Collection and processing of hematopoietic progenitor cell products at risk of presenting with cold agglutination.

BACKGROUND AIMS: Cold agglutinins are commonly identified in transfusion laboratories and are defined by their ability to agglutinate erythrocytes at 3-4°C, with most demonstrating a titer >64. Similarly, cryoglobulins can precipitate from plasma when temperatures drop below central body temperature, resulting in erythrocyte agglutination. Thankfully, disease associated from these autoantibodies is rare, but unfortunately, such temperature ranges are routinely encountered outside of the body's circulation, as in an extracorporeal circuit during hematopoietic progenitor cell (HPC) collection or human cell therapy laboratory processing. When agglutination occurs ex vivo, complications with the collection and product may be encountered, resulting in adverse events or product loss. Here, we endeavor to share our experience in preventing and responding to known cases at risk of or spontaneous HPC agglutination in our human cell therapy laboratory. CASE REPORTS: Four cases of HPC products at risk for, or spontaneously, agglutinating were seen at our institution from 2018 to 2020. Planned modifications occurred, including ambient room temperature increases, tandem draw and return blood warmers, warm product transport and extended post-thaw warming occurred. In addition, unplanned modifications were undertaken, including warm HPC product processing and plasma replacement of the product when spontaneous agglutination of the product was identified. All recipients successfully engrafted after infusion. CONCLUSIONS: While uncommon, cold agglutination of HPC products can disrupt standard processes of collection and processing. Protocol modifications can circumvent adverse events for the donor and minimize product loss. Such process modifications should be considered in individuals with known risks for agglutination going to HPC donation/collection.

How do I warm HPC(A) products to maximize cell viability in the setting of cold agglutinin disease?

BACKGROUND: High titers of cold agglutinins jeopardize the quality of an apheresis product meant for autologous or allogeneic transplant. Management of transplant patients with cold agglutinin disease (CAD) is often experience-based and under reported, yet decisions must be made quickly to optimize product management and patient outcomes. There remains a lack of data quantifying cell recovery and viability when using various warming methodologies. STUDY DESIGN AND METHODS: To expand the published experimental data on this subject, our human cellular therapy lab compared cellular recoveries and viabilities after manipulation of cryopreserved apheresis products through various warming methodologies: (1) extended warming in a water bath, (2) warming via blood warmer and infusion pump, and (3) warming in a water bath followed by infusion pump as a control to assess potential shear stress effects. RESULTS: The presented studies demonstrate that all methods of product warming produce the same rates of recovery of total and viable cells across vital cell types prior to patient administration. Statistically, use of an extended water bath protocol provided a marginal benefit in recovery of total nucleated cells, though this effect is diminished when products are held for an extended period to simulate a delay in administration. DISCUSSION: These results can inform decisions to improve patient care and minimize product manipulation and loss. Centers are encouraged to use this information to guide proactive measures to establish a standard operating procedure to manage CAD cases.

Promises and challenges of a decentralized CAR T-cell manufacturing model.

Autologous chimeric antigen receptor-modified T-cell (CAR T) products have demonstrated un-precedent efficacy in treating many relapsed/refractory B-cell and plasma cell malignancies, leading to multiple commercial products now in routine clinical use. These positive responses to CAR T therapy have spurred biotech and big pharma companies to evaluate innovative production methods to increase patient access while maintaining adequate quality control and profitability. Autologous cellular therapies are, by definition, manufactured as single patient batches, and demand has soared for manufacturing facilities compliant with current Good Manufacturing Practice (cGMP) regulations. The use of a centralized production model is straining finite resources even in developed countries in North America and the European Union, and patient access is not feasible for most of the developing world. The idea of having a more uniform availability of these cell therapy products promoted the concept of point-of-care (POC) manufacturing or decentralized in-house production. While this strategy can potentially decrease the cost of manufacturing, the challenge comes in maintaining the same quality as currently available centrally manufactured products due to the lack of standardized manufacturing techniques amongst institutions. However, academic medical institutions and biotech companies alike have forged ahead innovating and adopting new technologies to launch clinical trials of CAR T products produced exclusively in-house. Here we discuss POC production of CAR T products.

Alterations of mesenchymal stromal cells in cerebrospinal fluid: insights from transcriptomics and an ALS clinical trial.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been studied with increasing intensity as clinicians and researchers strive to understand the ability of MSCs to modulate disease progression and promote tissue regeneration. As MSCs are used for diverse applications, it is important to appreciate how specific physiological environments may stimulate changes that alter the phenotype of the cells. One need for neuroregenerative applications is to characterize the spectrum of MSC responses to the cerebrospinal fluid (CSF) environment after their injection into the intrathecal space. Mechanistic understanding of cellular biology in response to the CSF environment may predict the ability of MSCs to promote injury repair or provide neuroprotection in neurodegenerative diseases. METHODS: In this study, we characterized changes in morphology, metabolism, and gene expression occurring in human adipose-derived MSCs cultured in human (hCSF) or artificial CSF (aCSF) as well as examined relevant protein levels in the CSF of subjects treated with MSCs for amyotrophic lateral sclerosis (ALS). RESULTS: Our results demonstrated that, under intrathecal-like conditions, MSCs retained their morphology, though they became quiescent. Large-scale transcriptomic analysis of MSCs revealed a distinct gene expression profile for cells cultured in aCSF. The aCSF culture environment induced expression of genes related to angiogenesis and immunomodulation. In addition, MSCs in aCSF expressed genes encoding nutritional growth factors to expression levels at or above those of control cells. Furthermore, we observed a dose-dependent increase in growth factors and immunomodulatory cytokines in CSF from subjects with ALS treated intrathecally with autologous MSCs. CONCLUSIONS: Overall, our results suggest that MSCs injected into the intrathecal space in ongoing clinical trials remain viable and may provide a therapeutic benefit to patients.

A Comprehensive Method To Quantify Adaptations by Male and Female Mice With Hot Flashes Induced by the Neurokinin B Receptor Agonist Senktide.

Vasomotor symptoms (VMS; or hot flashes) plague millions of reproductive-aged men and women who have natural or iatrogenic loss of sex steroid production. Many affected individuals are left without treatment options because of contraindications to hormone replacement therapy and the lack of equally effective nonhormonal alternatives. Moreover, development of safer, more effective therapies has been stymied by the lack of an animal model that recapitulates the hot-flash phenomenon and enables direct testing of hypotheses regarding the pathophysiology underlying hot flashes. To address these problems, we developed a murine model for hot flashes and a comprehensive method for measuring autonomic and behavioral thermoregulation in mice. We designed and constructed an instrument called a thermocline that produces a thermal gradient along which mice behaviorally adapt to a thermal challenge to their core body temperature set point while their thermal preference over time is tracked and recorded. We tested and validated this murine model for VMS by administration of a TRPV1 agonist and a neurokinin B receptor agonist, capsaicin and senktide, respectively, to unrestrained mice and observed their autonomic and behavioral responses. Following both treatments, the mice exhibited a VMS-like response characterized by a drop in core body temperature and cold-seeking behavior on the thermocline. Senktide also caused a rise in tail skin temperature and increased Fos expression in the median preoptic area, a hypothalamic temperature control center. This dynamic model may be used to fully explore the cellular and molecular bases for VMS and to develop and test new therapeutic options.