cell processing
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2022 ◽  
Vol 20 (1) ◽  
Author(s):  
David F. Stroncek ◽  
Robert P. T. Somerville ◽  
Steven L. Highfill

AbstractThe use of cellular therapies to treat cancer, inherited immune deficiencies, hemoglobinopathies and viral infections is growing rapidly. The increased interest in cellular therapies has led to the development of reagents and closed-system automated instruments for the production of these therapies. For cellular therapy clinical trials involving multiple sites some people are advocating a decentralized model of manufacturing where patients are treated with cells produced using automated instruments at each participating center using a single, centrally held Investigational New Drug Application (IND). Many academic centers are purchasing these automated instruments for point-of-care manufacturing and participation in decentralized multiple center clinical trials. However, multiple site manufacturing requires harmonization of product testing and manufacturing in order to interpret the clinical trial results. Decentralized manufacturing is quite challenging since all centers should use the same manufacturing protocol, the same or comparable in-process and lot release assays and the quality programs from each center must work closely together. Consequently, manufacturing cellular therapies using a decentralized model is in many ways more difficult than manufacturing cells in a single centralized facility. Before an academic center decides to establish a point-of-care cell processing laboratory, they should consider all costs associated with such a program. For many academic cell processing centers, point-of-care manufacturing may not be a good investment.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2847-2847
Author(s):  
Yasna Behmardi ◽  
Laurissa Ouaguia ◽  
Laura Jean Healey ◽  
MinJung Kim ◽  
Cole Jones ◽  
...  

Abstract CAR-T autologous cell therapies are delivering impressive results in the clinic. However, there are still significant manufacturing challenges impeding the rapid adoption of these advanced therapies. On the first day of cell processing, most manufacturing approaches require ~5 steps (~4 hours) to obtain a white blood cell (WBC) preparation sufficiently depleted of red blood cells (RBCs) for T-cell selection and activation steps; and involves large cell losses and a great deal of inconsistency. Here we present a single-step procedure that yields >2 fold more cells that centrifugal processing with comparable or better quality in <1 hour. We previously reported a small-scale microfluidic approach using deterministic cell separation (DCS) to effectively isolate and separate WBCs with high recoveries, no loss of WBC subtypes, no cell damage, and greater numbers of central memory T cells than traditional Ficoll-based processing. Extending this work, we now present the results of our fully scaled-up processing of 23 normal donor leukopaks and 4 disease samples using a full-scale DCS prototype. All samples were processed in <45 minutes, with only an additional 10 minutes hands-on time. On average, inclusive of aggregate removal by prefiltering, DCS achieved 88% WBC recovery, 94% RBC removal, and 98% platelet ( PLT) removal from the undiluted leukopak samples (n=23). Furthermore, DCS resulted in a RBC/WBC ratio of 0.1 compared with a ratio of 1.4 for Ficoll. Similarly, the PLT/WBC ratios were 0.89 versus 7.17 for DCS and Ficoll, respectively (n=20). In addition, DCS preparations contained 2-fold more CD3+ T cells (n=17), and, importantly, the CD4+ cells were less differentiated (more cells in naïve and central memory stages) than those recovered by Ficoll. Similarly, DCS processed blood from cancer patients had a ratio of RBC/WBC = 7.0 versus 20.1 for Ficoll, and a PLT/WBC ratio = 0.7 versus 15.6 for Ficoll (n=4). These results demonstrate the capabilities of DCS in processing not only samples from normal donors but also blood from cancer patients with similar efficiencies. Further, with DCS we achieved wash efficiencies of more than 3 log, without the typically associated cell loss, as demonstrated by the removal of viral particles, soluble proteins and cytokines and growth factors present in plasma. Therefore, cells from leukopaks processed by DCS can be washed and collected directly into cell culture media, or other solutions, to ready them for downstream applications without pelleting and repeated washes, greatly simplifying workflows. We integrated our DCS technology into a full scale parallelized, disposable, closed fluid path solution and automated platform prototype, the Curate ® Cell Processing System, capable of processing undiluted leukopacks at rates in excess of 300mL/hour. Designed to process blood products in bags using a single-use cassette containing microfluidic components, the Curate ® delivers a debulked WBC product to a bag. With a hands-on time of only 10 minutes, the Curate ® reduces the time to activation- and expansion-ready cells from leukopaks by 6-fold as compared with centrifugation and elutriation methods (Bowles, et al. Cytotherapy 2018;20(5):S109). The system can process a full leukopak (200-300 mL containing up to 1.2x10 10 WBC) within 40 minutes with a maximal cell throughput of 1.8x10 10 WBC per hour. Additionally, the same Curate ® device can be used to achieve up to 200x10 6 cell/mL in as little as 40 mL of media and without requiring pelleting. In summary, we believe our technology enables a significant breakthrough in the production of CAR-T cells by efficiently recovering more and cleaner total and naÏve T cells, for CAR-T cell production. Furthermore, the closed-system Curate ® will simplify cell processing workflows by reducing the number of cell washing steps, as well as the hands-on time and resources. Supported in part by NIH Grant No 5R42CA228616-03 Disclosures Behmardi: GPB Scientific, Inc: Current Employment. Ouaguia: GPB Scientific, Inc: Current Employment. Healey: GPB Scientific, Inc: Current Employment. Jones: GPB Scientific, Inc: Current Employment. Rahmo: GPB Scientific, Inc: Current Employment. Skelley: GPB Scientific, Inc: Current Employment. Gandhi: GPB Scientific, Inc: Current Employment. Campos-Gonzalez: GPB Scientific, Inc: Current Employment, Current holder of stock options in a privately-held company. Civin: GPB Scientific, Inc: Current holder of individual stocks in a privately-held company. Ward: GPB Scientific, Inc: Current Employment.


