FOR RE SEARCH USE ON LY . NO T I NTE ND ED FOR HUMAN OR AN IMA L DIAGNO ST IC OR T HER APEUT IC USES.

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SUGGESTED PROTOCOL: Feeder-free Maintenance & Expansion of hiPSCs/hESCs using StemBeads® FGF2

StemBeads® FGF2

This protocol uses StemBeads® FGF2, feeding only 2-3 �mes a week, and spli�ng cells once a week to effec�vely maintain/expand human pluripotent stem cells (hPSCs). It was developed using mul�ple lines of human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs). Some op�miza�on may be required due to variability between hPSC lines. StemBeads® FGF2 can be used in whichever FGF2-dependent hPSC culture medium you normally use.

Preparation of Media with StemBeads® FGF2 1) Mix vial of StemBeads® FGF2 thoroughly by vortexing or pipe�ng prior to use. 2) Add 8 μl of StemBeads® FGF2 per 1 mL of medium. This will provide cells with stable 10 ng/mL FGF2.

Culturing hPSCs with StemBeads® FGF2

Day -3: Remove medium from cultures and replace with StemBeads® FGF2

*Note: Washing is highly recommended prior to each feed to remove cell debris and remaining beads.

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B

Figure 1. Long term growth of hPSCs in StemBeads® FGF2 maintains pluripotency. hPSCs were cultured over 3 months in medium supplemented with StemBeads® FGF2 A) Cells maintain typical hPSC colony morphology and B) express pluripotency markers SSEA4/Tra-1-60 and lack the differen�a�on marker SSEA-1, quan�fied by FACS.

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10 11 12 13 14 15 16 17 18 19 20 21 22StemBeads Size (μm)®

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StemBeads® FGF2 Release Profile

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StemBeads® FGF2 Conc.:4 μl/mL

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Day 0: Split hPSCs using preferred enzyma�c method, then plate into pre-coated culture dish in medium supplemented with StemBeads® FGF2.

supplemented medium.

Day 1 Wash 2x with medium e.g. DMEM/F12 if large amount of (optional) una�ached cells are observed and replace with StemBeads® FGF2 supplemented medium.

Day 4: Remove culture medium, *wash 2x with medium e.g. DMEM/F12 and replace with StemBeads® FGF2 supplemented medium.

Day 7: Repeat split and feed as described above, Day 0 - Day 4.

Figure 2. StemBeads® FGF2 Sustained Release Profile and Standard Size Distribu�on. A) StemBeads® FGF2 maintain a stable FGF2 concentra�on in hPSC cultures over 3 days. 4 μL/mL or 8 μL/mL StemBeads® FGF2 generate a stable release of 5 ng/mL or 10 ng/mL FGF2 protein respec�vely. B) Phase image of StemBeads® FGF2 under 32X objec�ve. C) Size distribu�on of StemBeads® FGF2; Analysis performed using ImageJ (NIH).

hPSCs Growing with StemBeads® FGF2

StemBeads® FGF2 Size Distribu�on

Conven�onal Culture Feeding 7x per WeekMedium Supplemented with StemBeads® FGF2 Feeding 2-3x per Week

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