Introducing the Gibco CTS Rotea System

The Gibco CTS Rotea Counterflow Centrifugation System applies a proven counterflow centrifugation method for a broad range of cell processing applications such as chimeric antigen receptor T cell (CAR-T) therapy, stem cell therapy, and peripheral blood mononuclear cell (PBMC) isolation. This system offers exceptional flexibility in cell therapy development and manufacturing systems. The Rotea system is designed to handle a wide range of input volumes from 50 mL to 20 L and output volumes as low as 5 mL. The system is powerful but gentle on cells, compared to other separation methods. It enables over 95% cell recovery while maintaining cell viability and achieving high throughput. The single-use kit interface enables an easy transition to commercial manufacturing and helps enable good manufacturing practice (GMP) compliance with industry standards.

Whilst viral delivery has been used for some time, viral methods have limitations such as safety concerns, immunogenicity, mutagenesis, increasing test burden, payload limitations, and cost. Viral engineering of cells can lead to poor and inconsistent regulation of CAR expression. Non-viral engineering approaches are attractive because they allow more specific control of engineering. Electroporation (EP) is an interesting alternative to viral delivery due to its simplicity of use and ease of large-scale production.

The Gibco CTS Xenon Electroporation System is a closed and scalable EP system for GMP-compliant cell therapy manufacturing. The system can transfect up to 2.5×10⁹ T cells/25 mL in less than 25 min. It shows up to 95% gene knockout with CTS TrueCut™ Cas9 protein and 80% cell viability. The user program enables the creation and optimization of EP protocols for various cell types and payloads, from process development through to commercial manufacturing. It can be used to deliver DNA, RNA, and protein payloads. The Xenon MultiShot Electroporation Cartridge helps enable sterile welding to PVC or C-Flex^® tubing. The system can be integrated with other Thermo Fisher Scientific instruments and consumables into a complete closed-cell therapy manufacturing workflow.

Thermo Fisher Scientific also offers reagents, buffers, and consumables for the CAR-T workflow. The SingleShot chamber designed for processing development can transfect 2.2×10⁹ cells in one batch. The 5–25 mL MultiShot chamber can transfect 0.1–2.5 billion cells in a continuous process, with an intuitive rapid user interface. The Gibco CTS Xenon editing buffer is designed to improve performance with gene editing-specific payloads, such as CRISPR/Cas9, for knockout or knock-in applications in a variety of human primary cells. Bottles (100 mL) or bags (100 mL) are available. The non-viral workflow is shown in Figure 1The non-viral CAR-T cell workflow..

EP system testing for CAR-T cells

The Invitrogen™ Neon™ Transfection and Xenon Electroporation systems were compared in an investigation. Flow cytometry was used to assess the gene editing efficiency and phenotype. The V5 antibody was used to detect part of a CAR antigen on T cells to quantify how many cells expressed the CAR on their membrane.

Superior efficiency was observed with the CTS Xenon system (22–44% knock-in efficiency) compared to the Neon system (15–23% knock-in efficiency), which suggests that the CTS Xenon system can be used to easily scale and optimize the transfection process in a closed system (Figure 2Transfection efficiency (top) and CD4/CD8 phenotype (bottom).).

T cell phenotype was assessed on the Invitrogen™ Attune™ NxT Flow Cytometer. Compared to no EP controls, there is minimal or no phenotypic change across the EP volumes tested.

CAR-T cells generated by the Xenon EP from donors A and B were tested for functionality in a cytotoxic assay. Effector CAR-T cells or control cells were seeded into a 96-well plate containing GFP and nalm-6 target cells. Effector:target ratio ranged from 10:1–0:1. The effector and target cell mixtures were incubated for 6 h and then analyzed for present cytotoxicity using the Invitrogen™ EVOS™ M5000 Imaging System and flow cytometer. The results showed that 48.2–60.8% of CAR-T cells demonstrated the ability to efficiently kill the GFP neighbors and nalm-6 target cells in a dose-dependent manner compared to the control cells in vitro.

Optimizing CAR-T workflows with the CTS Rotea system

Optimization of the cell and gene therapy workflows can be complex due to the number of process steps and variables included. Here, variable conditions were tested with the modification of the workflow. First, buffer exchange was performed by Rotea system prior to EP. Second, to test the impact of activation time on editing efficiency, T cell activation was performed over 2–3 days with CTS DynaBeads™ CD3 and CD28. Three different donors were used for the EP steps. The comparisons are the closed and semi-automated process on the Rotea system versus the open and manual process for buffer exchange. Additionally, the Xenon system was compared to the Neon system for EP, and time to T cell activation was assessed.

