Bioprocessing considerations for generation of iPSCs intended for clinical application: perspectives from the ISCT Emerging Regenerative Medicine Technology working group.

Approval of induced pluripotent stem cells (iPSCs) for the manufacture of cell therapies to support clinical trials is now becoming realized after 20 years of research and development. In 2022 the International Society for Cell and Gene Therapy (ISCT) established a Working Group on Emerging Regenerative Medicine Technologies, an area in which iPSCs-derived technologies are expected to play a key role. In this article, the Working Group surveys the steps that an end user should consider when generating iPSCs that are stable, well-characterised, pluripotent, and suitable for making differentiated cell types for allogeneic or autologous cell therapies. The objective is to provide the reader with a holistic view of how to achieve high-quality iPSCs from selection of the starting material through to cell banking. Key considerations include: (i) intellectual property licenses; (ii) selection of the raw materials and cell sources for creating iPSC intermediates and master cell banks; (iii) regulatory considerations for reprogramming methods; (iv) options for expansion in 2D vs. 3D cultures; and (v) available technologies and equipment for harvesting, washing, concentration, filling, cryopreservation, and storage. Some key process limitations are highlighted to help drive further improvement and innovation, and includes recommendations to close and automate current open and manual processes.

Meningoencephalomyelitis and brachial plexitis in a dog infected with louping ill virus.

A foxhound from a hunting kennel in the United Kingdom was euthanized after being hospitalized with progressive neurologic signs, including tremors, seizures, and obtunded mentation. No abnormalities were appreciated on gross postmortem examination. Histologically, severe meningoencephalomyelitis and mild neuritis of the brachial plexus were present. Molecular analysis of brain tissue detected louping ill virus. In addition, louping ill virus-specific antigens were detected in neurons within the brainstem, the entire length of the spinal cord, as well as in rare cells in the brachial plexus using immunohistochemistry. The genetic sequence of the virus appears most closely related to a previously detected virus in a dog from a similar geographic location in 2015. This is the first characterization of the inflammatory lesions and viral distribution of louping ill virus in a naturally infected dog within the spinal cord and brachial plexus.

Performance of an artificial intelligence automated system for diabetic eye screening in a large English population.

AIMS: A diabetic eye screening programme has huge value in reducing avoidable sight loss by identifying diabetic retinopathy at a stage when it can be treated. Artificial intelligence automated systems can be used for diabetic eye screening but are not employed in the national English Diabetic Eye Screening Programme. The aim was to report the performance of a commercially available deep-learning artificial intelligence software in a large English population. METHODS: 9817 anonymised image sets from 10,000 consecutive diabetic eye screening episodes were presented to an artificial intelligence software. The sensitivity and specificity of the artificial intelligence system for detecting diabetic retinopathy were determined using the diabetic eye screening programme manual grade according to national protocols as the reference standard. RESULTS: For no diabetic retinopathy versus any diabetic retinopathy, the sensitivity of the artificial intelligence grading system was 69.7% and specificity 92.2%. The performance of the artificial intelligence system was superior for no or mild diabetic retinopathy versus significant or referrable diabetic retinopathy with a sensitivity of 95.4% and specificity of 92.0%. No cases were identified in which the artificial intelligence grade had missed significant diabetic retinopathy. CONCLUSION: The performance of a commercially available deep-learning artificial intelligence system for identifying diabetic retinopathy in an English national Diabetic Eye Screening Programme is presented. Using the pre-defined settings artificial intelligence performance was highest when identifying diabetic retinopathy which requires an action by the screening programme.

Impact considerations of post-production processes on cell and gene drug products.

