Cell Culture and Maintenance
You will now begin to learn to handle cells under sterile conditions. YOU will be responsible for maintaining your cell lines throughout the semester, so good sterile technique is important.
Each student will be given a dish with growing Dictyostelium discoideum cells. (A detailed background of this organism is found in the supplemental reading attached). You will harvest the cells from the dish and determine the “titer” (concentration) of cells. Then you will set up a fresh “culture” in which you will maintain.
Harvesting Cells from a plate (using Sterile Technique)
1.
Option A: AT THE BENCH
Pick up the plate in your non-pipetting hand so that your thumb is in the front holding the plate and lid and your middle finger is in the back holding the plate only. Now use your index finger to lift the lid at the rear so that you can insert the pipette through the opening. This lifts the lid away from you so that you protect the plate from you as the source of contamination
Option B: INSIDE THE HOOD
If you are working in a hood, you can either use the technique above or actually lift the lid off the plate and set it down, sterile side up, inside the hood. Now pick up the plate making sure your fingers do not touch near the sterile top of the dish.
2. Pipet up most (you don’t want to suck up bubbles) of the media from the dish and rapidly expel it onto the cells on the surface. The force of the liquid stream will blow the cells off the surface, but not damage them. You can actually see the change in the transparency of the plate as the cells come off. Move the pipette in a pattern to cover all parts of the dish so that you remove all cells. It will take you 3-4 times for one half of the dish, and then 3-4 times for the other half of the dish to get all the surface washed.
Before you remove the media and cells, check with the microscope to see that you a have removed most of the cells.
IMPORTANT: Keep an eye on both the liquid in the pipet and the location of the tip at the same time. Stop the flow before all the liquid has exited the pipette so you don’t make bubbles which make further pipetting much more difficult.
3. After you remove most of the cells from the surface of the plate, you now want to remove them from the plate to use them for your experiment. Suck up most of the media and dispense it into a sterile 15 ml centrifuge tube. Take the empty plate and tube back to your bench and take a look under the microscope. See how well you did in terms of getting all the cells off the surface. You can now add 10ml of fresh HL-5 back to the plate and place it in your box (if you find your Dicty have new “friends” next lab meeting you may want to keep an eye on your sterile technique a bit more).
Note: Cells will settle and reattach to the plate within 10 minutes or so. Do not let the plate sit long before harvesting or else you will get no cells.
Maintaining Cultures of Dictyostelium
1.General Facts
a.Dictyostelium cells grow with a generation time of about 3-12 hours depending upon the conditions.
b.Regardless of how many cells you start with, the cells will grow until they reach stationary phase (106-107 cells/ml).
c.They will stay in stationary phase for a while, and then begin to die
d.Doing experiments with stationary phase cells will not work as well as using cells in log phase of growth.
e.You can fool cells at any density into thinking they are in log phase by giving them fresh media and they will return to growth for a time. The more cells in the dish,the shorter the time.
2.Handling cells
a.You should keep a 100mm dish of cells in log phase at all times. This means keeping an eye on the cells over time, learning to recognize what a dish that is nearing stationary phase looks like and knowing how and when to transfer cells to a new dish
b.The cells can be maintained in two ways. Each is useful depending on when you need cells and how many you need. Sometimes you will want to start multiple dishes of cells to have enough for an experiment.
3.Transfering Cells- If a dish is nearing stationary phase you have two options
a.Triturate the cells to remove them from the bottom, aspirate as much media as possible, then add back fresh media.
b.Swirl the dish to get some cells in suspension, and pipette a small amount to a new dish containing media.
c.Triturate the cells and add some to a new dish.
d.The choice between b and c depends on how many cells you have. If the number is small and most are stuck to the bottom, you will get very few using method 2. If you need to have a certain number of cells on a given day, then you need to use the hemocytometer to measure the cell number and calculate the number you need in the dish.
Passaging Cells:
Dictyostelium discoideum (or Dicty) is a soil amoeba that lives by eating bacteria and other microorganisms via phagocytosis. However, there are strains of Dicty that can survive without ingesting solid particles as source of nutrients. They live by a process called pinocytosis or cell drinking. One of the strains called NC4A2, which is an axenic strain (which means nothing alive-i.e., no bacteria) will be the organism that we will use for most of our experiments. NC4A2 grows well in liquid broth medium, HL5. Cells will grow until they become saturated and then begin to die, and so you need to passage your cells periodically to keep them happy. Happy cells make happy experiments!
