peristaltic pump inside incubator

The oil immersion objective used has a working distance of about 0.1mm and cannot focus across standard Petri dishes and is designed for imaging across a cover-slip. We have designed, fabricated, and tested a perfusion culture system consisting of miniature bioreactors and miniature peristaltic pumps for doing perfusion culture completely inside standard incubators. The distribution of shear stress is asymmetric with regions of maximum shear stress occurring close to the bioreactor inlet and further local maxima near to the center of the bioreactor (Fig. The versatility of our bioreactor was further assessed by growing primary rat hepatocytes in them. 7A). National Library of Medicine The cell viability on the second, fourth, and sixth days were estimated by using Calcein AMEthidium Bromide livedead assay kit (Life Technologies). sec).14 The media is assumed to be an incompressible and Newtonian fluid. 10). The cover-slip was then prebaked at 95C for 10min to ensure that SU-8 does not stick to the mask during UV exposure. The authors would like to acknowledge Deepa Prasanna for designing the peristaltic pump control board, Maniprasad of Keerthana Industries, Bengaluru, for fabricating the rotor of the peristaltic pump, and the Department of Biotechnology, Govt. Careers, This Open Access article is distributed under the terms of the Creative Commons License (, 3D printing, bioreactor, live imaging, microfabricated scaffolds, perfusion culture, peristaltic pump, VeroWhite, A new approach to neural cell culture for long-term studies, A simple flow-through micro-chamber for handling fragile, small tissue explants and single non-adherent cells, A thin-walled polydimethylsiloxane bioreactor for high-density hepatocyte sandwich culture, An integrated environmental perfusion chamber and heating system for long-term, high resolution imaging of living cells, Perfused multiwell plate for 3D liver tissue engineering. High-magnification time-lapse imaging of HuH7 cells incubated with livedead assay mixture (Calcein AMEthidium Bromide). 7B, bottom). Furthermore, the material can be autoclaved which helps in effective and convenient sterilization of the bioreactors. The asymmetry in the shear stress distribution is due to the inlet being at a lower height than the outlet. The scaffold was then washed thoroughly with PBS and sterilized by exposing to UV overnight.

The bioreactor is also suitable for high-magnification imaging, single cell imaging studies since the cells are grown on cover-slips and hence they can be viewed from below on an inverted microscope. 8). This is significant because any stable structure can be fabricated easily using 3D printing and can be used as tissue engineering scaffolds or containers for cell culture. Scaffolds made by microfabrication on cover-slips can be incorporated directly into the bioreactor and cells grown under perfusion on these scaffolds. 8600 Rockville Pike The maximum shear stress is around 0.29mPa and occurs close to the inlet. Blue, day 2; red, day 4; green, day 6. 7B, top). The maximum shear stress on the cells is around 0.29mPa, while the reported shear stress level that is harmful for cell viability is about 33mPa16about three orders of magnitude higher. In addition, our system can also be used for high-resolution live cell imaging. Flow inside the bioreactor was assumed to be laminar. Live imaging of NIH 3T3 fibroblasts growing on ring scaffolds. Percentage of live cells in 24-well plate, 24-well plate coated with collagen, static and perfusion bioreactors are shown. The postbaked cover-slip was then immersed in SU-8 developer solution for 7min to remove the unexposed regions and further dehydrated at 200C for 2min to get a dry sample. The bioreactors were coated with collagen before seeding cells by incubating the bioreactor with 700L of 30g/mL of type I collagen (rat tail, Gibco by Life Technologies) for 4h at 37C.

The numerical simulation was done using the Laminar Flow module in COMSOL 4.2 (COMSOL AB). This shows that collagen does not affect the viability of MCF7 cells. Time-lapse images of the HuH7 cells incubated with the livedead assay mixture are shown in Figure 11. Kostov Y, Harms P, Randers-Eichhorn L, et al..

The cover-slip containing the scaffold was assembled into the bioreactor and further coated with collagen by immersing it in a solution of 30g/mL of type I collagen (rat tail, Gibco by Life Technologies) for 4h at 37C to aid cell attachment. 11). Bioreactor and pump characterization. The cells were found to be viable under perfusion (>80%) even after 6 days without significant difference in cell viability from those in static bioreactors. The flow from the pump was assumed to be uniform and any pulsatile action was neglected. Since the cells in the bioreactors (static and perfusion) were grown on top of collagen-coated cover-slips, we wanted to rule out the effect of collagen on the cell viability. Cells were found to grow on the glass substrate inside the rings as well as on top of the rings. The bioreactors were made of VeroWhite material and fabricated by 3D printing.

