Interview Questions. K: Marketing is not in the current plan. 6. Who will be using it? S: Undergrad students. 3rd-5 th years. Two professors.

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1 Interview Questions 1. What issues are we currently encountering with cell culturing? S: It is a brand new device. Want under flow instead of 2D plastic surface. So far, developed advanced cell culture techniques. Can mechanically and electrically stimulate cells. Flow is more complicated. K: This is a brand new area, culturing under flow. New tech. elective called Advance Cell Culture Techniques and would like to add culturing cells under flow to the curriculum. It is more complicated than the basic 2d or 3d static cultures. 2. What experiments are you planning to do? S: No specifics. Variables we would change are cell type, shear stress on cells (velocity/flow rate), chemical stimulation. K: No specific exps. Variables include cell type (just a few relevant for flow), shear stress (velocity/ flow rate), and chemical stimulation (media additives) 3. What is the goal of the advanced cell culture class? S: To teach different cell culture techniques. To learn about advanced tissue engineering. To utilize and carry out [missed it]. Basic migration assays. Electrical and mechanical stimulation. K: To teach different culture techniques. More advanced tissue engineering. Builds on skills learned in bio class like aseptic techniques and mammalian cell culture. Want to go into electrical stimulation and migration assays. 4. What is the device s purpose/plan? S: Use for students experiments. Learn about technique of performing experiments and analyzing data. Collect protein secretions, staining cells, function and phenotype. Morphology? K: Use the device for experiments to allow the students to learn about culturing cells under flow. Students will learn from the technique of culturing under flow and from the data collected from the experiment. Data examples are protein secretion and cell staining, to be carried out after the flow experiment has finished. 5. Are you looking to learn from the system or market a product? S: Learn from the system. For experimentation, not for research. Only for teaching labs. K: Marketing is not in the current plan. 6. Who will be using it? S: Undergrad students. 3rd-5 th years. Two professors. K: 3-5th year students primarily

2 7. How frequently will it be used? S: Used in class 2-5 times a year. Or up to 20 times a year. Depending on resources. Spend 2 weeks on each one. Used 6 weeks by 3 different groups. Class size is 12. So 24 students a year. Groups of 3-4 at once. K: 2-5 times a year, up to 20 times potentially.. Range from once a semester to multiple lab groups per semester as they rotate through different labs. 8. How many people do you think would work with the device at one time? S: Ideally want to run 4-7 at a time. Students could take turns using one. However, it could take up an entire incubator by itself and we can only use one chamber at a time. Pump limits cost (pump is expensive). K: There is a cap of 12 students per class, so 24 a semester. Split into groups of 3 or Where will this system be kept? (hopefully will have a better idea of size by her answer, as opposed to How large are you expecting the system to be? which will lead to a solution oriented answer.) S: Store anywhere. Modular aspect is for analysis. Unhook flow to view cells immediately. Ideally flow should run 24/7. Cells spend 23.5 sitting in incubator, except when check on growth and status. Cell culture flow while viewing it is ideal, but if cannot, it s okay. Long tubes are hooked up to CO2 tanks. Split tubes near incubator. Can unhook from CO2 if look in microscope. System should last 5 years. K: The culture chamber needs to fit on general class microscope. Off of the scope could be the pump or the media reservoir. Stored on a shelf it would take up a lot of space, so a modular set up may be ideal where the chamber can be unhooked from flow to view the cells. Ideally cells would be under flow 24/7 but to unhook to view would be fine. 10. What level of human interaction with the system are you expecting? S: Temperature and CO2 need to be automated. Hands on: needs to be checked on microscope once a day/as needed. Media is changed by hand. Color of media indicates it is old. It becomes acidic-turns yellow as cells eat media. Depends on volume of media, # of cells. Media gets changed 2-3 days. Depends on size of reservoir. Perhaps once a week.