There are several ways of passaging cells:
1.When the titer gets too high, just wash down the surface of your Petri dish, pipet out all the cells you can, throw them away, and add media back (you will find there will always be plenty of cell left in the dish to keep the culture going). Using this method you don’t need to start a new dish every time your cells need passage.
2.If you need many cells for an experiment, then don’t remove all the cells. You can remove 5 ml of the media and add 5ml fresh media to refeed them. Alternatively you could remove 9 ml and add back 9 to give a 1/10 dilution of the cells.
3.If you need to leave the cells for a long time before you need them (a week or so) then you can put 10ml of media in a sterile dish and add a very small volume of cells (a few microliters). Once you know the growth rate of the cells and their titer, you can predict quite accurately how long it will take cells to overgrow the plate.
4.You can also change the media. In this case you leave the cells attached to the plate and remove the media to give the cells fresh media. Cells secrete factors into the media that allow them to assess their growth state. Cells will also deplete the media of necessary components. Changing the media resets the clock on cell growth without having to harvest the cells. This is a good thing to do the day before an experiment.
Note on Mammalian Cell lines and passaging
Some mammalian cell lines can be handled exactly like Dictyostelium. Some are more fastidious in their handling requirements. Most are much more adherent than Dicty to plastic so you can’t simply blow them off. Two methods are commonly used: First is to scrape them off with a sterile disposable plastic scraper. Second is to treat them with a solution containing EDTA which chelates calcium that is necessary for adhesion. Some are stuck so hard that you have to add the protease trypsin and EDTA. This protease will chew off exposed surface proteins and so break the links with the plastic.
Counting Cells Using a Haemocytometer
Now you want to determine the cell titer using a haemocytometer. The bottom glass part of the unit has two counting chambers. When you put the coverslip onto the supports, it creates a chamber of a precisely known volume. You then count the number of cells using a grid ruled onto the glass surface and since the counting area is a known size, and the volume is a known size, you can derive the titer of the cells you added.
This simple looking device is very expensive because of the precision necessary to create the chamber. BE VERY CAREFUL HANDLING IT! It should never move farther than your benchtop and the drawer it is stored in!!! If you never hold it over the floor, you will never drop it on the floor.
Procedure:
1.Make sure cells are fully detached from plate. Using 20-200 ul micropipette, take up 10-12 ul of culture making sure the coverslip is on the hemocytometer.
2.Gently expel the liquid into the “V” (this should move across the measuring field via capillary action).
3.There are two identical chambers so you could count two samples at one time. Use both and count both to check your reproducibility.
4.Carefully lift the hemocytometer to the microscope and start counting the cells. Don’t forget to have your cell counter at hand. This will make the counting easier.
5. Focus the field of view of the microscope to the space beyond the “V” on the hemocytometer using a low power objective (10X). You will see a grid of lines set up like a tic tac toe board. In each corner there is a 4x4 grid (circled in one corner) and a central area with a 5x5 grid as shown in the figure below. All are the same exact size!
6. Move field of view to center measuring field (see figure).
7. Count the cells in the central 25 squares to give you the most accurate cell titer.
➤ this number is the number of cells x 104 = cells/ mL
For example,
If you count 45 cells, you have 45 x 104 cells/ml or 4.5 x 105 cells/ml
If the volume of culture is 10 mL, then you have
45 x 104 cells/mL x 10mL = 45 x 105 cells = 4.5 x 106 cells
You may count one square and multiply by 25, but this is a very rough estimate and should not be used to determine an accurate titer.
If you have very few cells, you may use the central area plus the outer 4x4 grids and then divide by 5 to average. To estimate a higher titer, you may count one row of cells and multiply by 5.
In general, to get good statistics, you should count about 100 cells.
Experiment 2: Growth of Dictyostelium discoideum in Shaking Suspension and Plates
You will set up cultures at about 1x105 cells/ml in petri dishes for attached cell growth & flasks for shaking suspension growth, and follow the increase in titer of the cells over the rest of the week and weekend and calculate the growth rate (doubling time) of your cells for both conditions.
Setting up Cell Cultures
(**Recall everything you do from here is done using sterile technique**)
Starting with the right number of cells, the key is to start off with enough cells to allow them to grow in log phase for several days since it will be those data points needed for the generation time (time it takes the population to double).
First, wash the cells from your stock culture and place them in a 15 ml sterile tube. Titer the cells using the hemocytometer.
