Growing Cells in Culture

1. Growing Cells in Culture Part 1: Terminology

2. Pros Use of animals reduced Cells from one cell line are homogenous and have same growth requirements, optimizing growing patterns. In vitro models allow for control of the extracellular environment Able to monitor various elements and secretions without interference from other biological molecules that occurs in vivo Cell Culture The maintenance of cells outside of the living animal (in vitro) for easier experimental manipulation and regulation of controls.

3. Cons • Removal of cells from their in vivo environment means removing the cells, hormones, support structures and various other chemicals that the cells interact with in vivo. • It is nearly impossible to recreate the in vivo environment. The artificial conditions could cause cells to de-differentiate which will cause them to behave differently and produce proteins other than it would in vivo. • Genotype: the genetic make-up of the cell • Phenotype: the appearance and behavior of a cell as a result of their genotype. Most often, scientists are looking at phenotypic changes in their analysis of cells in culture

4. Classification of Cell Cultures • Primary Culture • Cells taken directly from a tissue to a dish • Secondary Culture • Cells taken from a primary culture and passed or divided in vitro. • These cells have a limited number of divisions or passages. After the limit, they will undergoapoptosis. • Apoptosis is programmed cell death

5. Primary culture from Poeciliopsis lucida (the desert topminnow)

6. Making a Primary Culture

7. Cell Lines • Cell Line • Cells that have undergone a mutation and won’t undergo apoptosis after a limited number of passages. They will grow indefinitely. • Transformed cell line • A cell line that has been transformed by a tumor inducing virus or chemical. Can cause tumors if injected into animal. • Hybrid cell line (hybridoma) • Two cell types fused together with characteristics of each

8. Our Cell Line • PLHC-1 Poeciliopsis lucida (topminnow) • Hepatocellular carcinoma • Originially from the liver so they are “hepatocytes” • Epithelial cells • ATCC CRL-2406 • http://www.atcc.org/ • Lawrence E. Hightower’s lab, in culture since 1985.

9. Our Cell Line • An immortal cell line, but not tumorogenic, will reach contact inhibited state • Originally used to study heat shock response • These cells maintain a number of differentiated cell functions of hepatocytes. The cells possess inducible and stable cytochrome P450 (CYF) activity. • Not known to harbor an agent known to cause disease in humans

10. Growing Cells in Culture Part 2: Understanding Cell Behavior

11. Confluency • How “covered” the growing surface appears • This is usually a guess • Optimal confluency for moving cells to a new dish is 70-80% • too low, cells will be in lag phase and won’t proliferate • Too high and cells may undergo unfavorable changes and will be difficult to remove from plate.

12. Contact Inhibition • When cells contact each other, they cease their growth. • Cells arrest in G0 phase of the cell cycle • Transformed cells will continue to proliferate and pile upon each other

13. Anchorage Dependence • Cells that attach to surfaces in vivo require a surface to attach to in vitro. • Other cells or specially treated plastic or other biologically active coatings • Blood cells are primary exception. • Transformed cells may not require attachment.

14. Passage number • The number of times the cells have been removed (or “split”) from the plate and re-plated. • Always write this on your plate or flask as P#

15. Growing Cells in Culture Part 3: Solutions used in cell culture

16. Phosphate Buffered Saline - Ca2+ Mg2+ Free (PBS) • Used to wash/remove excess serum that inhibits the function of Trypsin-EDTA. • Calcium will also inhibit the function of TRED. • Must be warmed in the water bath before use so cells are not shocked by cold liquid.

17. Trypsin EDTA • An enzyme used to detach the cells from a culture dish. • Trypsin cleaves peptide bonds (LYS or ARG) in fibronectin of the extracellular matrix. • More about fibronectin and the ECM next week • EDTA chelates calcium ions in the media that would normally inhibit trypsin. • Trypsin will self digest and become ineffective if left in water bath more than 20 minutes. • Trypsinizing cells too long will reduce cell viability

18. Trypan Blue • An exclusion dye • Living cells cannot take up the dye and will appear bright and refractile. • Dead cells with broken membranes will absorb the dye and appear blue. • Usually add 200 ml of trypan blue to 200 ml of cell suspension in eppendorf tube

19. Bleach • Used to destroy any remaining cells in dishes and tubes before they are tossed in the trash can. • Add enough to change media to clear, • wait 5 minutes, • rinse solution down sink • throw away the dish/flask/plate in the trash can.

