This work is supported by the Victorian Government&#39;s Operational Infrastructure Support Program, and the Victorian Government Technology Voucher provided by the Department of Economic Development, Jobs, Transport and Resources. RL is the recipient of a National Health and Medical Research Council Career Development Fellowship. AL is the recipient of an Australian Postgraduate Award.

1. Preparation of reagents and cells for buffer exchange
<ol>
	<li>Prepare buffers (see Table of Materials) in a class 2 laminar flow hood. Using a syringe and needle assembly, remove 50 mL of saline solution from a 500 mL saline bag. Replace this with 50 mL of 20% human serum albumin (HSA) to make 2% HSA in saline, which will serve as the wash buffer.</li>
	<li>Remove the cells from the culture vessels and perform a cell count to determine the starting cell quantity and viability by trypan blue exclusion. Load the cells into a transfer bag. 
	NOTE: In this protocol Jurkat cells were cultured in RPMI and MSCs were cultured in DMEM:F12. Both media were supplemented with 10% fetal bovine serum (FBS) and 1% Antibiotic-Antimycotic solution. Cells were cultured at 37 &#176;C with 5% CO2. For MSCs or adherent cells, add a digestion enzyme (e.g., trypsin) into the culture vessels in order to detach the cells, and quench with culture media once the cells are detached. A transfer bag was used in this protocol because the cells were expanded in culture vessels. However, if the cells are cultured in a cell culture bag, it can be directly connected to the single-use kit via sterile tube welding. In this protocol, either 3 x 107 Jurkat cells or 1 x 107 MSCs were used for each run, where cells were suspended in 40 mL of culture media.
	<ol>
		<li>Load cells into a transfer bag (see Table of Materials) using a syringe and needle assembly if a sterile tube welder is available to connect the transfer bag to the single use processing kit.</li>
		<li>Alternatively, use a pair of autoclaved scissors to cut the connecting tube with around 10 cm remainining attached to the transfer bag and add a female Luer connector to the end of the tubing. Use a syringe to aspirate cells and media, then connect the syringe to the Luer connector and load cells to the transfer bag.</li>
	</ol>
	</li>
	<li>Connect the bag containing the cells, wash buffer bag containing 500 mL of wash buffer prepared in step 1.1, waste bag, and collecting syringe to the single-use kit (Figure 1B). 
	NOTE: The connecting position of each bag depends on the settings in the program. In this protocol, we connected the waste bag at position A, wash buffer at position B, cell bag at positions D and F, and collection syringe at position H (Figure 1C).</li>
</ol>
2. Program for automated buffer exchange protocol
<ol>
	<li>Open the Graphic User Interface (GUI) of the device and press the &#34;START&#34; button on a laptop PC or tablet. Then &#34;Open&#34; an existing protocol or press &#34;New&#34; to create a new one.</li>
	<li>Use the &#34;Forward&#34; and &#34;Backward&#34; button to navigate through each step, select the valves to be opened/closed, centrifugal speed, pump speed, pump direction, and action triggers. The settings for each step are summarized in Table 1. Then save the protocol.</li>
</ol>
3. Setting up the machine
<ol>
	<li>Place the single-use processing kit on the machine and hang the connected bags accordingly. Press the red &#34;Stop/Reset&#34; button to reset all valves into the default closed position. 
	NOTE: The machine will perform a test when the door is closed to ensure the kit has been placed correctly and that all the valves are functioning appropriately.</li>
	<li>Connect the GUI to the processing device and press the &#34;Connect&#34; button. Then download the saved program. When the green &#34;Play&#34; button lights up, it indicates that the protocol is ready to start.</li>
	<li>Open the manual clamps for the transfer bag tubing. 
	NOTE: If the operator forgets to open the clamps, the device will stop and the pressure sensor warning will appear. Press the &#34;Stop&#34; button to reset the device and open the clamps to start again.</li>
</ol>
4. Automated buffer exchange
<ol>
	<li>Press &#34;Start&#34; to initiate the buffer exchange program. 
	NOTE: To manually alter the automated process, press the &#34;Next&#34; button to move to the next step before reaching the &#34;Trigger&#34; or press &#34;Pause&#34; to put the process on hold. This will recirculate the cells through the kit while keeping only valves J and K open (Figure 1C).</li>
	<li>At the end of automation step 6 (Table 1), the process will pause when air in the now emptied bag triggers the bubble sensor. Press &#34;Next&#34; to move the process to the next step if the bag is indeed empty, or press &#34;Pause&#34; to resume the processing if the sensor was triggered by a bubble in the line.</li>
</ol>
5. Collecting and sampling the cells
<ol>
	<li>When the automated process is finished, close all tubing clamps. Open the door and take the single-use kit out of the device. Disconnect the syringe to collect the cells for subsequent processes. Take a sample of the collected cells for a cell count and viability check (see step 6.2).</li>
</ol>
6. Process validation
<ol>
	<li>Perform control experiments using a manual buffer exchange protocol.
	<ol>
		<li>Centrifuge cell suspension at 200 x g for 5 min. Discard the supernatant and resuspend the cells with 30 mL of cell wash buffer. Centrifuge the cell suspension again at 200 x g for 5 min. Discard the supernatant and resuspend the cells with 10 mL of cell wash buffer.</li>
	</ol>
	</li>
	<li>Perform cell counting via the trypan blue exclusion assay to assess cell viability using an automated cell counter.</li>
	<li>Perform an MTS assay to quantify cell proliferation at 2, 24, and 48 h. 
	NOTE: The MTS assay was performed on a 96 well tissue culture plate according to the manufacturer&#39;s instructions. A 100 &#181;L aliquot of cell culture media (containing 10,000 Jurkat cells or 1,500) per well were plated after the buffer exchange process. At each time point, 20 &#181;L of MTS reagents were added into each well and incubated for 2 h at 37 &#176;C. The absorbance was assessed at 490 nm using a plate reader.</li>
	<li>Perform functional assays to compare the impact of the automated process versus the manual process on the Jurkat cells and MSC function.
	<ol>
		<li>Culture Jurkat cells in a 96-well plate at a density of 1 x 106 cells/mL stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 &#181;g/mL ionomycin (cell stimulation cocktail) in DMEM:F12 media containing 10% fetal bovine serum (FBS) for 24 h. Incubate the cells at 37 &#176;C with 5% CO2.</li>
		<li>Measure interleukin-2 production from culture supernatant using bead-based ELISA assay (see Table of Materials) according to the manufacturer&#39;s instructions.</li>
		<li>Culture MSCs in a 12 well plate at the density of 10,000 cells/cm2 in 1 mL of DMEM:F12 media containing 10% FBS supplemented with interferon-&#947; 50 units/mL for 48 h. Collect supernatant for the kynurenine concentration assay to measure indoleamine 2,3-dioxygenase (IDO) enzyme activity of MSCs as previously described21.</li>
	</ol>
	</li>
</ol>

