Cell Bank Research Articles

Quality and Regulatory Requirements for the Manufacture of Master Cell Banks of Clinical Grade iPSCs: The EU and USA Perspectives.

The discovery of induced pluripotent stem cells (iPSCs) and protocols for their differentiation into various cell types have revolutionized the field of tissue engineering and regenerative medicine. Developing manufacturing guidelines for safe and GMP-compliant final products has become essential. Allogeneic iPSCs-derived cell therapies are now the preferred manufacturing alternative. This option requires the establishment of clinical-grade master cell banks of iPSCs. This study aimed at reviewing the Quality and Regulatory requirements from the two main authorities in the world-Europe (EMA) and the United States (FDA)-regarding the manufacture of clinical grade master cell banks (iPSCs). The minimum requirements for iPSCs to be used in first-in-human clinical trials were also reviewed, as well as current best practices currently followed by iPSC bank manufacturers for final product characterisation. The methodology used for this work was a review of various sources of information ranging from scientific literature, published guidance documents available on the EMA and FDA websites, GMP and ICH guidelines, and applicable compendial monographs. Manufacturers of iPSCs cell banks looking to qualify them for clinical use are turning to the ICH guidelines and trying to adapt their requirements. Specifically with the impact of the field of iPSC cell banks, the following areas should be subject to guidance and harmonisation: i) expression vectors authorized for iPSC generation; ii) minimum identity testing; iii) minimum purity testing (including adventitious agent testing); and iv) stability testing. Current ICH guidelines for biotechnological/biological products should be extended to cover cell banks used for cell therapies.

IPSC stem cell banks: is it really necessary if we already have cord blood banks?

iPSC stem cell banks: is it really necessary if we already have cord blood banks?

Mechanism of THBS1 Regulation of MDCK Cell Proliferation and Apoptosis Through TGF-β/Smad Signalling.

Madin-Darby Canine Kidney (MDCK) cells are a key cell line for influenza vaccine production, due to their high viral yield and low mutation resistance. In our laboratory, we established a tertiary cell bank (called M60) using a standard MDCK cell line imported from American Type Culture Collection (ATCC) in the USA. Due to their controversial tumourigenicity, we domesticated non-tumourigenic MDCK cells (named CL23) for influenza vaccine production via monoclonal screening in the early stage of this study, and the screened CL23 cells were characterised based on their low proliferative capacity, which had certain limitations in terms of expanding their production during cell resuscitation. It was thus our objective to enhance the proliferation efficiency of MDCK cells for influenza vaccine production following cell resuscitation, with a view to improving the production of non-tumourigenic MDCK cells for vaccines and enhancing the production of influenza virus lysate vaccines from MDCK cells through genetic intervention. We concentrated on the protein thrombosponin-1 (THBS1), which was markedly differentiated in the proteomics data of the two MDCK cells. By integrating this finding with related studies, we were able to ascertain that THBS1 exerts a significant influence on the level of cell proliferation and apoptosis. Consequently, our objective was to investigate the impact of THBS1 expression on MDCK cell apoptosis by verifying the differences in THBS1 expression between the two MDCK cells and by interfering with THBS1 expression in the MDCK cells. We found that the knockdown of THBS1 significantly increased the proliferation and apoptosis of CL23 cells without causing significant changes in cell migration and invasion, and its overexpression significantly decreased the proliferation of M60 cells and increased cell migration, invasion, and apoptosis. In addition, the TGF-β/Smad pathway target genes transforming growth factor-β1 (TGF-β1), mothers against decapentaplegic homolog 2 (Smad2), and mothers against decapentaplegic homolog 3 (Smad3), were significantly down-regulated in CL23 cells after THBS1 knockdown and up-regulated in M60 cells after overexpression, with consistent expression identified at both the mRNA and protein levels. The treatment of cells with TGF-β activators and inhibitors further demonstrated that THBS1 regulated MDCK cell proliferation and apoptosis through the TGF-β/Smad signalling pathway. Finally, we found that THBS1 also regulated H1N1 influenza virus replication. These findings enable a comprehensive understanding of the regulatory mechanisms of THBS1 regarding MDCK cell proliferation and apoptosis functions and the effects of influenza virus replication.

