days is required. With it, Dr. Zambon will be able to cheaply and quickly procure embryoid bodies for experiments, allowing more time and resources to be focused on the research of mESCs. Design Goals

Safety measures will address the electrical components of the design as well as the pressurized gas and spinning impeller. Each of these components can potentially cause harm to the user, in the form of electrical shock, explosion, and physical abrasion, and their design will be extensively reviewed to greatly minimize this risk or eradicate it completely. Furthermore, measures will be established that focus on protecting the environment including: an efficient bioreactor system to reduce the amount of media/cells/reagents used with the ability to recycle media through an automated design and use of only minimal levels of required nutrients, O2, and CO2 for optimal cell growth. Additionally, disposal of biohazardous waste will follow strict guidelines established by UCSD. In order to generate repeatable conditions for the derivation of cardiomyocytes from EBs, the culture environment and set parameters of the bioreactor must be consistent and endure multiple experiments (for up to 5 to 14 days) without the need of repair or adjustments. This will meet the goals of reliability and durability, and will be accomplished by a monitoring system that measures O2, pH and CO2 levels during the entire process. Also, the use of proper materials and controls, such as stainless steel and glass, and the proper sterilization protocols will ensure the reliability and durability of the bioreactor. Meanwhile, the performance goals



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for the bioreactor reflect how the user will operate the system. Therefore the bioreactor design must include an easy assembly, durability, must be monitored and controlled remotely, and be compact to minimize the required lab space for application. A minimum of 0.5% of the produced cells in the final solution should be cardiomyocytes in order to meet the performance goal. The biocompatibility goal of the bioreactor is currently focused on recapitulating an environment for successful derivation of cardiomyocytes for research use only. This includes use of biocompatible materials and reagents that promote differentiation. In addition, if the bioreactor had applications for developing a cell line for Tissue Engineered Medical Products (TEMPs), components of the design such as material, culture conditions, and contamination risk factors would need to be addressed in order to properly assess the biocompatibility of the cells produced. Currently, the bioreactor consists primarily of stainless steel components, which minimize an effect on cell phenotype which may in turn compromise their biocompatibility for medical application. Goals for Ethical Considerations

Current considerations for intellectual property are not a high priority. Only through novel discovery of an EB mixing model or an engineered gas exchange system, will intellectual property become relevant to the design of the stem cell bioreactor. Also, ethical considerations for possible failure of our design would not be as readily apparent due to its isolation from the public and inability of causing lasting harm to others. In addition, design failure would be based on technical and design process error, not from a breakdown in ethics.

One highly relevant ethical consideration for the bioreactor system is an ethical standard involving stem cell use which will be understood and fulfilled. The immediate intentions for the bioreactor are to use Mouse Embryonic Stem Cells (mESCs) for the aggregation of EBs, which will then differentiate into cardiomyocytes. The governing body which oversees the research application of animal products, including human ESCs in conjunction with animal testing, is the Institute Animal Care and Use Committee (IACUC)

15. IACUC investigates the ethical issue, rather than the morality behind, the

research of ESCs; they pose guidelines that regulate how the cells are acquired, transferred, and stored16

. Additionally, if the bioreactor were intended for expansion of hESCs additional committees

would be involved in reviewing the ethical and legal use of the cells, which include the Institute Review Board (IRB) and Embryonic Stem Cell Research Oversight (ESCRO). These advisory boards regulate materials used for handling hESCs, oversee the request of generating hESCs derivatives and banking them, and also investigate compliance with university policy. In all cases, review and approval by one of these governing bodies must be carried out prior to initiating research. There will be adherence to these well-established scientific regulations and ethical standards to successfully design and test the bioreactor. Goals for Public Acceptance, Laws and Regulations Public acceptance is an important aspect of the bioreactor design so that research may be carried out with less resistance from the community and will ensure higher sales if the product were to become commercially available. Relevant issues arise from the bioreactor’s association with stem cells and the ethical concern with their use in science. Therefore, it would remain as a high priority to clarify the differences in stem cell types based on their origin, for instance hMSCs versus hESCs. In addition, our team plans to follow the strict ethical guidelines enforced by ESCRO, IACUC, and IRB. This would increase the level of public acceptance and help guarantee its success in both the research and community settings. The bioreactor’s design and immediate application for expanding a homogeneous population of EBs from MESCs does not currently require FDA regulation. However, if the bioreactor was applied to expanding a cell population for protein production and other biological factors for a therapeutic treatment, for example, then it would require FDA approval. In this case, extent of work necessary to gain FDA approval includes: full characterization of the host cell and genetic construct; details in how the cell line was established and maintained; how the proteins/factors were produced, extracted, and purified; and finally an extensive list of manufacturing measures to be taken

17.

Relevant laws would apply primarily to the use of stem cells for expansion in the bioreactor. In addition to the previously noted advisory boards which oversee the bioethics, there are also laws and governmental organizations in place to facilitate research on ESCs. One recent law passed in the U.S. was Executive Order 13505, which enabled further embryonic stem cell research. After President Obama signed the order, it had two important components that dealt with removing barriers to hESC research and a revocation which terminated the federal funding restriction set by former President Bush in 2001

18. This

law will enable both the funding and legal permission to expand hESC using this bioreactor.



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Furthermore, governmental organizations such as the NIH have considerable power in regulating the use of hESCs for research purposes. According to the NIH, only specific hESC cell lines may be used under NIH regulations in order to receive funding support for research. These 137 approved cell lines are listed with their registration number, and include the provider, plus the date of approval

19.

Functional Requirements

There are several functional requirements the bioreactor must meet in order to realize our design objective. These are conditions favorable to mouse embryoid body formation, proliferation, and maintenance.

A mouse’s body temperature is approximately 36.9°C20

, and keeping the bioreactor at 37°C ± 2°C using a water jacket should be sufficient to culture the stem cells and EBs at an agreeable temperature

21.

The percent oxygen (pO2) should range from 0.0%- 20.0% ± 0.1%21

. The pH in the bioreactor tank should be set at a slightly alkaline 7.2, with little variance

22. This

may be achieved by maintaining CO2 levels at 5.0%-10.0% with ±0.1% accuracy21, 23

. The volume of the

tank ought to be 2.0L ± 0.1L22

. The maximum allowable level of shear stress on the cells ought to be capped at 6.1 dyn/cm

2 and

preferably should not exceed 4.5 dyn/cm2; in one study, these stresses corresponded to 80-100 rpm

24.