Cell and gene therapies are demonstrating clinical efficacy, but prohibitive product costs and operational complexity bottlenecks may limit expanded patient access to these innovative and transformative products. An initial survey and subsequent article published through the International Society for Cell & Gene Therapy in 2017 presented a roadmap on how specific steps, from tissue procurement and material acquisition to facility operation and production, contribute to the high cost of cell and gene therapies. Herein the authors expanded the investigation to provide considerations to better understand how post-production procedures can impact a product's accessibility to patients. The administration of a drug product to and follow-up in a patient involve key decisions in several post-production process areas, such as product storage, distribution and handling logistics and compliance, across the value chain through integrated data management solutions. Understanding as well as carefully evaluating these specific components is not widely considered during early process development but is critical in developing a viable product life cycle.

Transitioning from development to commercial: risk-based guidance for critical materials management in cell therapies.

A key hurdle to ensuring patient access to cell and gene therapies (CGTs) and continued growth of the industry is the management of raw materials. The combination of rapid growth, individual product and process complexity and limited industry-specific guidance or awareness presents non-obvious risk mitigation challenges for transitioning from development to clinical application. Understanding, assessing and mitigating the varied raw material risks for CGT products during product and clinical development are critical for ensuring smooth transitions into commercialization and for preventing interruption of product supply to patients. This article presents a risk-based approach driven by concerns for patient safety that can help focus and coordinate efforts to address the most critical risk factors. Highlighted are some of the highest risk materials common to the manufacture of many CGTs, including the primary starting material, culture media, reagents and single-use components. Using a hypothetical gene-edited cell therapy as an example, we describe the general manufacturing process and subsequently incorporate the described methodology to perform a sample risk assessment. The practical approach described herein is intended to assist CGT manufacturers and suppliers in actively assessing materials early in development to provide a basic starting point for mitigating risks experienced when translating CGT products for clinical and long-term commercial application.

Mechanosensory hairs in bumblebees (Bombus terrestris) detect weak electric fields.

Bumblebees (Bombus terrestris) use information from surrounding electric fields to make foraging decisions. Electroreception in air, a nonconductive medium, is a recently discovered sensory capacity of insects, yet the sensory mechanisms remain elusive. Here, we investigate two putative electric field sensors: antennae and mechanosensory hairs. Examining their mechanical and neural response, we show that electric fields cause deflections in both antennae and hairs. Hairs respond with a greater median velocity, displacement, and angular displacement than antennae. Extracellular recordings from the antennae do not show any electrophysiological correlates to these mechanical deflections. In contrast, hair deflections in response to an electric field elicited neural activity. Mechanical deflections of both hairs and antennae increase with the electric charge carried by the bumblebee. From this evidence, we conclude that sensory hairs are a site of electroreception in the bumblebee.

Peter Barlow's insights and contributions to the study of tidal gravity variations and ultra-weak light emissions in plants.

Background: A brief review is given of Peter W. Barlows' contributions to research on gravity tide-related phenomena in plant biology, or 'selenonastic' effects as he called them, including his early research on root growth. Also, new results are presented here from long-term recordings of spontaneous ultra-weak light emission during germination, reinforcing the relationship between local lunisolar tidal acceleration and seedling growth. Scope: The main ideas and broad relevance of the work by Barlow and his collaborators about the effects of gravity on plants are reviewed, highlighting the necessity of new models to explain the apparent synchronism between root growth and microscale gravity changes 107 times lower than that exerted by the Earth's gravity. The new results, showing for the first time the germination of coffee beans in sequential tests over 2 months, confirm the co-variation between the patterns in ultra-weak light emission and the lunisolar tidal gravity curves for the initial growth phase. For young sprouts (<1 month old), the rhythm of growth as well as variation in light emission exhibit the once a day and twice a day periodic variations, frequency components that are the hallmark of local lunisolar gravimetric tides. Although present, this pattern is less pronounced in coffee beans older than 1 month. Conclusions: The apparent co-variation between ultra-weak light emission and growth pattern in coffee seedlings and the lunisolar gravity cycles corroborate those previously found in seedlings from other species. It is proposed here that such patterns may attenuate with time for older sprouts with slow development. These data suggest that new models considering both intra- and intercellular interactions are needed to explain the putative sensing and reaction of seedlings to the variations in the gravimetric tide. Here, a possible model is presented based on supracellular matrix interconnections.