A risk-based approach can guide safe cell line development and cell banking for scaled-up cultivated meat production.

For commercial viability, cultivated meats require scientifically informed approaches to identify and manage hazards and risks. Here we discuss food safety in the rapidly developing field of cultivated meat as it shifts from lab-based to commercial scales. We focus on what science-informed risk mitigation processes can be implemented from neighbouring fields. We case-study pre-market safety assessments from UPSIDE Foods, GOOD Meat and Vow Group using publicly available dossiers. Quality control and safety assurance practices need to be established and standardized for cell lines and food-grade cell banks.

Establishment and characterization of a novel patient-derived cell line from conventional central grade 3 chondrosarcoma, NCC-CS1-C1.

Chondrosarcoma (CS) is a malignant tumor that produces cartilaginous matrix and is the second most common primary bone sarcoma. CS encompasses a range of histological subtypes, with high-grade conventional central CS being particularly rare, occurring at a rate of 1.81 cases per 1 million person-years. Complete surgical resection is the standard curative treatment for this subtype, as radiation therapy and chemotherapy have proven ineffective. High-grade conventional central CS is highly metastatic and prone to recurrence, resulting in a poor prognosis. Therefore, effective multidisciplinary treatment strategies are urgently needed. Patient-derived cell lines offer promising tools for exploring new therapeutic approaches. However, only two cell lines of high-grade CSs are currently available in public cell banks. In this study, we aimed to establish a novel cell line for high-grade conventional central CS. We successfully developed the NCC-CS1-C1 cell line using surgically resected tumor tissues from a patient with conventional central grade 3 CS. This cell line harbored an IDH1 mutation (p.R132S), commonly found in 50% of CS cases, and exhibited complex copy number variants. A high-throughput screening of 221 anti-cancer drugs identified five candidates-bortezomib, carfilzomib, doxorubicin, panobinostat, and romidepsin-that demonstrated low IC50 values, indicating potential efficacy in treating CS. These findings suggest that NCC-CS1-C1 is a valuable tool for both preclinical and basic research on high-grade conventional central CS.

Xenogeneic-free culture of human intestinal stem cells on functional polymer-coated substrates for scalable, clinical-grade stem cell therapy

The need for basement membrane extract (BME) with undefined constituents, such as Matrigel, for intestinal stem cell (ISC) culture in traditional systems poses a significant barrier that must be overcome for the development of clinical-grade, scalable, ready-to-use ISCs. Here, we propose a functional polymer-based xenogeneic-free dish for the culture of intestinal stem cells (XF-DISC), ensuring substantially prolonged maintenance of ISCs derived from 3-dimensional human intestinal organoids (ISCs3D-hIO). XF-DISC enables remarkable expandability, exhibiting a 24-fold increase in cell numbers within 30 days, with long-term maintenance of ISCs3D-hIO for more than 30 consecutive passages (>210 days). In addition, XF-DISC is fully compatible with a cell banking system. Notably, human pluripotent stem cell-derived ISCs3D-hIO cultured on XF-DISC are successfully transplanted into intestinal injury and inflammation mouse models, leading to engraftment and regeneration of damaged mouse intestinal epithelium. As a reliable and scalable xenogeneic-free ISC3D-hIO culture method, XF-DISC is highly promising for the development of regenerative ISC therapy for human intestinal diseases.