3 K: The temp and CO2 need to be automated. Media changes and viewing the cells will be more hands on. As cells digest media it becomes acidic and turns yellow (from pink) Media digestion is dependent on media volume, cell density, and cell growth. 11. Are the experiments with the cells terminal? S: Experiments are terminal. Won t pull cells out. Can collect cell parts/protein RNA. No freezing of cells. K: Terminal. Passage seed run exp remove from chamber run analysis 12. What different types of cells will be analyzed using the system? I think this is answered already, endothelial cells? Was there a different motivation behind this question? I think it gave that as one example we can run with. Might there be other cells with which it could be used? S: Relevant ones for flow (endothelial). Number of cells: just a few cells. K: Cells that react to flow, mostly endothelial. 13. What process is to be used to sterilize the system? S: Sterilization is done to remove bacteria. Done in culture hoods in petri dishes. Do not want to pipette material into system. Have to figure out sterilize system before you use it. Autoclave it. High pressure steam 220 deg F. Pressure?==22 PSI. All autoclavable equipment. 10% bleach. 70% ethanol. Gamma radiation requires special equation. Have all options. Except gamma radiation. Chamber and media container, the insides have to remain sterile. Tubing, etc. have to be sealed. K: Autoclave ideal- high pressure and temperature steam. Plastics can melt, so we need to be careful. There are autoclavable bags to use. The chamber inside needs to remain sterile, the outside becomes unsterile as you walk around with it, but thats fine. Other sterilization options are 10% bleach or 70% ethanol. Gamma radiation isn t really an option for us, but is another sterilization technique in general. 14. Why can we not filter media through 0.2 micron filter? S: Media can t be filtered through 0.2 micron filter. Media is sterile common way to do this. Media for endothelial cells requires serum (larger particles) don t want to have to sterilize it through filter, filter takes nutrients with it. K: The media will be sterile and needs to remain so in the system. One way that is usually used to sterilize the media is by forcing the fluid through a 0.2 micron filter. In

4 our case, this will also filter out the large proteins in teh serum that is used as a supplement in the media. 14. What are the ideal materials? may be a solution oriented question Answered above. 15. What disposables and how many of them are available for use? S: Pipettes, petri dishes, media, cells to test system with, flasks, spatulas. K: pipettes, petri dishes, media, cells, beakers/flasks, spatulas 16. What parts of the chamber are to be disposable? S: Cell culture surface may be disposable if it gets [missed it]. Cells sit on protein, not plastic. Petri dishes are treated so they are chemically active to promote adhesion. Tubing may be replaceable. K: None. Potentially the cell culturing surface. Cells do not sit on plastic, they sit on proteins, so they will not stick to general plastics. Another disposable/replaceable part could be the tubing. Not a significant portion of the device is to be disposable. 17. What labs or equipment are available for use? S: Student projects lab (Michelle s office). Need access to biosafety cabinets. Have teaching lab prep space with biosafety cabinets. K: The students project lab near Michele's office. There is no Bio Safety Cabinet (BSC) in that lab so for cell work we will move to the teaching lab prep space. 18. What equipment needs to be compatible with the system? 19. What does appropriate cell culture equipment mean? 20. What is the typical process for cell culture? 21. What are typical culturing conditions? 22. Describe the dynamics of the flow. S: Shear stress ranges (she needs to double check numbers) fluid mechanics gives you flow rate range. Viscosity of the media (pretty close to water, varies depending on serum). K: Shear stress ranges are the only thing the cells care about. No ranges were given in the PRP, she will follow up with the required info. 23. What sensory data needs to be collectable and to what degree of accuracy/precision? 1. % from 5%. 24. Are you expecting a finished, working model or a prototype for another group to advance? 25. Are you intending to produce multiple systems based upon this one?

5 26. Do you know of any other devices similar to this one already in existence? Probably, but they are expensive 27. Which needs should take the most priority? S: Sterility, CO2 and temperature. Microscopes have flurorescent capabilities--- will arrive in 1-2 months. We need dimensions on the microscope parts. K: Sterility. Then CO2 and temperature. Then compatibility with the microscope dimensions available in class. There will be a different fluorescent machine that will also need compatibility. 28. When are you typically available? If have any questions, can ask anytime. Bi- and tri-weekly. 29. How frequently would you like to meet with us? Available generally 12:30 P.M. every day. Not available: Labs Wed & Fri at noon. Thurs evening 4-7 How tight is the budget? Ask Dan Phillips. S: Culture chamber has to rest on microscope. Media reservoir to the side.