The bee, the flower, and the electric field: electric ecology and aerial electroreception.

Bees and flowering plants have a long-standing and remarkable co-evolutionary history. Flowers and bees evolved traits that enable pollination, a process that is as important to plants as it is for pollinating insects. From the sensory ecological viewpoint, bee-flower interactions rely on senses such as vision, olfaction, humidity sensing, and touch. Recently, another sensory modality has been unveiled; the detection of the weak electrostatic field that arises between a flower and a bee. Here, we present our latest understanding of how these electric interactions arise and how they contribute to pollination and electroreception. Finite-element modelling and experimental evidence offer new insights into how these interactions are organised and how they can be further studied. Focussing on pollen transfer, we deconstruct some of the salient features of the three ingredients that enable electrostatic interactions, namely the atmospheric electric field, the capacity of bees to accumulate positive charge, and the propensity of plants to be relatively negatively charged. This article also aims at highlighting areas in need of further investigation, where more research is required to better understand the mechanisms of electrostatic interactions and aerial electroreception.

Off the shelf cellular therapeutics: Factors to consider during cryopreservation and storage of human cells for clinical use.

The field of cellular therapeutics has immense potential, affording an exciting array of applications in unmet medical needs. One of several key issues is an emphasis on getting these therapies from bench to bedside without compromising safety and efficacy. The successful commercialization of cellular therapeutics will require many to extend the shelf-life of these therapies beyond shipping "fresh" at ambient or chilled temperatures for "just in time" infusion. Cryopreservation is an attractive option and offers potential advantages, such as storing and retaining patient samples in case of a relapse, banking large quantities of allogeneic cells for broader distribution and use and retaining testing samples for leukocyte antigen typing and matching. However, cryopreservation is only useful if cells can be reanimated to physiological life with negligible loss of viability and functionality. Also critical is the logistics of storing, processing and transporting cells in clinically appropriate packaging systems and storage devices consistent with quality and regulatory standards. Rationalized approaches to develop commercial-scale cell therapies require an efficient cryopreservation system that provides the ability to inventory standardized products with maximized shelf life for later on-demand distribution and use, as well as a method that is scientifically sound and optimized for the cell of interest. The objective of this review is to bridge this gap between the basic science of cryobiology and its application in this context by identifying several key aspects of cryopreservation science in a format that may be easily integrated into mainstream cell therapy manufacture.

Current perspectives on the use of ancillary materials for the manufacture of cellular therapies.

Continued growth in the cell therapy industry and commercialization of cell therapies that successfully advance through clinical trials has led to increased awareness around the need for specialized and complex materials utilized in their manufacture. Ancillary materials (AMs) are components or reagents used during the manufacture of cell therapy products but are not intended to be part of the final products. Commonly, there are limitations in the availability of clinical-grade reagents used as AMs. Furthermore, AMs may affect the efficacy of the cell product and subsequent safety of the cell therapy for the patient. As such, AMs must be carefully selected and appropriately qualified during the cell therapy development process. However, the ongoing evolution of cell therapy research, limited number of clinical trials and registered cell therapy products results in the current absence of specific regulations governing the composition, compliance, and qualification of AMs often leads to confusion by suppliers and users in this field. Here we provide an overview and interpretation of the existing global framework surrounding AM use and investigate some common misunderstandings within the industry, with the aim of facilitating the appropriate selection and qualification of AMs. The key message we wish to emphasize is that in order to most effectively mitigate risk around cell therapy development and patient safety, users must work with their suppliers and regulators to qualify each AM to assess source, purity, identity, safety, and suitability in a given application.

Managing particulates in cell therapy: Guidance for best practice.