The study of the antiviral effect of a soft dosage form

Aim. To study the antiviral activity of a soft medicine with mangiferin against the herpes simplex virus. Materials and methods. The studies used samples of the soft dosage form developed with mangiferin, a xanthone belonging to the class of polyphenols. Mangiferin is a light yellow fine crystalline powder obtained from the leaves of the mango tree (Mangifera indica), (manufacturer Shaanxi, China). The substance contains 98.5 % of mangiferin. The antiviral activity was studied on Vero kidney cells of an African green monkey obtained from the Cell Bank of the Institute of Experimental Pathology, Oncology and Radiobiology named after R. E. Kavetsky of the National Academy of Sciences of Ukraine. The cell cultivation was performed according to standard methods. The cultivation and accumulation of the virus was carried out on Vero cell culture. The infectious titer of the virus was 7.4 log10 TCD 50/ml. The MTT modification analysis was used to study the cell viability. The antiviral activity was studied according to standard methods. The virulicidal activity against HSV-1 was determined by decreasing the cytopathic effect of the virus on cells and the virus titer using the MTT method. Results and discussion. The determination of the direct effect of the test sample on the extracellular herpes virus (virucidal effect) of an undiluted sample and samples in a dilution of 1:2.5 and 1:5 showed their ability to exhibit a significant virucidal effect against HSV-1 regardless of the dilution used and the exposure time with the virus. It was found that the decrease in the titer of the herpes virus was within 1.7-2.2 log10. When studying the ability of the test sample to influence the development of the cytopathic effect of the herpes virus on cells, it was determined that in dilutions (1:20-1:320), the sample inhibited the reproduction of the virus and the development of HSV-1 on Vero cells compared to the control virus. The greatest efficiency was found when using the sample in dilutions of 1:80-1:320; the reduction in the development of the cytopathic effect of the virus on cells was in the range of 59-64 %. When studying the infectious titer of the herpes virus synthesized de novo after the treatment with various dilutions of the test sample, it was shown that the test sample in the dilutions used did not significantly affect the synthesis of the infectious progeny of the virus, the decrease in the viral titer did not exceed 1.1 log10. Cytotoxicity assays showed that in the dilutions of 1:40 or more, the maximum reduction in cell viability was 18 %, indicating low toxicity of the sample. Conclusions. According to the results of the study, the ability of the test sample containing 5 % mangiferin to effectively inactivate the extracellular herpes simplex virus Type 1 and significantly inhibit the development of the cytopathic effect of the virus on cells has been found.

Optimized protocol for the isolation and expansion of menstrual blood-derived mesenchymal stem cells for combination with oncolytic adenoviruses in cancer treatment

Oncolytic viruses (OVs) are a promising therapeutic approach for cancer, although their systemic administration faces significant challenges. Mesenchymal stem cells have emerged as potential carriers to overcome these obstacles due to their tumor-tropic properties. This study investigates the use of menstrual blood-derived mesenchymal stem cells (MenSCs) as carriers for oncolytic viruses in cancer therapy, focusing on enhancing their efficacy through different culture conditions. MenSCs were isolated from donors of different ages and cultured under normoxic and hypoxic conditions, with varying adherence capacities. Hypoxic conditions significantly improved MenSCs proliferation and tumor migration capabilities, as demonstrated by proliferation assays and RNA-Seq analysis, which revealed upregulation of genes related to cell division and tumor tropism. In vivo studies using a lung adenocarcinoma mouse model confirmed that hypoxia-conditioned MenSCs had superior tumor homing abilities. The study also demonstrated the feasibility of establishing a master and working cell bank from a single menstrual blood donation. These findings suggest that hypoxia-conditioned MenSCs could be highly effective as OV carriers, potentially leading to better clinical outcomes in cancer treatment by enhancing tumor targeting and therapeutic efficacy.

Unlocking Potential: A Comprehensive Overview of Cell Culture Banks and Their Impact on Biomedical Research.

Cell culture banks play a crucial role in advancing biomedical research by providing standardized, reproducible biological materials essential for various applications, from drug development to regenerative medicine. This opinion article presents a comprehensive overview of cell culture banks, exploring their establishment, maintenance, and characterization processes. The significance of ethical considerations and regulatory frameworks governing the use of cell lines is discussed, emphasizing the importance of quality control and validation in ensuring the integrity of research outcomes. Additionally, the diverse types of cell culture banks-primary cells, immortalized cell lines, and stem cells-and their specific contributions to different fields such as cancer research, virology, and tissue engineering are examined. The impact of technological advancements on cell banking practices is also highlighted, including automation and biobanking software that enhance efficiency and data management. Furthermore, challenges faced by researchers in accessing high-quality cell lines are addressed, along with proposed strategies for improving collaboration between academic institutions and commercial entities. By unlocking the potential of cell culture banks through these discussions, this article aims to underline their indispensable role in driving innovation within biomedical research and fostering future discoveries that could lead to significant therapeutic breakthroughs.

