Primary mesenchymal stromal cells (MSCs) represent an important cell source with
great potential for use in regenerative medicine, tissue engineering, and advanced cell therapies
due to their proliferative capacity, immunomodulatory properties, and differentiation potential.
MSCs derived from perinatal tissues, like the amniotic membrane, umbilical cord, and placenta,
represent a convenient source of MSCs, as these tissues are considered biological waste after
childbirth. Their procurement is therefore non-invasive and ethically acceptable, in contrast to
some of the other sources of MSCs. Despite the considerable therapeutic potential of MSCs,
the optimization of isolation procedures remains one of the key challenges. The choice of
isolation method, reagents, and conditions can significantly influence the quality of the isolated
cells, their proliferative capacity, and their ability to form colonies.
The aim of this master’s thesis was to compare the efficiency of primary MSC isolation from
different perinatal tissues using two enzymatic methods: isolation with collagenase and
isolation with an enzyme mixture containing collagenase as well as DNase I and dispase II.
This research includes 15 donors from the Department of Perinatology, Division of
Gynaecology and Obstetrics, University Medical Centre Ljubljana. We obtained three perinatal
tissues per donor that we used for primal cell isolation using both methods. The isolated cells
were cultured under standard conditions for primary human cell cultures. The efficiency of
isolation was evaluated based on following three parameters: their colony-forming ability, time
until trypsinization at passage 0 (p0) and with cell count of the trypsinizated cells at p0. Based
on the collected data, we determined the success rate for the isolation with both methods. We
tested the following hypotheses: 1. the efficiency of primary cell isolation is the same for both
methods, 2. the time to trypsinization at p0 is the same for both methods and 3. the number of
trypsinized cells at p0 is the same for both isolation methods. The results showed no significant
differences in isolation efficiency between the two selected methods based on these parameters.
For both methods, an overall low efficiency was observed for primal cell isolation. The
effectiveness of the isolation is influenced by several other factors besides the enzymes
concentrations or activities, including the time of incubation with enzymes, the time from tissue
sampling to cell isolation, and others that should be more closely examined in the future.
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