Different bars represent different days of culture. After 9 days, the cells were apparently found to lose viability and were mostly in the suspension. Around 30,000MCF7 cells were seeded in the bioreactors and controls. The flow rate of the pump was measured for different voltages at zero pressure head across the pump and also for different pressure heads at a constant voltage of 3V across the pump (Fig. The cells were mounted on the microscope at room temperature (25C) and hence the cells slowly started to die. Scaffolds were made on a 22mm circular cover-slip using photolithography using SU-8, a negative photoresist used in lithography-based patterning in microfabrication. The cells were allowed to attach for 24h before starting perfusion.

BioResearch Open Access 4:1, 343357, DOI: 10.1089/biores.2015.0024. In this study, we demonstrate live imaging of NIH 3T3 mouse fibroblast cells (Fig. Halldorsson S, Lucumi E, Gmez-sjberg R, et al.. Cells are stained using Calcein AM (live) and Ethidium Bromide (dead). NIH 3T3 cells were added into the bioreactor containing the scaffold immediately after collagen coating and allowed to attach for 24h. The bioreactor was transferred to a microscope with a live-cell stage for long-duration live imaging. FOIA Other assumptions like no-slip boundary condition on the boundaries, uniform velocity at the inlet of the bioreactor, and zero gauge pressure at the outlet of the bioreactor were made. This could be useful for cell migration assays, drug toxicity studies, characterizing cell response to chemical stimuli, etc. Hepatocytes were found to be viable inside the bioreactor and could continue growing up to 9 days.

Our study shows that this material and fabrication technique is conducive for ex vivo cell culture. The bioreactor was then transferred to an inverted fluorescence microscope (Leica DMI6000B) and imaged using a high numerical aperture oil immersion objective (HCX PL APO 40/1.250.75OIL) for around 30min. Cell viability of MCF7 cells in perfusion bioreactor is compared with the cell viability in static bioreactor (i.e., no perfusion) in Figure 8. The days are counted from the day when cells were seeded. Tayalia P, Mendonca CR, Baldacchini T, et al.. 3D cell-migration studies using two-photon engineered polymer scaffolds, Membrane-based PDMS microbioreactor for perfused 3D primary rat hepatocyte cultures, Long-term culture and coculture of primary rat and human hepatocytes, http://creativecommons.org/licenses/by/4.0. The hepatocytes grew into cell aggregates and exhibited cuboidal and polygonal morphology inside the bioreactor, as has been reported in various previous studies5,21 (Fig. The cover-slips were heated to about 200C for 2min to evaporate water that might be there on the cover-slip and allowed to cool to room temperature. While many previously reported bioreactor systems (reviewed in Shulman and Nahmias22) have achieved longer term hepatocyte cultures, we have just shown that the basic miniature bioreactor that we have developed also supports primary cell culture. Interestingly, the absolute numbers of cells were lower in the bioreactors (both static and perfusion) in comparison with the 24-well plates, but the cell viability was similar (Fig. Viability of cells under perfusion in the bioreactor.

All images were taken at 10 magnification. The cover-slip was spin-coated with SU-8 2005 at 4,000rpm for 40sec to get a uniform layer of SU-8 of thickness 5m over the cover-slip. Once inserted into the bioreactors high-resolution live cell imaging and/or perfusion of media can be performed on the cells growing on these scaffolds. There is no special significance about the ring geometry here; this readily available geometry is representative of many other geometries that can be fabricated with SU-8. Only those preparations of hepatocytes with viability greater than 90% were used for the experiments. The geometry was created, meshed, and solved using COMSOL and the resultant shear stress on the bottom of the bioreactor was calculated and plotted. Low-cost microbioreactor for high-throughput bioprocessing, Design of a high-throughput flow perfusion bioreactor system for tissue engineering, Design of a flow perfusion bioreactor system for bone tissue-engineering applications. We have also demonstrated high-resolution imaging of liver carcinoma (HuH7) cells. This could be due to slower growth rate in the bioreactor. We have tested it up to a pressure of 2m high column of water (20kPa) and the pump worked without any discernible difference in the flow rate. Many researchers use microfabricated structures as moulds for making soft polymer (like PDMS) scaffolds.19 In such cases, the final soft polymer scaffold can be attached to a cover-slip by oxygen plasma treatment and then inserted into our bioreactors. Figure 9 shows that such microfabricated scaffolds can be conveniently inserted into our bioreactor and the effect of the scaffold on the cells can be studied. Our pump, like most other peristaltic pumps, can work at much higher pressure heads. The live and dead cells were manually counted on a set of 10 images which were randomly selected from the total number of images for each tile scan. Microfabrication is a popular choice for making scaffolds of exact geometries for growing cells.17,18 Microfabricated scaffolds have been used for 3D cell culture,19 studying cell migration,20 etc.

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