Therefore the shear stress on the cells should not be greater than 0.45-0.61 N/m. The cells must also be able to better withstand collisions with obstacles in the tank such as the sensor probes and impeller (A. Zambon, personal communication, September 30, 2011). To estimate the shear stress on the cells, several formulae may be used. The simplest is to use the integrated shear factor:

Where N is the bioreactor agitation rate, Di is the diameter of the impeller, and Dv is the diameter of the bioreactor tank.

More robust formulae would require knowledge of parameters such as: the diameter of the vortex zone; the bulk modulus of the impeller; Kolmogorov scale eddies; and the kinematic viscosity of the media; dimensionless power number; and the dissipation volume

24. Computer modeling and simulation

software may be used to compute an estimate of the shear stress. It is unlikely that the shear stress on the cells in this project bioreactor will be measured, and so success in staying within the set bounds of allowable stress will be determined by a sufficient number of healthy cardiomyocytes, as determined by our defined need.

The pores for the stem cell gas diffusion filter must be smaller than 10-20µm, the size of a typical mESC. Therefore, a pore diameter of 0.45µm should suffice

21.

The target for cell viability is defined as: more than 70% of the EBs are in good condition after culture in the bioreactor. If feasible given time and monetary constraints, the bioreactor should compete with other models capable of roughly 95% cell viability (A. Zambon, personal communication, October 7, 2011). It is hoped that roughly 12.5x10

6 cells/ml will be produced at the end of the bioreactor run from a

starting culture of 2.0x105 cells/ml

22.

The stem cells should be E14 mESCs derived from the 126/Ola laboratory mouse strain (A.

Zambon, personal communication, October 27, 2011). They ought to begin differentiating into

Table 1: Functional Requirements Temperature 37°C ± 2°C pH 7.2; (5.0% ± 0.1% < pCO2 < 10.0% ± 0.1%) pO2 0% - 20% ± 0.1% Volume of Media 2.0L ± 0.1L Shear Stress/rpm Minimum 35 rpm Target 65 rpm Maximum 80 - 100 rpm Number of Cells at Start 2x10

5 cells/ml

Number of Cells at Termination 12.5x106 cells/ml

Time to Produce Cardiomyocytes 12-13 days Duration of Bioreactor Run ~14 days Media Replacement Rate 2 days, preferably continuous Measure of Healthy Cells 30%-40% of EBs exhibit vigorous contraction



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cardiomyocytes by day 12 or 13 (A. Zambon, personal communication, October 7, 2011), and the bioreactor must run for a duration of ~14 days, the time at which healthy cardiomyocytes should contract (A. Zambon, personal communication, October 24, 2011)

22. The project should be considered successful

if 30%-40% of the EBs contract vigorously22

. The media must be replaced every two days in order to remove waste materials from solution as

well as introduce fresh nutrients, and other reagents (A. Zambon, personal communication, October 24, 2011). It would be preferable to have a continuous flow of media constantly removing waste while provide the opportunity to introduce new reagents (A. Zambon, personal communication, October 24, 2011). Part II: Design Alternatives and Analysis Design Alternative 1: Modified Schroeder Spinner Flask Bioreactor (Existing Design)

This is an existing bioreactor. Figure 1A/B shows a basic spinner flask type bioreactor, the cells are kept in suspension with the aid of a pitched blade impeller. The reactor vessel is kept at a constant temperature by means of a glass water jacket, and O2 and CO2 are pumped in to maintain oxygen and pH levels. Oxygen and pH sensors are placed in the vessel lid and descend into the cell media. Oxygen is delivered by a porous filter at the bottom of the reactor vessel. This design utilizes a tried-and-true method of

culturing cells, which should be able to produce sizable quantities of cells. However, when tested, it has not performed up to expectations. As it would require no change from what we already have, this would be the cheapest and easiest option.

21

This bioreactor is different from the other alternatives in several respects. Notably, this is the only model under consideration that includes an impeller circulating the media and potentially coming into contact with the EBs. Like the STLV design, it does not require many new parts—indeed, it requires none. Like the continuous flow model, it utilizes filters to prevent media from becoming polluted. However, unlike either of the other two models, this design produces considerable shear stresses that can impair cardiomyocyte survival and development. For a list of parts and their costs, one is referred to the MAE 156B paper, “Embryonic Stem Cell Bioreactor”.

21 Patent related issues

may arise due to the fact that this is heavily based on the design made by Schroeder, et al. and the model in its current form may use LabView, and uses oxygen and pH sensor manufactured by Mettler Toledo, while the impeller is similar to one produced by Stelzer Ruhrtechnik International GmbH.

21,1

Design Alternative 2: Slow Turning Lateral

Vessel (STVL)

Vessel diameter: 130mm Vessel height: 260mm Vessel capacity: 2L Platform dimensions: 12” x 8.5” Velocity: 0-210 rpm Orbit diameter: 1” Orbital shaker weight: 7lbs Heating tape: 37°C

Figure1B. Modified Shroeder Design

Specifications (SolidWorks)

Figure2. STLV Inspired Design (orbital shaker method) (CAD

and Inventor).

Figure1A. Modified Shroeder

Design(MAE Group)



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(Source: Chang Bioscience) As seen from Figure 2, the STLV technique consists of an orbital shaker placed at the base of the

reactor vessel which rotates at a constant angular velocity to mix the media and cells.25,4,5

The Bioreactor sensors will be suspended in the media. There will also be inlet and outlet tubing in order to input serum and media as well as replacing them. The bioreactor is securely attached to the shaker, possibly with an adhesive, straps or a fastener. This design is based on prior research indicating that the STLV method is useful for large scale production of stem cells.

4,5,3,36 Thus, this could fulfill the need for a 100-fold

increase of stem cells versus the current design, as stated in the needs assessment. Another advantage is that the dead space previously found in other models would be eliminated.

36 In essence, this circulation

of cells via rotation would avoid putting stress on the cells caused by an impeller. For example, a study by Collignon, et al. showed that the fluid mechanics of the fluid due to the blade created a circular flow below the impeller causing a greater stress on the cells.

36 Therefore, by using the rotatory orbital shaker

this limitation is addressed, since the entire bioreactor would be moving rotationally, still in an upright position.

