BI258Dr. Joshua Slee

DeSales UniversityNovember 2 – December 7, 2017

Making it Stick: A CURE Designed to Introduce Students to Culturing THP-1 Suspension Cells and the Host Response to Implantable Biomaterials

This lab was designed by Dr. Joshua B. Slee in collaboration with Dr. Jacqueline S. McLaughlin and is an adaptation of the four step-step pedagogical framework

demonstrated in: (McLaughlin & Coyle, 2016)

Objective and Introduction:In this laboratory, you will be utilizing a four-step process to design and execute an authentic research project. This experimental framework is consistent with the manner in which professional research scientists devise, design, execute, interpret, and communicate their experimental results. You will use the knowledge and skill set you previously acquired in the THP-1 Cell Attachment Assay Lab to design an experiment which determines the efficacy of different material coatings at preventing the Foreign Body Response in vitro. The experimental question which you should be able to answer by the end of this lab series is “Will ________ decrease the number of adhered THP-1 cells to polyurethane, a commonly used polymer in implantable devices? To accomplish this goal, you will complete the following four tasks.

1. Select a passive coating that you believe has the potential to affect the adhesion of THP-1 cells to polyurethane films. You will perform a search of published literature to assist you in choosing your variable. Your instructor will help guide you through the process of performing a literature search.

2. Formulate a hypothesis regarding the effects of this passive coating on THP-1 cell adhesion, basing it on information you find in a search of the published scientific literature on this topic. You will design an experiment which incorporates your passive coating into a THP-1 Attachment Assay, using an appropriate control. You will write up this experiment as a traditional research protocol, with numbered steps, data tables, and recipes included throughout. Your instructor will review your experimental design before you implement it, to ensure that the protocol you develop is relevant and executable within the time frame of the course.

3. Execute this experiment and gather data. This will require coordinated work with you and your lab partners, and you will be expected to make good use of open laboratory time, in addition to scheduled class time. Your instructor will be available to you for assistance or questions, but the expectation is that your work will be self-directed and paced appropriately.

4. Review your results and interpret your data. If your data is puzzling or unclear, your instructors can help you make sense of it. Once you are able to draw the conclusions of your experiment, you will present it to your instructors and classmates in the form of a presentation.

Figure 1: The four-step process of this laboratory (McLaughlin & Coyle, 2016)

Background Information: Often overlooked in many cell biology laboratory and cell culture courses, suspension cells represent an important aspect of cell biology and cell culture. Most primary cell cultures and cell lines are adherent cells which grow in monolayers on surfaces. However, other cells such as hematopoietic cells, certain tumor cells, and cells of the immune system are suspension cells which are anchorage-independent and grow and divide in solution. THP-1 cells are an excellent model for suspension cell culture. THP-1 cells are a spontaneously immortalized monocyte-like cell line derived from the peripheral blood of a one year old male infant with acute monocytic leukemia (Tsuchiya et al., 1980). They are excellent models of monocytes and macrophages (Bosshart & Heinzelmann, 2016; Qin, 2012) and are a valuable model for studies of the immune system (Chanput, Mes, & Wichers, 2014). Researchers have used THP-1 cells to study the host response to implantable devices and biomaterials in vitro. Tissue contacting surfaces of implantable materials initiate a host inflammatory response characterized by inflammatory events, one of which includes macrophage attachment to the biomaterial, which ultimate leads to degradation and failure of the material (Slee, Christian, Levy, & Stachelek, 2014). Using a THP-1 adhesion assay (Slee et al., 2016), researchers can begin to elucidate mechanisms to prevent the host response to implantable medical devices and potentially ensure the long-term success of the device.

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Materials: THP-1 cells RPMI-1640 media

o + 5% FBSo + 0.05 µM 2-MEo Differentiated with 1.6 x

10-6 M PMA (Phorbol 12-myristate 13-acetate)

Student-selected coatings 1% Formaldehyde DAPI Polyurethane films PPEs (smock, gloves, safety

glasses) 70% EtOH 37 °C incubator 1/5/10 mL pipettes

Pipettor Fresh culture medium 37 °C incubator, 5% CO2

Cells in T flask Waste beaker Trypan blue Cytometer and coverslip 1X PBS 15 mL conical tube 1.5 mL microcentrifuge tube

Microcentrifuge T-25 flask 24-well TC dish Micropipette and tips (10 µL) Inverted microscope Tally counter

Protocol Overview:

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Protocol:1. Warm RPMI media in 37 °C incubator.

2. Clean hood with ethanol. Spray ethanol liberally over surfaces and wipe clean.

3. Sterilize all materials, bottles, etc. which are loaded into the hood. Spray hands with ethanol. Spray jars of liquid with ethanol. Place sterile pipettes directly in the hood.

4. Label new T flasks and 24-well dish with group name, date, passage #, cell line, and treatment. (control vs. coated)

5. Cut polyurethane films into 1 cm x 0.5 cm rectangles and place in wells of a 24-well dish.

6. Check cells in the T flask under the microscope to confirm that the cells are healthy and densely populated.

7. Using a micropipette, transfer 10 µL of cell suspension from the T flask into a sterile 1.5 mL microcentrifuge tube. Replace the T flask into the incubator during cell counting.

8. Add 10 µL of trypan blue to the same 1.5 mL microcentrifuge tube (from #7) and triturate.

9. Add 10 µL of trypan blue/cell suspension to a hemocytometer and visualize the hemocytometer grid under the microscope.

a. Trypan blue is a "vital stain"; it is excluded from live cells.i. Live cells appear colorless.

ii. Dead cells stain blue.iii. To aid accuracy and consistency of cell counts use the counting

system illustrated in Figure 2C.

b. Count viable (live) and dead cells in the four large corner squares and record cell counts.

