Establishment of a new cytotoxicity assay using mesenchymal stem cells; comparison of flow cytometric and fluorimetric methods

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Supervisor:
Dr. Szlávecz Ákos
Department of Control Engineering and Information Technology

As a first biological step of pharmacological development is in vitro testing in cell cultures. During this process, cells are cultured in the presence or absence of the tested compound. Cell proliferation, or in certain cases, special cell functions are determined, mostly on cancer cell lines, which are the main tools of testing cytotoxicity. Similar methods can be applied for the detection of the toxicological-, or side effect of drugs. In this case, human cells of healthy origin should be used, this stage of drug testing is called toxicology.

During my M.Sc. work, I developed and compared cytotoxicity assays using a cancer cell line, while similar measurements were performed on human mesenchymal stem cells. A well characterized chemotherapeutic compound, mitoxantrone (Mx) was used in the experiments, as a control substance.

I cultured PLB985 cell line, established 15 years ago from a patient, suffering from myeloid leukemia. This cells stay in the early progenitor stage of hematopoietic differentiation, they are cancer cells. As a model of healthy tissues, mesenchymal stem cells from adipose tissue have been used (Zs-MSC). Both cell lines were engineered to express a fluorescent protein: PLB985 express mCherry (red), while MSCs express eGFP (green). I developed the conditions, how to perform determination of fluorescence measurement to follow cell proliferation. I introduced some minor modification in a well-controlled assay, ‘Alamar Blue’ or with another name ‘resazurin assay’ for my purposes. Comparing the results of the two assays, determining cell proliferation, in vitro drug concentration can be decided (IC50), which kills cancer cells without influencing healthy tissues.

Results show that Zs-MSCs are less sensitive to the chemotherapeutic drug mitoxantrone, then the applied myeloid leukemia cell line, PLB985. According to my observations, low dose Mx kills 50% of cancer cells (IC50: 11 nM), while MSCs are fare less sensitive (IC50: 59 nM). The toxic effect of Mx appeares much slower in MSC (at 6 days), reflecting the longer population doubling time of stem cells.

In the second stage of my work interaction of stem cells and the cancer cell line was investigated. The methodology established earlier was applied. In the co-culture of MSC and PLB985, cancer cell growth was decreased significantly, while MSC multiplied in the presence of PLB985 cells similar to the control.

Influence of both cell’s supernatant was monitored on the growth of the partner cell and the toxicity of Mx. Supernatant, collected from cancer cells induced drug resistance of MSC (IC50: from 73 nM to 160 nM), while MSC’s supernatant was less effective (IC50 32 nM to 41 nM). The later result needs further confirmation.

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