To calculate the number of cells to add to your new culture, you can use the equation
C1 x V1 = C2 x V2
Where C1= the number of cells you calculated from your titer
V1= the volume of your initial culture to put into your final volume
C2= the cell titer you want
V2= the volume of HL-5 you will start your growth curve culture in
Your calculations may look something like this if you have a culture that is at 6x107 cells/ml and you want to start a new culture that is at 5x104 cells/ml in a total of 10ml:
(hint-it is sometimes useful to write down all your data before you begin)
C1 = 6 x 107 cells/ml
V1 = unknown (need to calculate)
C2 = 5 x 104 cells/ml
V2 = 10 mL of HL-
Now calculate your unknown (how much of your starter culture to add to the new culture using this algebraic equation: (6x107)(V1)=( 5x104)(10mL)
V1 = .0083mL Convert to uL (.0083 mL)(1000µl/mL) = 8.3µL
Vortex the tube to resuspend all the cells and add this volume of cells to 10mL of fresh HL-5 in a dish and begin your growth curve. For shaking suspension, add this calculated volume to 10mL of fresh HL-5 in a flask, and place the flask on a shaker. (At time 0, the first Y-value for concentration will be 1 X 105, right? Wrong- you need to measure the titer in the flask and plate and see if you did it right. What you measure is the starting titer).
Alternative Method: You want a culture of 1x105 cells/ml in 10ml which means you need 1x106 cells. So how much volume of your 6x107 culture do you need for 1x106 cells?
(6x107 cells/ml) X = (1x106 cells) X=(1x106)/(6x107)ml
Determining Growth rate (generation time)
Once cultures are set up, take an immediate sample to check your starting cell density to make sure you calculated and pipetted correctly. For the Petri dish, if you do this immediately after adding the cells, you can simply swirl and take a sample. If you wait, you will need to detach them again from the surface by triturating.
Titer the cells in your petri dish & flask twice per day using the hemocytometer.
For flasks, take a sterile Pasteur pipette and simply dip it into the solution. Touch the tip tot the hemocytometer and capillary action will pull some into the counting chamber. Do not leave the flasks on the bench while counting. Take your sample and immediately return the flasks to the shaker. If you don’t the cells will settle to the bottom and begin attaching to the glass
For plates, triturate the cells to remove them from the surface and then use the residual small volume on the end of the pipette to add to the hemocytometer.
Record the concentration and time when you took the titer in your notebook.
Growth curves are usually done over the course of five to seven days. This will allow you many data points which when plotted on a log scale should give you a pattern of log growth then a plateau. For the growth curve, you will be most interested in the data points which are part of the logarithmic part of the growth curve. It is from this data that you will be able to calculate generation time.
Presentation of Data
1.For your result, you will be handing graphing the data from shaking suspension and plates as time vs. titer. This could be done using Excel or other spreadsheet programs.
2.Make sure you use a XY scatter so that the X values represent numbers and are correctly spaced.
3.Plot both a linear graph and a semilog graph of the data and note the difference. You may also want to graph the shaking vs. the plate separately or the mutant vs. the wild-type.
4.In the semilog graph, the linear portion of the data represents exponential growth. Therefore if your cells were not in log phase (exponential growth) throughout the experiment, you will need to replot the portion that is exponential alone, so that your trend line is only for that portion of the data. Cells will eventually go into stationary phase, and you don’t want that affecting your calculation of the growth rate. So in your writeup, note the difference between the log plot and the linear and what portion you chose for your calculation.
5. Number your graphs (Figure 1 etc.) and refer to them by number in the Discussion.
Biologically, you are starting with a certain number of cells and over time the cell number will increase as an exponential function. Mathematically it looks like Nt = N0 2tf
So the cell number at time t (Nt) is equal to the starting cell number increased by a factor of 2 each cell generation. t= time (hr) and f=generation frequency (generations/hr).
What you want to figure out is the generation time (hr/generation) which is 1/f. So basically when one generation is passed you have 21 times as many cells etc.
The simplest way to estimate out the generation time is to estimate based upon the raw data. Look for the period when the cells are growing exponentially (so-called mid log phase). Then figure out how long it took to get twice as many cells simply by looking at the numbers. If the cells increased 2 fold over two days then they are dividing once per day (or about a 24 hour generation time). If they increase 6 fold then you calculate from 2x=6 telling you how many generations they did in that amount of time.
Plotting in Excel [Office 2008]
Here is a detailed protocol how to make your graph in Excel 2008.
1.First enter all your data into the spreadsheet. It does not matter if it is in rows or in columns.