Harnessing Global HLA Data for Enhanced Patient Matching in iPSC Haplobanks

Several countries have either developed or are developing national induced pluripotent stem cell (iPSC) banks of cell lines derived from donors with HLA homozygous genotypes (two identical haplotypes) prevalent in their local populations to provide immune matched tissues and cells to support regenerative medicine therapies. This ‘haplobank’ approach relies on knowledge of the HLA genotypes of the population to identify the most beneficial haplotypes for patient coverage, and ultimately identify donors or cord blood units carrying two copies of the target haplotype. A potentially more efficient alternative to a national bank approach is to assess the haplotypes required to provide global patient coverage and to produce a single, global haplobank. Toward that end, we have developed an algorithm to prioritize HLA haplotypes that optimize coverage across the global population.We analyzed data from eighteen countries participating in the Global Alliance for iPS Therapy (GAiT). A representative pool of HLA genotypes, reflecting the HLA of patients, was derived by sampling from each country's WMDA hematopoietic stem cell donor registry, or surrogate population. An algorithm was created based on HLA-A, -B and -DRB1 haplotype homozygous types with population HLA matching coverage defined by the absence of Host versus Graft (HvG) mismatches at these loci. HLA matching coverage was determined by iteratively selecting HLA haplotypes that provide the largest coverage against patient HLA genotypes sampled from the same population, excluding genotypes compatible with previous iterations. The top 10 haplotypes for each of the 18 countries were identified with patient coverage ranging from 19.5% in Brazil to 63.8% in Japan, with a mean coverage of 33.3%. In a ‘global’ model, utilizing the 180 most frequent haplotypes across all 18 populations (equivalent to 10 lines per country), the patient coverage ranged from 54.6% in India to 81.7% in Sweden, with a mean of 68.4%. Our findings demonstrate that global collaboration could more than double the potential for patient HLA matching coverage.Interrogation of unrelated hematopoietic stem cell donor registry and cord blood bank HLA data demonstrated that HLA-A, -B, and -DRB1 homozygous donors for the top 180 global haplotypes are widely available. These results show that a globally coordinated strategy for haplobanking would reduce redundancy and allow more patients to be treated with the same investment.

Sterility of cord blood units: Evaluation of the collection, preparation and conservation process with a view to establishing a bank of stem cells from cord blood at the Biological Resources Center of the Institut Pasteur de Côte d’Ivoire (2024)

Introduction: Cord blood stem cells are used as an alternative to bone marrow and peripheral blood cell transplantation. For this purpose, cord blood banks have been established worldwide for the collection and cryopreservation of cord blood units. These banks must implement rigorous protocols to ensure the microbiological safety of grafts. The aim of this study is to evaluate the process of selecting mothers and collecting and processing these samples developed for this purpose. Methodology: Pregnant women were enrolled in the study on the basis of selection criteria. Maternal blood and cord blood samples were collected by the in utero method. Serological analyses of maternal blood for the detection of vertically transmitted infections and microbiological examinations of cord blood units were carried out. Results: 31 women out of 46 (68.4%) were enrolled. All had negative serology for the targeted pathogens. Similarly, microbiology revealed no microbial contamination of the cord blood units. Conclusion: This work demonstrates that the procedures for selecting mothers and processing implementations are effective in ensuring the microbiological safety of cord blood units and could be suggested in the context of banking cord blood stem cells at the Institut Pasteur de Côte d’Ivoire.

