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JoVE Journal Bioengineering

Bioengineering

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Published: December 12, 2019 doi: 10.3791/60423
Automatic Translation
1,2, 3, 3, 1,2, 1, 1,2, 1,2, 3, 1,2,4
1The Ritchie Centre, Hudson Institute of Medical Research, 2Department of Obstetrics and Gynaecology, Monash University, 3Scinogy, 4Australian Regenerative Medicine Institute, Monash University

Summary

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.

Abstract

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.

Introduction

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 "GMP-in-a-box", 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.

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Protocol

1. Preparation of reagents and cells for buffer exchange

  1. 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.
  2. 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 °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.
    1. 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.
    2. 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.
  3. 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).

2. Program for automated buffer exchange protocol

  1. Open the Graphic User Interface (GUI) of the device and press the "START" button on a laptop PC or tablet. Then "Open" an existing protocol or press "New" to create a new one.
  2. Use the "Forward" and "Backward" 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.

3. Setting up the machine

  1. Place the single-use processing kit on the machine and hang the connected bags accordingly. Press the red "Stop/Reset" 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.
  2. Connect the GUI to the processing device and press the "Connect" button. Then download the saved program. When the green "Play" button lights up, it indicates that the protocol is ready to start.
  3. 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 "Stop" button to reset the device and open the clamps to start again.

4. Automated buffer exchange

  1. Press "Start" to initiate the buffer exchange program.
    NOTE: To manually alter the automated process, press the "Next" button to move to the next step before reaching the "Trigger" or press "Pause" to put the process on hold. This will recirculate the cells through the kit while keeping only valves J and K open (Figure 1C).
  2. 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 "Next" to move the process to the next step if the bag is indeed empty, or press "Pause" to resume the processing if the sensor was triggered by a bubble in the line.

5. Collecting and sampling the cells

  1. 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).

6. Process validation

  1. Perform control experiments using a manual buffer exchange protocol.
    1. 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.
  2. Perform cell counting via the trypan blue exclusion assay to assess cell viability using an automated cell counter.
  3. 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's instructions. A 100 µL 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 µL of MTS reagents were added into each well and incubated for 2 h at 37 °C. The absorbance was assessed at 490 nm using a plate reader.
  4. Perform functional assays to compare the impact of the automated process versus the manual process on the Jurkat cells and MSC function.
    1. 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 µg/mL ionomycin (cell stimulation cocktail) in DMEM:F12 media containing 10% fetal bovine serum (FBS) for 24 h. Incubate the cells at 37 °C with 5% CO2.
    2. Measure interleukin-2 production from culture supernatant using bead-based ELISA assay (see Table of Materials) according to the manufacturer's instructions.
    3. 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-γ 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.

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Representative Results

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 the image in Figure 2A, where cells accumulate in the middle and towards the front of the cone with a small space at the tip of the chamber, where cells do not accumulate and the opening of the cell loading inlet is visible. The fluidized cell bed may be compressed (Figure 2B) when introducing new buffer that is at a different viscosity or density. In this protocol, the pump speed was lowered from 30–35 mL/min to 20 mL/min at the start of the washing step. Once the chamber was filled with the new buffer, the pump speed was returned to normal to prevent pelleting cells at the tip of the chamber. A high flow rate (Figure 2C) may be applied to select for live cells because dead cells are smaller and lighter and can be forced out of the chamber by increasing the flow rate.

The automated process of buffer exchange was achieved by first concentrating the cells, then introducing the wash buffer, which was around 10x of the volume of the fluidized cell bed. The cells were then formulated to the desired volume. These three processing steps were designed to follow the same principles as a manual buffer exchange. Typically, a two-cycle centrifugation (200 x g, 5 min) is used to perform manual buffer exchange, in which cells are pelleted to concentrate, resuspended to wash, then centrifuged again and resuspended to the final volume. The processing time of the automated processes was shorter compared to the manual ones (Figure 3). The recovery rate between manual and automated processes were similar for both Jurkat cells and MSCs, and cell viability was not affected by the process (Figure 4). The cell quality was verified by cell proliferation (MTS assay) and cytokine/enzyme production. The recovered cells showed similar proliferation rates between the manual and the automated processes (Figure 5A). The level of interleukin-2 production from Jurkat cells and IDO activity of MSCs were also comparable between the two groups (Figure 5B and 5C).