Author(s):  
Pui Sha Victoria Ling ◽  
Anders Hagfeldt ◽  
Sandy Sanchez

Author(s):  
Ryosuke Nonoyama ◽  
Makoto Jinno ◽  
Koichiro Yori ◽  
Keiichi Sugiura ◽  
Tadashi Sameshima
Keyword(s):  

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Shinji Hayashi ◽  
Rieko Yagi ◽  
Shuhei Taniguchi ◽  
Masami Uji ◽  
Hidaka Urano ◽  
...  

AbstractCell-assisted lipotransfer (CAL) is an advanced lipoinjection method that uses autologous lipotransfer with addition of a stromal vascular fraction (SVF) containing adipose-derived stromal stem cells (ASCs). The CAL procedure of manual isolation of cells from fat requires cell processing to be performed in clean environment. To isolate cells from fat without the need for a cell processing center, such as in a procedure in an operation theater, we developed a novel method for processing SVF using a closed cell washing concentration device (CCD) with a hollow fiber membrane module. The CCD consists of a sterilized closed circuit, bags and hollow fiber, semi-automatic device and the device allows removal of >99.97% of collagenase from SVF while maintaining sterility. The number of nucleated cells, ASCs and viability in SVF processed by this method were equivalent to those in SVF processed using conventional manual isolation. Our results suggest that the CCD system is as reliable as manual isolation and may also be useful for CAL. This approach will help in the development of regenerative medicine at clinics without a cell processing center.


2021 ◽  
pp. 29-70
Author(s):  
Saleem Hussain Zaidi
Keyword(s):  

Author(s):  
Suman C. Nath ◽  
Lane Harper ◽  
Derrick E. Rancourt

Cell-based therapy (CBT) is attracting much attention to treat incurable diseases. In recent years, several clinical trials have been conducted using human pluripotent stem cells (hPSCs), and other potential therapeutic cells. Various private- and government-funded organizations are investing in finding permanent cures for diseases that are difficult or expensive to treat over a lifespan, such as age-related macular degeneration, Parkinson’s disease, or diabetes, etc. Clinical-grade cell manufacturing requiring current good manufacturing practices (cGMP) has therefore become an important issue to make safe and effective CBT products. Current cell production practices are adopted from conventional antibody or protein production in the pharmaceutical industry, wherein cells are used as a vector to produce the desired products. With CBT, however, the “cells are the final products” and sensitive to physico- chemical parameters and storage conditions anywhere between isolation and patient administration. In addition, the manufacturing of cellular products involves multi-stage processing, including cell isolation, genetic modification, PSC derivation, expansion, differentiation, purification, characterization, cryopreservation, etc. Posing a high risk of product contamination, these can be time- and cost- prohibitive due to maintenance of cGMP. The growing demand of CBT needs integrated manufacturing systems that can provide a more simple and cost-effective platform. Here, we discuss the current methods and limitations of CBT, based upon experience with biologics production. We review current cell manufacturing integration, automation and provide an overview of some important considerations and best cGMP practices. Finally, we propose how multi-stage cell processing can be integrated into a single bioreactor, in order to develop streamlined cGMP-compliant cell processing systems.


2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A11-A11
Author(s):  
Liping Yu ◽  
Silin Sa ◽  
Alice Wang

BackgroundAdvancements in fields of multi-omics analysis and cell-based therapies depend upon efficient cell processing tools to isolate rare cancer and immune cells from complex biologic samples as an initial step in sample preparation. Conventional technologies are limited in automation, recovery and purity. We present an integrated system based on multiple physics principles with built-in novel technologies to achieve cell purification, concentration and target cell isolation, with high recovery at an unprecedented flow rate. This platform, the Multi-physics Automated Reconfigurable Separation (MARS), combines tunable, acoustic cell processing and in-flow immuno-magnetic separation technologies, enabling automation of the entire cell sample preparation workflow for proteomics and genomics analysis.MethodsCirculating tumor cells (CTC) are present in extreme low frequency in blood stream (1–100 in billions of blood cells) thus it has been a challenge to isolate CTCs with high recovery. We have developed protocols on MARS to isolate CTCs from whole blood for multi-color flow cytometry analysis. To demonstrate the extent of enrichment of tumor cells in whole blood, PC3 cells were used for spike recovery. RBC lysed blood sample was then loaded on MARS and automatically processed through cell washing, concentration, and magnetic depletion. Enriched tumor cells were collected and analyzed by flow cytometry.ResultsResults show > 4 log enrichment of tumor cells and average recovery of spiked CTC > 85% in the clinical relative range <100 cells per ml of whole blood (R2=0.929) with a throughput of 60 ml/hr. Isolated cells were confirmed to be cancer cells with imaging analysis and single cell genomic sequencing. The protocol was also validated with other cell line cells such as A549. The purity of the cells prepared by MARS are ideal for single cell genomics platforms.ConclusionsThe fluidics of MARS is also replaceable and can be sterilized to minimize sample to sample contamination. The high molecular debris removal achieved by MARS is ideal for single cell genomics platforms, as is the first-to-market automated and integrated sample preparation and cell separation system designed to be a versatile tool for downstream cell analysis.


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