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Microfabricated modular scale-down device for regenerative medicine process development.

Reichen M, Macown RJ, Jaccard N, Super A, Ruban L, Griffin LD, Veraitch FS, Szita N - PLoS ONE (2012)

Bottom Line: The capacity of milli and micro litre bioreactors to accelerate process development has been successfully demonstrated in traditional biotechnology.Inactivated mouse embryonic fibroblasts (iMEF) and human embryonic stem cell (hESC) colonies were successfully seeded on gelatine-coated tissue culture polystyrene (TC-PS) using standard static seeding protocols.Cells were cultured for two days with media perfused at 300 µl.h(-1) resulting in a modelled shear stress of 1.1×10(-4) Pa.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Engineering, University College London, London, United Kingdom.

ABSTRACT
The capacity of milli and micro litre bioreactors to accelerate process development has been successfully demonstrated in traditional biotechnology. However, for regenerative medicine present smaller scale culture methods cannot cope with the wide range of processing variables that need to be evaluated. Existing microfabricated culture devices, which could test different culture variables with a minimum amount of resources (e.g. expensive culture medium), are typically not designed with process development in mind. We present a novel, autoclavable, and microfabricated scale-down device designed for regenerative medicine process development. The microfabricated device contains a re-sealable culture chamber that facilitates use of standard culture protocols, creating a link with traditional small-scale culture devices for validation and scale-up studies. Further, the modular design can easily accommodate investigation of different culture substrate/extra-cellular matrix combinations. Inactivated mouse embryonic fibroblasts (iMEF) and human embryonic stem cell (hESC) colonies were successfully seeded on gelatine-coated tissue culture polystyrene (TC-PS) using standard static seeding protocols. The microfluidic chip included in the device offers precise and accurate control over the culture medium flow rate and resulting shear stresses in the device. Cells were cultured for two days with media perfused at 300 µl.h(-1) resulting in a modelled shear stress of 1.1×10(-4) Pa. Following perfusion, hESC colonies stained positively for different pluripotency markers and retained an undifferentiated morphology. An image processing algorithm was developed which permits quantification of co-cultured colony-forming cells from phase contrast microscope images. hESC colony sizes were quantified against the background of the feeder cells (iMEF) in less than 45 seconds for high-resolution images, which will permit real-time monitoring of culture progress in future experiments. The presented device is a first step to harness the advantages of microfluidics for regenerative medicine process development.

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Schematic representation of a longitudinal section across the culture device.(a) Without a lid for coating with extra-cellular matrix compounds, and seeding feeder cells and hESC colonies with a pipette according to standard laboratory procedures. (b) With a lid and tubing for media perfusion.
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pone-0052246-g007: Schematic representation of a longitudinal section across the culture device.(a) Without a lid for coating with extra-cellular matrix compounds, and seeding feeder cells and hESC colonies with a pipette according to standard laboratory procedures. (b) With a lid and tubing for media perfusion.

Mentions: To address this goal our device includes a re-sealable lid. The lid provides a simple means of opening and closing the culture chamber. Thus, the device can be operated on both open and closed configuration during culture protocols (Figure 7). In the closed configuration, the height of the culture chamber is repeatably defined by the re-sealable lid. Additionally, the hard material of the lid does not deform during medium perfusion ensuring reproducible fluid flow patterns. In the open configuration, the culture chamber is directly accessible with laboratory pipettes facilitating pipette-based methods typically employed in laboratory scale stem cell maintenance including static seeding, static cell recovery and immunostaining. A further advantage of our device is that, in open configuration, the depth of media in the culture chamber is similar to the depths used in T25-flasks or culture dishes. Thus, during cell settling and attachment, the cells experience a similar microenvironment to traditional culture systems, addressing our objective of maintaining a link to conventional culture methods for validation. Previously presented re-sealable systems required seeding before assembly [47], which is cumbersome and results in poorly defined culture areas, or limited the height of the culture chamber to the total thickness of the device [48], potentially leading to excessive media hold-up times during perfusion.