This model is distinct from the other designs proposed in that it uses a rotary orbital shaker to circulate the media and diffuse oxyge n, carbon dioxide, and nitrogen into the media. The reactor vessel is the same as the Schroeder model, but without the need for an impeller and a gas delivery system. This should address concerns about high shear stress and inadequate delivery of oxygen to the cells. The glass water jacket will be replaced with a Velcro heating jacket. Unlike the continuous flow model, this STLV design will require the periodic removal and replacement of media—thus it will be necessary to stop the reactor at intervals in order to exchange media. It will also require few new parts to create from the existing bioreactor. Additionally, and unlike the other two designs, this model will involve the physical movement of the glass bioreactor vessel itself; therefore it is important that the vessel is secured well to the orbital shaker platform. Many of the parts in this model are commercial products which have been patented, and the bioreactor may use LabView as a monitor/controller. Additionally, there are several patents for STLV bioreactors, but these designs differ from the model proposed.

37,38

Parts used for the STLV model will include the existing bioreactor minus its impeller, glass water jacket, and oxygen and gas delivery system. Additionally, an orbital shaker, adhesive tape, security straps, and a silicone heating jacket will be used to create this design. None of these items are custom made, and so the items can arrive soon after ordering.

Table 2: STLV parts list.

Manufacturer Part and Catalog # Quantity Cost Estimate HTS/Amptek 2”x8’ heating tape #AWH-252-080 1 $284.00

ChangBioscience.com Orbital Shaker #KJ-201BD 1 $164.90 Security Chain Company Security Strap #CC1415 1 $14.45

3M Molding Tape #03614 1 $6.86 Total: $470.21 (without heating tape: $ 186.21)

Design Alternative3: Continuous Flow Bioreactor

Reactor vessel volume: 32 oz. Media tank volume: 2 L Both vessels maintained at 37°C Sensors placing in media tank Reactor vessel ID: 4.25” OD: 4.5” Reactor vessel height: 12” Hose ID: 0.197” Hose length: 13”

(Sources: McMaster-Carr; Millipore; Fisher Scientific) (Figure 3 made with AutoCAD and Autodesk Inventor)

This design is a continuous flow type bioreactor that removes much of the sensing equipment from the reactor vessel in order to reduce collision trauma to the cells. The reactor vessel itself contains a filter attached to a hose that continually siphons off a small amount of media without removing EBs. As seen in Figure 3, the siphoned media goes into a separate well where measurements can be taken and re-oxygenation and alteration of the pH of the media can occur. Freshly oxygenated media is then pumped back into the bioreactor at an angle in order to create a whirlpool effect that keeps the cells suspended



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without the use of a propeller to agitate the solution. Media to be filtered is taken from the middle of the reactor vessel while fluid replacement occurs at the bottom.

The continuous flow model is radically different from either the existing model or the STLV alternative. Unlike either of the others, it uses continuous flow to provide fresh media to the cells while simultaneously removing depleted media containing waste products. This design also is more complex than either other design, and may take up more space. Like the STLV model, the continuous flow design should theoretically produce a greater yield of EBs than the existing bioreactor as it would produce less shear stress on the cells. A disadvantage is that unlike the modified Schroeder or STLV models, this design would have a lag time between detecting a change in pH, oxygen, or temperature and actually changing those values in the reactor vessel. A large number of components increases the cost and risk that one part breaking down will cause a failure in the entire bioreactor. This bioreactor would use many commercial parts which have been patented, and may use LabView as a monitor/controller. Although other there are patents for other vaguely similar bioreactors using continuous flow, this one is sufficiently distinct from them.

25 Parts list can be seen in Table 5

Patenting Issues Specific Comparisons to current patents with the two design alternatives

The current bioreactor model shown in Figure1, was taken and inspired by Shroeder’s design which uses an impeller from the top to mix nutrients and stem cells, has pH, CO2 and O2 sensors and uses a water jacket to heat up the vessel

21. The modified Shroeder’s design in Figure1. utilizes an altered

blade, which was rounded at the edges and custom made to fit the need of the MAE group 21

. The group also changed the way gas was delivered and sensed to the cells, by removing a connection to the tank and adding an internal oxygen sparger

21. Some patents already exist for blade impeller based bioreactors,

such as U.S Patent 5075234, which has a double impeller sticking out from the base with flat blades 26

. This patent uses both flat blade and bulb shaped impellers from the base to mix the cells and nutrients

26.

However, since the Shroeder’s modified design has a customized blade, this could be unique to that design as well as the oxygen diffusion system which was placed at the bottom of the bioreactor to reduce oxygen expenditure

21. There is another patent that uses a similar concept, this is patent W.O 2010/08951

A1, which is more of an industrial type, has a larger volume (250L), used for mammalian stem cells, and uses two impellers that are lowered into the solution from the top lid

27.The modified Shroeder’s design

Step2: Put cells

and Media in

corresponding

tanks

Step3: Autoclave Tanks and Secure all Tubing with O-rings

Step1: Sterilize all

tubing and flasks

Step4: Check gas supplies and connection to the controller

Step5: Secure O2, CO2 and NO2 supply and Pump

Step6: Take preliminary sensor data readings

Step7: Start the pump and observe fluid motion in and out of both tanks

Step8: Run experiment for 14 days or until cardiomyocytes are observed

Figure3. Continuous flow Set-up and Components (Inventor)



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will thus be accepted more easily among the public since the design is similar, but uses a modified impeller and tries to minimize oxygen waste by utilizing a sparger

26.

General Patent Requirements and FDA regulations for Tissue Culture Bioreactors

In general, the United States Patent and Trademark Office(USPTO) patent for a variety of devices used in the tissue culture area. Usually a bioreactor would be classified as a utility and machine or device. In order to obtain a patent, the device or machine has to be new and useful according to the US patent office standards

28. From their novelty and use, this would apply to the modified Schroeder bioreactor,

STLV, and Continuous Flow bioreactor design alternatives. One of the advantages of patenting the proposed design, is that Chapter 18 of the USPTO patent

laws (Patent rights in inventions made with federal Assistance) would not be upheld since intellectual property would belong to the students themselves and not to the government or in this case the UC system 29

. Nevertheless, since the UC system has a lot of credibility within the scientific community, the USPTO may be able to patent the device more easily under the UC system. In any case, if an invention is true, testable, new and useful, and complying with all the patent laws, the patent could be reached.

