Step 6

Summary

March 2010

Initial flask of HMC-1 cells was moved to CO2 incubator.  Observations mention that layers of rounded cells grouped in clusters developed.  Cells in suspension were split into two new flasks, and adherent cells in the first flask were replenished with fresh media.  This resulted in a base layer with full coverage and clumps of cells on top.  The initial formulation was the base media plus 1ng/ml IL-3.

April 2010

In the two new flasks, cells exhibited a high cell density and an even distribution no longer in clumps according to written observations.

May 2010

Each of the two new flasks was split into two more flasks early in the month.  Near the end of the month, cells were very dispersed in the culture, with a few areas of small cell clumps, as written observations noted.

June 2010

In the four flasks, cells existed as well-dispersed small clusters of cells with much space in between.  In two of the flasks, the IL-3 level was increased from 1 to 10 ng/ml.  This resulted in somewhat improved cell density, with some clusters and layering.  The two flasks without the IL-3 increase showed not much in the way of clusters.  The IL-3 concentration in the two selected flasks was further increased to 20 ng/ml.  Observations indicated better cell density and small clusters of a few new, small cells.  The two control flasks exhibited some small clustering which was less noticeable than in the two enhanced flasks.

July-August 2010

The four flasks, two at 1ng/ml IL-3 and two at 20ng/ml IL-3, were left in the incubator at 5% CO2 and 37.0◦C to evaluate long-term differences in cell growth.

September 2010

The approximate distribution and size of the cell clusters in each flask was examined.  The two control flasks exhibited sparse scattering of clusters with about 5 to 20 cells per cluster.  The two enhanced flasks showed variation in clusters, with about 5 to about 50 or more cells per cluster.  After this point, IL-6 was added to each flask such that all four combinations of 1 and 20 ng/ml for IL-3 and IL-6 existed.

October 2010

The four combinations were observed at increasing intervals of time.  The two flasks with IL-6 at 20 ng/ml exhibited the most cell density and cell division taking place, with cells suspended in dispersed clusters.  The concentrations of IL-3 and IL-6 were further increased in these two flasks to yield a flask with 20 ng/ml IL-3 and 50 ng/ml IL-6 and another with the reverse concentrations.

November 2010

The VWR VistaVision used to observe the cells during the experiment at frequent intervals was connected to an AmScope MD900 digital camera to photograph the end results of cell growth and proliferation.

HMC-1 cells cultured in 1 ng/ml of IL-3 and IL-6.

HMC-1 cells cultured in 20 ng/ml of IL-3 and 1 ng/ml of IL-6.

HMC-1 cells cultured in 50 ng/ml IL-3 and 20 ng/ml IL-6.

HMC-1 cells cultured in 20 ng/ml IL-3 and 50 ng/ml IL-6.