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G., Subramanian, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Continuous Processing in Pharmaceutical Manufacturing. , (2014).</li><li>. SMALL BATCH CELL SEPARATION, WASH &amp; CONCENTRATION Available from: <a target="_blank" href="https://www.scinogy.com/projects">https://www.scinogy.com/projects</a> (2019)</li><li>Yu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Jurkat+T+Lymphocyte+Leukemia+Cells+by+an+Engineered+Interferon-Alpha+Hybrid+Molecule.">Targeting Jurkat T Lymphocyte Leukemia Cells by an Engineered Interferon-Alpha Hybrid Molecule.</a> Cellular Physiology and Biochemistry. 42 (2), 519-529 (2017).</li><li>Moharram, S. A., Shah, K., Kazi, J. 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A., Mulyasasmita, W., Su, J., Lampe, K. J., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+viability+of+stem+cells+during+syringe+needle+flow+through+the+design+of+hydrogel+cell+carriers.">Improving viability of stem cells during syringe needle flow through the design of hydrogel cell carriers.</a> Tissue engineering. Part A. 18 (7-8), 806-815 (2012).</li><li>Zhu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyethyl+starch+as+a+substitute+for+dextran+40+for+thawing+peripheral+blood+progenitor+cell+products.">Hydroxyethyl starch as a substitute for dextran 40 for thawing peripheral blood progenitor cell products.</a> Cytotherapy. 17 (12), 1813-1819 (2015).</li><li>Schwandt, S., Korschgen, L., Peters, S., Kogler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cord+blood+collection+and+processing+with+hydroxyethyl+starch+or+non-hydroxyethyl+starch.">Cord blood collection and processing with hydroxyethyl starch or non-hydroxyethyl starch.</a> Cytotherapy. 18 (5), 642-652 (2016).</li><li>Stroncek, D. 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SW, IF, and DJ are COO, CTO, and CEO of Scinogy Pty. Ltd. The access to the CFC device was also provided by Scinogy.