The CTS Rotea system can be programmed to perform effective washout of media and buffer components. See the application note for additional information: Residual washout on the CTS Rotea Counterflow Centrifugation System [1]. Wash buffer can be washed through the fluidized cell bed, enabling over 95% removal of original medium components with minimal cell loss and maintenance of cell viability.

The Gibco CTS Rotea single-use kit was primed, then cells were washed, concentrated, and harvested. The viability and recovery percentage of T cells were measured on days two and three. The cells were then debeaded before being washed and concentrated either manually, or using a Rotea system. The viability of the cells recovered by both methods was over 89%. On day two, the Rotea system and manual methods showed similar recovery, although on day three, the Rotea system outperformed the manual method in terms of recovery rate. With the Rotea system, the results were well over 85% for all conditions tested.

EP efficiency knock-in versus knockout was assessed 3 days post-EP (Figure 3Electroporation efficiency 72 h post-electroporation.). Manual versus the Rotea system processes were tested for buffer exchange, and the Neon system versus the Xenon system were tested for EP.

As expected, there was donor-to-donor variation with a knock-in efficiency of up to 36.8%. Knock-in efficiency of the Xenon system was consistently higher than the Neon system for all three donors across all conditions.

The result was analyzed by evaluating the total number of adhesive cells for donor three. Activations for 2 days showed higher EP efficiency compared to 3 day activations across all donors and conditions. For the 2 day activation, the protocol efficiency was similar using manual versus the Rotea system process for buffer exchange. The 3 day activation resulted in lower efficiency for donors one and two on Rotea system with similar efficiency for donor three.

Phenotypic characterization of CD4/CD8 ratio and CD69 and CD25 activation markers was also assessed. No significant difference in the phenotypic analysis was seen between day two and day three activation. Testing on either day two or three resulted in sufficiently activated CD4/CD8 T cells. We observed no significant difference in activation markers between the days.

Cells were evaluated for viability and growth after EP on the Xenon system. Good viability of more than 80% was observed for all conditions compared to no EP controls. Cells from the 2 day activation protocol showed a slightly improved growth over those from the 3 day activation protocol, but overall, growth scores showed a similar trend in both groups.

In conclusion, the 2 day activation protocol showed higher knock-in efficiency, the CTS Rotea system outperformed the manual buffer exchange, and the CTS Xenon system outperformed the Neon system. The CTS Rotea system and the CTS Xenon system are powerful modular tools in the quest towards creating a closed cell therapy manufacturing process by providing exceptional performance and helping to reduce contamination in a cell therapy manufacturing workflow.

Genome editing of natural killer cells using the CTS Xenon Electroporation System

Natural killer (NK) cells are innate immune effector cells that can rapidly identify and kill abnormal, virally infected, and tumor cells. They can be genetically modified to obtain capable effector cells for adoptive cellular treatment of cancer patients. CAR-NK cells may represent a valuable complementary tool to the use of CAR-T cells in the treatment of adoptive immunotherapy of leukemia and solid tumors. However, gene transfer or gene editing of human NK cells is a
challenging task.

NK cells for cell therapy applications can originate from multiple sources including peripheral blood, cord blood, induced pluripotent stem cells (iPSCs), and NK cancer cell lines. To improve the immune cell function against cancer or other diseases, cells must
be engineered.

Engineering of NK cells is challenging using conventional methods because plasmid transfection has limited efficiency to express the transgene, and retroviral transduction requires a high viral titer and poses concerns around insertional mutagenesis and oncogenesis. Furthermore, lentiviral transduction is inconsistent for NK cells, even at a high multiplicity of infection (MOI). A robust and precise toolkit is urgently needed for NK cell engineering and expansion.

CTS NK-Xpander™ Medium is designed to meet the needs of transitional- and clinical-stage cell therapy developers by expanding human NK cells without the need for feeder cells. With this medium, cells have been proven to expand and maintain CD56 and CD16 expression as well as having robust cytotoxic capability. The NK cell process workflow is shown in Figure 4NK cell workflow..

In this experimental design, on day six of post-isolation, NK cells were counted and suspended in genome editing (GE) buffer. The CTS TrueCut guide cas9 was used with the target of B2M knockout. Re-suspended NK cells were electroporated using either a Neon (10 mL) or a Xenon (1 mL) EP system to assess scalability. The same EP parameters were used for both Neon and Xenon systems. After 3 days of EP, editing efficiency was analyzed using flow cytometry.

First, PBMC were isolated using the CTS Rotea system and then characterized for CD56, CD16, and CD3 populations (Figure 5Phenotypic analysis by flow cytometry of PBMCs isolated using a CTS Rotea Counterflow Centrifugation System.).