In addition, this gives rise to the discussion about the commercialization of the proposed Continuous Flow Bioreactor since a patent does not necessarily mean commercially ready. The FDA is the regulatory agency that would be able to say whether the product can be marketed or not. Usually, bioreactors are classified as PMAs, which are devices requiring preapproval quality inspection and post-market demands as well

30. This is a disadvantage of the design in the sense that the PMA is a slow

process, and would require testing and extensive studies. Unfortunately, the proposed design would not be able to be filed as a 510(k) for commercially availability because it would require a similar device that has been legally approved for the market and did not require a PMA study

28. After an extensive patent

research, it was concluded that the chance of finding such a device is small, since even if there were parts of the proposed design in a 510(k) classification, the split predicates is not well received within the FDA regulators

30.

Nevertheless, if the device performed well, it would be wise to pursue a commercialization alternative with a Biotechnology company in order to sell it to laboratory settings that would require a high cardiomyocyte yield from an EB culture. Ranking of Design Goals and Ranking Matrix

Table 3: Ranking Matrix

Saf

ety

Envir

onm

ent

al P

rote

ctio

n

Rel

iabil

ity/

Dura

bil

ity

Per

form

ance

Bio

com

pat

i-bil

ity

Eas

e of

Oper

atio

n

Sta

ndar

d

Par

ts

Min

imum

C

ost

Eth

ical

S

tandar

ds

Sust

ainab

ilit

y

Publi

c A

ccep

tance

Tota

l

WF

Safety X 1 0 0 1 1 1 1 0 1 1 7 80 Environmental Protection

0 X 0 0 0 0 0 0 0 1 0 1 10

Reliability/ Durability

1 1 X 1 0 1 1 0 0 1 1 7 85

Performance 1 1 0 X 0 1 1 1 1 1 1 8 90 Biocompatibility 0 1 1 1 X 1 1 1 1 1 1 9 100 Ease of Operation 0 1 0 0 0 X 1 0 0 1 1 4 50 Standardized Parts 0 1 0 0 0 0 X 0 0 0 1 2 25 Minimum Cost 0 1 1 0 0 1 1 X 0 1 1 6 65 Ethical Standards 1 1 1 0 0 1 1 1 X 0 1 7 70 Sustainability 0 0 0 0 0 0 1 0 1 X 0 2 35 Public Acceptance 0 1 0 0 0 0 0 0 0 1 X 2 20 Rationale for the Ranking Matrix

The goals were ranked based on pairwise comparisons of each goal and determining which of the two were more important. This process was repeated for each goal of the matrix. Biocompatibility was ranked highest, due to the fact that in order for the design to meet the stated need, the cells must remain alive. Similarly, performance was given a high ranking because the bioreactor must produce



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cardiomyocytes. Reliability and durability, considered a single goal, were the third most highly ranked because the bioreactor must run for around 14 days, and is designed to be used multiple times. Safety for humans was deemed important because the potential for injury would deter researchers from using the device. Likewise, ethical standards were ranked highly due to the fact that poor ethical standards might lead to opposition to the use of the bioreactor. Next was minimum cost, which was considered of moderate importance, based on the allotted budget of $1,500 and a desire to not exceed this amount. Ease of operation followed and was so ranked on account of the likelihood that researchers would prefer to use an easy-to-operate bioreactor over one requiring a more complex assembly and operation. It was determined that sustainability would largely involve minimizing the use of reagents. As those reagents are easily obtained and biodegradable, sustainability was considered less of a concern. Standardized parts were also considered a minor goal based on the fact that many of the parts needed are in the existing model. Public acceptance was ranked lowly due to the bioreactor’s use in a laboratory that already conducts cultivation of mESCs—acceptance of this bioreactor may be assumed. For the general public, as the bioreactor’s designed purpose is to culture mESCs, there would be less opposition than if the bioreactor was made to culture hESCs. The least important goal was determined to be environmental protection. The rationale behind this is that the bioreactor is made of biocompatible parts and the reagents and cells used are biodegradable and do not contain any infectious diseases, and therefore there is not much risk to the environment.

These rankings were then used to determine the weighting factors (WF) for each goal. The most important goal was given a weighting factor of a hundred, and the lowest a factor of ten. Goals deemed critical were given weighing factors of 70-100; important-but-not-critical goals were assigned weighting factors of 40-69; and goals considered optional received weighting factors of 10-39. Ease of operation, biocompatibility, performance, safety, and cost were ranked highest, the rationale being they are most likely to induce researchers to use the bioreactor. For goals that received identical rankings—for instance, standardized parts, sustainability, and public acceptance—were further distinguished through pairwise comparisons. Reliability/durability was given a WF higher than safety because the bioreactor should be quite safe to any operator, especially the alternatives without an impeller. Sustainability received a weighting factor of 35 based on the fact that minimizing reagent use would also result in a lower operating cost. The goal of standardized parts was therefore considered of less importance than sustainability. Public acceptance received an even smaller weighting factor, again based on the reasoning that researchers would readily accept any of the designs. Rating of Designs and Decision Matrix

Table 4: Decision Matrix Modified

Schroeder Continuous Flow

STLV Weighting Factor

Safety – safety to humans 5 7 6 80 Environmental Protection 10 7 8 10 Reliability/Durability 7 6 9 20 Performance – yield of cardiomyocytes (more shear lowers yield; more continuity will increase yield)

6 9 8 85

Biocompatibility 6 10 8 90 Ease of Operation 8 6 9 100 Standard Parts 10 8 8 50 Ethical Standards 7 7 7 25 Sustainability – not using too much resources 6 9 7 65 Minimum Cost – considers Schroeder’s model to be free

9 7 6 70

Public Acceptance – public is other labs that may use this bioreactor

10 6 8 35

Total Ranking Σ[(Design Goal Score, 1-10)*WF]

4275 4900 4810

Analysis

The three bioreactor designs were then rated on how well they fulfilled the goals, receiving a score from 0-10, with ten being a very high fulfillment of the goal. The goal of safety is met by the