The automated buffer exchange protocol described is simple and user friendly. Nevertheless, there are a few key steps in this protocol that are critical and require particular attention. In our experience, when processing larger cells such as MSCs (average diameter 10&#8211;15 &#181;m) each run should include at least 1 x 107 cells to achieve optimal cell recovery (Figure 4B). Processing smaller cells, such as Jurkat cells (average ~10 &#181;m diameter), requires ...

The landscape of modern medicine has transformed rapidly through recent developments in gene and cell-based therapies (GCT). As one of the fastest growing fields in translational research, the GCT sector also faces unique and unprecedented challenges. In addition to robust clinical outcomes, efficient and cost-effective manufacturing processes are essential for the commercial success of GCT, which is particularly difficult to achieve in small-scale manufacturing1. The cost of time, labor, and quality assurances are magnified when each batch of cells only produces a few doses for one patient instead of hundreds or thousands. Unlike allogeneic cell therapies in which the manufacturing processes are more akin to the production of antibodies and recombinant proteins, autologous cell therapies are typically produced as small-scale operations1. As a relatively new phenomenon in biopharmaceutical manufacturing2, options for small-scale cell processing are currently quite limited.
Buffer exchange is essential to cell manufacturing. It is one of the downstream processes where cells are removed from culture media and concentrated for cryopreservation or infusion. Currently, small-scale cell manufacturing often applies processes similar to those in the academic research setting and relies on specialized clean rooms to maintain sterility3. Manual downstream processes often use benchtop centrifuges to pellet and resuspend cells for volume reduction and buffer exchange. These open processes are costly (i.e., labor and clean room maintenance) and have limited manufacturing capacity, which are not ideal for commercial production2,3.
Implementing automation has been proposed as a solution to improve manufacturing efficiency and achieving commercial scale productions2. Sterility cannot be achieved in cell-based products through traditional methods used for biologics, such as gamma irradiation or terminal end filtration. Instead, an automated closed system is deployed to reduce risks of contamination and operators relying on clean rooms to maintain sterility4. Process automation also addresses the issue of scalability by either having multiple systems running in parallel (scale-out) or increasing the processing capacity of an individual device (scale-up), which in turn minimizes the variability between operators. Furthermore, cost modelling analysis of autologous therapies suggests that automation may reduce the cost of manufacturing5,6. However, no cost benefit was found in an autologous stem cell clinical trial where an automated manufacturing platform was used7, suggesting that the cost benefit of automation may depend on the individual manufacturing process.
There are different strategies in which automation can be introduced into an existing manufacturing process. This can be achieved either by implementing a fully integrated platform or a modular-based processing chain. There are several fully integrated platforms commercially available for autologous cell manufacturing, such as CliniMACS Prodigy (Miltenyi Biotec), Cocoon (Octane Biotech), and Quantum (Terumo BCT). These integrated platforms, which are often described as &#34;GMP-in-a-box&#34;, have low demands on infrastructure and are easy to operate. However, the manufacturing capacity of a fully integrated setup may be restricted by the incubator attached to the system. For example, the culturing capacity of Prodigy is limited to its 400 mL chamber8 and the Quantum cartridge has a limiting surface area set to 2.1 m2 (equivalent to 120 T175 flasks)7, which may not be sufficient for patients requiring higher cell doses9,10. Additionally, Prodigy and Quantum have a common attribute that limits their use: the operational unit is occupied by a single batch of cells throughout the cell expansion period, thus limiting the number of batches that can be manufactured by each unit11. The modular approach to automation is to create a manufacturing chain with multiple modular units that simulates the commercial manufacturing process12,13. This approach, which separates the culture device from the cell washing device, can thereby maximize manufacturing efficiency. An ideal processing device would be one that is adaptable and scalable to manufacturing needs12.