The intent of this article is to provide guidance and recommendations to cell therapy product sponsors (including developers and manufacturers) and their suppliers in the cell therapy industry regarding particulate source, testing, monitoring and methods for control. This information is intended to help all parties characterize the processes that generate particulates, understand product impact and provide recommendations to control particulates generated during manufacturing of cell therapy products.

Actopaxin (α-parvin) phosphorylation is required for matrix degradation and cancer cell invasion.

Dysregulation of cell adhesion and motility is known to be an important factor in the development of tumor malignancy. Actopaxin (α-parvin) is a paxillin, integrin-linked kinase, and F-actin binding focal adhesion protein with several serine phosphorylation sites in the amino terminus that contribute to the regulation of cell spreading and migration. Here, phosphorylation of actopaxin is shown to contribute to the regulation of matrix degradation and cell invasion. Osteosarcoma cells stably expressing wild type (WT), nonphosphorylatable (Quint), and phosphomimetic (S4D/S8D) actopaxin demonstrate that actopaxin phosphorylation is necessary for efficient Src and matrix metalloproteinase-driven degradation of extracellular matrix. Rac1 was found to be required for actopaxin-induced matrix degradation whereas inhibition of myosin contractility promoted degradation in the phosphomutant-expressing Quint cells, indicating that a balance of Rho GTPase signaling and regulation of cellular tension are important for the process. Furthermore, actopaxin forms a complex with the Rac1/Cdc42 GEF ß-PIX and Rac1/Cdc42 effector PAK1, to regulate actopaxin-dependent matrix degradation. Actopaxin phosphorylation is elevated in the invasive breast cancer cell line MDA-MB-231 compared with normal breast epithelial MCF10A cells. Expression of the nonphosphorylatable Quint actopaxin in MDA-MB-231 cells inhibits cell invasion whereas overexpression of WT actopaxin promotes invasion in MCF10A cells. Taken together, this study demonstrates a new role for actopaxin phosphorylation in matrix degradation and cell invasion via regulation of Rho GTPase signaling.

Managing particulates in cellular therapy.

he concept of particulates, while common to many in the pharmaceutical and blood transfusion disciplines, represents a distinct challenge in the field of cellular therapy. With newly discovered products advancing through clinical trials, the focus has shifted to ensuring products are manufactured in a reliable and safe manner. Given the unique manufacturing processes and resulting products (i.e. the cell being the active ingredient of the product), the way in which particulates are viewed and subsequently tested needs to be reviewed. No specific test or method for particulates will apply to all products, and guidance documents will be generated over time as more cell therapy products are approved. The details of the processes, testing methods used and acceptance criteria established for particulates will play a major role in generating the guidance documents. This will ultimately allow for the manufacture and administration of safe and effective products without thwarting advancement of the cellular therapy field. The intent of this review is to bring awareness to the topic of particulates with respect to cell therapy, and encourage a more open dialog and exchange of examples within the industry. We have reviewed the concept of particulates, where they originate and how they are introduced to cell therapy products, and the current methods available for their detection. We have also reviewed the relevance of current guidance documents and present potential strategies to move forward and address and control unwanted contaminating particulates in cell therapy products.

Cryopreservation of umbilical cord blood with a novel freezing solution that mimics intracellular ionic composition.