Development of a quantitative prediction algorithm for human cord blood-derived CD34+ hematopoietic stem-progenitor cells using parametric and non-parametric machine learning models

The transplantation of CD34+ hematopoietic stem-progenitor cells (HSPCs) derived from cord blood serves as the standard treatment for selected hematological, oncological, metabolic, and immunodeficiency disorders, of which the dose is pivotal to the clinical outcome. Based on numerous maternal and neonatal parameters, we evaluated the predictive power of mathematical pipelines to the proportion of CD34+ cells in the final cryopreserved cord blood product adopting both parametric and non-parametric algorithms. Twenty-four predictor variables associated with the cord blood processing of 802 processed cord blood units randomly sampled in 2020–2022 were retrieved and analyzed. Prediction models were developed by adopting the parametric (multivariate linear regression) and non-parametric (random forest and back propagation neural network) statistical models to investigate the data patterns for determining the single outcome (i.e., the proportion of CD34+ cells). The multivariate linear regression model produced the lowest root-mean-square deviation (0.0982). However, the model created by the back propagation neural network produced the highest median absolute deviation (0.0689) and predictive power (56.99%) in comparison to the random forest and multivariate linear regression. The predictive model depending on a combination of continuous and discrete maternal with neonatal parameters associated with cord blood processing can predict the CD34+ dose in the final product for clinical utilization. The back propagation neural network algorithm produces a model with the highest predictive power which can be widely applied to assisting cell banks for optimal cord blood unit selection to ensure the highest chance of transplantation success.

From banked human cord blood to induced pluripotent stem cells: New opportunities and promise in induced pluripotent stem cell banking (Review).

Umbilical cord blood (CB) is a valuable source of haematopoietic stem/progenitor cells (HSCs) and is known for the therapeutic use of these cells in treating blood disorders. However, challenges such as a high running cost and the increasing availability of treatment alternatives have made the effort to sustain CB banks difficult. This prompts the need to revisit the current CB banking initiatives to retain the relevance in this ever‑changing era parallel to the fast‑pacing development of cell‑based therapeutic technology. Cellular reprogramming has shown to have successfully converted adult somatic cells into human induced pluripotent stem cells (hiPSCs), which promise wider applications in regenerative medicine, personalized treatment and tissue engineering. CB is the youngest, primitive adult cell source that has not been affected by any prior, acquired disorders. Hence, using CB as a source of candidate cells for generating hiPSCs may be a new opportunity for banking, albeit with challenges. The present review summarizes the rise and fall of CB usage and banking for clinical therapy, the considerations in reprogramming CB into hiPSCs, the safety concerns regarding the use of hiPSC‑derived cells in clinical transplantation and the prospect of using CB‑derived hiPSCs.

Dental stem cell banking: a promising future for regenerative medicine applications

Dental stem cells originating from different oral tissues in and around dental structures have recently gained attention as a potential alternative for regenerative medicine applications. To date, many dental stem cells are identified specific to the tissue from which they originate. They exhibit many valuable advantages including high proliferation ability, self-renewal capacity, and multiple differentiation potentials that make them an important candidate for clinical applications, especially in treating degenerative and inflammatory diseases. The fact that they can be easily obtained from an individual’s waste tooth without any ethical concern provides them an excellent opportunity for autologous treatment with a low risk of immune rejection. Nowadays, the storage of autologous dental stem cells isolated from wisdom teeth or healthy extracted teeth in biobanks without ethical concerns has become a very important approach for the regeneration of damaged and diseased tissue and for the treatment of life-threatening diseases that may be encountered in the future life of the donor. This study provides a comprehensive overview of dental stem cells, recent advances in their clinical use, long-term preservation processes, and the latest advances in Dental Stem Cell Banking.

Paradigm Shift in Eye Banking: From Tissue Retrieval to Cellular Harvesting and Bioengineering.

An integrated cell, tissue, and eye bank is vital to meet the evolving needs of ocular transplant therapies. In addition to traditional corneal transplant tissues, it encompasses processing and delivery of transplant materials for newer treatments like cell-based therapies and gene-modified products, adhering to rigorous standards, optimizing tissue utilization with comprehensive services for surgeons.

