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Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates.

Ungrin MD, Joshi C, Nica A, Bauwens C, Zandstra PW - PLoS ONE (2008)

Bottom Line: Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions.Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis.These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues, and accelerate the pre-clinical evaluation of hESC-derived cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT

Background: Human embryonic stem cells (hESC) should enable novel insights into early human development and provide a renewable source of cells for regenerative medicine. However, because the three-dimensional hESC aggregates [embryoid bodies (hEB)] typically employed to reveal hESC developmental potential are heterogeneous and exhibit disorganized differentiation, progress in hESC technology development has been hindered.

Methodology/principal findings: Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions. These aggregates exhibited coordinated bi-domain structures including contiguous regions of extraembryonic endoderm- and epiblast-like tissue. A silicon wafer-based microfabrication technology was used to generate surfaces that permit the production of hundreds to thousands of hEB per cm(2).

Conclusions/significance: The mechanisms of early human embryogenesis are poorly understood. We report an ultra high throughput (UHTP) approach for generating spatially and temporally synchronised hEB. Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis. These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues, and accelerate the pre-clinical evaluation of hESC-derived cells.

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Related in: MedlinePlus

Micropatterned surfaces allow ultra-high-throughput (UHTP) production of size-specified aggregates.Surfaces patterned with arrays of microwells were generated (A) in poly(dimethylsiloxane) via serial replica moulding from a pattern etched into a silicon wafer. Imaging of the silicon master, the PDMS negative cast, and the PDMS positive cast show conservation of form across these steps with wells 100, 200, 400 and 800 microns square (B). Note that the 800 micron wells were generated in the form of a truncated pyramid. Dashed yellow square represents one square millimeter. Sections of PDMS textured with 400 micron wells (C) were inserted into individual wells in a 24-well plate. A single-cell suspension of hESC grown on MEF, predifferentiated with serum for 48 hours was dispensed into the well such that each microwell was predicted to capture the desired number of cells. After 24 hours, the well contents were imaged, extracted, and re-imaged. Scale bar represents 400 microns. D. Centrifugation is not required in the presence of ROCK inhibition. Cells grown on Matrigel in defined conditions were dispensed over the microwells in the presence or absence of 10 µM of the ROCK inhibitor Y-27632. Aggregates formed only in the presence of 10 µM of the ROCK inhibitor Y-27632, in the presence or absence of centrifugation. Scale bar represents 400 microns.
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pone-0001565-g004: Micropatterned surfaces allow ultra-high-throughput (UHTP) production of size-specified aggregates.Surfaces patterned with arrays of microwells were generated (A) in poly(dimethylsiloxane) via serial replica moulding from a pattern etched into a silicon wafer. Imaging of the silicon master, the PDMS negative cast, and the PDMS positive cast show conservation of form across these steps with wells 100, 200, 400 and 800 microns square (B). Note that the 800 micron wells were generated in the form of a truncated pyramid. Dashed yellow square represents one square millimeter. Sections of PDMS textured with 400 micron wells (C) were inserted into individual wells in a 24-well plate. A single-cell suspension of hESC grown on MEF, predifferentiated with serum for 48 hours was dispensed into the well such that each microwell was predicted to capture the desired number of cells. After 24 hours, the well contents were imaged, extracted, and re-imaged. Scale bar represents 400 microns. D. Centrifugation is not required in the presence of ROCK inhibition. Cells grown on Matrigel in defined conditions were dispensed over the microwells in the presence or absence of 10 µM of the ROCK inhibitor Y-27632. Aggregates formed only in the presence of 10 µM of the ROCK inhibitor Y-27632, in the presence or absence of centrifugation. Scale bar represents 400 microns.

Mentions: To accomplish this goal, we have used a novel approach to generate textured surfaces consisting of numerous collecting volumes, or micro-wells, at the base of a single common liquid volume. Our design parameters included angled collecting surfaces sloped towards a common collecting point, complete surface tiling (to avoid interference and inefficiencies arising from cells landing in the dead space between micro-wells), and biocompatibility. Microfabrication of a master mold, followed by replica molding in poly(dimethylsiloxane) (PDMS) is a technique capable of duplicating extremely fine features [43]. PDMS replica molding has been used to cast arrays of widely spaced vertical-walled micro-wells for ESC culture from templates generated via soft-lithographic techniques [44], [45]. Importantly however, soft lithography is not well suited to the generation of the angled collecting surfaces required for this application, and the use of vertical sidewalls also places mechanical-strength limitations on how closely wells can be spaced. We took advantage of the susceptibility of crystalline silicon to anisotropic etching[46] to generate square-pyramidal pits in the surface of a 1-0-0 silicon wafer. As a result of the orientation-specific resistance of internal crystal planes to the etchant, angled sidewalls of near atomic-level perfection parallel to these planes are formed. These sidewalls converge to a point if the etching is allowed to proceed to completion; if the reaction is stopped before completion, a truncated pyramidal well is formed. We then proceeded through two serial rounds of PDMS casting, generating first a negative cast of the silicon wafer, an array of pyramids, and then a positive cast from the negative cast, replicating the array of micro-wells from the original silicon wafer in biocompatible PDMS. Figure 4A depicts a schematic of this process, while panel B contains the corresponding micrographs of the production of 800, 400, 200 and 100 micron square micro-wells (yellow box denotes one square millimetre). The upper row shows a top view of the silicon master, the middle row a section through the PDMS negative casts, and the lower row a section through the final PDMS wells (Figure 4A and B). Note that the 800 micron wells were not etched to completion, demonstrating a truncated pyramidal shape. The apparent rounding of the points of the smaller wells in the lateral sections is an artefact of imperfect alignment between the well point and the plane of the cut.


Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates.

