Transition from the manual processes that are performed during the initial research and development (R&D) stage to automated processes for later and commercial stage cell therapy manufacturing can be challenging. It often requires significant effort, time, and costs - which hinders the therapy's access to the clinic. To ease this transition, we have developed a novel and flexible manufacturing platform, Bioreactor with Expandable Culture Area (BECA), that aims to support both R&D and manufacturing to accelerate cell therapies from bench to bedside. This report introduces two models in this manufacturing platform: BECA-S for manual small-scale operation at R&D phase and BECA-Auto for functionally closed and automated scaled-out operation at manufacturing phase. We employed these two models to streamline transition of the T cell culture process from manual to automated and reported insignificant differences in the culture outcome between the two. Our work represents the first detailed development and demonstration of a standalone cell manufacturing platform that facilitates a seamless transition between manual and automated processing for autologous T cell therapy manufacturing.

Third-degree burns result in extensive damage to the skin's epidermal and dermal layers, with limited treatment options available. Currently, xenogeneic collagen-based skin grafts are used as scaffolds to integrate into the wound bed and provide a template for neodermis formation. Existing commercial products like Integra dermal templates rely on a time-consuming and variable dehydrothermal (DHT) crosslinking process. This study presents a novel crosslinking process for collagen sponges, utilizing UV irradiation followed by glutaraldehyde (GA) crosslinking. This UV method allows to fine-tune the template's crosslink density and degradation profile while significantly reducing the total crosslinking time from 48 to 24 h compared to DHT/GA crosslinking. In vitro characterization and in vivo validation are conducted using a full-thickness skin wound mouse model. The collagen template supports the human dermal fibroblast cell line WS-1 proliferation more effectively than the Integra template after 2 weeks in culture. Additionally, in vivo data indicate a similar level of regeneration of full-thickness skin wounds in mouse models between the sponge and Integra templates. Furthermore, the sponge template does not elicit any abnormal angiogenic or immune responses. The crosslinking approach offers a promising alternative production process for collagen sponge scaffolds.

Technological advances have increasingly provided more and better treatment options for patients with severe burns. Here, we provide a bird's-eye view of the product development process for third-degree burn wounds with considerations of the critical interaction with regulatory bodies, existing technological gaps, and future directions for skin substitutes.

EDITORIAL article Front. Bioeng. Biotechnol., 26 September 2022Sec. Biomaterials Volume 10 - 2022 | https://doi.org/10.3389/fbioe.2022.1035601

The ex-vivo expansion of antigen-specific T-cells for adoptive T-cell immunotherapy requires active interaction between T-cells and antigen-presenting cells therefore culture density and environment become important variables to control. Maintenance of culture density in a static environment is traditionally performed by the expansion of the culture area through splitting of culture from a single vessel into multiple vessels-a highly laborious process. This study aims to validate the use and efficacy of a novel bioreactor, bioreactor with an expandable culture area-dual chamber (BECA-D), that was designed and developed with a cell chamber with expandable culture area (12-108 cm2) and a separate media chamber to allow for in-situ scaling of culture with maintenance of optimum culture density and improved nutrient and gas exchange while minimizing disturbance to the culture. The performance of BECA-D in the culture of Epstein-Barr virus-specific T-cells (EBVSTs) was compared to the 24-well plate. BECA-D had 0.9-9.7 times the average culture yield of the 24-well plates across 5 donor sets. BECA-D was able to maintain the culture environment with relatively stable glucose and lactate levels as the culture expanded. This study concludes that BECA-D can support the culture of ex-vivo EBVSTs with lower manufacturing labour and time requirements compared to the use of the 24-well plate. BECA-D and its adaptation into a closed system with an automated platform (currently being developed) provides cell therapy manufacturers and developers with a closed scale-out solution to producing adoptive cell therapy for clinical use.