The NK cells were isolated from three different PBMC donors and enriched. On day zero, the CD56, CD16, and CD3 NK cells were expanded. After the NK cells were isolated, NK expander media was added, supplemented with 5% human serum, and 500 units per mL IL-2. The cells were fed every 2–3 days. At the beginning of day five, the total fold expansion viability and phenotypes were analyzed (Figure 6NK cell expansion and characterization.). Across the three donors, CTS NK-Xpander expanded the cells by an average of 70-fold in 2 weeks.

For NK cells to be successful in allogeneic therapy, they must maintain their functionality post-expansion. All three donors maintained CD56⁺ at more than 80–90% and maintained 70–80% CD16⁺. This means they all maintained their functionality.

Additionally, a different Neon program was used to identify optimal conditions for NK editing efficiency. Consequently, on day six of the NK cell culture, cells were electroporated using Neon programs 1–24.

After 72 h of EP, a genomic cleavage detection (GCD) assay was performed to measure the knockout efficiency. Data suggested that program five showed the best editing efficiency. The NK editing efficiency between GE buffer and EP buffer was also compared. It was observed that GE buffer has better editing efficiency than EP buffer.

For NK cell therapy requiring a large number of NK cells, high efficiency of NK cell editing on a larger scale is required. For this, the same CTS Xenon Electroporation system can help to edit various cell types on a larger scale, including NK cells. Data for NK cells are shown in Figure 7Comparative analysis of gene editing efficiency in expanded NK cells using different electroporation systems.. The NK cells were isolated at day six and expanded on both Neon and Xenon systems. On average, the Xenon System showed greater knockout efficiency than the Neon system.

To summarize, PBMCs were isolated using a Rotea system. Pre-isolation, the CD56 NK cell population was 13.9%. NK cells were isolated from the PBMCs of three different donors, and 88.3% of CD56 cells were purified. NK cells were expanded using NK-Xpander medium method to achieve 65-fold expansion. NK cells were edited using non-viral methods, with B2M as a knockout target. When compared, the Xenon system demonstrated greater knockout efficiency than the Neon system, with the Xenon system achieving approximately 85% knockout efficiency on average across three donors.

For more information about the Xenon system, visit thermofisher.com/xenon, and for additional information about the Rotea system, please visit thermofisher.com/rotea.

Reference

1. Application note: residual washout on the CTS Rotea Counterflow Centrifugation System. Crossref

Q&A

David McCall, Editor, BioInsights speaks to (pictured left to right) Sung Lee, Scientist, Thermo Fisher Scientific and Deepak Kumar, Scientist, Thermo Fisher Scientific

Can you comment further on the donor-to-donor variability observed with CAR-T workflows?

SL: The donor-to-donor variability is due to the difference in cell types. The cell cycle dynamic usually significantly affects cas9 efficiency. In our case, primary T cell editing efficiency could be affected by T cell donor-to-donor factors, such as genetic factors, recent infection, T cell activation stages, and/or the changing of gene locus due to chromatin stages. Characterizing these variables and further optimizing genomic engineering efficacy can increase the therapeutic editing of the T cells using cas9 protein.

Can the CTS Xenon system edit NK cells at a large scale?

DK: In cell manufacturing for cell therapy, a large scale is often required. We observe that the Xenon system is a powerful tool and can perform larger-scale NK editing. The Xenon system can be used to edit up to 50–100 million NK cells. We can also edit different cell types, such as T cell samples.

What is the recovery time post-EP?

SL: Recovery time is the time that the cells rest post-EP in our EP buffer prior to adding the media. Typically, a shorter recovery time is better. After 0–60 min, we try to put the cells back into the media after EP.

Can CTS NK-Xpander Medium be used to expand pluripotent stem cell-derived NK cells in a feeder-free system?

DK: Our team has found that for an induced pluripotent stem cell (iPSC)-derived natural killer (NK) cell expansion, we can use our NK cell Xpander medium with good results.

Do NK cells maintain their phenotype after editing using a Xenon Electroporation System?

DK: Yes, we observed that after EP, the NK cells maintained their phenotype. After EP, we measured the percentage of CD56 and CD16 and observed that they maintained their cell surface markers. After using the Xenon Electroporation System or editing the NK cell by using the Xenon system, we observed more than 90% CD56 was maintained.

Why does the Xenon system show higher editing efficiency than the Neon system?

DK: The Xenon system is a closed system while the Neon is an open system. This could explain the higher editing efficiency of the Xenon system.

What are the payload considerations for EP?

SL: You must consider whether the payload is toxic to the cells, as well as payload purity and quality. You also must optimize your concentration of the payload and consider the size of the donor DNA and what kind of buffer you need to use.

What is the maximum number of cells that can be washed and concentrated using a CTS Rotea System?