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10. To Calculate Cell Viability:

No . LiveCellsTotal No.Cells

X100 %=%Viability

11. To Calculate Cell Concentration per ml:

( AverageNo .Viable Cells ) ¿

*Dilution Factor in our case is 2 (1:1 dilution with trypan blue)**10,000 = conversion factor to convert mm2 to mL

12. To Calculate Specific Seeding Density (specific # of cells):

Seeding Denisty∗¿CellConcentration∗¿

=Volume of Cells ¿ Add(mL)

* Seeding Density = 300,000 cells in each well. Multiply 300,000 cells by the number of wells you are making. The number you calculate becomes the numerator in the equation. **Calculated in Step #11

For example, seeding density is 300,000 cells/well. Cell concentration is 500,000 cells/mL. We have plans for 3 control wells with films and 3 experiment wells with films. 300,000 cells/well ÷ 500,000 cells/mL = 0.6mL/well. So, we need 0.6mL/well x 6 wells= 3.6mL from original T flask.

13. Trypan blue/cell mixture is biohazardous and should be disposed in biohazardous waste.

14. Add the necessary volume determined in step #12 to X mL of cell solution in a new 15 mL conical tube.(Any excess cell solution in the original T flask should be returned to the incubator.)

15. Centrifuge cells at 3,000 rpm for 3 min to pellet the cells.

16. Remove supernatant from pellet using a sterile 10 mL pipette and place in waste beaker as soon as possible.

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17. Re-suspend (triturate) the cell pellet in X mL of new pre-warmed media (1ml/well).

For example, if we have a plan for using 6 wells, add 6mL of new pre-warmed media into a 15mL tube with cell pellets.

18. Activate/differentiate the THP-1 cells by adding the 1 µL /well of stock PMA (1.6 x 10-3 M) to achieve a final concentration of 1.6 x 10-6 M(1600nM) PMA.

For example, if we have a plan for using 6 wells, add 6 µL PMA into a 15mL tube.

19. Add 1 mL of the PMA-activated cell solution from the new conical tube to the required well of the 24-well dish and gently swirl to mix.

20. Place TC dishes in 37 degree and 5% CO2 incubator.

21. Dispose of liquid and solid biohazardous wastes properly.

22. Clean hood with ethanol. Spray ethanol liberally over surfaces and wipe clean.

23. After incubating the TC dishes for 2-3 days, rinse the dishes with 1X PBS three times.

24. Add 1 mL of 1% formaldehyde to each well and incubate for 10 min at room temperature to fix the cells.

25. Rinse the dishes with 1X PBS three times. Remember that up to three rinses contain formaldehyde and need to be placed in an appropriately labeled waste beaker.

26. Add a few drops (enough to cover polyurethane film) of Vectashield plus DAPI(1.5ug/ml) and incubate at room temperature in the dark for 30 min.

27. Remove the polyurethane films from the dishes and place them on labelled microscope slides. The polyurethane films cannot be mounted to the microscope slides.

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a. Polyurethane is not fully optically clear, but it is clear enough to visualize through most fluorescent microscopes. Depending on the orientation of the optics of the microscope, the polyurethane films may need to be placed cell-side up or cell-side down on the microscope slide.

28. Randomly select 10 fields of view under 200X total magnification and count the number of nuclei you see. Record these numbers for each polyurethane film you have.

a. It is important to randomly select the fields to count, or you may be biasing your data. The best way to do this, is to close your eyes, move the stage to another location, open your eyes, and count what you see.

b. By counting the nuclei on each film, you are counting the number of adhered THP-1 cells to the films.

29. Create bar graphs by plotting the type of polyurethane (modified vs. unmodified/control) on the X-axis and the average number of cells/200X field on the Y-axis.

30. Do this experiment at least three times, so you can run statistical analyses on your data to determine significance.

References:

Bosshart, H., & Heinzelmann, M. (2016). THP-1 cells as a model for human monocytes. Annals of Translational Medicine, 4(21), 438–438.

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http://doi.org/10.21037/atm.2016.08.53

Chanput, W., Mes, J. J., & Wichers, H. J. (2014). THP-1 cell line: an in vitro cell model for immune modulation approach. International Immunopharmacology, 23(1), 37–45. http://doi.org/10.1016/j.intimp.2014.08.002

McLaughlin, J. S., & Coyle, M. S. (2016). Increasing Authenticity & Inquiry in the Cell & Molecular Biology Laboratory. The American Biology Teacher, 78(6), 492–500. http://doi.org/10.1525/abt.2016.78.6.492

Qin, Z. (2012). The use of THP-1 cells as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature. Atherosclerosis, 221(1), 2–11. http://doi.org/10.1016/j.atherosclerosis.2011.09.003

Slee, J. B., Alferiev, I. S., Nagaswami, C., Weisel, J. W., Levy, R. J., Fishbein, I., & Stachelek, S. J. (2016). Enhanced biocompatibility of CD47-functionalized vascular stents. Biomaterials, 87, 82–92. http://doi.org/10.1016/j.biomaterials.2016.02.008

Slee, J. B., Christian, A. J., Levy, R. J., & Stachelek, S. J. (2014). Addressing the Inflammatory Response to Clinically Relevant Polymers by Manipulating the Host Response Using ITIM Domain-Containing Receptors. Polymers (Basel), 6, 2526–2551. http://doi.org/10.3390/polym6102526

Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., & Tada, K. (1980). Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). International Journal of Cancer, 26(2), 171–6. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/6970727

Figure 2 – A Typical Cytometer

Top view (A) and side view (B) are shown with dimensions. The squares you should count are magnified and numbered in (C). Proper cytometer loading is illustrated in (D)

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(B)

(A)

(C)

Top View

Side View

(D)