Index
Cell Studies
on PLLA Films
You will be required to demonstrate the viability, attachment and proliferation of HDF (human dermal fibroblast) cells on PLLA films. You must apply the techniques learned in BIOE 342 and adapt the protocols, as necessary, to explore a number of questions. Specific protocols for conducting these experiments will not be given. You will need to adapt and develop protocols by trial and error. You have only six class periods (plus 3 sessions on Friday or Saturday, as needed) to complete your experiments.
The following topics should be addressed during your experimental work.
· Demonstrate the viability of HDF cells on PLLA films during 2-3 days of culture.
· Demonstrate the attachment of HDF cells on PLLA films as a function of time during 4-6 hrs of culture.
· Demonstrate the proliferation of HDF cells on PLLA films during 5-7 days of culture.
· Describe how HDF cells attached to and proliferating on PLLA films are similar to and different from HDF cells attached to and proliferating on control surfaces.
The following materials and equipment are available:
DMEM with 10% serum and antibiotic
PBS
Ethanol
Trypsin
All materials and equipment necessary for cell passaging procedure
TC-treated and untreated 12-well plates
T-75 flasks
Tally counter
Light and fluorescent microscopes
Coulter Counter and hemocytometer
Vacuum grease
Timer
The following materials can be prepared if requested. Please notify Dr. Saterbak several hours - 1 day before you need these reagents:
DMEM with 1% serum and antibiotic
DMEM with 5% serum and antibiotic
DMEM without serum and/or antibiotic
Live/Dead reagent stock solution
Fibronectin
Reagents for MTT viability test
Let Dr. Saterbak know if you need any additional materials (e.g. reagents and antibody for Anti-PCNA staining protocol). If the item needs to be ordered, expect a 3 day delay.
You will be
responsible for making enough PLLA films for all your cells tests. Confluent flasks of HDF cells will be given
to you in Session 5. You will be
responsible for maintaining that HDF cell line throughout your
experiments. You will also be
responsible for planning your experiments and passaging to appropriate
dilutions so that your flask(s) is confluent when you wish to begin an
experiment. As a rule of thumb, a
You may conduct a protocol that was not conducted in BIOE 342. However, you need to discuss the protocol with Dr. Saterbak before you begin. If reagents need to be ordered, expect a 3 day delay.
Like the rest of the experiments in the Tissue Engineering Module, partners may share tasks. For example, both partners do not need to conduct a successful attachment assay; only one partner needs to conduct each assay.
General Experimental Information
1. The surface area of 12-well plates is 4 cm2.
2. The media volume requirements for 12-well plates are 1.5-2 mL/well.
3. The PLLA films have been cast on 18 mm round glass coverslips. The coverslips easily fit into 12-well plates. However, the coverslips must be secured to the bottom of the wells for the cell experiments. A protocol for this procedure is given below.
4. The films and the 12-well plates must be sterilized. A protocol for this procedure is given below.
5. Before trypsinizing cells from a PLLA film, the film/coverslip must be removed from the original plate and transferred to another. A protocol for this procedure is given in the “HDF Proliferation on PLLA Films” protocol.
Securing Coverslips with Films to 12-Well Plates
· Materials include PLLA films on glass coverslips, 1 mL micropipette tips, vacuum grease, TC-treated 12-well plates, and tweezers.
· Wear disposable nitrile gloves.
· You may want to practice this procedure using glass coverslips without films and 12-well plates.
· This procedure should NOT be done in the laminar flow hood.
OPTION 1
1. Place a small amount of vacuum grease onto the back of a glass coverslip (i.e., not the side with the PLLA film).
· Use the pointed end of a 1 mL micropipette tip to add vacuum grease to the center of the coverslip.
· Spread the grease around in a circle smaller than the coverslip; usually, a 10 mm diameter circle is good. Grease should not be within 2-4 mm of the edge of the coverslip.
2. Place the PLLA film/coverslip into a well of a 12-well plate using tweezers or forceps.
· Ensure that the PLLA film side is up and the side with vacuum grease is down.