20. Growing Cells in Culture Part 4 : Equipment

21. CO2 incubator • maintains CO2 level (5-10%), humidity and temperature (37o C) to simulate in vivo conditions.

22. Water bath • To warm media, TRED and PBS before placing on cells • Can harbor fungi and bacteria, spray all items with 70% ethanol before placing in the hood. • Usually takes 10 -15 minutes for media to warm, 5-10 for TRED to thaw

23. Vacuum pump • For permanent aspiration of liquids (media, PBS and TRED). • Use unplugged glass pasteur pipets, throw into sharps box when done.

24. Inverted Phase Microscope • A phase contrast microscope with objectives below the specimen. • A phase plate with an annulus will aid in exploiting differences in refractive indices in different areas of the cells and surrounding areas, creating contrast

25. Mechanics of phase microscopy Shifting of phase by ½ a wavelength Add and subtract amplitudes to create more contrast

26. A comparison Phase contrast microscopy Light microscopy Can be used on living cells requires stain, thus killing cells

27. Basic cell culture instructions

28. Aseptic Technique • For best results in tissue culture, we want to work to keep microbial (bacteria, yeast and molds) contamination to a minimum. To do this, there are certain things you must be aware of and guidelines to follow. • Work in a culture hood set-aside for tissue culture purposes. Most have filtered air that blows across the surface to keep microbes from settling in the hood. Turn off the UV/antimicrobial light and turn on the hood 30 minutes prior to entering the hood. • Wear short sleeves or roll your sleeves up. Turn your baseball caps back if you MUST wear them, tie long hair back and remove rings and watches.

29. Wash hands with soap and water before beginning the procedure and rewash if you touch anything that is not sterile or within the hood. • Spray down your hands, work surface, and anything that will go into the hood with 70% ethanol. Rewipe at intervals if you are working for a long time in the hood. This will reduce the numbers of bacteria and mold considerably. • Do not breathe directly into your cultures, bottles of media, etc. This also means to keep talking to a minimum. No singing or chewing gum.

30. Work as quickly as you can within limits of your coordination. Also, keep bottles and flasks closed when you are not working with them. Avoid passing your arm or hand over an open bottle. • Use only sterilized pipets, plates, flasks and bottles in the hood for procedures. • Take special precautions with the sterile pipets. Remove them from the package just before use. Make certain to set up the numbers on the pipet so that they face you. Never mouth-pipet, use the pipetting aid. Change pipets for each manipulation. If the tip of the pipet touches something outside of the flask or bottle, replace with a new one. Never use a pipet twice.

31. Basic Cell Culture Procedure for Anchorage Dependent Cells • View cells using inverted phase microscope • Aseptically aspirate media • Rinse media with PBS • Add Trypsin-EDTA to cells • Aspirate Trypsin-EDTA • Incubate cells with layer of Trypsin-EDTA at 37° C • Resuspend cells with fresh media • Take sample and count cells • Calculate how many cells are needed to add to new plate or flask

32. Remember • Some volumes don’t need to be exact in cell culture • Rinsing volume of PBS (as long as it fits in the dish and is sufficient to rinse the serum). • Volume of trypsin EDTA as long a bottom of plate or flask can be covered. • Volume of media used to resuspend your cells. The same number of cells will be there despite the volume of media used. • Too little resuspension media will result in very high cell count and would require more dilution (and higher dilution factor). The volume needed to seed your next plate would then be very small, maybe too small to work with. • Too much media would result in low cell count/ml and you may need a large volume to add to your new plate.

33. Volume of cells removed for cell counting. • You want enough to work with, but not take all of your cells from your plate. If you want a dilution factor of 2, just add an equal amount of trypan blue. • Exact # of cells to be plated • If you want to plate 2 x 10 5 cells onto your plate, but you have 2.1 x 10 5 cells/ml, plating 1ml will be easier than plating .953 ml.

