Comparison of explant-derived and enzymatic digestion-derived mesenchymal stem cells from Wharton’s jelly
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1
Universiti Malaysia Sabah, Biotechnology Research Institute, Malaysia
Mesenchymal stem cells (MSCs) with self-renewal and multi-lineage differentiation potencies have emerged as a promising candidate for tissue engineering and regenerative therapeutics. MSCs can be isolated from various tissue sources, but umbilical cord Wharton jelly (WJ)-derived MSCs are easily attainable and contain higher proliferative and differentiation capabilities, which make them a potent MSCs source over the last decade. Moreover, umbilical cord derived MSCs have not been impeded with any ethical issues. Recently, explant culture is developed to isolate MSCs to overcome the shortage of enzymatic-digestion method that destroys the meshwork surrounding the cells. The objective of this study is to examine the cell morphology and cell proliferation within MSCs isolated using different methods, which are enzymatic digestion and explant culture method.
Cell culture reagents and chemicals were purchased from Gibco unless otherwise stated. For enzymatic digestion method, human WJ-derived MSCs were digested with 0.1% collagenase for 2 hours at 37oC. For explant culture method, the WJ was cut into pieces with an approximate diameter of 3-4mm each, were transferred onto the surface of petri dishes and the tissues were settled for 2-3 minutes until attachment onto dish (Yoon et al., 2013). Then, DMEM-F12 supplemented with 10% fetal bovine serum, antibiotic-antimycotic, and L-ascorbic acid (Sigma) was added. Media was changed every 4-5 days until the cells migrated out from the tissues and reached 70-80% confluence. Cell morphology was compared between 2 isolation methods where images were captured using inverted microscopy. Proliferation assay was compared and assessed for up till 6 days using Presto BlueTM Cell Viability reagent (Invitrogen). MSCs were seeded at 5 x 103 per well in 96 well plate and fluorescence reading were measured.
The results showed that the WJ-MSCs derived from both methods showed similar morphological features such as fibroblast-like, flat, round, spindled-shape cells during primary culturing with fibroblast-like cells being dominant (Salehinejad et al., 2012). In later passages, the MSCs were transformed into elongated spindled-shape and fibroblast-like cells. From Fig.1B and 1C, more round cells were seen in explant group and fibroblast-like cells started to grow longer during passage 2 in both enzymatic digestion and explant group. In passage 5, fibroblast-like cells were found dominant in enzymatic group and spindled-shape cells were found dominant in explant group. In addition, they showed differences in terms of proliferation. As seen in Fig.2, enzymatic WJ-MSCs took 6 days to reach 100% confluence, while explant WJ-MSCs took a longer period of time, which was 9 days. This suggested that the proliferation level of MSCs isolated using enzymatic digestion method is higher than the explant culture method, thus grew faster to over confluence until detachment in a shorter length of time. The results were consistent to previous studies where explant culture method could acquire MSCs, but the growing conditions of explant WJ-MSCs are still under optimization. Moreover, MSCs still need to be characterized using cell surface markers by flow cytometry.
In conclusion, the Wharton jelly contains rich source of MSCs that are easily obtainable without any ethical issues. It is important to investigate which is the most appropriate isolation method for WJ-MSCs. Our preliminary results showed that explant culture method could use to obtain MSCs however its application in regenerative medicine still require extensive studies.
Acknowledgements
The project is sponsored by Ministry of Higher Education under TRGS0003-SG-2/2014.
Keywords:
Mesenchymal Stem Cells,
Isolation,
Explant culture,
Enzymatic-digestion,
Cells
Conference:
6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting, Seberang Jaya, Penang, Malaysia, 17 Nov - 18 Nov, 2016.
Presentation Type:
Poster
Topic:
Stem cells and Regenerative Medicine
Citation:
Vun
HV,
Simat
F and
Lin
TP
(2016). Comparison of explant-derived and enzymatic digestion-derived mesenchymal stem cells from Wharton’s jelly.
Front. Bioeng. Biotechnol.
Conference Abstract:
6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting.
doi: 10.3389/conf.FBIOE.2016.02.00030
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Received:
08 Dec 2016;
Published Online:
19 Dec 2016.
*
Correspondence:
Ms. Hiew V Vun, Universiti Malaysia Sabah, Biotechnology Research Institute, Kota Kinabalu, Sabah, 88400, Malaysia, vunvun0524@hotmail.com