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continuous flow and STLV methods which remove the impeller from the original Schroeder design, which itself is generally safe assuming a person does not touch the moving impeller; the continuous flow model was deemed to be safer than the STLV based on potential injuries caused by the orbital shaker’s movement and the reactor vessel falling. Environmental protection is met by all designs in that they isolate the media and EBs from the outside environment; as the Schroeder model would not use many new materials, it was ranked highest. The goal of reliability and durability is met by all designs in that they could each produce a large yield of cardiomyocytes. However, the Schroeder design would result in cells dying due to shear stresses above 6.1 dyn/cm2, while the continuous flow design is so novel that it is far from certain the yield would consistently be high, in spite of the fact that continuous flow might lead to more uniform conditions and therefore more uniform yield—hence the STLV model is ranked highest due to the low shear stresses involved and an orthodox design. Meanwhile, although all designs meet the performance goal in that they succeed in producing cardiomyocytes, the continuous flow model is ranked highest due to its potential for producing up to 12.5x106 cells/ml, as stated in the functional requirements. The STLV should also produce a high yield, while the Schroeder design’s yield was predicted to be more modest. The goal of biocompatibility is addressed by each design, with the continuous flow model having the highest score. This derives from the flow alternatives use of a Fisher Science pump, intended for processing media, stainless steel parts and biocompatible tubing and vessels

31. These components:

would be heat resistant (up to 175oF)

32; would eliminate toxins in the media; and would not leach agents

into the media which may interfere with EB development. The Schroeder and STLV model also have the cells held in a vessel made of biocompatible materials, with the Schroeder model having reduced points due to its impeller. All the models considered were deemed easy to operate in that there would be little need for much technical expertise to operate the bioreactors and that they all could run with limited human oversight. However, the STLV model was ranked highest based on its simplicity of design, whereas the continuous flow model would require more time to assemble and repair. All models make use of standardized parts, but the Schroeder design is ranked highest since all of its parts are currently assembled and readily usable. Each of the designs also meet the goal of ethical standards equally in that none is more ethically controversial than any of the others. Although the three designs meet the sustainability goal, in that their use of resources is far from excessive, the continuous flow model is scored highest on account of its reduction in media use throughout the culturing period. Each design’s cost is estimated to fall within the $1,500 budget allotted to this project, yet the Schroeder model clearly scored highest for the minimum cost goal because it requires essentially no new materials. For the goal of public acceptance, the ‘public’ was defined to be scientists and researchers who would use one of these bioreactors in their laboratory. Based on this definition, all designs meet this goal in that they culture cells and take up a conservative amount of space. However, the Schroeder design is ranked highest due to the popularity and reputation of the batch reactor/spinner flask method of cell cultivation to which the Schroeder model belongs.

These ratings were then multiplied by the corresponding WF and these eleven values in turn were summed in order to obtain a final score. The continuous flow model received the highest score, 4900. As can be seen, the continuous flow model’s score only slightly surpasses the STLV design’s. The continuous flow alternative was confidently selected as the best option because it is ranked higher than STLV in all of the critical goals (WFs 70-100) except for ease of operation, as well as it being ranked highest for sustainability. Additionally, although it may be cumbersome to set up, the continuous flow model would be easier to operate after its initial assembly.

Part III: Design Solution Rationale for Final Design Choice

The final design choice of the continuous flow bioreactor design was primarily derived from the results of the decision matrix as well as its technical advantages over the design alternatives. As seen from the matrix, the continuous flow bioreactor outscored the design alternatives for the goals of safety, performance, biocompatibility, and sustainability. Though all alternative designs would continue to utilize pressurized gas for the cell environment as well as electrical components for data analysis, the continuous flow system is a safer option for users due to the removal of a spinning impeller and shake table, features of the Schroeder and STLV designs, respectively. The continuous flow also scored highly for performance. Using a controllable peristaltic pump with adjustable flow rates between 0.4ml/min to 600ml/min, media can be moved through 1/4" tubing to generate a vortex which both enhances mixing of gases and nutrients for the developing EBs while also reducing shear. This feature poses an advantage over the Schroeder design, which utilizes a impeller to facilitate mixing that inflicts damaging shear force



December 1, 2011

13

upon the developing EBs, compromising their viability. The adjustable vortex will promote healthy EB aggregation and development, while maintaining low shear forces within the range of 4.5 dyn/cm

2 to 6.1

dyn/cm2, as specified in the functional requirements. Biocompatibility was scored a perfect 10 for the

continuous flow design. The score was chosen primarily due to its ability to operate throughout the EB developmental time period in a closed, self-sustained fashion through continual cycling of media. Through the removal of sensors and access ports from the tank holding the EBs, risk of foreign contaminants will be greatly reduced. Also, by implementing the Sterivac filtration system, up to 10 liters of media can be processed at a time to remove waste which may otherwise harm the cells. In addition, all standardized parts used to assemble the design are primarily composed of polycarbonate, glass, and stainless steel which are compatible with the media conditions. These biocompatibility attributes of the model provide an advantage to the cells the alternative designs do not, and will help meet the functional requirement of producing 12.5x10

6 cells/ml after a 14 day period. The fourth goal in which the

continuous flow design outscored the alternatives was in sustainability. Both the Schroeder design and STLV require removal of media every two days, which exposes the EBs to non-sterile conditions and depletes laboratory resources. The continuous flow design solves these problems, by isolating the cells from the media storage tank where media can be extracted without compromising the cells. In addition, the use of filters will offset the frequency of changing media, reducing the probability of contamination as well as reducing the demand for resources. The high scores the continuous flow design earned for these 4 design goals adds to its overall total ranking score of 4900, surpassing the STLV and Schroeder designs by 90 and 625 points, respectively. Therefore, based on results from the ranking table and the design’s ability to meet established technical requirements, the chosen design is the continuous flow bioreactor. Design description and resources required

The following description can be compared with Figure 3. The readily available tubing from McMaster-Carr is made from polycarbonate, which has been shown to be chemically compatible with culture media. The tubing can also withstand pressures of up to 1,250 psi and endure temperature fluctuations between 32

oF and 250

oF, making it ideal for moving heated media between the EB holding

tank and the monitored tank at high pressures.32

The tubing is also clear, which gives an advantage for continuous visual observations to changes in the hue of the phenol-red pH indicator. The tube fittings have an inside diameter of 3/8”, allowing for a snug fitting of the polycarbonate tube to successfully deliver and export media between the two tanks.

