Step 7

Summary

Initial results involved cells in a well-dispersed, non-clumped distribution. When the incubator experienced a humidity failure, adherent cells of two flasks were salvaged through the use of full volume media changes.  After splitting cells into four flasks, cells became very dispersed in culture, with only small cell clumps present.  When increasing IL-3 did not provide any significant changes over long-term culture, IL-6 was introduced in a controlled manner into the four flasks, reaching combinations of 1 and 20 ng/ml.  This yielded increases in the number of cells within the clusters of the IL-6 flasks at 20 ng/ml, allowing for the seeding of new flasks after centrifuging the cells in suspension. In each case, either IL-3 or IL-6 was increased to reach the two new 20/50 combinations.  The following images indicate changes in types of HMC-1 morphology captured at 40X with a VWR VistaVision cell culture microscope through an AmScope MD900 digital camera.

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

Cells are found in small clusters scattered sparsely through the culture.  This indicates that low levels of growth factors are sufficient to keep cells alive but do not stimulate noticeable cell division and proliferation.  As the summer months long-term study indicated, 1 ng/ml IL-3 alone did not stimulate significant cell.  Adding 1 ng/ml IL-6 did not appreciably increase the amount of cell proliferation.

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

This indicates that focusing on the IL-3 concentration provides somewhat irregular growth limited to clusters.  Increasing the IL-3 concentration to 20 ng/ml for the duration of the summer had some effect on the growth of the cells, though it was not to the extent that the cells needed a media change or to be passaged into new flasks.  This cell division was limited to clusters, and the cells remained in roughly spherical cell clumps that were in suspension in the culture.

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

This demonstrates a disorderly growth pattern of stacked layers of cells congregating into irregular clusters. It is remarkably unlike the small, rounded, individually distinguishable cells characteristic of HMC-1.  Instead, cells grew in disorderly layers upon each other.  This emphasis on IL-3 again demonstrated undesirable cell growth patterns resulting in a phenotype unsuitable for the study of exosomal communication.

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

This was found to be a successful formulation supporting HMC-1 growth in the form of small, rounded cells at confluent concentrations.  Because studying exosomal transfer requires that cells be able to exchange vesicles with each other, it is desirable for the cells to not grow in clusters or disorderly layers.  Instead, this ideal pattern of cell growth shows cells as individually distinguishable and arranged in an orderly fashion.