Characterization of Adipose-

Derived Stem Cells (ASCs)

Student: Mary Beth
Timeline: Summer 2018
Learning Goals and Objectives
• Read the relevant literature to the project
• Observe and note the techniques and methodology applied in the lab
• Perform own experiments and record data
• Presentation of the research work
• Write manuscript based on this study
 Project layout
• Overall objective: Characterization of Adipose-Derived Stem Cells
isolated from porcine and human sources
• Techniques: Basic cell culture, flow cytometry, staining, microscopy,
XTT
Culture Multilineage
and
Observe Chondrogen
Differentiatio
Expansion proliferati n: esis ASCs vs
chondrocyte:
of porcine on (XTT) Adipogenesis,
osteogenesis RNA
and human and and isolation and
ASCs populatio chondrogene qPCR (ACAN,
(Microscop n sis (Staining COL2A1 and
y and flow and SOX9)
cytometry)
doubling microscopy)
Data
Flow Cytometry
Marker Type BD Cat. No. Format
CD90 Mouse Anti-Human 555596 PE
CD105 Mouse Anti-Human 562408 APC
CD73 Mouse Anti-Human 561258 PE-Cy™7
HLA-DR Mouse Anti-Human Dr. Brand Alexa Fluor®488
CD34 Mouse Anti-Human 562383 PE-CF594
CD45 Mouse Anti-Human 564105 PerCP-Cy™5.5
pASCs

1.05 ± 0.10 98.50 ± 0.51 0.66 ± 0.07

3.56 ± 0.22 0.15 ± 0.04 1.46 ± 0.10

hASCs

73.73 ± 0.15 82.23 ± 0.20 81.60 ± 7.45

0.34 ± 0.04 1.84 ± 0.24 0.01 ± 0.01

Population Doubling
12

pASCs hASCs
10

Population Doubling Time (Day) 8

0
P6 P7 P8 P9 P10 P11 P12
Passage
 LDH & Total NO
LDH Assay Total NO Assay
90 250
10% FBS 2% FBS Serum-free 10% FBS 2% FBS Serum-free
80

200
70
LDH Release (%Control)

60

Total NO (pmol)
150
50

40
100
30

20
50

10

0 0
pASCs hASCs pASCs hASCs
 Viability: Trypan blue & XTT

Trypan Blue Exclusion Assay XTT Assay

120 0.7
10% FBS 2% FBS Serum-free 10% FBS 2% FBS Serum-free
0.6
100

Viability (OD: 450-690 nm)

0.5
80
%Cell Viability

0.4
60
0.3

40
0.2

20
0.1

0 0
pASCs hASCs pASCs hASCs
Colony Forming Efficiency (CFE)