32 Assembly of the tubing and fittings will require

minimal time and resources, attachment ensured through application of glue. The fittings are also pre-sterilized and cheap, allowing for cost effective replacement during media changes. The cost component of repairing the design is also an advantage over the design alternatives. The fittings can also endure temperatures up to 200

oF, easily enduring media temperatures of 37°C ± 2°C.

32 The oxygen delivery

system involves two main components. The first is a re-engineered tire valve which lacks a valve stem for continuous input of oxygen and utilizes step by step instructions.

31 The second component is a

stainless steel tee connector which joins the tire valve body for oxygen delivery to the 1/4" tubing for media transport.

33 This junction delivers fresh oxygen in a continuous manner to the cycling media to be

delivered to the developing EBs within the holding tank. This feature is an advantage over the STLV and Schroder design since it reduces shear and need for an oxygen sparger. To drive the flow of media from the filter tank to the EB culture tank, a peristaltic pump from Fisher Scientific will be used. The pump features a highly controllable flow rate from 0.4ml/min to 600ml/min.

34 This large range in power will allow for an optimal flow rate that will both generate a

vortex within the EB culture tank for increased mixing as well as reduce shear forces on the developing EBs. The pump also features bidirectional flow for easy media drainage and cleaning, is made from chemical resistant material, accommodates 1/4" ID tubing, and can withstand temperatures of up to 175

oF.

34 Incorporation of the pump into the design will only require tube attachments for assembly. The holding tank for EB development will be a 32oz adjustable oil reservoir from McMaster-

Carr.32

The reservoir features both plastic and aluminum material that can withstand temperatures up to 140° F, a vented fill cap, and two outlet ports with manual toggle switches.

32 One outlet port will gravity

feed media back to the monitored media tank and the second outlet port will allow for continuous and noninvasive media sampling. The vented fill cap will also serve as the inlet port for oxygenated media. Assembly may require additional O-rings and fasteners to ensure a seal and proper alignment; no machining of the tank will be necessary. The Sterivac Filters from Millipore will allow for quick media filtration to remove waste and reduce the demand for media replacement. The filters have 1/4" tube adapters, operate through a vacuumed stimuli (which can be generated by the peristaltic pump), and can remain functional at temperatures up to 113

oF.

35 The recycling procedure can be performed by



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14

Figure4. Bioreactor Lid Machine Shop Specifications

(Inventor Creation)

systematically restricting media flow to the monitored media tank, preventing contamination to the growing EBs, and then removing the collected media by way of a vacuum. The filtered media can then be directly added back into the EB culture tank by placing the filter outlet over the opened oil reservoir fill cap.

Using the existing Schroeder bioreactor as the media tank, the impeller and shaft will be removed as well as the oxygen spargers. Remaining will be the O2, and pH sensors. In place of the removed components, an inlet and outlet tube will be inserted into the media tank to transport media following real time data acquisition and carbonation of the solution. Through utilization of the exposed openings, no additional machining of the lid will be required, saving cost and time. Once the media is examined for suitable conditions, it is pumped out of the tank, oxygenated, and then moved into the EB culture tank to supply the cells with nutrients and oxygen.

The estimated total cost of the design parts will be $746.55. No machine shop services, custom synthesis, or specialized equipment will be required. Anticipated additional cost for assembly will be estimated at $50.00 for various screws, glue, and other components to facilitate attachment of the design parts.

Machined Items and Additional Resources Lab space availability is crucial, as well as lab personnel to set up the initial runs. Also, the availability of the client’s graduate students to bring cells into Dr. McCulloch’s lab is important to make design changes at an early stage of the design process. This will allow the necessary time to modify the design if needed. Finally, the machine shop might be used to create a back-up Media Tank (Figure3). However, this is not necessary at this moment since the group already counts with an actual tank with the needed specifications. Parts List Table 5 : Parts List Continuous Flow

Part Supplier Function Catalog Number

Q Cost

Per Unit

Total

Tubing McMaster Transport Media Between Media Tanks 9176T12 4 $2.33 $9.32

Tube Fittings McMaster Connect Tubes to Media Tanks 5116K114 10 $0.25 $2.50

Tire Valve Camel Continuously delivers O2 30-463 1 $3.26 $3.26

Tube Tee McMaster Input O2 into media flow 51065K12 1 $86.60 $86.60

Pump Fisher Scientific Move Media between tanks 13-876-1 1 $286.82 $286.82

Culture Tank McMaster Hold EBs and Support Growth 1067K4 1 $150.15 $150.15

Sterivac Filter Millipore Filters Media SKGPM-10RJ 5 $41.60

$208.00

Media Tank MAE Group Monitor Media Conditions n/a 1 n/a n/a

Total Cost: $746.55

Summary of Strength and Weakness

The manufacturing requirements of the continuous flow bioreactor are minimal. Once the parts have arrived, the assembly of the design can be completed within one day. There are no parts which require custom assembly or machining. The EB culture tank’s vented cap will be used for importing media to generate the vortex and one of its outlet ports will be used to export media to the media tank. The removal of the impeller shaft and oxygen sparging equipment from the existing bioreactor will allow



