Rodekruis Vlaanderen Gent
Abstract Bachelor Project FBT 2020-2021: Optimization of eukaryotic cell culture with platelet lysates
Cell culturing is an increasingly used method with multiple downstream applications such as cell therapy, vaccine development,… To stimulate the cell growth and differentiation, a supplement is added, most commonly fetal bovine serum (FBS). The use of FBS has a risk of zoonotic infection and patient immunization. This resulted in the search of a human derived alternative that can be applied in cell culturing, namely human platelet lysate (hPL). The existing production methods showed problems, so the research department of Red Cross Flanders developed a new method that can avoid these disadvantages. Despite being the preferred option, even this method still has a reoccurring problem, calcium based precipitation.
The aim of this study is to optimize the cell culturing protocols to avoid the forming of the precipitation and to further validate the use of older platelet concentrates in the production of hPL. The study investigates precipitation by culturing mesenchymal stem cells (MSC) in medium with different concentrations of hPL or calcium. The precipitation was ascertained by microscopic images. To study the proliferative capacity, the cell doubling time was calculated. For the validation of the new method, platelet concentrates of different ages (day 7, 10, 14 and 18 post donation) were used in the production of hPL. The quality of the hPL was then further tested on the composition and efficiency. The results of day 7 were used as a standard to compare results with.
The results are given as follows, lowering the calcium concentration in the basal medium did result in fewer calcium complex precipitation without vast influence on cell doubling time. The use of 1,2 mM calcium resulted in at least 7 weeks precipitation-free culturing without influencing the cell growth. The age of platelet concentrates show no significant difference on the quality and functionality of the hPL.
In conclusion, to obtain less precipitation, a lower calcium concentration can be used. Platelet concentrates older than 7 days can be used without causing any influence on the hPL. A bacterial test should be performed in the future to ensure the patients safety. This study contributes to the knowledge and use of hPL and a solution can now be given to solve the current problems of hPL in cell culture.