Step 5

Summary

    My experiment tests the ability of HMC-1 cells to grow and proliferate in the context of different media conditions.  My independent variables were the concentrations of the recombinant human interleukins IL-3 and IL-6 in the media for the HMC-1 cells.  The dependent variable was the pattern of cell growth and proliferation exhibited by the HMC-1 cells.  My controlled variables were the base media formulation and the growth environment of the cells in a 37.0◦C, 5% CO2 incubator.

    A single 25-cm^2 tissue culture flask containing HMC-1.1 cells was a gift of Dr. Joseph Butterfield of the Mayo Clinic in Rochester. MN.  The established medium used in culturing the cells was Iscove's medium [(+) 25 mM Hepes, (+) sodium bicarbonate, (+) L-glutamine; without alpha thioglycerol, without beta mercaptoethanol] with 10% defined, iron-supplemented calf serum . . . and 1.2 mM alphathioglycerol (Sigma).  Upon arrival, the flask was left in a 37.0◦C and 5.0% CO2 incubator for two weeks, as suggested.

    The adapted serum free medium formulation used initially was based on the StemPro®-34 SFM Complete Medium by Invitrogen generated through the addition of the 40X nutrient supplement to the medium. Of the initial 500ml supply, 50ml batches of medium were made with the addition of 1.3ml of the 13ml of provided 40X nutrient supplement, which was then aliquoted.  This StemPro®-34 SFM Complete Medium was supplemented with 1-thioglycerol (Sigma-Aldrich) to reach a concentration of 1.2mM.  Next, the medium was supplemented with L-glutamine from Invitrogen to reach a concentration of 2mM.  These concentrations were used to match an existing additive system for IMDM/FBS media used in HMC-1 research.  Finally, the recombinant human interleukin IL-3 from ProSpec was reconstituted in filter-sterilized 18mΩ-cm water and aliquoted to allow for the addition of 5μl of 10ng/μl IL-3 into 50ml of media to reach a concentration of 1ng/ml. 

    Aliquots of the supplies were also made for future usage, and all chemicals were stored at the temperatures recommended by the manufacturers.  These procedures were carried out in a Class II Biological Safety Cabinet by Labconco using established techniques for wiping down all materials entering the BSC hood with 70% isopropanol alcohol to prevent contamination.  BacDown disinfectant, a thorough 70% IPA wipe-down, and ultraviolet light were used each time the BSC was set up in accordance with Florida State College at Jacksonville standard operating procedures.  A 37◦C water bath was used to bring materials to the same temperature as the cell culture incubator whenever media and/or additives were added to flasks or a media bottle.  Standard eppendorf micropipettors ranging from the .5-10μl volume up to the 100-1000μl volume were used with corresponding boxed eppendorf micropipette tips throughout the course of the research.  For larger volumes, Drummond rechargeable pipet-aids were used with individually wrapped pipets opened under the BSC hood.

    IL-3 and IL-6 concentrations ranging from 1 to 50 ng/ml were tested in various combinations through the course of the experiment, and the effects on cell growth and proliferation were observed.