Microfabricated modular scale-down device for regenerative medicine process development.

Reichen M, Macown RJ, Jaccard N, Super A, Ruban L, Griffin LD, Veraitch FS, Szita N - PLoS ONE (2012)

Schematic representation of a longitudinal section across the culture device.(a) Without a lid for coating with extra-cellular matrix compounds, and seeding feeder cells and hESC colonies with a pipette according to standard laboratory procedures. (b) With a lid and tubing for media perfusion.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526573&req=5

pone-0052246-g007: Schematic representation of a longitudinal section across the culture device.(a) Without a lid for coating with extra-cellular matrix compounds, and seeding feeder cells and hESC colonies with a pipette according to standard laboratory procedures. (b) With a lid and tubing for media perfusion.
Mentions: To address this goal our device includes a re-sealable lid. The lid provides a simple means of opening and closing the culture chamber. Thus, the device can be operated on both open and closed configuration during culture protocols (Figure 7). In the closed configuration, the height of the culture chamber is repeatably defined by the re-sealable lid. Additionally, the hard material of the lid does not deform during medium perfusion ensuring reproducible fluid flow patterns. In the open configuration, the culture chamber is directly accessible with laboratory pipettes facilitating pipette-based methods typically employed in laboratory scale stem cell maintenance including static seeding, static cell recovery and immunostaining. A further advantage of our device is that, in open configuration, the depth of media in the culture chamber is similar to the depths used in T25-flasks or culture dishes. Thus, during cell settling and attachment, the cells experience a similar microenvironment to traditional culture systems, addressing our objective of maintaining a link to conventional culture methods for validation. Previously presented re-sealable systems required seeding before assembly [47], which is cumbersome and results in poorly defined culture areas, or limited the height of the culture chamber to the total thickness of the device [48], potentially leading to excessive media hold-up times during perfusion.

Bottom Line: The capacity of milli and micro litre bioreactors to accelerate process development has been successfully demonstrated in traditional biotechnology.Inactivated mouse embryonic fibroblasts (iMEF) and human embryonic stem cell (hESC) colonies were successfully seeded on gelatine-coated tissue culture polystyrene (TC-PS) using standard static seeding protocols.Cells were cultured for two days with media perfused at 300 µl.h(-1) resulting in a modelled shear stress of 1.1×10(-4) Pa.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Engineering, University College London, London, United Kingdom.

ABSTRACT
The capacity of milli and micro litre bioreactors to accelerate process development has been successfully demonstrated in traditional biotechnology. However, for regenerative medicine present smaller scale culture methods cannot cope with the wide range of processing variables that need to be evaluated. Existing microfabricated culture devices, which could test different culture variables with a minimum amount of resources (e.g. expensive culture medium), are typically not designed with process development in mind. We present a novel, autoclavable, and microfabricated scale-down device designed for regenerative medicine process development. The microfabricated device contains a re-sealable culture chamber that facilitates use of standard culture protocols, creating a link with traditional small-scale culture devices for validation and scale-up studies. Further, the modular design can easily accommodate investigation of different culture substrate/extra-cellular matrix combinations. Inactivated mouse embryonic fibroblasts (iMEF) and human embryonic stem cell (hESC) colonies were successfully seeded on gelatine-coated tissue culture polystyrene (TC-PS) using standard static seeding protocols. The microfluidic chip included in the device offers precise and accurate control over the culture medium flow rate and resulting shear stresses in the device. Cells were cultured for two days with media perfused at 300 µl.h(-1) resulting in a modelled shear stress of 1.1×10(-4) Pa. Following perfusion, hESC colonies stained positively for different pluripotency markers and retained an undifferentiated morphology. An image processing algorithm was developed which permits quantification of co-cultured colony-forming cells from phase contrast microscope images. hESC colony sizes were quantified against the background of the feeder cells (iMEF) in less than 45 seconds for high-resolution images, which will permit real-time monitoring of culture progress in future experiments. The presented device is a first step to harness the advantages of microfluidics for regenerative medicine process development.

Show MeSH