Step Number Description Open valves Centrifuge speed (g) Pump speed (ml/min) Triggers
1 Prime tubing from B to A A, B, K 10 50 Volume: 45 ml
2 Fill bubble Trap from A to B A, B, K 100 50 (reverse) Volume: 10 ml
3 Prime tubing A to D A, D, K 100 50 (reverse) Volume: 2 ml
4 Prime tubing J to K J, K 100 50 Volume: 3 ml
5 Load cells – initial start D to G D, K, G 1600 (Jurkat) 1500 (MSCs) 25 (Jurkat) 30 (MSCs) Volume: 100 ml
6 Load cells with bubble detect [At the detection of bubble/ empty tubing, the program will pause and wait for operator's command, press 'pause' or 'next'] A, D, K = last step 30 (Jurkat) 35 (MSCs) Bubble sensor D
7 Empty remaining media on port D tube A, D, K = last step = last step Volume: 1.5 ml
8 Wash cells 1 A, B, K = last step 20 Volume: 20 ml
9 Ramping up washing speed A, B, K = last step 35 Timer: 5 seconds speed ramping
10 Wash Cells 2 A, B, K = last step = last step Volume: 20 ml
11 Prepare to harvest J, K = last step = last step Timer: 2 seconds
12 Recover cells to output and dilute to target volume B, J, K = last step 60 (Reverse) Volume: 10 ml
Note: '= last step' is a setting option for centrifuging speed and pump speed in the GUI.

Table 1: Automated buffer exchange GUI setup for Jurkat cells and MSCs.

Figure 1
Figure 1: Counterflow centrifugal cell processing system. (A) A schematic diagram illustrating the principle of counterflow centrifugation. The counterflow force is present in a gradient within the cell chamber. While centrifuging (grey arrow), cells with larger diameters receive a higher sedimentation force, in which the cells reach force equilibrium towards the narrow end of the chamber, forming a fluidized cell bed. Cell debris and small particles that are too small to remain in the chamber are washed away. (B) The counterflow centrifugal processing system consists of the processing device and the single-use processing kit. (C) The single-use kit configuration for the buffer exchange protocol. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Fluidized cell bed in the cell chamber. Representative images of a fluidized cell bed under (A) medium, (B) low, and (C) high flow rate. The dotted line indicates the area of the fluidized cell bed within the chamber. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Comparison of cell processing time of Jurkat cells and MSCs with manual and automated processing. (n = 3–4 in each group, data are presented as mean ± SD). Please click here to view a larger version of this figure.

Figure 4
Figure 4: Comparison of cell viability and live cell recovery of Jurkat cells and MSCs with manual and automated processing. Cell viability (A) and live cell recovery (B) were measured by trypan blue exclusion assay using an automated cell counter. Live cell recovery was reduced in the absence of serum or when only 3 x 106 or 1 x 106 cells were processed. (n = 4–9 in each group, data are presented as mean ± SD). Please click here to view a larger version of this figure.

Figure 5
Figure 5: Cell proliferation and cell function from manual and automated processing. (A) MTS assay of Jurkat cells and MSCs were performed at 2, 24, and 48 h after buffer exchange. The quality of recovered cells was quantified by Interleukin-2 production from Jurkat cells (B), and IDO activity in MSCs (C). (n = 4–8 in each group, data are presented as mean ± SD). Please click here to view a larger version of this figure.

Figure 6
Figure 6: Fluidized cell bed stability at various flow rate. Two processes were performed for each cell type. In one process, either 3 x 107 Jurkat cells or 1 x 107 MSCs. In the second process, 10x the number of cells were used. In both processes, 10 mL of cell elutriate was collected from port A at various flow rates using the centrifuging speed indicated in this protocol (1,600 x g for Jurkat cells and 1,500 x g for MSCs). The number of cells in the elutriate was determined and presented as percentage of the total amount of cells loaded in the chamber. (n = 3 for each group, data are presented as mean ± SD). Please click here to view a larger version of this figure.