SL: From our study, we used up to 1.9 billion cells and then concentrated the cells in 50 million per mL for the output. We achieved a 76–80% recovery rate. Internally, the Rotea team used a single-use kit and showed the capability of 5–500 billion cells per mL for the maximum output.

How do the expansion rates compare between primary NK and iSPC-derived NK cells in NK Xpander media?

DK: When we isolated the NK cells and compared these, we monitored the expansion rate every 3 days. We found that both iPSC-derived NK cell and primary NK cells have the same expansion rate.

What is the viability of cells post-EP and what impact does dead cellular material have on the final product from a safety perspective?

SL: The shorter the processing time, the better. We are trying to optimize conditions to better serve the patient. EP is harsh, but we see that 3 days post-EP, we have high viability of about 80–90%. Depending on how you optimize the condition, you can achieve higher viability of cells. Post-EP, we have observed some cell debris, but we try to remove as much as possible and we are currently working on that further.

For routine assays in GMP labs, is the Xenon electroporation device reliable for generating reproducible transfection efficiency?

DK: In our group currently, we are not currently performing any good manufacturing practice (GMP) runs. However, this EP system can also be used for GMP.

What were the target cells used in the T cell cytotoxicity assay?

SL: The target cells used were NALM6, which are GFP-positive cells that also express CD19. We are using the CD19 CAR-T as our effector cell and then the target cell is GFP-expressing NALM6.

What is the expansion rate with NK-Xpander plus modified feeder cells?

DK: I have not used any feeder cells in these experiments as our NK cell expander media is a feeder-free media.

Is your process a closed system from start to finish?

SL: From beginning to end, this is not yet a fully closed system, but we are working to try to connect each instrument to transfer to each step in a closed way. Recently, we also launched the Gibco™ CTS™ DynaCellect™ Magnetic Separation System. We are working on minimizing any open steps.

Have you used any human serum in your media and what types of cytokines did you use for cell survival?

SL: We are not using any human serum – instead, we use Gibco™ CTS™ Immune Cell Serum Replacement which is a xeno-free formulation. We use animal-free components in the system. The cytokines in this experiment used are interleukin (IL)-2, although we are also testing IL-7 and IL-15 in different studies. You can expect to learn more about that in the future. With IL-7 and IL-15, it is important to maintain the stemness of the cells.

What optimal voltage strength can enhance EP without killing NK cells?

DK: Here, we use 1700 volts. After trying different voltages, we found that this was the best way to not kill the cells and still have the best editing efficiency for EP systems.

Do you check for viability of cells after EP?

DK: Yes. Checking for viability is important. We waited for 30 min, as there is some repair of the cell membrane after you perform EP. The cells electroporated with the Neon or Xenon systems both showed more than 90% cell viability.

Biographies

Sung Lee, PhD, is a Scientist at Thermo Fisher Scientific where he focuses on non-viral delivery for cell and gene therapy applications. Dr Lee received his PhD in Medical Immunology from University of California, Irvine and performed his postdoctoral work at City of Hope, Brigham and Women’s Hospital (Harvard Medical School) and UCSF Medical Center. He also worked in two start-ups (Pro-Drug/Immuno-Oncology related) prior to joining Thermo Fisher.

Deepak Kumar, PhD, is a Scientist at Thermo Fisher Scientific where he focuses on NK Cells gene editing using viral and non-viral approach to support cell and gene therapy workflow applications. Dr Kumar received his PhD in Molecular Biology and Genetic Engineering from the Institute of Life Sciences (Department of Biotechnology, Government of India), India. After his PhD, he was a Postdoctoral Fellow at the Moores Cancer Center, University of California, San Diego and then worked as a Project Scientist at the Department of Medicine, University of California, San Diego.

Affiliations

Sung Lee, PhD
Scientist,
Thermo Fisher Scientific

Deepak Kumar, PhD
Scientist,
Thermo Fisher Scientific

Authorship & conflict of interest

Contributions: All named authors take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.

Acknowledgements: None.

Disclosure and potential conflicts of interest: The authors have no conflicts of interest. C-Flex^® is a registered trademark of Saint-Gobain.

Funding declaration: The authors received no financial support for the research, authorship and/or publication of this article.

Article & copyright information

Copyright: Published by Cell and Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0 which allows anyone to copy, distribute, and transmit the article provided it is properly attributed in the manner specified below. No commercial use without permission.

Attribution: Copyright © 2023 Thermo Fisher Scientific. Published by Cell and Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0.

Article source: This article is a transcript of a webinar, which can be found here.

Webinar recorded: Dec 8 2022; Revised manuscript received: Jan 11 2023; Publication date: Feb 6 2023.