Microfabricated Modular Scale-Down Device for Regenerative Medicine Process Development

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.

Bioprocess engineering strategies for autologous human MSC-based therapies: one size does not fit all

“…Reichen et al [68] have shown that the quantification of cell specific attributes can be measured in high throughput microfluidic devices, however the behaviour of cells on flat surfaces may not be necessarily predictive of that on surfaces possessing curvature such as for instance on microcarriers. Shear stress is a factor that can be encountered in multiple units of operation from impeller shear to acute capillary shear during flow through orifices and capillaries.…”

condiCharacterisation of an Adhesive-free Packaging System for Polymeric Microfluidic Biochemical Devices and Reactors

“…32 Briefly, reusable components and the PDMS chip were sterilised separately by autoclave before assembly in a biological safety cabinet. Culture surface coating and cell seeding were carried out by pipette before placing the system in an incubator during cell attachment.…”

“…2 and has been described previously. 32 It consisted of a disposable PDMS microfluidic layer (containing a culture chamber volume of 25 µL and microfluidic channels) placed on a microscope-format TC-PS slide. Polycarbonate frames and interconnects provided packaging, sealing and connection with external pumping systems.…”

“…Secondly, two different cell culture PDMS chips were fabricated from precision milled aluminium moulds to demonstrate biological relevance. Additionally, perfusion culture of human embryonic stem cell colonies 32 was demonstrated. Finally, a hydrodynamic vertical flow-focusing device showed the compatibility of our packaging approach with multilayer structures comprised of thin slabs of cast PDMS.…”

Soluble compound screening using microfluidic gradients for regenerative medicine research

“…Micronit, microLIQUID) or are mainly meant for PDMS cell culture devices. [250][251][252] To overcome these issues, a platform was developed in which entirely closed microfluidic systems made from gas-impermeable materials can be used for cell culture under varying physiological conditions (Fig. 3.1).…”