2.Highlight all your data then click the gallery icon. This icon will open a huge gallery divided into four sections. Click the Chart gallery, and select XY scatter. This will open different types of XY scatter charts and you can select the one you want by clicking on the chart. Once you click a chart, the chart you selected with you data on it will appear in a separate box.
3.To label Axes, click on the toolbox icon and a box will appear. Now, select your graph by highlighting it and go to chart options in the toolbox. You can choose any category from the dropdown menu, and below that, there is a box where you can type the Chart title, vertical axis and horizontal axis of your chart. Once you are done typing, you can immediately see it in your chart.
4.Chart legends. Legends are important in some cases but not in others. If you want to include the growth of two different cell types in a graph, then legend should be shown to distinguish between the two populations. The legend box is by default shown everytime you make a graph. If you don’t want to show the legend, right-click the legend box and choose delete. If you want to edit the legend, double click the legend box and a box labeled format legend will appear. You can play around how your legend would look like using this box.
5.Formatting Axes. In case you want to change the scale (i.e., minimum and maximum number) in the X or Y axes, double click on the values of the axis you want to change then a box named format axis will appear. Now you can enter values you want for your minimum and maximum numbers. Not only that, you can also change major and minor units that separate the values you indicated and will appear as lines in your graph. You can also play around with graph and line color,weight, etc... using the format axis box.
6.To create a semilog chart, double click the values in the Y axis. In the format axis box, there are three choices given below the box. Check the logarithmic scale box and now, your Y axis is in logarithmic scale while your axis is still in linear scale.
7.Assuming that your semilog chart only contains the log phase data extracted from the growth profiles of your cells, you want to determine the doubling time or generation time of these cells. To get the generation time, click the data in the graph and from the chart dropdown menu, click add trendline. A box for format trendline will appear and on the side menu, click type. This will open different types of trendlines. Click on exponential then go back to the side menu and click options and then check the box for display equation on chart and then click OK.
8.The equation given in the trendline can be used to calculate for generation time indicated in method 1 below.
Method1. On the linear plot in Excel, you will get a curve that starts low and then steeply curves upward. This is the essence of exponential growth. You have few cells for a long time and then all of a sudden the population explodes as doubling gives you a greater increase with each generation. If you apply an exponential curve fit to the data:
(Chart/Apply Trendline (exponential) and then in options check the show equation box)
You should see a nice fit to your data. To check yourself, create an imaginary dataset with perfect numbers doubling at some arbitrary rate of your choosing.
Excel fits an exponential with the function
Nt = N0 etf
Where N= number of cells at time t
N0= number of cells at initial time
t=time in hr and f=generation/hr
In order to find the generation time, you need to solve the equation for the case you want: that is when Nt =2 N0 or what is t when you have twice the number of cells you started with (doubling time).
So: 2= etf
Ln2=tf x Ln(e)
.693=tf
Excel will give you f from its curve fit and you solve for t which is the hours for one generation.
Discussion: You will also hand in a 1-2 pages long discussion along with your Excel graph.
Consider the following questions when you write your discussion. These are suggested issues. They don’t all have to be covered and you may think of others but they are a good guideline for summarizing your experiment.
1.From your data, what is the generation time for the Dictyostelium discoideum strain NC4A2 under both growth conditions?
2.Based on these numbers, did the two growth conditions make a difference in growth rates? If so, explain what you observed (ie: was there a decrease, increase in generation time, was there a higher density, lower density observed)?
3.Any hypotheses as to why or why not this was observed? You might remember from MCB2210 a discussion of the relationship between adhesion of cells and growth and that cancer cells lose their substrate dependence. What do your results tell you concerning this issue.
4.Stationary Phase: What is the maximum titer the cells grow to under the two conditions?
5. Now that you know the generation time, calculate how many cells you would need to plate (start with) if you wanted 106 cells in the dish 3 days after plating.
If your plates/flasks became contaminated describe some of the causes and what you would do to prevent future contaminations in new cultures. Did you get enough data for a conclusion even if you didn’t get all you wanted?
__________________________________________________________________________
Reagents/Media:
HL5 media
10 g BBL Thiotone E peptone
10 g Glucose
0.35 g Na2HPO4
0.35 g KH2PO4
Add dd-H20 to final volume of 1L
adjust pH to 6.5-6.7, autoclave
Strains:
NC4A2
HK321
Other things needed for this experiment:
1.sterile flasks [125ml]
2.haemocytometer with cover slips [6 pieces for 6 groups of two students]
3.sterile Pasteur pipet
4.Make sure shaker is in good condition