Human mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture's viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical [Formula: see text]-galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA-[Formula: see text]-galactosidase activity using the fluorogenic substrate, C[Formula: see text]FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in [Formula: see text]-galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p [Formula: see text] 0.001 for the fluorogenic C[Formula: see text]FDG method, and r = 0.72, p [Formula: see text] 0.05 for the forward scatter method), and good fold difference ranges (1.120-4.436 for total autofluorescence mean and 1.082-6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

Regenerative MedicineVol. 15, No. 12 CommentaryDevelopment of a closed and automated bioreactor technology for cell therapy manufacturing – a sharing of our journeyYing Y Wu‡, Dan Liu‡ & May W NaingYing Y Wu‡ https://orcid.org/0000-0003-0176-7791Bioprocessing Technology Institute, A*STAR Research Entities, 20 Biopolis Way, #06-01, Centros, 138668, Singapore, Dan Liu‡ *Author for correspondence: E-mail Address: Liu_Dan@bti.a-star.edu.sghttps://orcid.org/0000-0002-5192-695XBioprocessing Technology Institute, A*STAR Research Entities, 20 Biopolis Way, #06-01, Centros, 138668, Singapore & May W Naing https://orcid.org/0000-0003-3762-4725Bioprocessing Technology Institute, A*STAR Research Entities, 20 Biopolis Way, #06-01, Centros, 138668, SingaporeSingapore Institute of Manufacturing Technology, A*STAR Research Entities, 2 Fusionopolis Way, #08-04, Innovis, 138634, SingaporePublished Online:7 Jan 2021https://doi.org/10.2217/rme-2020-0142AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: automated systemsbioreactorscell therapyclosed manufacturingclosed systemsintellectual propertylegal/regulatorymanufacturingtechnology platformsReferences1. June CH, O'Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science 359(6382), 1361–1365 (2018).Crossref, Medline, CAS, Google Scholar2. Chia WK, Teo M, Wang WW et al. Adoptive T-cell transfer and chemotherapy in the first-line treatment of metastatic and/or locally recurrent nasopharyngeal carcinoma. Mol. Ther. 22(1), 132–139 (2014).Crossref, Medline, CAS, Google Scholar3. Alliance for Regenerative Medicine. Quarterly regenerative medicine sector report (2019).Google Scholar4. Kino-oka M, Mizutani M, Medcalf N. Cell manufacturability. Cell Gene Ther. Insights 5(10), 1347–1359 (2019).Crossref, Google Scholar5. Masri F, Hoeve MA, De Sousa PA, Willoughby NA. Challenges and advances in scale-up of label-free downstream processing for allogeneic cell therapies. Cell Gene Ther. Insights 3(6), 447–467 (2017).Crossref, Google Scholar6. Wu YY, Yong D, Win Naing M. Automated cell expansion: trends & outlook of critical technologies. Cell Gene Ther. Insights 4(9), 843–863 (2018).Crossref, Google Scholar7. ASME. Crawford M. Validation and verification for medical devices (2015). www.asme.org/topics-resources/content/validation-verification-for-medical-devicesGoogle ScholarFiguresReferencesRelatedDetailsCited ByIn-situ scalable manufacturing of Epstein–Barr virus-specific T-cells using bioreactor with an expandable culture area (BECA)29 April 2022 | Scientific Reports, Vol. 12, No. 1 Vol. 15, No. 12 Follow us on social media for the latest updates Metrics Downloaded 110 times History Received 22 September 2020 Accepted 10 December 2020 Published online 7 January 2021 Published in print December 2020 Information© 2021 Future Medicine LtdKeywordsautomated systemsbioreactorscell therapyclosed manufacturingclosed systemsintellectual propertylegal/regulatorymanufacturingtechnology platformsFinancial & competing interests disclosureThis research is supported by the Agency for Science, Technology and Research (A*STAR), Singapore. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