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15

for ports for media exchange. This will remove any requirements for machining the bioreactor lids or need for custom made parts. The design as a whole can be constructed from readily available, standardized parts. The tire valve can be purchased at an auto part store. A majority of the parts including the tubing, tube fittings, tube tee, and oil reservoir (the EB culture tank) are available from McMaster-Carr which specializes in next day delivery. In addition, the variable flow pump from Fisher Scientific can be shipped the same day of purchase. Lastly, the filters from Millipore can be rush delivered and come in a pack of 5 for long term use. All parts can be easily replaced at low cost except for the pump, filter, and culture tank. The design’s use of tubes to transport media provides a method for systematically sealing off sections of the bioreactor as needed for part replacement in sterile conditions. Hazards and risks of manufacturing would not be a factor in the design due to all parts being readily applicable without further machining. Only minor risks of leaking media may pose as a problem during initial assembly of the bioreactor and minimal materials will be required to seal openings in the system. Use of hazardous materials might include pressurized gases, biohazardous waste, and potentially unsafe chemicals. Included with the pressurized gas is the oxygen delivery system, which can generate a flammable environment from pockets of trapped oxygen or leaks. In addition, spent media, culture vessels, and parts which are in contact with the cycled media must be handled with care and disposed of by following the strict guidelines enforced by UCSD. Lastly, if any potentially hazardous chemicals are used throughout the design process, correct clothing and precautions would be used in order to ensure safe conditions for both the personnel and experiment. Malfunction risk and failure of the continuous flow bioreactor system would primarily result from: the movement of fluid; amount of parts involved; and integration of liquid, gas and electrical components. Though the risks may be significant throughout the EB developmental process, the level of danger to the personnel operating the bioreactor would not be high. Structural malfunctions in the system would more commonly result in leakage of media, clogging of the pump or tubes, and progressive wear on components from stress. The level of danger for the developing EBs due to malfunction in the bioreactor would be significantly more. Failure in the oxygen delivery system, malfunction in the regulation of pH, temperature, and carbon dioxide from damaged sensors; and inadequate mixing of nutrients would all result in a dramatic drop in cell viability. Malfunctions in the flow speeds may increase shear forces on the cells and low speeds may not be sufficient to generate the vortex for mixing. Therefore, overall the design poses minimal risk in case of malfunction to the personnel operating the system. However, such failures would be detrimental to cell survival. Gantt Chart The Gantt Chart below shows the timeline that the group is intended to fulfill by the end of the design process. The chart is broken down into the main stages of the design process and with subtasks pertaining to the specific stage. Throughout the Fall quarter, the group has been on track with the main subtasks and is fully intending to complete those steps of the design that were belated such as the testing of the old design. This will be done during the second week of December expecting results ~14 days later, being supervised by Dr. Zambon and Dr. McCulloch. Some of the limiting steps were the safety training courses and the lab availability early in the quarter. The testing of the old design/current model includes; building by protocol, understanding the details of the current model, observation of the experiment runs, and recording of environment and media changes. This will allow to have a base data to compare the new design runs and quantify its performance.



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DUR Who

TASK

(16-2

2)

(23-2

9)

(30-3

1)

(1-5

)

(6-1

2)

(13-1

9)

(20-2

6)

(27-3

0)

(1-3

)

(4-1

0)

(11-1

7)

(18-2

4)

(1-7

)

(8-1

4)

(15-2

1)

22-2

8

29-3

1

1-4

)

(5-1

1)

(12-1

8)

(19-2

5)

(26-2

9)

(4-1

0)

(11-1

7)

(18-2

4)

(25-3

1)

Interview Client 1w TEAM a a

Read Shroeder Paper 1w TEAM a

Write draft 1w CM, CA, AM a

Literature research 2w CA, CM a a

Write final paper 1w TEAM a

Literature research 2w TEAM a a

Brainstorm Meeting 4d TEAM a a

Write Draft 1w AM, CM, CA a

Write final paper 2w TEAM a a

Patents research 4w CA a a a a

Draw CAD Figures CA, CM a a a a

Risk analysis 2w TEAM a a

Detailed Protocol 1w TEAM a a

Parts List 2w AM a a

Write final paper 3w TEAM a a a aSafety Training 1d CM, EY a

Testing old design M M M

b. Setup 4d JEN M

c. Building by

protocol EY, CA M M

d. Experimentation 3w EY, CA M M

e. Result collection 1w EY, CA MAnalysis of Current Model

Results 2w TEAM

Order Parts 2w AM

Additional Safety Training 3w TEAM

Prototype building 4w AM, CA, CM

Prototype testing 4w TEAM

Data collection 4w AM, CM

Data Analysis 3w TEAM

Write Draft Report 2w TEAM

Write Final Report 3w TEAM

JAN FEB MAR

ANALYSIS/CONCEPT SELECTION

IMPLEMENTATION

PROBLEM FORMULATION

BRAINSTORMING

FALL QUARTER WINTER QUARTER

OCT NOV DEC

KEY Description

Fall Quarter Milestones

Winter Quarter Milestones

a Done

To do (on track)

M Moved to date in orange square

To do (belated)

CA Constance Ardila

CM Christopher Meeks

AM Aaron Mitchell

EY Edward Yragui

JEN Dr. McCulloch's Lab Personnel

TEAM All Team Members



December 1, 2011

17

References 1. Schroeder M, Niebruegge S, Werner A, Willbold E, Burg M, Ruediger M, Field LJ, Lehmann J,

Zweigerdt R. “Differentiation and Lineage Selection of Mouse Embryonic Stem Cells in a Stirred Bench Scale Bioreactor With Automated Process Control” Biotechnol Bioeng. 2005 Dec 30;92(7):920-33.

2. Rungarunlert S, Klincumhom N, Bock I, Nemes C, Techakumphu M, Pirity MK, Dinnyes A. “Enhanced cardiac differentiation of mouse embryonic stem cells by use of the slow-turning, lateral vessel (STLV) bioreactor” Biotechnol Lett. 2011 Aug;33(8):1565-73. Epub 2011 Apr 8.

3. Sargent CY, Berguig GY, Kinney MA, Hiatt LA, Carpenedo RL, Berson RE, McDevitt TC. "Hydrodynamic Modulation of Embryonic Stem Cell Differentiation by Rotary Orbital Suspension Culture" Biotechnol Bioeng. 2010 Feb 15;105(3):611-26.

4. Carpenedo RL, Sargent CY, McDevitt TC. "Rotary Suspension Culture Enhances the Efficiency, Yield, and Homogeneity of Embryoid Body Differentiation" Stem Cells. 2007 Sep;25(9):2224-34. Epub 2007 Jun 21.

5. Kehoe DE, Jing D, Lock LT, Tzanakakis ES. "Scalable Stirred-Suspension Bioreactor Culture of Human Pluripotent Stem Cells" Tissue Eng Part A. 2010 Feb;16(2):405-21.

6. International Stem Cell Research . In Stem Cell Information [World Wide Web site]. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services, 2011 [cited Sunday, October 16, 2011] <http://stemcells.nih.gov/research/intlresearch>

7. Shrink Nanotechnology . In Biological Research Tools [World Wide Web site]. Shrink NanotechnologiesInc. , 2011 [cited Sunday,October16,2011] Available at <http://www.shrinknano.com/products/product-tools/>.

8. Uthayashanker R. Ezekiel, Mariappan Muthucham, Jan S. Ryers, Rita M. Heuert. “Single embryoid body formation in a multi-well plate” Electronic Journal of Biotechnology ISSN: 0717-3458

9. Kurosawa H, Imamura T, Koike M, Sasaki K, Amano Y. “A simple method for forming embryoid body from mouse embryonic stem cells.” J Biosci Bioeng. 2003;96(4):409-11.