· Lightly press coverslip with the blunt end of a 1 mL micropipette tip to ensure good contact.
· Grease should not ooze out around the sides. If it does, you have too much grease; discard this well.
3. Repeat until you have added as many films/coverslips as necessary.
OPTION 2
1. Place a small amount of vacuum grease into each well.
· Use the pointed end of a 1 mL micropipette tip to add vacuum grease to the center of the well.
· Spread the grease around in a circle smaller than the coverslip; usually, a 10 mm diameter circle is good.
2. Place the PLLA film/coverslip into a well of a 12-well plate using tweezers or forceps.
· Ensure that the PLLA film side is up.
· Make sure that the coverslip covers the grease.
· Lightly press coverslip with the blunt end of a 1 mL micropipette tip to ensure good contact.
· Grease should not ooze out around the sides. If it does, you have too much grease; discard this well.
3. Repeat until you have added as many films/coverslips as necessary.
Sterilizing 12-well Plates Containing PLLA Films and
Glass Coverslips
1. Place the 12-well plate containing PLLA films into the vacuum oven for 2 hours.
2. Place the 12-well plate containing PLLA films under UV light in a laminar flow hood for 24 - 48 hours. Do NOT leave the films under the UV light for more than 48 hours. Leave the lid OFF the plate. The plate and its lid may turn slightly yellow/brown during this sterilization procedure.
HDF Viability
on PLLA Films
Goal
· Demonstrate the viability of HDF cells on PLLA films during 2-3 days of culture.
Safety
1. Wear disposable latex gloves at all times
2. Wear safety glasses at all times.
3. Dispose of biohazardous materials properly.
Question to Consider for the Viability of HDF Cells on
PLLA Films
1. Are HDF cells that are attached to PLLA films viable? What fraction of the HDF cells is viable?
2. How does the fraction of viable HDF cells on the PLLA films compare to the fraction of viable HDF cells on a control surface?
Hints on Viability Study
1. Films/coverslips must be securely attached to 12-well plates. The films and 12-well plates must be sterile.
2. Think about and conduct appropriate controls. What surface(s) is a true control? Is a negative control (killing the cells with some agent such as ethanol) important to conduct?
Dr. Saterbak will prepare the Live/Dead Reagent when you ask.
HDF Attachment
on PLLA Films
Goal
· Demonstrate the attachment of HDF cells on PLLA films as a function of time during 4-6 hrs of culture.
· Describe how HDF cells attached to PLLA films are similar to and different from HDF cells attached to control surfaces.
1. Wear disposable latex gloves at all times
2. Wear safety glasses at all times.
3. Dispose of biohazardous materials properly.
Question to Consider for the Attachment of HDF Cells on
PLLA Films
1. Do HDF cells attach to PLLA films?
2. What is the morphology of HDF cells attached to PLLA films during the attachment process? How does the morphology of HDF cells attached to PLLA films compare to HDF cells attached to a control surface?
3. Are the cells attached on the top of the film or beneath it? Are many cells attached to perimeter of well (i.e. the ring of the well not covered by the film/coverslip) that is exposed to the media and cells?
4. How many films should be assessed at each time point?
5. What are the kinetics of cell attachment on PLLA films over a 5-6 hr period?
6. How do the kinetics of cell attachment on PLLA films compare with the kinetics of cell attachment on a control surface?
7. Assess the variability between samples taken at the same time point.
8. Is the overall attachment fraction (cells attached divided by cells seeded) different between the conditions of seeding on PLLA films and on a control surface?
9. Does the number of cells attached to PLLA films plateau during the time course of your experiments? How will this result affect the design of your proliferation assay?
10. How do surface properties such as the hydrophobicity and roughness of the PLLA films affect the attachment of HDF cells?
11. With what materials or proteins could you treat the surface to enhance cell attachment rate?
Hints on Attachment Study
1. Films/coverslips must be securely attached to 12-well plates. The films and 12-well plates must be sterile.
2. To assess attachment to PLLA films, seed cells at a concentration two to 10 times higher than what was used in the Quantitative Cell Attachment Assay from BIOE 342.