Extracellular matrix (ECM) is a natural biomaterial scaffold that provides biochemical and structural support to its surrounding cells, forming tissue and respective organs. These ECM proteins can be extracted from organs and tissues through decellularization, which is the process of removing cellular content and nuclear material from the organs to obtain decellularized ECM (dECM). dECM is a versatile and functional biomaterial that can be used as the base component of bioinks for rebuilding tissue and organs. Intact dECM of whole organs can be used as a scaffold for recellularization with human stem cells to produce a functioning organ. As decellularization is a relatively new lab process, the associated technologies and devices are largely non-standardized and only available in small, lab-specific scales. Additionally, there is a lack of standardized protocols to analyze the quality and consistency of harvested dECM for medical applications. This review discusses the relevant decellularization systems and devices currently available to facilitate further development of this process for larger scales with the intention to commercialize dECM materials. Statement of Significance Extracellular matrix (ECM) is a natural cocktail of biomaterials that provides biochemical and structural support to its surrounding cells. ECM proteins are extracted from organs and tissues through decellularization. Being a versatile and functional biomaterial, decellularized extracellular matrix (dECM) is being used as base component of bioinks/hydrogels for rebuilding of tissue and organ constructs. Decellularization is a relatively new lab process with associated technologies/devices being largely non-standardized and only available in lab-specific scales. We discuss categories of decellularization systems and devices for the first time being used in academic and commercial settings. We highlight inherent challenges with the current systems and suggest possible solutions. We comment on further development of these processes for large-scale and commercial applications of dECM. (C) 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Collagen is a natural polymer found abundantly in the extracellular matrix (ECM). It is easily extracted from a variety of sources and exhibits excellent biological properties such as biocompatibility and weak antigenicity. Additionally, different processes allow control of physical and chemical properties such as mechanical stiffness, viscosity and biodegradability. Moreover, various additive biomanufacturing technology has enabled layer-by-layer construction of complex structures to support biological function. Additive biomanufacturing has expanded the use of collagen biomaterial in various regenerative medicine and disease modelling application (e.g., skin, bone and cornea). Currently, regulatory hurdles in translating collagen biomaterials still remain. Additive biomanufacturing may help to overcome such hurdles commercializing collagen biomaterials and fulfill its potential for biomedicine.

Cell-based meats have arose substantial interest due to the promotion of these products as cleaner and sustainable alternatives to conventional meats. We identify the most notable ambiguities surrounding the nomenclature of cell-based meats and summarize current discussions around it. Notable ambiguities include: whether cell-based meat can be called “meat”; identifying the most descriptive and appealing name for cell-based meat; the nomenclature of cell-based meat products with different levels of complexity; and the standards cell-based meats need to meet before claiming to be cleaner and more sustainable than conventional meat. The need to clarify ambiguities is crucial to facilitate discussions, enhance investor interests, build consumer confidence and prevent any future strife with traditional farming.

Organoids have been successfully exploited for drug screening, disease modeling, pathogenesis, and regenerative medicine. Herein, we discuss the progress achieved in the commercialization of organoids in the last few years. We further elaborate on the concept of organoid biobank and highlight ethical and regulatory issues surrounding organoid research and commercialization.

Spheroid culture provides cells with a three-dimensional environment that can better mimic physiological conditions compared to monolayer culture. Technologies involved in the generation of cell spheroids are continuously being innovated to produce spheroids with enhanced properties. In this paper, we review the manufacturing capabilities of current cell spheroid generation technologies. We propose that spheroid generation technologies should enable tight and robust process controls to produce spheroids of consistent and repeatable quality. Future technology development for the generation of cell spheroids should look into improvement in process control, standardization, scalability and monitoring, in addition to advanced methods of spheroid transfer and characterization.