Ungrin MD, Joshi C, Nica A, Bauwens C, Zandstra PW - PLoS ONE (2008)

Micropatterned surfaces allow ultra-high-throughput (UHTP) production of size-specified aggregates.Surfaces patterned with arrays of microwells were generated (A) in poly(dimethylsiloxane) via serial replica moulding from a pattern etched into a silicon wafer. Imaging of the silicon master, the PDMS negative cast, and the PDMS positive cast show conservation of form across these steps with wells 100, 200, 400 and 800 microns square (B). Note that the 800 micron wells were generated in the form of a truncated pyramid. Dashed yellow square represents one square millimeter. Sections of PDMS textured with 400 micron wells (C) were inserted into individual wells in a 24-well plate. A single-cell suspension of hESC grown on MEF, predifferentiated with serum for 48 hours was dispensed into the well such that each microwell was predicted to capture the desired number of cells. After 24 hours, the well contents were imaged, extracted, and re-imaged. Scale bar represents 400 microns. D. Centrifugation is not required in the presence of ROCK inhibition. Cells grown on Matrigel in defined conditions were dispensed over the microwells in the presence or absence of 10 µM of the ROCK inhibitor Y-27632. Aggregates formed only in the presence of 10 µM of the ROCK inhibitor Y-27632, in the presence or absence of centrifugation. Scale bar represents 400 microns.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2215775&req=5

pone-0001565-g004: Micropatterned surfaces allow ultra-high-throughput (UHTP) production of size-specified aggregates.Surfaces patterned with arrays of microwells were generated (A) in poly(dimethylsiloxane) via serial replica moulding from a pattern etched into a silicon wafer. Imaging of the silicon master, the PDMS negative cast, and the PDMS positive cast show conservation of form across these steps with wells 100, 200, 400 and 800 microns square (B). Note that the 800 micron wells were generated in the form of a truncated pyramid. Dashed yellow square represents one square millimeter. Sections of PDMS textured with 400 micron wells (C) were inserted into individual wells in a 24-well plate. A single-cell suspension of hESC grown on MEF, predifferentiated with serum for 48 hours was dispensed into the well such that each microwell was predicted to capture the desired number of cells. After 24 hours, the well contents were imaged, extracted, and re-imaged. Scale bar represents 400 microns. D. Centrifugation is not required in the presence of ROCK inhibition. Cells grown on Matrigel in defined conditions were dispensed over the microwells in the presence or absence of 10 µM of the ROCK inhibitor Y-27632. Aggregates formed only in the presence of 10 µM of the ROCK inhibitor Y-27632, in the presence or absence of centrifugation. Scale bar represents 400 microns.
Mentions: To accomplish this goal, we have used a novel approach to generate textured surfaces consisting of numerous collecting volumes, or micro-wells, at the base of a single common liquid volume. Our design parameters included angled collecting surfaces sloped towards a common collecting point, complete surface tiling (to avoid interference and inefficiencies arising from cells landing in the dead space between micro-wells), and biocompatibility. Microfabrication of a master mold, followed by replica molding in poly(dimethylsiloxane) (PDMS) is a technique capable of duplicating extremely fine features [43]. PDMS replica molding has been used to cast arrays of widely spaced vertical-walled micro-wells for ESC culture from templates generated via soft-lithographic techniques [44], [45]. Importantly however, soft lithography is not well suited to the generation of the angled collecting surfaces required for this application, and the use of vertical sidewalls also places mechanical-strength limitations on how closely wells can be spaced. We took advantage of the susceptibility of crystalline silicon to anisotropic etching[46] to generate square-pyramidal pits in the surface of a 1-0-0 silicon wafer. As a result of the orientation-specific resistance of internal crystal planes to the etchant, angled sidewalls of near atomic-level perfection parallel to these planes are formed. These sidewalls converge to a point if the etching is allowed to proceed to completion; if the reaction is stopped before completion, a truncated pyramidal well is formed. We then proceeded through two serial rounds of PDMS casting, generating first a negative cast of the silicon wafer, an array of pyramids, and then a positive cast from the negative cast, replicating the array of micro-wells from the original silicon wafer in biocompatible PDMS. Figure 4A depicts a schematic of this process, while panel B contains the corresponding micrographs of the production of 800, 400, 200 and 100 micron square micro-wells (yellow box denotes one square millimetre). The upper row shows a top view of the silicon master, the middle row a section through the PDMS negative casts, and the lower row a section through the final PDMS wells (Figure 4A and B). Note that the 800 micron wells were not etched to completion, demonstrating a truncated pyramidal shape. The apparent rounding of the points of the smaller wells in the lateral sections is an artefact of imperfect alignment between the well point and the plane of the cut.

Bottom Line: Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions.Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis.These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues, and accelerate the pre-clinical evaluation of hESC-derived cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT

Background: Human embryonic stem cells (hESC) should enable novel insights into early human development and provide a renewable source of cells for regenerative medicine. However, because the three-dimensional hESC aggregates [embryoid bodies (hEB)] typically employed to reveal hESC developmental potential are heterogeneous and exhibit disorganized differentiation, progress in hESC technology development has been hindered.

Methodology/principal findings: Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions. These aggregates exhibited coordinated bi-domain structures including contiguous regions of extraembryonic endoderm- and epiblast-like tissue. A silicon wafer-based microfabrication technology was used to generate surfaces that permit the production of hundreds to thousands of hEB per cm(2).

Conclusions/significance: The mechanisms of early human embryogenesis are poorly understood. We report an ultra high throughput (UHTP) approach for generating spatially and temporally synchronised hEB. Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis. These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues, and accelerate the pre-clinical evaluation of hESC-derived cells.

Show MeSH
Related in: MedlinePlus