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. 2019 Fall;8(4):283-294.
doi: 10.22088/IJMCM.BUMS.8.4.283.

Proliferation, Characterization and Differentiation Potency of Adipose Tissue-Derived Mesenchymal Stem Cells (AT-MSCs) Cultured in Fresh Frozen and non-Fresh Frozen Plasma

Wahyu Widowati  1 , Rachmawati Noverina  2   3 , Wireni Ayuningtyas  2   4 , Dedy Kurniawan  2 , Hanna Sari Widya Kusuma  5 , Seila Arumwardana  5 , Dwi Surya Artie  5 , Ika Adhani Sholihah  5 , Rr Anisa Siwianti Handayani  5 , Dian Ratih Laksmitawati  6 , Ratih Rinendyaputri  7 , Rilianawati Rilianawati  8 , Ahmad Faried  9
Affiliations

Proliferation, Characterization and Differentiation Potency of Adipose Tissue-Derived Mesenchymal Stem Cells (AT-MSCs) Cultured in Fresh Frozen and non-Fresh Frozen Plasma

Wahyu Widowati et al. Int J Mol Cell Med. 2019 Fall.

Abstract

Mesenchymal stem cells (MSCs) have unique properties, including high proliferation rates, self-renewal, and multilineage differentiation ability. Their characteristics are affected by increasing age and microenvironment. This research is aimed to determine the proliferation, characteristics and differentiation capacity of adipose tissue-derived (AT)-MSCs at many passages with different media. The cell proliferation capacity was assayed using trypan blue. MSCs characterization (CD90, CD44, CD105, CD73, CD11b, CD19, CD34, CD45, and HLA-DR) was performed by flow cytometry, and cell differentiation was determined by specific stainings. Population doubling time (PDT) of AT-MSCs treated with fresh frozen plasma (FFP) and non-FFP increased in the late passage (P) (P15 FFP was 22.67 ± 7.01 days and non-FFP was 19.65 ± 2.27 days). Cumulative cell number was significantly different between FFP and non-FFP at P5, 10, 15. AT-MSCs at P4-15 were positive for CD90, CD44, CD105, and CD73, and negative for CD11b, CD19, CD34, CD45, and HLA-DR surface markers. AT-MSCs at P5, 10, 15 had potential toward adipogenic, chondrogenic, and osteogenic differentiation. Therefore, PDT was affected by increased age but no difference was observed in morphology, surface markers and differentiation capacity among passages. Cumulative cell number in FFP was higher in comparison with non-FFP in P5, 10, 15. Our data suggest that FFP may replace FBS for culturing MSCs.

Keywords: Adipose tissue-MSCs; multilineage differentiation; population doubling time; proliferation; surface marker.

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Figures

Fig. 1.
Fig. 1.
Cumulative cell number after FFP and non-FFP treatment at P3, P5, P10 and P15. Different superscripts of small letters (a,b) in the same color charts (blue color for FFP, red color for non-FFP treatment) were significantly different at P < 0.05 (Tukey post hoc test), each column represents the mean ± standard error of three independent experiments (*: P < 0.05). FFP: fresh frozen plasma; Non FFP: non-fresh frozen plasma
Fig. 2
Fig. 2
Morphology of AT-MSCs from one representative donor at early passage (P5), moderate passage (P10), and late passage (P15). A: P5 FFP; B: P10 FFP; C: P15 FFP; D: P5 non-FFP; E: P10 non-FFP; F: P15 non-FFP. Magnification: 100×. FFP: fresh frozen plasma; Non FFP: non-fresh frozen plasma
Fig. 3
Fig. 3
Dot blot representative of AT-MSCs surface markers CD105, CD90, CD44, CD105, CD73. A: passage 5 (P5); B: passage 10 (P10); C: passage 15 (P15) in FFP supplementation
Fig. 4.
Fig. 4.
Morphological appearance of osteogenic, adipogenic and chondrogenic differentiation of AT-MSCs. Result of AT-MSCs differentiation (after staining) was indicated by A, B and C. Undifferentiated AT-MSCs (negative control) were indicated by D, E, and F. A: osteogenic (A1: P5; A2: P10; A3: P15); B: adipogenic (B1: P5; B2: P10; B3: P15); C: chondrogenic (C1: P5; C2: P10; C3: P15); D: control of osteogenic ; E: control of adipogenic; F: control of chondrogenic
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