Extracellular matrix (ECM) is a natural biomaterial scaffold that provides biochemical and structural support to its surrounding cells, forming tissue and respective organs. These ECM proteins can be extracted from organs and tissues through decellularization, which is the process of removing cellular content and nuclear material from the organs to obtain decellularized ECM (dECM). dECM is a versatile and functional biomaterial that can be used as the base component of bioinks for rebuilding tissue and organs. Intact dECM of whole organs can be used as a scaffold for recellularization with human stem cells to produce a functioning organ. As decellularization is a relatively new lab process, the associated technologies and devices are largely non-standardized and only available in small, lab-specific scales. Additionally, there is a lack of standardized protocols to analyze the quality and consistency of harvested dECM for medical applications. This review discusses the relevant decellularization systems and devices currently available to facilitate further development of this process for larger scales with the intention to commercialize dECM materials. Statement of Significance Extracellular matrix (ECM) is a natural cocktail of biomaterials that provides biochemical and structural support to its surrounding cells. ECM proteins are extracted from organs and tissues through decellularization. Being a versatile and functional biomaterial, decellularized extracellular matrix (dECM) is being used as base component of bioinks/hydrogels for rebuilding of tissue and organ constructs. Decellularization is a relatively new lab process with associated technologies/devices being largely non-standardized and only available in lab-specific scales. We discuss categories of decellularization systems and devices for the first time being used in academic and commercial settings. We highlight inherent challenges with the current systems and suggest possible solutions. We comment on further development of these processes for large-scale and commercial applications of dECM. (C) 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Recently, particle concentration and filtration using inertial microfluidics have drawn attention as an alternative to membrane and centrifugal technologies for industrial applications, where the target particle size varies between 1 µm and 500 µm. Inevitably, the bigger particle size (>50 µm) mandates scaling up the channel cross-section or hydraulic diameter (DH > 0.5 mm). The Dean-coupled inertial focusing dynamics in spiral microchannels is studied broadly; however, the impacts of secondary flow on particle migration in a scaled-up spiral channel is not fully elucidated. The mechanism of particle focusing inside scaled-up rectangular and trapezoidal spiral channels (i.e., 5-10× bigger than conventional microchannels) with an aim to develop a continuous and clog-free microfiltration system for bioprocessing is studied in detail. Herein, a unique focusing based on inflection point without the aid of sheath flow is reported. This new focusing mechanism, observed in the scaled-up channels, out-performs the conventional focusing scenarios in the previously reported trapezoidal and rectangular channels. Finally, as a proof-of-concept, the utility of this device is showcased for the first time as a retention system for a cell-microcarrier (MC) suspension culture.

Background & Aim With the recent successes of autologous immunotherapy, with 2 therapeutic products approved by the Food & Drugs Administration (FDA) since 2017 and more in the clinical pipeline, there is a need for cell and gene therapy companies to transition from lab scale production to mass manufacture. Traditionally, bioreactors have been routinely used in process development and scale up manufacturing of biologics. However, such bioreactor systems are not suitable for immunotherapy production as, beyond standard external factors such as shear stresses, immune cells are also sensitive to factors such as density and require cell-to-cell contact to proliferate well. Methods, Results & Conclusion A novel single use bioreactor for non-adherent cell culture has been developed in SIMTech (Patent Application WO2018/182533 A1) that allows for culturing cells at constant density while increasing volume of culture media to support cell proliferation. The bioreactor features two different chambers, one for media reserves and the other for cells. The two chambers are separated by a membrane that allow for fluidic exchange between these chambers. The cell chamber has a built-in expansion mechanism that can be adjusted as cells proliferate, thus allowing the user to adjust at any time point and maintain consistent culture density. The bioreactor has been validated with Jurkat cell line and T cells derived from commercial PBMCs with promising results; it was observed that the cultured cells exhibited high viability and selectivity. Jurkat cells had a 240-fold increase in cell number after 14 days in culture while T cells (from commercial PBMCs), had a 47-fold increase in cell number after 21 days in culture. A direct comparison study (1 bioreactor vs multiple 24-well plates for the same increase in cell number) also demonstrated a significant reduction in protocol steps and culture handling time. The bioreactor is currently being tested on donor PBMCs and other cell types, in collaborations with clinicians and cell therapy laboratories, in order to develop a GMP workflow for clinical use.