Substrate Elasticity |
Human |
ESC |
Rigidity of PDMS micropost arrays |
1.92–1218.4 kPa |
24 hours |
Traction force measurements; pluripotency and cytoskeletal changes (IF) |
[93] |
Human |
iPSC |
Elastomer pillars of different heights on a rigid substrate |
3–168 kPa |
Up to 8 days |
Morphology; viability; proliferation; pluripotency (IF); cardiac differentiation (IF, FACS, Ca2+ assays, electrical stimulation) |
[85] |
Substrate Topography |
Human |
iPSC |
Polyimide substrate with submicrometer groove-ridge structure |
340 nm, 650 nm, 1400 nm periodicity, 200 nm depth; compared to unstructured |
3 days |
Morphology; pluripotency (IF, qRT-PCR); YAP/TAZ expression (WB), gene expression profiles |
[94] |
Colony Size |
Human |
ESC, EB |
Size of initial 2D colony and EB size via micropatterning |
200, 400, 800 µm |
4–22 days |
Morphology; pluripotency (FACS) and germ layer differentiation (qRT-PCR, IF) |
[70] |
Mouse |
ESC, EB |
Size of initial 2D colony and cell density via laser direct-write cell printing |
Colony size: 200–3000 µm; cell density: <25,000 cells, 25,000–125,000 cells, >125,000 cells |
3–8 days |
Morphology |
[71] |
Mouse, human |
ESC, EB |
Size of EBs; substrate hydrophobicity |
Colony size: <100 µm, 100–300 µm, >300 µm; surface chemical properties: agarose, PEG, pHEMA, PDMS, TCP, LAC |
4–20 days |
Morphology; viability; proliferation; (germ layer) differentiation potential (qRT-PCR, FACS) |
[72] |
Mouse |
ESC, EB |
Size of EBs via adhesive stencils with different diameter |
100–500 µm diameter |
20 days |
Germ layer differentiation (IF, qRT-PCR) |
[73] |
Shear Stress |
Mouse |
ESC |
Laminar shear stress |
1.5–10 dyn/cm2 |
3 days |
Cell density; cell cycle (FACS, ELISA); endothelial differentiation (IF, WB, qRT-PCR) |
[75] |
Mouse |
ESC, EB |
Shear stress in rotary suspension culture |
Comparison of static and rotary suspension culture (25–55 rpm) |
12 hours to 7 days |
Morphology; viability; proliferation; cyst formation; germ layer formation (qRT-PCR) |
[76] |
Mouse |
ESC, EB |
Y-channel microfluidic system with two different media |
Laminar flow 50–200 µL/min |
5 days |
Differentiation potential (WB, IF) |
[78] |
Mouse |
EB |
Microfluidic chip system with continuous laminar flow and shear stress |
Comparison of static culture and laminar flow 2 µL/min |
21 days |
Viability; proliferation; steroidogenic differentiation (hormone release, IF, ELISA) |
[77] |
Mouse |
ECS, EB |
Comparison of static conditions and shear stress as pre-condition |
0–5 dyn/cm2 |
48 hours before EB formation; up to 10 days of EB culture |
Morphology; pluripotency and endothelial differentiation (IF, qRT-PCR, FACS) cellular organization |
[79] |
Mouse |
ESC, EB |
Shear stress in rotary orbital suspension culture |
0.7–2.5 dyn/cm2; 20–60 rpm |
EB culture for 7 days; up to day 12 of differentiation |
Morphology (IF); pluripotency and germ layer differentiation (qRT-PCR, FACS); global gene expression (PCR array analysis) |
[80] |
Mechanical Strain |
Mouse |
ESC, EB |
Mechanical strain by short-term magnetization via incorporated RGD-conjugated paramagnetic beads |
Stimulation pulses using short-term magnetization; 0.128–0.4 Tesla |
1 hour stimulation for up to 7 days |
Morphology; viability; protein expression and cardiomyogenesis (β1 integrin inhibition, FACS, WB, IF) |
[82] |
Mouse |
ESC, EB |
Mechanical strain by (cyclic) stretching and compression between two microtips with a magnetic tissue stretcher |
Stretching amplitude of 50% of original size (+ cyclic stretching 1 Hz, 10% amplitude, twice daily for 2 hours) |
3 days; with three additional days of cyclic stretching |
Morphology; viability; proliferation; pluripotency and germ layer differentiation (qRT-PCR, IF) |
[39] |
Mouse |
ESC, EB |
Mechanical strain by stretching device (Flexercell Strain Unit) |
10% elongation of undifferentiated EBs |
2 hours stretching on day 4 of EB generation |
Monitoring of intracellular [Ca2+]i; Expression of angiogenesis guidance molecules; Expression of pro-angiogenic growth factors; ROS generation |
[83] |
Mouse |
ESC, EB |
Mechanical strain by stretching device (Flexercell Strain Unit) |
5%, 10%, or 20% elongation of undifferentiated EBs |
2 hours stretching on day 4 of EB generation |
Staining of capillary-like structures; counting of beating bodies; staining of sarcomeric α-actinin; upregulation of NADPH oxidase subunits; ROS generation; inhibition of mechanical-strain stimulated MAPKs |
[84] |
3D Culture in Hydrogel |
Human |
ESC, EB |
Agarose 3D culture system |
Comparison of agarose 3D culture and suspension and hanging droplet culture |
7 days up to 8 weeks |
In vivo teratoma assay; morphology; germ layer differentiation (IF) |
[95] |
Human |
ESC |
Hydrogel based material for switching between alginate and collagen via ionic de-crosslinking |
Change hydrogel composition and in matrix elasticity from 21.37 ± 5.37 kPa (alginate) to 4.87 ± 1.64 kPa (collagen) |
21 days |
Viability; proliferation; pluripotency and germ layer differentiation (qRT-PCR) |
[96] |
Human |
ESC, EB |
Dextran-acrylate and PEG hydrogel +/- RGD and VEGF |
Comparison of dextran-acrylate and PEG hydrogel +/- RGD and VEGF |
10 days |
Viability; vascular differentiation (FACS, IF, qRT-PCR) |
[88] |
Human |
ESC, EB |
Biodegradable polymer scaffolds (50:50 PLGA:PLLA) |
Medium supplemented with different growth factors |
14 days in vitro; 14 days in vivo |
Proliferation; germ layer differentiation (qRT-PCR); transplantation into SCID mice (IF) |
[89] |
Human |
iPSC, EB |
Concave PEG hydrogel microstructures via 3D projection printing; initial cell number |
Comparison between concave and flat gels; low (250,000/mL) and high (750,000/mL) cell density |
Up to 10 days |
Morphology; culture duration; pluripotency and germ layer differentiation (IF); cyst formation |
[90] |
Mouse |
ESC, EB |
Hybrid hydrogels (GelMA, PEG) with varying matrix elasticity |
Analysis of a hybrid GelMA (3 wt%)/PEG (10 wt%) hydrogel |
Up to 7 days |
Vasculogenic and cardiogenic differentiation (qRT-PCR, IF) |
[91] |
Mouse |
ESC, EB |
PEG hydrogel with and without RGD |
Comparison of different PEG gels; 150 µm EBs and 450 µm EBs |
Up to 15 days |
Morphology; endothelial and cardiac differentiation (contraction behavior, IF, qRT-PCR) |
[92] |