BIO2101 Comprehensive Biology Laboratory

Exercise 3:  Cell Culture

Purpose: To understand the conditions that limit cells' ability to survive and proliferate in vitro.

Introduction:

Cell Growth Curve

Techniques for growing cells in the laboratory were originally developed by microbiologists. The  rapid  generation  time  of  most  microorganisms  as  well  as  their autonomous growth and relatively few nutritional requirements made it easy to provide the cells with an optimum environment and to avoid contamination by other organisms. In the past 50 years these techniques have been extended to the culture of cells from higher plants and animals. Using cell culture, one can address questions relating not only to  cell  growth  per  se  but  also  to  nutritional  requirements,  metabolic  activity,  gene expression,  motility,  and  features  of differentiation,  aging,  and  regulation.  The  chief advantage of animal or plant cell culture is the control the investigator can exert over the cells' environment in the absence of influences from other systems of the multicellular organism. It is important to keep in mind, however, that the laboratory culture cannot reproduce  every  detail  of  the  cell's  natural  surroundings,  and  some  of  the  missing properties may be significant to the process under study. Thus, cell culture is a model system, a simplified version of what is understood to be "real life."

When growth is not limited by the composition of the medium, cells are said to be in "exponential" or "logarithmic" phase: in each successive time period the size of the population increases by a constant factor.  Represented mathematically,

where N is the number of cells present at time t and k is a growth constant that depends on that cell type and those growth conditions.

Rearranging:

Integrating between (No, to) and (N,t): In (N/No) = kt

The population doubling time is the value of t for which N/No  = 2.  Moreover, a

plot of ln (N/No) as a function of twill be a straight line whose slope is k.  (Even simpler is to use semi-logarithmic graph paper, plotting t along the evenly spaced axis and N/No on the axis perpendicular to it.)  If you know the initial number of cells and the growth rate constant, the size of a culture in log phase can be predicted at any subsequent time.

Exponential  phase  is  usually  limited  by  the   availability  of  nutrients  or  the environment's carrying capacity for waste.  At that point the population growth slows until the culture is in stationary phase, where cell death balances cell division and even cell division may be slowed.  Cells in adult organs of most higher animals are in stationary phase.  If conditions become sufficiently adverse, a death phase may ensue.  A growth curve is the result of an experiment to measure the characteristics of the various phases of a population of a certain cell type under a defined set of conditions (see Figure 1).

Figure 1. Culture growth phases

Cell Culture Technique

When working with  cell  culture,  it  is  absolutely  essential  to  maintain  sterility. Typical bacterial contaminants double in number every 30 minutes, and 1 to 2-hour for molds.  Several measures are customarily taken to ensure sterility:

1.  Sterilize everything likely to come into contact with the culture.  Media and glassware may  be  autoclaved;  disposable  plastic  ware  has  been  sterilized  by  radiation  or treatment with gas, and should remain wrapped before use; tools and implements can be dipped in alcohol and flamed.

2.  Work in a clean protected area whenever possible.   The ideal situation is to use a biological  safety  cabinet  (“tissue  culture  hoods”),  whose  air  environment  is filtered to remove particles.

3.  Avoid opening sterile containers for longer than necessary to sample or inoculate.

4.  In some cases, it is advisable to include antibiotics such as penicillin or streptomycin in the culture medium.

Cell Number Measurement

Cell number in the culture medium can be accessed by the followings,

•    Hemocytometer

A device for cell number counting as described in Exercise 2

•    Electronic cell counting

In which a particle passing over a charged orifice interrupts the charge and is recorded as a pulse.  The number of pulses is a measure of the number of particles in the volume that flowed past the orifice.  This procedure is useful if you have large numbers of cells and many samples to count, as in a clinical hematology laboratory.

•    Using protein assay to monitor cell growth

It is sometimes necessary to determine how much protein a cell contains in order to learn something about the characteristics of the cell or to compare one cell type to another.  Once that value is obtained, one can use the amount of protein in a cell lysate as a measure of the number of cells lysed.  It is possible to measure the amount of protein per cell using the Bradford procedure, providing that you have enough cells to produce more than 10 μg of protein, and you know the number of cells.  Cells in suspension can be collected by centrifugation, lysed in detergent, and assayed for protein calorimetrically.

Demonstration:

Key features of sterile technique will be briefly demonstrated at the beginning of the lab session.

Procedure:

A.   Construction of Growth Curve of HL-60 cell culture

A stock culture of HL-60 cells in log phase will be provided in a 37oC incubator containing 5% CO2 .  Each group is required to prepare two growth curves on these cells, one under optimum conditions (RPMI 1640 medium containing 20% fetal bovine serum (FBS) and one under sub-optimum conditions (RPMI 1640 medium containing 4% FBS).

1.   Using aseptic technique, transfer 16 mL of 1640 with 20% FBS to a T75 sterile flask, labeled 20%; and 16 mL of 1640 with 4% FBS to another T75 flask, labeled 4%.

2.   Using aseptic technique, transfer 4 mL of the stock cell culture to both flasks from Step 1.

3.   Gently shake the flasks several times.  Label the flasks with your group number and session L01 or L02.

4.   Using  a  micropipette and tip with aseptic techniques, take 0.2 mL of “20%” to a microfuge tube.  Similarly, take 0.2 mL of “4%” to another microfuge tube.