BACKGROUND: Cryopreservation protocols have remained relatively unchanged since the first umbilical cord blood banking program was established. This study evaluated the preservation efficacy of a novel intracellular-like cryopreservation solution (CryoStor, BioLife Solutions, Inc.), the rate of addition of two cryopreservation solutions to cord blood units (CBUs), and reduced final dimethyl sulfoxide (DMSO) concentration of 5%. STUDY DESIGN AND METHODS: Split-sample CBUs were cryopreserved with either an in-house 20% DMSO-based cryopreservation solution or CryoStor CS10 at a rate of 1 mL/min (n = 10; i.e., slow addition) or as a bolus injection (n = 6; i.e., fast addition). Infrared images of exothermic effects of the cryopreservation solutions were monitored relative to the rate of addition. Prefreeze and postthaw colony-forming unit assays, total nucleated cells, and CD34+ cell counts were compared. RESULTS: Maximum temperature excursions observed were less than 6°C, regardless of the rate of solution addition. Fast addition resulted in peak excursions approximately twice that of slow addition but the magnitude and duration were minimal and transient. Slow addition of CryoStor CS10 (i.e., final concentration ≤ 5% DMSO) resulted in significantly better postthaw CD34+ cell recoveries; no other metrics were significantly different. Fast addition of CryoStor resulted in similar postthaw metrics compared to slow addition of the in-house solution. CONCLUSION: Slow and fast addition of cryopreservation solutions result in mean temperature changes of approximately 3.3 to 4.45°C. Postthaw recoveries with CryoStor were equivalent to or slightly better than with the in-house cryopreservation solution. CryoStor also provides several advantages including reduced processing time, formulation consistency, and reduced DMSO in the frozen product (≤ 5%).

Direct concentration measurements of the unfrozen portion of solutions under freezing.

Phase diagrams of solutions consisting of cryoprotective agents (CPA) are very useful in cryobiology research. Those diagrams depict the points of solution concentrations at corresponding temperatures: one of essential inputs that can be utilized to compute the volume response of cell under freezing process. However, generating such plots is costly and time-consuming. A direct method is proposed in this study to determine the solution concentration of unfrozen parts at multiple sub-zero temperatures. Measurements of binary solutions, composed of water and sodium chloride, were performed and compared with published data. Ternary solutions, consisting of water, sodium chloride and dimethyl sulfoxide, were also measured. The uniqueness and advantage achieved through the usage of this method are demonstrated when phase diagrams of complex cryopreservation solutions (CryoStor solutions including CryoStor Base and CryoStor 10) are generated. The temperature range where the method is utilized is either limited by the osmometry (0-3200 mmol/kg) or by the availability of liquid samples at sub-freezing temperatures. Modified methods will be required to address the limitation of osmolality measurements and the availability of sub-freezing liquid samples at lower temperatures.

Improved post-thaw recovery of peripheral blood stem/progenitor cells using a novel intracellular-like cryopreservation solution.

BACKGROUND AIMS: Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products, which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate-freezing and liquid nitrogen storage have become 'routine' practice in many cell-processing facilities, there is clearly room for improvement as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment, and can also expose the patients to relatively undefined serum components and larger volumes of dimethylsulfoxide (DMSO) that can contribute to the morbidity and mortality of the transplant therapy. METHODS: This study compared cryopreservation of PBSC in a novel intracellular-like, fully defined, serum- and protein-free preservation solution, CryoStor (BioLife Solutions Inc.), with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly, human PBSC apheresis specimens were collected and 5 x 10(7) cells/1 mL sample vial were prepared for cryopreservation in the following solutions: (a) FHCRC standard, Normosol-R, 5% human serum albumin (HAS) and 10% DMSO; and (b) CryoStor CS10 (final diluted concentration of 5% DMSO). A standard controlled-rate freezing program was employed, and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of 1 week. Vials were then thawed and evaluated for total nucleated cell count (TNC), viability, CD34 and granulocytes by flow cytometry, along with colony-forming activity in methylcellulose. RESULTS: The PBSC samples frozen in CryoStor CS10 yielded significantly improved post-thaw recoveries for total viable CD34(+), colony-forming units (CFU) and granulocytes. Specifically, relative to the FHCRC standard formulation, cryopreservation with CS10 resulted in an average 1.8-fold increased recovery of viable CD34(+) cells (P=0.005), a 1.5-fold increase in CFU-granulocyte-macrophage (GM) numbers (P=0.030) and a 2.3-fold increase in granulocyte recovery (P=0.045). CONCLUSIONS: This study indicates that use of CryoStor for cryopreservation can yield significantly improved recovery and in vitro functionality of stem/progenitor cells in PBSC products. In addition, it is important to note that these improved recoveries were obtained while not introducing any extra serum or serum-derived proteins, and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary; however, these findings imply use of CryoStor for cryopreservation could result in improved engraftment for those patients with a lower content of CD34(+) cells in their PBSC collections, along with reducing the requirement for additional apheresis collections and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO.

