Details
| Original language | English |
|---|---|
| Pages (from-to) | 2463-2471 |
| Number of pages | 9 |
| Journal | BIOMATERIALS |
| Volume | 34 |
| Issue number | 10 |
| Publication status | Published - Mar 2013 |
Abstract
In most pluripotent stem cell differentiation protocols the formation of embryoid bodies (EBs) is an important step. Here we describe a rapid, straightforward soft lithography approach for the preparation of hydrophilic silicon masters from different templates and the subsequent production of patterned agarose-DMEM microwell surfaces for scalable well standardized stem cell aggregation and EB formation. The non-adhesive agarose microwell plates represent an accurate replication of the templates' topography and were used for aggregation of murine induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs). Direct microscopic assessment by time-lapse analysis demonstrated rapid formation of uniformly shaped EBs from murine iPSCs with similar or even more consistent results concerning size distribution and harvesting efficiency compared to the commonly used but time-consuming hanging drop technique. For human ESCs, homogenous aggregates were obtained after single cell inoculation on agarose microwells with efficient differentiation into the cardiac lineage using state-of-the-art protocols for directed differentiation via small molecules. With this soft lithography-based strategy, sufficient and reproducible numbers of stem cell-derived cardiomyocytes necessary for tissue engineering purposes can be realized in a highly controllable manner. Moreover, it might be useful for different cell types in any application that requires scalable and highly standardized aggregation.
Keywords
- Agarose, Cardiomyogenic differentiation, Embryoid body, Microwell, Pluripotent stem cells, Soft lithography
ASJC Scopus subject areas
- Chemical Engineering(all)
- Bioengineering
- Materials Science(all)
- Ceramics and Composites
- Biochemistry, Genetics and Molecular Biology(all)
- Biophysics
- Materials Science(all)
- Biomaterials
- Engineering(all)
- Mechanics of Materials
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In: BIOMATERIALS, Vol. 34, No. 10, 03.2013, p. 2463-2471.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - The use of agarose microwells for scalable embryoid body formation and cardiac differentiation of human and murine pluripotent stem cells
AU - Dahlmann, Julia
AU - Kensah, George
AU - Kempf, Henning
AU - Skvorc, David
AU - Gawol, Anke
AU - Elliott, David A.
AU - Dräger, Gerald
AU - Zweigerdt, Robert
AU - Martin, Ulrich
AU - Gruh, Ina
N1 - Funding information: We thank Holm Zaehres and Hans Schöler for providing us with a batch culture of reprogrammed fibroblasts from OG2 mice and Monica Jara Avaca for the establishment and characterization of an iPSC-clone selected thereof. We thank Andrew G. Elefanty and Edouard G. Stanley for encouraging discussion and support concerning the human ESCs. The inhibitors CHIR-99021 and Y-27632 were a kind gift of Andreas Kirschning. Thomas Scheper kindly provided human FGF-2. We thank Ingrid Schmidt-Richer and Johanna Nolte for excellent technical assistance. This work was supported by the Cluster of Excellence REBIRTH ( DFG EXC 62/1 ), the National Health and Medical Research Foundation (NHMRC) of Australia and the Qatar National Research Fund (QNRF) .
PY - 2013/3
Y1 - 2013/3
N2 - In most pluripotent stem cell differentiation protocols the formation of embryoid bodies (EBs) is an important step. Here we describe a rapid, straightforward soft lithography approach for the preparation of hydrophilic silicon masters from different templates and the subsequent production of patterned agarose-DMEM microwell surfaces for scalable well standardized stem cell aggregation and EB formation. The non-adhesive agarose microwell plates represent an accurate replication of the templates' topography and were used for aggregation of murine induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs). Direct microscopic assessment by time-lapse analysis demonstrated rapid formation of uniformly shaped EBs from murine iPSCs with similar or even more consistent results concerning size distribution and harvesting efficiency compared to the commonly used but time-consuming hanging drop technique. For human ESCs, homogenous aggregates were obtained after single cell inoculation on agarose microwells with efficient differentiation into the cardiac lineage using state-of-the-art protocols for directed differentiation via small molecules. With this soft lithography-based strategy, sufficient and reproducible numbers of stem cell-derived cardiomyocytes necessary for tissue engineering purposes can be realized in a highly controllable manner. Moreover, it might be useful for different cell types in any application that requires scalable and highly standardized aggregation.
AB - In most pluripotent stem cell differentiation protocols the formation of embryoid bodies (EBs) is an important step. Here we describe a rapid, straightforward soft lithography approach for the preparation of hydrophilic silicon masters from different templates and the subsequent production of patterned agarose-DMEM microwell surfaces for scalable well standardized stem cell aggregation and EB formation. The non-adhesive agarose microwell plates represent an accurate replication of the templates' topography and were used for aggregation of murine induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs). Direct microscopic assessment by time-lapse analysis demonstrated rapid formation of uniformly shaped EBs from murine iPSCs with similar or even more consistent results concerning size distribution and harvesting efficiency compared to the commonly used but time-consuming hanging drop technique. For human ESCs, homogenous aggregates were obtained after single cell inoculation on agarose microwells with efficient differentiation into the cardiac lineage using state-of-the-art protocols for directed differentiation via small molecules. With this soft lithography-based strategy, sufficient and reproducible numbers of stem cell-derived cardiomyocytes necessary for tissue engineering purposes can be realized in a highly controllable manner. Moreover, it might be useful for different cell types in any application that requires scalable and highly standardized aggregation.
KW - Agarose
KW - Cardiomyogenic differentiation
KW - Embryoid body
KW - Microwell
KW - Pluripotent stem cells
KW - Soft lithography
UR - http://www.scopus.com/inward/record.url?scp=84872679014&partnerID=8YFLogxK
U2 - 10.1016/j.biomaterials.2012.12.024
DO - 10.1016/j.biomaterials.2012.12.024
M3 - Article
C2 - 23332176
AN - SCOPUS:84872679014
VL - 34
SP - 2463
EP - 2471
JO - BIOMATERIALS
JF - BIOMATERIALS
SN - 0142-9612
IS - 10
ER -