10. Chen M, Lin YQ, Xie SL, Wu HF, Wang JF.“Enrichment of cardiac differentiation of mouse embryonic stem cells by optimizing the hanging drop method.” Biotechnol Lett. 2011 Apr;33(4):853-8. Epub 2010 Dec 17.

11. Sargent CY, Berguig GY, McDevitt TC. “Cardiomyogenic Differentiation of Embryoid Bodies Is

Promoted by Rotary Orbital Suspension Culture” Tissue Eng Part A. 2009 Feb;15(2):331-42. 12. James A. King, William M. Miller. ”Bioreactor Development for Stem Cell Expansion and

Controlled Differentiation” Curr Opin Chem Biol. 2007 August; 11(4): 394–398. 13. Cameron CM, Hu WS, Kaufman DS. ”Improved development of human embryonic stem cell

derived embryoid bodies by stirred vessel cultivation.” Biotechnol Bioeng. 2006 Aug 5;94(5):938-48.

14. Kenneth R. Bohele et al. Differentiation of Pluripotent Embryonic Stem Cells Into Cardiomyocytes. 2002

15. VandeWoude, Susan, and Bernard E. Rollin. (2010) Practical Considerations in Regenerative Medicine Research: IACUCs, Ethics, and the Use of Animals in Stem Cell Studies. ILAR, 51.1: 82-84. http://dels-old.nas.edu/ilar_n/ilarjournal/51_1/PDFs/v5101VandeWoude.pdf

16. UCSD Guidelines For Human Embryonic Stem Cell Research. (2011) Stem Cell Research. UCSD Research Affairs. , http://escro.ucsd.edu/UCSD_guidelines.html

17. Cartwright, Terence. Regulatory Aspects of Using Cells as Bioreactors. Animal Cells as Bioreactors. Cambridge: Cambridge UP, 1994. 129-35. Print.

18. Exec. Order No. 13505, 3 C.F.R. 10667 (2009). Print. 19. NIH Human Embryonic Stem Cell Registry . (2011) US Department of Health and Human Services.

NIH. Retrieved from http://grants.nih.gov/stem_cells/registry/current.htm 20. “Mouse Facts.” Mouse Genome Informatics. (2011, October 19). General format.

http://www.informatics.jax.org/mgihome/projects/aboutmgi.shtml 21. Chun, Christina, et al. (March 2009). “Embryonic Stem Cell Bioreactor.” MAE 156B, UCSD. 22. Schroeder, et al. (June 30, 2005). “Differentiation and Lineage Selection of Mouse Embryonic Stem

Cells in a Stirred Bench Scale Bioreactor With Automated Process Control.” Biotechnology & Bioengineering. 97(7), 920-933.

23. Cormier, Jaymi T., et al. (November 2006). “Expansion of Undifferentiated Murine Embryonic Stem Cells as Aggregates in Suspension Culture Bioreactors.” Tissue Engineering. 12(11), 3233-3245.

http://www.shrinknano.com/products/product-tools/

http://dels-old.nas.edu/ilar_n/ilarjournal/51_1/PDFs/v5101VandeWoude.pdf

http://escro.ucsd.edu/UCSD_guidelines.html

http://www.informatics.jax.org/mgihome/projects/aboutmgi.shtml



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24. Kehoe, Daniel E., et al. (February 2010). “Scalable Stirred-Suspension Bioreactor Culture of Human Pluripotent Stem Cells.” Tissue Engineering: Part A. 16(2), 405-421.

25. Surapaneni, K. (2010) U.S. Patent No. 20100144022. Washington, D.C: U.S. 26. Tunac, Josefino . “Fermentor/bioreactor systems having high aeration capacity.” Patent 5075234. 24

December 1991. 27. Khan, Moshan. “Bioreactor for the cultivation of mamillian cells” International Patent 000783. 9

Feburary 2010. 28. Lecture, FDA Regulation, Dr. Micou. 22 Sept 2011. 29. Patent Laws, Appendix L, http://www.uspto.gov/web/offices/pac/mpep/consolidated_laws.pdf, 6

September 2007. 30. Yock, Paul. PMA regulatory Strategy. http://www.stanford.edu/group/biodesign/cgi-

bin/ebiodesign/index.php/component/content/article/353?template=skeleton-video 31. Fisher Scientific. www.fishersci.com. 32. McMaster-Carr. www.mcmaster.com. 33. Richard J. Kinch http://www.truetex.com/carbonation.htm 34. Drillspot Inc. http://www.drillspot.com/products 35. Millipore Inc. www.millipore.com 36. Collignon, M.L., et al. “A Study of the Mixing by PIV and PLIF in bioreactor of cells animals

culture.” Sixth International Symposium on Mixing in Industrial Process Industries. 2008 37. Gerecht-Nir, et al. U.S. Patent No. 20110027887. Washington, D.C: U.S. Nov. 30, 2003. 38. Cailleret, et al. U.S. Patent No. 20100197007. Washington, D.C: U.S. August 5, 2010.

Work Breakdown Constance Ardila: Created design renderings using SolidWorks, AutoCAD and Inventor. Design Alternatives Description Edition, Gantt Chart scheduling and description. Additional resources. Researched Patents and FDA Regulations. Discussed Patenting Issues and Laws. Advantages and Disadvantages of New Model. References and Citations. Christopher Meeks: Design Goals, Ethical Considerations, Laws/Regulations. Goal Ranking, Weighting Factors, Ranking rationale, Decision Matrix, and Decision analysis. Table of Contents and cover page. Secondary initial editing and formatting. Initial modeler of STLV figure. Aaron Mitchell: Wrote new parts for: identification of the final design and rationale used, description of the design, summary of the designs strengths/weaknesses, additional resources needed and parts description. Researched, contacted suppliers, and formulated the parts list. Researched assembly methods and special requirements for the design. Set up meetings for the group. Organized and compiled the projects rough draft, performed primary initial editing, and formatting. Edward Yragui: Wrote new parts for the strengths and weakness of the final design and the similarities and differences between the two. Revised Needs Assessment, Intended Use, Target Audience, and Current State of the Technology. Cited references. Performed initial and final editing and formatting of the paper.

http://www.patentstorm.us/applications-by-date/2010/0805/1.html

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