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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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Related in: MedlinePlus

Modelling of flow conditions in the microfluidic chip.(a) represents the velocity field at half the height of the inlet channel. (b) represents the velocity field 15 um above the culture plane (ACP). (c) shows velocity profiles at x0 along the z-axis.
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pone-0052246-g003: Modelling of flow conditions in the microfluidic chip.(a) represents the velocity field at half the height of the inlet channel. (b) represents the velocity field 15 um above the culture plane (ACP). (c) shows velocity profiles at x0 along the z-axis.

Mentions: To evaluate the design, we analysed the velocity fields and shear stress produced at a flow rate of 300 µl.h−1. This flow rate corresponds to replacing 13.8 ml of media per day for each square centimetre of culture area, a rate 50 times higher than typical in hESC culture. It is therefore unlikely cells would ever be subjected to a higher shear stress. The uniformity of the velocity field in the culture device was investigated at various heights above the culture plane (i.e. above the TC-PS slide). 15 µm above the cell culture plane, the average fluid velocity is approximately a factor of 10 lower than at 200 µm above the cell culture plane, which is in line with the inlet and outlet channels (Figure 3 (a, b)). The microfluidic chip design produces a relatively even velocity field across the majority of the culture chamber (Figure 3(b, c)). An increased velocity at the boundaries of the culture chamber can be observed due to the larger gap between flow restrictor and the boundary. This effect was deliberate and intended to remove air bubbles, entrapped during closing or filling. Hydrodynamic shear stress was also calculated 15 µm above the cell culture plane for a flow rate of 300 µl.h−1. An average of 1.1×10−4 Pa and a standard deviation of 0.14×10−4 Pa were obtained from the model. The calculated value of 1.3×10−4 Pa, using an analytical solution for shear stress at the culture surface, supports the result obtained through finite element modelling.


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)

Modelling of flow conditions in the microfluidic chip.(a) represents the velocity field at half the height of the inlet channel. (b) represents the velocity field 15 um above the culture plane (ACP). (c) shows velocity profiles at x0 along the z-axis.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0052246-g003: Modelling of flow conditions in the microfluidic chip.(a) represents the velocity field at half the height of the inlet channel. (b) represents the velocity field 15 um above the culture plane (ACP). (c) shows velocity profiles at x0 along the z-axis.
Mentions: To evaluate the design, we analysed the velocity fields and shear stress produced at a flow rate of 300 µl.h−1. This flow rate corresponds to replacing 13.8 ml of media per day for each square centimetre of culture area, a rate 50 times higher than typical in hESC culture. It is therefore unlikely cells would ever be subjected to a higher shear stress. The uniformity of the velocity field in the culture device was investigated at various heights above the culture plane (i.e. above the TC-PS slide). 15 µm above the cell culture plane, the average fluid velocity is approximately a factor of 10 lower than at 200 µm above the cell culture plane, which is in line with the inlet and outlet channels (Figure 3 (a, b)). The microfluidic chip design produces a relatively even velocity field across the majority of the culture chamber (Figure 3(b, c)). An increased velocity at the boundaries of the culture chamber can be observed due to the larger gap between flow restrictor and the boundary. This effect was deliberate and intended to remove air bubbles, entrapped during closing or filling. Hydrodynamic shear stress was also calculated 15 µm above the cell culture plane for a flow rate of 300 µl.h−1. An average of 1.1×10−4 Pa and a standard deviation of 0.14×10−4 Pa were obtained from the model. The calculated value of 1.3×10−4 Pa, using an analytical solution for shear stress at the culture surface, supports the result obtained through finite element modelling.

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
Related in: MedlinePlus