Counterflow centrifugation (CFC) technology, which dates back to the 1970s, has had a long history in cell processing14. It achieves cell concentration and separation by balancing centrifugal force with a counterflow force. Typically, a cell suspension enters from the narrow end of a cell chamber under a constant flow rate while subjected to a centrifugal force (Figure 1A). The flow of the fluid is exerted in the opposite direction to the centrifugal force. This is referred to as the counterflow force, which forms a gradient within the cell chamber. The counterflow force then decreases as the cell chamber widens away from the tip of the cone-shaped cell chamber. Cells with higher density and larger diameter have a higher sedimentation rate, and thus they reach force equilibrium towards the tip of the cone-shaped cell chamber. Smaller particles may reach equilibrium towards the base of the chamber or be too small to be retained in the chamber and will be washed away. The CFC technology is mostly known for its application in processing blood apheresis products, such as isolating monocytes for dendritic cell therapies15,16. In terms of buffer exchange, the CFC technology has only been applied in large-scale manufacturing17 and has yet to be used for the smaller scale manufacturing of autologous cell therapies.
To address the need of a suitable device for small-scale cell manufacturing, an automated CFC device (See Table of Materials), was recently developed18. The automated cell processing device uses counterflow centrifugation technology to remove cell debris and facilitate buffer exchange. The device performs buffer exchange with a single-use kit that can be sterile-connected to a cell transfer bag, which allows the cells to be processed within a sterile, enclosed system. Here, we investigate the use of a counterflow centrifugal device to perform buffer exchange in mammalian cell cultures in automated protocols. In this study, we tested the buffer exchange protocol using Jurkat cells and mesenchymal stromal cells (MSCs) to model nonadherent and adherent cell types, respectively. Jurkat cells are immortalized T cells often used for the study of acute T cell leukemia19,20. MSCs are adult stem cells that have been studied in human clinical trials for a wide range of diseases9.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20 ml Luer lock syringes</td>
			<td>BD</td>
			<td>302830</td>
		</tr>
		<tr>
			<td>20% Human serum albumin (HSA)</td>
			<td>CSL Behring</td>
			<td>AUST R 46283</td>
		</tr>
		<tr>
			<td>4-(Dimethylamino)benzaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>156477-25g</td>
		</tr>
		<tr>
			<td>500ml IV saline bag</td>
			<td>Fresenius Kabi</td>
			<td>K690521</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240112</td>
		</tr>
		<tr>
			<td>Automated cell counter (Countess)</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Cell counting chamber slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10228</td>
		</tr>
		<tr>
			<td>Cell stimulation cocktail (500x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>00-4970-93</td>
		</tr>
		<tr>
			<td>Cell transfer bags</td>
			<td>Terumo</td>
			<td>T1BBT060CBB</td>
		</tr>
		<tr>
			<td>CellTiter AQueous One Solution Cell Proliferation Assay (MTS)</td>
			<td>Promega</td>
			<td>G3582</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
		</tr>
		<tr>
			<td>DMEM: F12 media</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320082</td>
		</tr>
		<tr>
			<td>EnVision plate Reader</td>
			<td>Perkin Elmer</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10099141</td>
		</tr>
		<tr>
			<td>Human Interleukin 2 (IL2) Kit</td>
			<td>Perkin Elmer</td>
			<td>Al221C</td>
		</tr>
		<tr>
			<td>Luer (female) fittings</td>
			<td>CPC</td>
			<td>LF41</td>
		</tr>
		<tr>
			<td>PC laptop or PC tablet device</td>
			<td>ASUS</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Plate reader (SpectraMax i3)</td>
			<td>Molecular Device</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Recombinant Human IFN-&#947;</td>
			<td>PeproTech</td>
			<td>300-02</td>
		</tr>
		<tr>
			<td>Rotea counterflow centrifuge cell processing device</td>
			<td>Scinogy</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Rotea single-use processing kit</td>
			<td>Scinogy</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>RPMI media</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875119</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>ProSciTech</td>
			<td>420SS</td>
		</tr>
		<tr>
			<td>Trichloroacetic acide</td>
			<td>Sigma-Aldrich</td>
			<td>T6399-250g</td>
		</tr>
		<tr>
			<td>Trypan Blue stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
		</tr>
		<tr>
			<td>Trypsin digestion enzyme (TrypLE Express Enzyme)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604013</td>
		</tr>
	</tbody>
</table>

automated counterflow centrifugal system for small-scale cell processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

In this protocol, we used Jurkat cells and MSCs as representative examples to demonstrate the automated buffer exchange process. During the process, Jurkat cells and MSCs shared the same processing steps with differences in centrifugal force and pump speed that control the flow rate (Table 1). Figure 2 shows representative images captured by the camera of how the fluidized cell bed may appear during the buffer exchange process. Typically, the fluidized cell bed will resemble...

Watch this Scientific Journal Video about Automated Counterflow Centrifugal System for Small-Scale Cell Processing at JoVE.com

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

automated-counterflow-centrifugal-system-for-small-scale-cell

Research

JoVE Journal

Bioengineering

9.1K Views.  Hudson Institute of Medical Research. Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Video: Automated Counterflow Centrifugal System for Small-Scale Cell Processing

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...