5.   Use the hemocytometer to determine the concentration of cells in the two cell suspensions from Step 4 (cell concentration on day 0), as you  know from Exercise 2.  Once the cells are taken from the culture flask, they no longer need be handled sterilely.

6.   Calculate the mean (the average number of cells/square) and standard deviation of your readings from the eight “A” squares on the hemocytometer.  The concentration of cells in the sample is given by the mean count x 104  cells/mL.   Remember to correct this concentration by the dilution factor if required, and to convert the unit to x 106 cells/mL.  Record all data on the Experimental Datasheet accordingly.

7.   On day 1, 3, 5 and 6, repeat Step 4 for sampling of your two cultures.   Further dilution of the sample is required if the cell concentration is > 70 x 104  cells/mL.

Pay close attention to aseptic technique when sampling to avoid the cell cultures being contaminated.

Note: Exact date of sampling would be announced by the instructor.

B.       Preparation of Cell Pellet for Protein Assay

1.   Transfer  1 mL of cell suspension from “20%” and “4%” culture flasks, by aseptic techniques, to two microfuge tubes, respectively.  Label the test tubes with “20%” or “4%”, your group no., session and the date with a marker pen.

2.   Using a swing out rotor and centrifuge, spin down the cells at 3,000 revolutions per minute (rpm) for 5-minutes. (This operation is done by instructor or TAs)

Note:  Whenever the centrifuge is used, the rotor must be balanced with tubes on opposite sides containing equal volumes.

3.   When the rotor comes to rest, remove tubes and carefully discard the supernatant (but keep the pellet!).  The pellet now contains the cells.

4.   Resuspend the cell pellets with 0.5 mL phosphate buffered saline (PBS). Repeat the centrifugation  step  as   in  Step  2  to  obtain  the  cell  pellets  again.   Discard  the supernatant and save the pellet.

5.   Store the samples (cell pellet) at -20oC.  (From your determination of cell density in Part A, you should know the number of cells in the pellets from the two cultures, respectively.)

6.   Repeat Step 1 to 4 on each sampling day of Part B and save the corresponding cell pellets for lab session of next week.

Part C and D should be done in the following week.

C.       Construction of Protein Standard Plot (Bradford Assay: 96-well plate)

We can use  Bradford  reagent to determine the  protein content of bovine serum albumin (BSA) in order to construct a standard plot as reference.  Bradford reagent contains a dye, Coomassie blue, which binds to protein.  The dye/protein complex produces  a  blue  color  whose  absorbance  is  directly  proportional  to  the  protein concentration.

1.    BSA has been prepared in 0.05% Triton X-100 at a concentration of 0.6 mg/mL.

2.    Using an aliquot of the 0.6  mg/mL protein standard  prepare a range of standards solutions in EP tubes. Add the following volumes of 0.6 mg/mL protein standard and 0.05% Triton X-100 (labeled as BSA dilution buffer) (Table 1) to the appropriate wells to construct a range of standards solutions between 0 and 0.6 mg/mL (e.g. 0, 0.0375, 0.075, 0.15, 0.3 and 0.6 mg/mL). Mix the content by vortex. A total volume of 50 μL of each standard is sufficient for all the required assays.

Table 1. Construction of Protein Standard Plot

Figure 2. Detailed presentation of dilution process

D.       Cell Lysis and Protein Assay

1.   To lyse the cells, suspend each cell pellet in 0.5 mL 0.1% Triton X-100.   Break up the clumps of cells by vortex.

Think about: How does Triton X-100 lyse cells?

2.  Immerse the tube in 95oC test tube heat blocks for a minute or two, to help the proteins go into solution.

Note: Use tweezers to avoid burns.

3.  Once the pellet is solubilized, dilute each sample 2 folds by adding 50 μL of each cell sample into 50 μL of ddH2O. Mix the content by vortex.

4.Add 10 μL of standard or cell lysate sample (2 repeats) to the appropriate wells on 96- well plate.

5.  Add 200 μL of Bradford reagent to each well.   Wait for 3-minutes. Full color development should occur within 3-minutes.

Figure 3. An example of how standard and samples can be presented.

Note: Because the Bradford reagent contains phosphoric acid, avoid contact with the mouth or skin.

6.  After 3-minutes, measure absorbance at 595 nm with microplate spectrophotometer (BioTek Epoch plate reader) .  Record the absorbance value on the datasheet (P. 9) .

Optional Step: If the protein amount of a sample exceeds the upper limit of the standard plot, dilute 50 μL of this sample with 50 μL of *0.05% Triton X-100, then use 10 µL of diluted sample for measuring absorbance, accordingly.  Record the dilution factor on datasheet, if necessary.

l  For operation of spectrophotometer, please consult TA.

For Data Processing on Laboratory Write-up:

Plot your data on graph paper, mg protein on the X axis and A595 on the Y axis (for example, See Figure 4).  Calculate the coefficient of determination and slope of the regression line.  The coefficient should be > 0.9.

Figure 4. A typical standard curve for the protein assay

Calculate the amount of protein per well.   Correcting for dilutions (if necessary), calculate the total amount of protein per ml in each lysate.   Knowing the number of cells that were collected to obtain each lysate, calculate the amount of protein per cell in terms of nano grams (ng) of protein per cell.  Show all calculations on your laboratory report.