Culture condition for maintain the

character of HCC cell line on sphere

formation

Kyung Sik Kim1,2, Seon Ok Min1,3, Sang Woo Lee1,3

1Dept of Surgery, Yonsei University College of Medicine, 2 Cell Therapy Center,

Severance Hospital, 3Graduate Program of Nano Science and Technology, Graduate School of Yonsei University

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Sphere Culture Reynolds and Weiss first cultured cells that exhibit stem cell properties as free-

floating spheres, called neurospheres, from the adult brain.

Recently, sphere culture has been increasingly used as a method for enriching

stem cells which relies on their property of anchorage independent growth.

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Generally accepted concepts It is generally agreed that, like all stem cells, the tumor sphere-forming cells are

capable of proliferation, self-renewal and possess higher tumorigenicity.

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Materials and Methods

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Monolayer 1. Huh7 cells were attach culture in Medium I (DMEM/High glucose with 10% FBS)

on 6well culture plates. 2. Cell harvest.

Cell cultures

Sphere culture 1. The cells were dissociated with trypsin-EDTA and the

resulting single cells were suspended cells at 10,000 cells/ml on Ultra Low attachment plates(corning) in A. Medium I (DMEM/High glucose with 10% FBS), B. Medium II (DMEM/High glucose without 10%

FBS) C. Medium III (DMEM/F12 with 20ng/ml EGF,

bFGF and 1X b27(GIBCO)) 2. Suspension cultures were incubated for 5 days.

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Number of cells determination 1. Inoculate cell suspension (100μl/well) in a 96-well plate.

2. Add 10μl of the CCK-8(Dojindo) solution to each well of the plate.

3. Incubate the plate for 3 hours in the incubator.

4. Measure the absorbance at 450nm using a microplate reader.

Gene detection for stemness and HCC cancer stem cells

Sox2 S 5'-AAC CCC AAG ATG CAC AAC TC-3'

152 A 5'-CGG GGC CGG TAT TTA TAA TC-3'

EpCAM S 5'-CTG GCC GTA AAC TGC TTT GT-3'

182 A 5'-AGC CCA TCA TTG TTC TGG AG-3'

Connexin32 S 5'-GTT TGA GGC CGT CTT CAT GT-3'

188 A 5'-CCA CAT TGA GGA TGA TGC AG-3'

Connexin43 S GGACATGCACTTGAAGCAGA

103 A GATGATGTAGGTTCGCAGCA

GAPDH S 5'-CAA TGA CCC CTT CAT TGA CC-3'

159 A 5'-TTG ATT TTG GAG GGA TCT CG-3'

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First antibody (1:300) Secondary antibody (1:400)

Rabbit Anti-human CD 73 antibody (Santa cruz) Anti- rabbit IgG HRL-F(ab)2-PE (Santa cruz)

Rabbit Anti-human VEGF antibody (Santa cruz)

Anti- rabbit IgG HRL-F(ab)2-PE (Santa cruz)

Rabbit Anti-human E-cadherin antibody (Santa cruz)

Anti- rabbit IgG HRL-F(ab)2-PE (Santa cruz)

Goat Anti-human CD45 antibody (Santa cruz) Anti-goat IgG - FITC

Goat Anti-human CD34 antibody (Santa cruz)

Anti-goat IgG - FITC

Goat Anti-human vimentin antibody (Santa cruz)

Anti-goat IgG - FITC

Rabbit Anti-human CD90 antibody (Santa cruz)

Anti- rabbit IgG HRL-F(ab)2-PE (Santa cruz)

Goat Anti-human CD133 antibody (Santa cruz)

Anti-goat IgG - FITC

Immunocytochemistry • Monolayer cells (6well plates)

fixed with 10% % formaldehyde solution for 30min at RT(room temperature)

• Sphere cells (ultra low attach 24well plates)

Cytospin and fixed with 10% % formaldehyde solution for 30min at RT(room temperature)

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Flow cytometry PE-conjugated mouse anti-human CD133. The cells from Huh7 cells line were suspended in PBS with 2% BSA and 0.5mM EDTA, sequentially labeled with anti-CD133 cells were evaluated by flow cytometry.

Marker/Method Frequency Minimal Cell Number

required for Tumor

Initiation

Other Solid Tumors

SP 0~28% 1000 Lung, breast, pancreas

CD133 0~65% 1000 Breast, colon, pancreas,

endometrium, prostate, brain

CD133/CD44 0.1~1.9% 100 Colon, breast

CD133/ALDH1 0~56% 500 Colon, breast

CD90 0~2.5% 500 Prostate stroma

EpCAM 0~99% 200 Breast, pancreas, colon

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Results

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A B

C D

Cell morphology of monolayer cultivation of Huh7 Medium I for 3days(A,B) and

5days(C,D) after seeding.

Scale bar(A, C=200μm B, D=100μm)

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DAY 0

M1 M2 M3

Fig. Morphology of sphere-forming culture of Huh7 cells .

Scale bar=100μm

DAY 1

DAY 5

DAY 7

DAY 10

DAY 15

0.000

0.500

1.000

1.500

2.000

2.500

3.000

3.500

4.000

DAY 1 DAY 3 DAY 5 DAY 7 DAY 10DAY 13DAY 14DAY 17DAY 20

M1

M2

M3

Fig. Proliferation assay of sphere-forming culture of Huh7 cells .

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Sox2 : 2,7 Cx32 : 4,9 Cx43 : 5,10

GAPDH : 1,6

EpCAM : 3,8

1 2 3 4 5 6 7 8 9 10

Day 3 Day 7

Huh7 (Monolayer culture - Day 3, 7)

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M1 M3

GAPDH

Sox2

EpCAM

Cx32

Cx43

M1 M3 M1 M3 M1 M3

DAY 3 DAY 7 DAY 11 DAY 13

M1 M3

DAY 19

Gene expression of sphere formation of Huh 7 cells

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0

20

40

60

80

100

sox2 EpCAM Cx32 Cx43

DAY 3

M1

M3

0

20

40

60

80

100

sox2 EpCAM Cx32 Cx43

DAY 7

M1

M3

0

20

40

60

80

100

sox2 EpCAM Cx32 Cx43

DAY 11

M1

M3

0

20

40

60

80

100

sox2 EpCAM Cx32 Cx43

DAY 13

M1

M3

0

20

40

60

80

100

sox2 EpCAM Cx32 Cx43

DAY 19

M1

M3

by Imagej

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Immunocytochemistry

LOGO M1-sphere M3-sphere M1-sphere M3-sphere M1-sphere M3-sphere

DAY 5 DAY 7 DAY 13

CD34

VEGF

CD45

CD73

Vimentin

E-cadherin

CD133

CD90

DAY 7

Monolayer

scale bar =200μm

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Flow cytometry(CD133)

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IgG CD133

( DAY 7)

Mono

layer M1

0.04%

M1

93.33%

M1

M3

M1

0.05%

M1

0.05%

M1

71.89%

M1

89.67%

( DAY 12)

M1

0.07% M1

48.73%

M1

50.41%

M1

61.34%

M1

M1

0.07%

0.07%

Huh7

IgG CD133

Fig. Flow cytometry using antibodies specifically targeting human CD133

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Conclusions

We established cultures medium for sphere formation of

hepatocellular carcinoma cells. we compared to cells in three

medium(M1, M2 and M3).

Our results suggest that growth factors may can change the cells

characterization.

So, We think that using the growth factors free medium for sphere

formation because of effective and no expensive.

However, we should try the activation with other cell lines in three

mediums and in vivo tumorigenesis from sphere-forming cells.

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