Phosphorylation of actopaxin regulates cell spreading and migration.

Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.

Development of a tissue engineered human prostate tumor equivalent for use in the evaluation of cryoablative techniques.

The study of the effectiveness of cryotherapy as a curative treatment for prostate cancer has often relied on the use of either in vitro cell culture monolayers or animal models. While the data gleaned from these studies have been valuable, each model has inherent limitations. In order to bridge the gap between in vitro studies and clinical applications, we developed a 3-dimensional, tissue engineered human prostate cancer model to simulate and assess the effects of cryotherapy and adjunctive treatments on cell viability and activation of cell death pathways throughout the thermally variable freeze zone. Human prostate cancer cells (PC3) were seeded into collagen based matrices and cryolesions were generated using an Oncura SeedNet Gold cryosurgical device with 17-gauge cryoprobes. Analyses revealed widespread necrosis diminishing towards the edge of the freeze zone, and a time-dependent wave of apoptosis starting as early as 1 hr post-thaw at low temperatures (< -40 degrees C) and moving toward the periphery (-20 degrees C) as recovery times reached 12 and 24 hr. Distal to the -10 degrees C isotherm, minimal cell death was apparent (< 20%) over controls. The adjunctive use of chemotherapeutic agents in conjunction with cryosurgery displayed a similar induction of cell death cascades, but with the zone of cryodestruction extending approximately 10 to 15 degrees C further into the freeze zone periphery. By providing an extracellular environment and a matrix to minimize innate variables, the tissue engineered model yielded a more in vivo-like, tumor-like environment supportive of a deeper understanding of the specific biological responses of cancer cells/tumors to cryotherapeutic intervention.

Cryoablation of renal cancer: variables involved in freezing-induced cell death.

The detection of renal tumors has increased significantly over recent years resulting in a greater demand for novel, minimally invasive techniques. Cryoablation has emerged as a valuable treatment modality for the management of renal cancer. In an effort to detail the effects of freezing in renal cancer, the human renal cancer (RCC) cell line, 786-O, was evaluated in vitro. 786-O cells were exposed to a range of freezing temperatures from -5 to -40 degrees C and compared to non-frozen controls. The data show that freezing to -5 degrees C did not affect 786-O cell viability, while -10 degrees C, -15 degrees C, and -20 degrees C results in a significant loss of viability (23, 70, and 91%, respectively). A complete loss of cell viability was evident at temperatures of -25 degrees C and colder. Following this analysis, variables involved in the success of cryoablation were investigated. For each of the temperatures tested, extended freeze hold times and passive thawing rates resulted in more extensive cell damage. Additionally, a double freeze-thaw cycle significantly increased cell death compared to a single cycle (62% vs. 22% at -10 degrees C; 89% vs. 63% at -15 degrees C, respectively). While these variables play an important part in the effective application of cryoablation, a molecular understanding of the cell death involved is critical to improving efficacy. Apoptotic inhibition afforded 12% (-10 degrees C), 25% (-15 degrees C), and 11% (-20 degrees C) protection following freezing. Using fluorescence microscopy analysis, the results demonstrated that apoptosis peaked at six hours post-thaw. Next, apoptotic initiating agents including 5-FU and resveratrol (RVT) applied prior to freezing exposure resulted in a significant increase in cell death compared to either application alone. Importantly, the combination of RVT and freezing was noticeably less effective when applied to normal renal cells. The results herein demonstrate the efficacy of freezing and describe a novel therapeutic model for the treatment of renal cancer that may distinguish between cancer and normal cells.