34. Troubleshooting Low Hemacytometer Counts

35. Trypsinization not complete • Trypsin is ineffective • too cold, be sure to warm sufficiently • self digested or expired check date, don't warm too long • too much serum left on plate rinse plate thoroughly with PBS

36. Trypsinization technique • Trypsin doesn't coat plate, completely add full 2 mls, lay flask down, count to 10, then remove • trypsin left on plate too long and then aspirated...cells removed along with trypsin • not left long enough in incubator depends on cell line 3T3-L1 can go 1-5 minutes • flask may need to be tapped or slapped to facilitate cell removal(this varies by cell line, but ok for 3T3s)

37. Resuspension technique • too much media added more media results in low cell/ml, but overall cells on plate should remain the same • cells not sprayed off surface properly • media and cells not pipetted (gently) up and down 3-4 times to break up clumps • too long of time before retrieving sample from flask (cells may settle). After mixing with trypan, don't wait too long before loading hemacytometer.  Get hemacytometer ready while trypsinizing cells in incubator

38. Stubborn cells • cells left on plate a long time (>4 days) will be more difficult to remove • very confluent plate will require more aggressive trypsinization because trypsin cannot recach plate surface effectively

39. Keeping a good lab notebook

40. Lab notebooks provide a convenient place for you to keep all of your procedures, data and observations in one place. • If written well, a lab notebook should contain everything you need to know to allow you or someone else to repeat any experiment you have ever performed. • It can be useful in finding the source of errors and unexpected results when problems arise. • Should your work ever be disputed, a lab notebook will provide testimony to your research. • By following the simple guidelines below, you will learn how to keep a good lab notebook.

41. The notebook should be bound (no spiral notebooks, please). • The pages should be numbered either by hand or preprinted before using the book. • Use only permanent ink. • Write your name, contact information, and dates the notebook covers on the first page. • Skip the next 2-3 pages for a Table of Contents. Fill in the experiment name and page numbers as they are completed. • Write the date, experiment title, and partner’s name at the top of each page.

42. The first time you use a procedure • Write the whole procedure in your own words into the notebook OR tape in the typed version • Include a reference to the lab manual page or the published procedure. • Note any changes made to the original procedure. • Do not just copy the lab manual or procedure word for word; restate each step simply and clearly. • If you repeat this procedure later, reference the page where it was first performed and write down any changes made.

43. All data and observations should be written in your notebook at the time you took the measurement. Do not write on scratch paper to be copied later into your notebook – little pieces of paper may be lost and data forever lost. • Remember your lab notebook is extemporaneous writing. Keep it neat but do not waste too much time making it perfect. Errors should be crossed out with a single line (example). Do not scribble out mistakes.

44. Write down all calculations, no matter how simple, in your notebook. For example, every time you perform a cell count, cell viability must be calculated and recorded. • Permanently attach (glue or tape) images, computer print outs, and other data in your notebook. Date and initial over the corner of the attachment. Be sure to label the image with any pertinent information. [For example, if you place a Western Blot image into your notebook, label the lanes with what was in each, and the gel composition. If the lysates were prepared on a date different from the date the gel was run make a reference to the page that contains information on how the lysates were made.] Partners may photocopy original data for inclusion in the lab notebook.

45. Including complete chemical equations, statistical equations, sample calculations, and sketches or block diagrams of any apparatus used is also good practice. • Record start and stop times. • Include conclusions from this data. What does it mean and did it work as expected? If unexpected results occur, explain why. Include expected values (with reference) where appropriate. • Do not skip pages. Use every page of the notebook. If you need to rewrite a page, draw a large X through the page, date, initial, and start over on the next page. The same applies if you don’t fill an entire page draw a line through the remaining space, date, and initial.

46. Six Essential Calculations

47. Hemacytometer • Specialized chamber with etched grid used to count the number of cells in a sample. • use of trypan blue allows differentiation between living and dead cells

48. Using the Hemacytometer • Remove the hemacytometer and coverslip (carefully) from EtOH and dry thoroughly with a kimwipe. • Center coverslip on hemacytometer • Barely fill the grid under the coverslip via the divet with your cell suspension. • Count cells in ten squares (5 on each side) by following diagram at station.

49. Looking at the grid under the phase contrast microscope

50. How the cells will appear • Bright refractile “spheres” are living cells, • Blue cells about the same size as the other cells are dead. • Keep a differential count of blue vs. clear for viability determination. • Sometimes there will be serum debris, and this will look red or blue and stringy or gloppy--don’t count it! These are blood cells, You will not have this many