Supplemental figures

Supplemental figure 1: Live/dead staining for the analysis of cell viability 24 hours after printing with

medium only and subsequently seeding of printed and non-printed hiPSCs on Geltrex coated cover

slips.; calcein AM stains living cells (green fluorescence), ethidium homodimer-1 (Eth-1) stains dead

cells (red fluorescence). (a) Overview (scale bars = 1 mm); (b) detailed view (scale bars = 250 µm).

Supplemental figure 2: Quantitative analysis of the effect of printing on hiPSCs’ pluripotency and the

effect of hyaluronic acid hiPSCs’ proliferation

(a) Effect of hyaluronic acid on hiPSCs’ proliferation, assessed without printing by analyzing the

amount of cells by LDH measurement. The proliferation of hiPSCs seems to be increased with

increasing hyaluronic acid concentration. However, this slight increase in proliferation is not

statistically significant.

(b) Alkaline phosphatase activity per cell one and two days after printing as a measure for hiPSCs’

pluripotency maintenance: Measured differences between printed, non-printed, and control cells are

small and not systematic, though statistical significant differences were observed between printed

and control cells one day after printing (p = 0.02) and between non-printed and control cells two days

after printing (p = 0.04). However, no significant difference was observed between printed and non-

printed cells (p = 0.42) or non-printed and control cells after one day (p = 0.09) and between printed

and non-printed cells (p = 0.48) or printed and control cells after two days (p = 0.25).

Supplemental figure 3: Maintenance of pluripotency three days after printing with medium only and

subsequently seeding of printed and non-printed hiPSCs on Geltrex coated cover slips.

Immunostaining with pluripotency markers (OCT4, NANOG, alkaline phosphatase (ALP), SSEA-4) and

proliferation marker Ki67 and cell nuclei staining with Hoechst 33342 as well as merged images

(immunostaining + Hoechst 33342) of hiPSCs, which were printed with medium as bio-ink, seeded on

Geltrex-coated glass slides, and cultivated for three days in complete E8 medium.

The maintenance of pluripotency is not observably reduced for printed hiPSCs compared non-printed

cells rinsed of the donor glass slide. Scale bars are 100 µm.

substrate

coatingbio-ink

hiPSCs printed

with bio-ink on

substrate coating

hiPSCs seeded

on

substrate coating

hiPSCs seeded

in

substrate coating

NIH3T3 seeded

on

substrate coating

NIH3T3 seeded

in

substrate coating

very

high viability,

spreaded

morphology

very

high viability,

spreaded

morphology

fibrinogen +

hyaluronic acid

very high viability,

very good pattern fid.,

form. of cell aggregates

alginate +

plasma

low viability,

low pattern fidelity,

form. of cell aggregates

plasma +

hyaluronic acid

low viability,

low pattern fidelity

plasmalow viability,

low pattern fidelity

high viability,

rounded

morphology

fibrinogen +

hyaluronic acidvery low viability

medium

high viability,

medium pattern fidelity,

form. of cell aggregates

very

high viability,

spreaded

morphology

Matrigel

medium +

hyaluronic acid

very high viability,

very good pattern fid.,

form. of cell aggregates

very

high viability,

spreaded

morphology

very

high viability,

spreaded

morphology

medium

very high viability,

good pattern fidelity,

form. of cell aggregates

Geltrex

medium +

hyaluronic acid

high viability,

medium pattern fidelity,

form. of cell aggregatesvery

high viability,

spreaded

morphology

low viability,

rounded

morphology

high viability,

spreaded

morphology

medium +

hyaluronic acid

medium viability,

medium pattern fidelity,

form. of cell aggregates

alginat +

plasma

low viability,

no pattern fidelity

medium

medium viability,

low pattern fidelity,

form. of cell aggregates

very

high viability,

spreaded

morphology

fibrin

fibrinogen +

hyaluronic acid

medium viability,

low pattern fidelity,

form. of cell aggregates

high viability,

formation

of cell

aggregates

low viability,

formation

of cell

aggregates

very

high viability,

spreaded

morphology

very

high viability,

formation

of cell

aggregates

medium +

hyaluronic acid

high viability,

no pattern fidelity,

form. of cell aggregates

fibrinogen +

hyaluronic acid

high viability,

no pattern fidelity,

form. of cell aggregates

high viability,

formation

of cell

aggregates

fibrinogen +

hyaluronic acid

low viability,

low pattern fidelity

medium +

hyaluronic acid

low viability,

low pattern fidelity

very

high viability,

formation

of cell

aggregates

collagen

collagenlow viability,

no pattern fidelity

high viability,

formation

of cell

aggregates

low viability

alginate

alginate +

plasma

low viability,

low pattern fidelity,

form. of cell aggregateshigh viability,

formation

of cell

aggregates

low viability,

formation

of cell

aggregates

Supplemental table 1: Effect of biomaterials (substrate coating and bio-ink) on hiPSCs viability,

morphology and maintenance of printed pattern. The results of printing experiments with hiPSCs, bio-

ink, and substrate coating are compared with those of pipetting hiPSCs or NIH3T3 fibroblasts into or

onto the substrate coating material. In some cases with low or very low viability no reliable

statements were possible on pattern fidelity or cell aggregation.

Supplemental video 1: Time-lapse microscopic video (duration of 18 hours) of hiPSCs suspended in a

bio-ink composed of alginate and blood plasma (1:1) and printed onto alginate as substrate coating.

At the beginning of the video, hiPSCs moving activity demonstrates their high viability shortly after

printing. However, after some hours, the movement gradually stops. Next day, nearly all cells were

dead.

Supplemental videos 2-6: Videos of beating hiPSC-derived cardiomyocytes: printed (Video2) and non-

printed (Video3) hiPSCs (medium-based approach), seeded in 12-well plates on Matrigel and cultured

for 12 days in cardiac differentiation medium; printed (Video4) and non-printed (Video5) hiPSCs

(medium-based approach), seeded with hyaluronic acid in ultra-low attachment plates and cultured

for 12 days with cardiac differentiation medium (The formation of cell aggregates occurred directly

after cell seeding.); hiPSCs, suspended in culture medium and hyaluronic acid, printed onto Matrigel

layers (sol/gel-based approach) and cultured for 20 days in cardiac differentiation medium (Video 6).