The days are counted from the day when cells were seeded. Long-term cell survival can be achieved by employing scaffolds, perfusion of media, and various other techniques such as collagen sandwich, etc., all of which can be conveniently incorporated into our system. The maximum shear stress is around 0.29mPa and occurs close to the inlet. Scaffolds used were rings of 300m inner diameter and about 5m height. About 4105 hepatocytes were seeded in each bioreactor and maintained in DMEM containing 100g/mL of penicillin and streptomycin. 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. 7B, bottom). We have measured the flow rates against pressure heads only up to 7kPa because when we are perfusing the media across a single bioreactor from the reservoir and back into the same, we do not expect the pressure head to be very large. Cell viability in 24-well plates and 24-well plates coated with collagen was also estimated to infer the contribution of collagen to cell growth and viability.

Blue, day 2; red, day 4; green, day 6. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices, Fundamentals of microfluidic cell culture in controlled microenvironments, A novel 3D mammalian cell perfusion-culture system in microfluidic channels, A practical guide to microfluidic perfusion culture of adherent mammalian cells, Fundamentals of Electric Circuit Analysis. teer chip encapsulation biochip transwell ali The numerical simulation was done using the Laminar Flow module in COMSOL 4.2 (COMSOL AB). 9) on SU-8 ring scaffolds. After the exposure, the cover-slip was postbaked at 95C for 2min. Different bars represent different days of culture. Various locations on the scaffolds were marked and pictures of these marked locations were taken every 20min for 40h. Primary rat hepatocytes were isolated from normal rat (Wistar strain) by collagenase perfusion.15 The viability of the cells was checked using Trypan blue (0.4%) exclusion.

Time-lapse images of the HuH7 cells incubated with the livedead assay mixture are shown in Figure 11. 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. This is clearly captured in the live imaging as the cells which were fluorescing green (Calcein AMstains live cells) slowly started to fluoresce red (Ethidium Bromidestains dead cells; Fig. 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. 7B, top). The cell viability on the second, fourth, and sixth days were estimated by using Calcein AMEthidium Bromide livedead assay kit (Life Technologies). Cell viability of MCF7 cells in perfusion bioreactor is compared with the cell viability in static bioreactor (i.e., no perfusion) in Figure 8. HHS Vulnerability Disclosure, Help Cell growth under perfusion for longer durations will be addressed in future experiments. 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. 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. 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. The cover-slip was then prebaked at 95C for 10min to ensure that SU-8 does not stick to the mask during UV exposure. We have also demonstrated high-resolution imaging of liver carcinoma (HuH7) cells. 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. Trachtenberg JE, Mountziaris PM, Miller JS, et al.. Open-source three-dimensional printing of biodegradable polymerscaffolds for tissue engineering, A three-dimensional scaffold with precise micro-architecture and surface micro-textures. 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. 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.

Furthermore, the material can be autoclaved which helps in effective and convenient sterilization of the bioreactors. 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. 8). High-magnification time-lapse imaging of HuH7 cells incubated with livedead assay mixture (Calcein AMEthidium Bromide). The temperature and CO2 were set to 37C and 5%, respectively, and allowed to stabilize for 5min before inserting the bioreactor. Our system is amenable to live-cell imaging, studying the growth of cells on scaffolds, and performing long-term perfusion culture completely inside an incubator. 7A).

Our pump, like most other peristaltic pumps, can work at much higher pressure heads. The results demonstrate that the design and implementation of the perfusion culture system allows cell growth and culture. Viability of cells under perfusion in the bioreactor.

Fluorescent images of a large portion (80% of the total area) of the bioreactors (static and perfusion) were obtained by using the tile scan option on a fluorescent microscope with a motorized stage. Our study shows that this material and fabrication technique is conducive for ex vivo cell culture. 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. Scaffolds were made on a 22mm circular cover-slip using photolithography using SU-8, a negative photoresist used in lithography-based patterning in microfabrication. We have demonstrated that the bioreactor is suitable for general and perfusion tissue culture studies by culturing primary (rat hepatocytes), immortalized (NIH 3T3), and cancerous (MCF7) cells. 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. Hepatocytes were found to be viable inside the bioreactor and could continue growing up to 9 days. 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. (A) Shear stress in the bioreactorThe total shear stress on the cells due to the flow of media from the peristaltic pump is shown. This could be useful for cell migration assays, drug toxicity studies, characterizing cell response to chemical stimuli, etc. The cells were mounted on the microscope at room temperature (25C) and hence the cells slowly started to die. The versatility of our bioreactor was further assessed by growing primary rat hepatocytes in them. Around 30,000HuH7 cells were seeded into the bioreactor and allowed to attach and grow for 24h. The cells were then incubated with the Calcein AMEthidium Bromide livedead assay (Life Technologies) mixture at 37C for 10min. FOIA

After 9 days, the cells were apparently found to lose viability and were mostly in the suspension. Kostov Y, Harms P, Randers-Eichhorn L, et al.. Around 30,000MCF7 cells were seeded in the bioreactors and controls. bioreactor electrospun fleeces of India, for funding. 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. This shows that collagen does not affect the viability of MCF7 cells.