Optimisation of cryopreservation conditions, including storage duration and revival methods, for the viability of human primary cells

BackgroundCryopreservation is a crucial procedure for safeguarding cells or other biological constructs, showcasing considerable potential for applications in tissue engineering and regenerative medicine.AimsThis study aimed to evaluate the effectiveness of different cryopreservation conditions on human cells viability.MethodsA set of cryopreserved data from Department of Tissue Engineering and Regenerative Medicine (DTERM) cell bank were analyse for cells attachment after 24 h being revived. The revived cells were analysed based on different cryopreservation conditions which includes cell types (skin keratinocytes and fibroblasts, respiratory epithelial, bone marrow mesenchymal stem cell (MSC); cryo mediums (FBS + 10% DMSO; commercial medium); storage durations (0 to > 24 months) and locations (tank 1–2; box 1–5), and revival methods (direct; indirect methods). Human dermal fibroblasts (HDF) were then cultured, cryopreserved in different cryo mediums (HPL + 10% DMSO; FBS + 10% DMSO; Cryostor) and stored for 1 and 3 months. The HDFs were revived using either direct or indirect method and cell number, viability and protein expression analysis were compared.ResultsIn the analysis cell cryopreserved data; fibroblast cells; FBS + 10% DMSO cryo medium; storage duration of 0–6 months; direct cell revival; storage in vapor phase of cryo tank; had the highest number of vials with optimal cell attachment after 24 h revived. HDFs cryopreserved in FBS + 10% DMSO for 1 and 3 months with both revival methods, showed optimal live cell numbers and viability above 80%, higher than other cryo medium groups. Morphologically, the fibroblasts were able to retain their phenotype with positive expression of Ki67 and Col-1. HDFs cryopreserved in FBS + 10% DMSO at 3 months showed significantly higher expression of Ki67 (97.3% ± 4.62) with the indirect revival method, while Col-1 expression (100%) was significantly higher at both 1 and 3 months compared to other groups.ConclusionIn conclusion, fibroblasts were able to retain their characteristics after various cryopreservation conditions with a slight decrease in viability that may be due to the thermal-cycling effect. However, further investigation on the longer cryopreservation periods should be conducted for other types of cells and cryo mediums to achieve optimal cryopreservation outcomes.

Banked Primary Progenitor Cells for Allogeneic Intervertebral Disc (IVD) Therapy: Preclinical Qualification and Functional Optimization within a Cell Spheroid Formulation Process.

Background/Objectives: Biological products are emerging as therapeutic management options for intervertebral disc (IVD) degenerative affections and lower back pain. Autologous and allogeneic cell therapy protocols have been clinically implemented for IVD repair. Therein, several manufacturing process design considerations were shown to significantly influence clinical outcomes. The primary objective of this study was to preclinically qualify (chondrogenic potential, safety, resistance to hypoxic and inflammatory stimuli) cryopreserved primary progenitor cells (clinical grade FE002-Disc cells) as a potential cell source in IVD repair/regeneration. The secondary objective of this study was to assess the cell source's delivery potential as cell spheroids (optimization of culture conditions, potential storage solutions). Methods/Results: Safety (soft agar transformation, β-galactosidase, telomerase activity) and functionality-related assays (hypoxic and inflammatory challenge) confirmed that the investigated cellular active substance was highly sustainable in defined cell banking workflows, despite possessing a finite in vitro lifespan. Functionality-related assays confirmed that the retained manufacturing process yielded strong collagen II and glycosaminoglycan (GAG) synthesis in the spheroids in 3-week chondrogenic induction. Then, the impacts of various process parameters (induction medium composition, hypoxic incubation, terminal spheroid lyophilization) were studied to gain insights on their criticality. Finally, an optimal set of technical specifications (use of 10 nM dexamethasone for chondrogenic induction, 2% O2 incubation of spheroids) was set forth, based on specific fine tuning of finished product critical functional attributes. Conclusions: Generally, this study qualified the considered FE002-Disc progenitor cell source for further preclinical investigation based on safety, quality, and functionality datasets. The novelty and significance of this study resided in the establishment of defined processes for preparing fresh, off-the-freezer, or off-the-shelf IVD spheroids using a preclinically qualified allogeneic human cell source. Overall, this study underscored the importance of using robust product components and optimal manufacturing process variants for maximization of finished cell-based formulation quality attributes.

Case Studies on Changes and Proposed Process Development Approaches Reflecting Applicability of PDA Technical Report No. 89: Strategies for Vaccine Development and Lifecycle Management.

