The Development of a Serum-Free Medium for Use in the Culture of Normal and Malignant Human Melanocytes

Gold, Jean Anderson (1986) The Development of a Serum-Free Medium for Use in the Culture of Normal and Malignant Human Melanocytes. MSc(R) thesis, University of Glasgow.

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Abstract

This project was initiated to study the use of serum-free medium in the culture of human malignant melanoma cell lines and normal human melanocytes. The development of a. suitable serum-free medium was carried out in two stages using the two melanoma cell lines. Firstly, various supplements were tested for their ability to improve cell growth in the basic culture medium (Ham's F-10) devoid of serum. From this work we established a group of five supplements which we termed the standard supplements and these were added at optimal concentrations to all serum-free media examined by us. This group consisted of human transferrin, bovine serum albumin, Intralipid, insulin and B-mercaptoethanol. Various other supplements also proved stimulatory to melanoma cell growth under serum-free conditions, however even in combination with the standard supplements the level of cell growth achieved was only a fraction of that achieved in medium supplemented with serum. Thus we decided to switch to a richer basic medium and found that our requirements were met by medium MCDB104, a modification of Ham's F-10. The standard supplements were re-optimised in this medium and again various potential supplements were tested. Although cell growth, under serum-free contiitions, was improved it still did not approach the level achieved in serum-containing medium. Thus it was decided that to improve cell growth under serum-free conditions the basic medium would have to bo optimised to suit our requirements. Thus, the second stage in the serum-free medium development was to re-optimise various components of the MCDB104 medium for the two melanoma cell lines. A batch of MCDB104 deficient in 15 components was prepared. Each of the omitted items was then tested over a concentration range and the optimum concentration for each determined for both cell lines. Using the determined concentrations a "new" MCDB104 medium was prepared for each cell line. Again the standard additives were added. Although this optimisation improved cell growth, the level achieved was still lower than that obtained with serum supplementation. To achieve a comparable level of cell growth it was felt that all components of the basic growth medium would require to be optimised for each cell line under study. The development of the serum-free medium was carried out using the two human melanoma cell lines, however it was hoped that any such medium would be useful in the culture of normal human melanocytes. We found that melanocytes would not grow in any of our serum-free media even when supplemented with the routine requirements for melanocyte growth. We also looked at the possibility of improving upon the two methods we elected to use for the culture of melanocytes. This was done by looking at ways in which the problem of fibroblast contamination and overgrowth could be dealt with and also by looking at factors as potential melanocyte stimulants . We found that calmodulin enhanced melanocyte numbers it used in conjunction with the phorbol ester and cholera toxin required routinely for melanocyte growth. Fibroblast contamination was not eliminated by any of our methods. Despite this however we were able to culture normal human melanocytes for periods of up to 6 months.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Keywords: Medicine
Date of Award: 1986
Depositing User: Enlighten Team
Unique ID: glathesis:1986-77384
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 11:53
Last Modified: 14 Jan 2020 11:53
URI: http://theses.gla.ac.uk/id/eprint/77384

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