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. The total number of cells in the bioreactors was extrapolated from the average of the number of cells in these 10 images by multiplying with the ratio of the area of the bioreactor to the area of the image. 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. 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. The media were changed every day. Halldorsson S, Lucumi E, Gmez-sjberg R, 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). lfdo a15 a13 labtron 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. National Library of Medicine 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. Since our bioreactor uses a cover-slip for growing cells and is open from below, an objective lens can easily approach the bioreactor from the bottom for imaging. incubator In this study, we demonstrate live imaging of NIH 3T3 mouse fibroblast cells (Fig. Bethesda, MD 20894, Web Policies 10). Representative fluorescent images of each culture are shown above with the days increasing from top to bottom. (B) Pump flow characterizationFlow rate (mL/min) of the pump at zero pressure head for various voltages (in Volts; top) and at a constant voltage (3V) for various pressure heads (in kPa; bottom). 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. In fact, the pressure drop across the bioreactor is only around 0.66Pa as was found from the computational fluid dynamics simulation. The current study shows that microfabricated scaffolds can be inserted into the bioreactor and that cells can be grown on them conveniently. Cells are stained using Calcein AM (live) and Ethidium Bromide (dead). Flow inside the bioreactor was assumed to be laminar. hotplate stirrer 115v The days are counted from the day on which cells were seeded. Only those preparations of hepatocytes with viability greater than 90% were used for the experiments. 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. 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.

Live imaging of NIH 3T3 fibroblasts growing on ring scaffolds. 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 rate increases almost linearly with the voltage for zero pressure head. sec).14 The media is assumed to be an incompressible and Newtonian fluid. lmi incubator microplate All images were taken at 10 magnification. BioResearch Open Access 4:1, 343357, DOI: 10.1089/biores.2015.0024. In the case of varying pressure heads (07kPa) at constant voltage of 3V, the flow rate is almost constant (Fig. 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. Scaffolds made by microfabrication on cover-slips can be incorporated directly into the bioreactor and cells grown under perfusion on these scaffolds.

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. 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. Primary rat hepatocytes in the static bioreactor. To investigate the effect of collagen coating on cell viability, we have performed the livedead assay on the cells cultured in 24-well plates with and without collagen coating.

The flow from the pump was assumed to be uniform and any pulsatile action was neglected.

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. As can be observed from Figure 9, the cells gradually populated the entire scaffold and by the end of 40h they had grown to almost full confluency. Further, the cover-slip was exposed to UV using an EVG 620 Mask Aligner with a peak exposure energy of 374mJ/cm2. Furthermore, it can be used for studying the response of cells to chemical stimulants and drugs at a single cell level (through high-magnification fluorescence imaging) and at a population level in long-term perfusion cultures. Ovsianikov A, Schlie S, Ngezahayo A, et al.. Two-photon polymerization technique for microfabrication of CAD-designed 3D scaffolds from commercially available photosensitive materials. 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. The cells were allowed to attach for 24h before starting perfusion. Accessibility In addition, our system can also be used for high-resolution live cell imaging. Lieberthal W, Wolf EF, Merrill EW, et al.. Hemodynamic effects of different preparations of stroma free hemolysates in the isolated perfused rat kidney, Effects of oxygenation and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor. 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. The cell viability or total numbers of cells were not different between 24-well plates with and without collagen coating (Fig. Cite this article as: Balakrishnan S, Suma MS, Raju S, Bhargav SDB, Arunima S, Das S, Ananthasuresh GK (2015) A scalable perfusion culture system with miniature peristaltic pumps for live-cell imaging assays with provision for microfabricated scaffolds. The geometry was created, meshed, and solved using COMSOL and the resultant shear stress on the bottom of the bioreactor was calculated and plotted. 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. 8). Bioreactor and pump characterization. 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. Percentage of live cells in 24-well plate, 24-well plate coated with collagen, static and perfusion bioreactors are shown. 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. This could be due to slower growth rate in the bioreactor. 8600 Rockville Pike