Vaccines are complex and a very diverse group of products with relatively long product life cycles. The manufacturing programs for these vaccines need to be continually updated to comply with evolving regulatory expectations. Members of the Parenteral Drug Association (PDA) Vaccines Interest Group (VIG) authored and published PDA Technical Report No. 89: Strategies for Vaccine Development and Lifecycle Management (TR 89), which seeks to provide context to vaccine developers and manufacturers regarding key aspects of new or legacy vaccines such as control strategy from process development to vaccine life cycle management, comparability and life cycle management including technical, validation, quality, and regulatory perspectives. To further explain and illustrate the concepts and topics discussed, seven relevant situations were selected as either case studies associated with changes implemented or proposed process development strategies, which are discussed in this article. The situations described are: working cell bank, modification or update of externally supplied product contact components for vaccine manufacturing, raw material change, new product at an existing site, vaccine development acceleration by leveraging existing platforms, selection and implementation of potency method, and modeling for stability forecast prediction. For each situation, the applicable key concepts from TR 89 are discussed as follows: Control Strategy, Prior Knowledge, Relying on Pharmaceutical Quality System (PQS), Classification of Parameters, Validation Approach, Use of a Risk-Based Approach, Comparability, Use of ICH Q12, and Additional Regulatory Considerations.

Design and Comparison of Fractional-Order Controllers in Flotation Cell Banks and Flotation Columns Used in Copper Extraction Processes

This work explores efficiency improvements in the copper flotation stage, a complex nonlinear, multivariable process subject to numerous perturbations. The primary objective is to design a fractional-order PID (FOPID) control strategy and a fractional-order model reference adaptive control (FOMRAC) system. The parameters for these controllers are optimized using the particle swarm optimization (PSO) algorithm with an objective function tailored to the control goals. This study employs models of both a bank series of five flotation cells and a flotation column. Their performance results are compared against traditional controllers, such as an integer-order PID and MRAC. The findings reveal that fractional-order controllers offer notable advantages over their integer-order counterparts, showing improved performance metrics with minimal changes to the existing control framework. This research highlights the effectiveness of fractional control in enhancing flotation processes and introduces a novel application of fractional control techniques in this area.

Establishing Primary and Stable Cell Lines from Frozen Wing Biopsies for Cellular, Physiological, and Genetic Studies in Bats.

Bats stand out among mammalian species for their exceptional traits, including the capacity to navigate through flight and echolocation, conserve energy through torpor/hibernation, harbor a multitude of viruses, exhibit resistance to disease, survive harsh environmental conditions, and demonstrate exceptional longevity compared to other mammals of similar size. In vivo studies of bats are challenging for several reasons, such as difficulty in locating and capturing them in their natural environments, limited accessibility, low sample size, environmental variation, long lifespans, slow reproductive rates, zoonotic disease risks, species protection, and ethical concerns. Thus, establishing alternative laboratory models is crucial for investigating the diverse physiological adaptations observed in bats. Obtaining quality cells from tissues is a critical first step for successful primary cell derivation. However, it is often impractical to collect fresh tissue and process the samples immediately for cell culture due to the resources required for isolating and expanding cells. As a result, frozen tissue is typically the starting resource for bat primary cell derivation, but cells in frozen tissue are usually damaged and have low integrity and viability. Isolating primary cells from frozen tissues thus poses a significant challenge. Herein, we present a successfully developed protocol for isolating primary dermal fibroblasts from frozen bat wing biopsies. This protocol marks a significant milestone, as this is the first protocol specifically focused on fibroblast isolation from bat frozen tissue. We also describe methods for primary cell characterization, genetic manipulation of primary cells through lentivirus transduction, and the development of stable cell lines. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Bat wing biopsy collection and preservation Support Protocol 1: Blood collection from bat venipuncture Basic Protocol 2: Isolation of primary fibroblasts from adult bat frozen wing biopsy Support Protocol 2: Primary fibroblast culture and subculture Support Protocol 3: Determination of growth curve and doubling time Support Protocol 4: Cell banking and thawing of primary fibroblasts Basic Protocol 3: Lentiviral transduction of bat primary fibroblasts Basic Protocol 4: Bat stable fibroblast cell line development Support Protocol 5: Bat fibroblast validation by immunofluorescence staining Basic Protocol 5: Chromosome counting.