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Discussion

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 10–15 µm) 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 µm diameter), requires around 3 x 107 cells to achieve a stable fluidized cell bed (Figure 4B). When the cell number is too low, cells are more likely to be removed from the fluidized bed, which will affect the final cell recovery. Additionally, we compared cell loss rates at different pump speeds when a constant centrifugal speed is applied. Here, we observed that the cell loss increased with the flow rate (Figure 6), which is due to the increasing instability of the fluidized bed at higher flow rates. However, the percentage of cell loss was lower when 10x the number of cells were loaded into the chamber, which suggests that a higher pump speed can be applied when processing a larger number of cells to allow faster processing. In step 5 of the process, 100 mL of culture media was recirculated from the chamber back to the cell transfer bag to allow the fluidized bed to form and stabilize prior to volume reduction and buffer exchange. If the cell concentration is low (e.g., below 0.2 x 106/mL), the recirculation step may need to be extended to allow enough cells to form the fluidized cell bed. Likewise, if the cell concentration is high, the recirculation step can be shortened.

The presence of serum in the wash buffer is also critical for cell recovery. Previous studies have shown that hydrodynamic stress can cause necrotic cell death in the absence of buffering proteins22,23. The flow dynamic of the counterflow centrifugal system is quite complex. In this protocol, cell recovery rates dropped to around 70% when the process was performed in the absence of serum (Figure 4B). Although the exact mechanism is not clear, the presence of serum in culture media or albumin in wash buffers was able to mitigate the potential mechanical damage to the cells. For applications requiring serum-free buffers, hydroxyethyl starch and dextran 40 are examples of non-protein additives that are often used in cell manufacturing to protect against hydrodynamic stress24,25.

Counterflow centrifugation technology has been used in large-scale biopharmaceutical manufacturing and blood product processing17. The applications of CFC technology in small-scale cell manufacturing (i.e., 0.1–10 L) are often limited to isolating mononuclear cells from apheresis products, such as the Elutra (Terumo BCT)26, in which additional manual intervention is required to perform wash and concentrate steps. Other small-scale cell processing platforms include membrane filtration-based buffer exchange systems such as the LOVO (Fresenius Kabi)27 and vertical centrifugation separation such as the Sepax (GE Healthcare)28. Although it is difficult to perform a direct comparison between devices, one of the advantages of this counterflow centrifugal device is its capacity to deliver very small output volumes (minimum 5 mL), which is the smallest among currently available cell processing platforms. The small output volume is suitable for applications such as formulating cells for local injections29 or infusions for neonates30. The camera embedded in the device is also a unique feature that provides visualization of the fluidized cell bed during the process development stage.

One of the limitations of the CFC technology is that it is sensitive to the size and density of the cells. When setting up a buffer exchange protocol for a different cell type, the protocol presented here should serve as a guideline for users to optimize according to their needs, keeping in mind the size of their cells of interest and the density of their cell suspension. Although the current protocol is usable for processing up to 5 L of media, the processing time will be substantially extended (i.e., 1–2 h). It is important to note that the impact of extended processing time on cell quality has not been examined in this current study.

Future studies will evaluate the impact of processing speed and processing time on cell function to determine the maximum processing capacity of the device. Furthermore, future studies may explore other applications such as the removal of dimethyl sulfoxide (DMSO) from cryopreserved products and selective removal of dead cells.

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Disclosures

SW, IF, and DJ are COO, CTO, and CEO of Scinogy Pty. Ltd. The access to the CFC device was also provided by Scinogy.

Acknowledgments

This work is supported by the Victorian Government'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.

Materials

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

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References

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Tags

Automated Counterflow Centrifugal System Small-scale Cell Processing Closed Automated Buffer Exchange Concentration Recovery Small Volume Automation Steps Programming Test Runs Medium Laminar Flow Head Syringe Needle Assembly Saline Solution Human Serum Albumin Culture Vessels Counting Cells Transfer Bag Luer Lock Wash Buffer Graphic User Interface Start Button Protocol Creation Valves Centrifugal Speed Pump Speed Pump Direction Action Triggers
Automated Counterflow Centrifugal System for Small-Scale Cell Processing
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Cite this Article

Li, A., Wilson, S., Fitzpatrick, I., … More

Li, A., Wilson, S., Fitzpatrick, I., Barabadi, M., Chan, S. T., Krause, M., Kusuma, G. D., James, D., Lim, R. Automated Counterflow Centrifugal System for Small-Scale Cell Processing. J. Vis. Exp. (154), e60423, doi:10.3791/60423 (2019).

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