3. Cell attachment on the PLLA films is slower than on TC-treated wells. Using the times from the attachment experiment in BIOE 342 may not be best choice. Check attachment at 5-6 hrs after seeding.
4. Conduct a “quick-and-dirty” experiment to determine an appropriate cell seeding concentration and appropriate time points for measurement before conducting a “full-blown” experiment.
5. Some practice is required to focus in on cells attached to the films. Removing the green filter may help. Some level of visual inspection and counting is necessary to answer some of the questions above. Work with Dr. Saterbak or a T.A. to confirm that you can visualize the cells attached to the films. Alternatively, devise another method to quantify the number of attached cells.
6. Think about and conduct appropriate controls.
If you choose to trypsinize cells during any type of attachment assay, see the protocol for trypsinization on the following pages.
HDF Proliferation
on PLLA Films
Goal
· Demonstrate the proliferation of HDF cells on PLLA films during 5-7 days of culture.
· Describe how HDF cells proliferating on PLLA films are similar to and different from HDF cells proliferating on control surfaces.
Safety
1. Wear disposable latex gloves at all times
2. Wear safety glasses at all times.
3. Dispose of biohazardous materials properly.
Question to Consider for the Proliferation of HDF Cells
on PLLA Films
1. Do HDF cells proliferate on PLLA films over a 5-7 day period? Are the cells viable at 5-7 days? Plot the cell number as a function of time.
2. How does cell proliferation on PLLA films compare with cell proliferation on a control surface?
3. Does the number of cells on the PLLA films plateau during the time course of your experiment? What surface concentration is “confluent” on a PLLA film?
4. How many films should be assessed at each time point?
5. Are the cell numbers at different days during your experiment statistically different from one another?
6. Assess the variability between samples taken at the same time point.
7. Determine the HDF cell doubling time on the PLLA films. How does this value compare to the HDF cell doubling time on a control surface?
Hints on Proliferation Study
1. Films/coverslips must be securely attached to 12-well plates. The films and 12-well plates must be sterile.
2. Select an attachment time for plating the cells that is conservative. Confirm that the cells have attached before rinsing.
3. Seed cells at a concentration of 1x104 - 5x104 cells/well for wells containing PLLA films. (Using a cell concentration in this range, however, does not guarantee success.) Note that this concentration is higher than the seeding concentration given for TC-treated wells in BIOE 342.
4. Conduct a “quick-and-dirty” experiment to determine an appropriate cell seeding concentration and appropriate time points for measurement before conducting a “full-blown” experiment. This is particularly important for the cells seeded on the PLLA films.
5. You should always look at your cells before you trypsinize them. Some practice is required to focus in on cells attached to the films. Removing the green filter may help. Work with Dr. Saterbak or a T.A. so you can see the cells and estimate their confluency.
6. Before trypsinizing cells from a PLLA film, the film should be removed from the original 12-well plate and transferred into another 12-well plate. A protocol for this procedure is given below.
7. Think about and conduct appropriate controls. Do the PLLA films degrade over time to form small particles that could be counted by the Coulter Counter?
NOTE: You do NOT need to show that proliferation is dependent on serum concentration.
Trypsinizing Cells Attached to PLLA Films/Coverslips
You must remove each film/coverslip from its initial well before beginning the trypsinization protocol. If you do not remove the film, cells which are attached at the perimeter of well (i.e. the ring of the well not covered by the film/coverslip) and cells attached under the coverslip will be trypsinized and counted - leading to an artificially high assessment of the cells that are actually attached to the film.
1. Rinse with PBS as necessary.
2. Carefully remove the film/coverslip from the well using tweezers and/or spatula.
· Slide the film/coverslip around in the bottom of the well to loosen.
· Be careful not to separate the film from the coverslip.
· Be careful not to break the coverslip.
3. Place the film/coverslip in a dry, clean well.
4. Rinse with PBS as necessary.
5. Add trypsin and proceed with your protocol.