Establishment and validation of in-house cryopreserved CAR/TCR-T cell flow cytometry quality control

Background Chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for the treatment of hematologic malignancies and solid tumors. Multiparametric flow cytometry-based assays play a critical role in monitoring cellular manufacturing steps. Since manufacturing CAR/TCR T-cell products must be in compliance with current good manufacturing practices (cGMP), a standard or quality control for flow cytometry assays should be used to ensure the accuracy of flow cytometry results, but none is currently commercially available. Therefore, we established a procedure to generate an in-house cryopreserved CAR/TCR T-cell products for use as a flow cytometry quality control and validated their use. Methods Two CAR T-cell products: CD19/CD22 bispecific CAR T-cells and FGFR4 CAR T-cells and one TCR-engineered T-cell product: KK-LC-1 TCR T-cells were manufactured in Center for Cellular Engineering (CCE), NIH Clinical Center. The products were divided in aliquots, cryopreserved and stored in the liquid nitrogen. The cryopreserved flow cytometry quality controls were tested in flow cytometry assays which measured post-thaw viability, CD3, CD4 and CD8 frequencies as well as the transduction efficiency and vector identity. The long-term stability and shelf-life of cryopreserved quality control cells were evaluated. In addition, the sensitivity as well as the precision assay were also assessed on the cryopreserved quality control cells. Results After thawing, the viability of the cryopreserved CAR/TCR T-cell controls was found to be greater than 50%. The expression of transduction efficiency and vector identity markers by the cryopreserved control cells were stable for at least 1 year; with post-thaw values falling within ± 20% range of the values measured at time of cryopreservation. After thawing and storage at room temperature, the stability of these cryopreserved cells lasted at least 6 h. In addition, our cryopreserved CAR/TCR-T cell quality controls showed a strong correlation between transduction efficiency expression and dilution factors. Furthermore, the results of flow cytometric analysis of the cryopreserved cells among different laboratory technicians and different flow cytometry instruments were comparable, highlighting the reproducibility and reliability of these quality control cells. Conclusion We developed and validated a feasible and reliable procedure to establish a bank of cryopreserved CAR/TCR T-cells for use as flow cytometry quality controls, which can serve as a quality control standard for in-process and lot-release testing of CAR/TCR T-cell products.

Basic Quality Controls Used in Skin Tissue Engineering

Reconstruction of skin defects is often a challenging effort due to the currently limited reconstructive options. In this sense, tissue engineering has emerged as a possible alternative to replace or repair diseased or damaged tissues from the patient’s own cells. A substantial number of tissue-engineered skin substitutes (TESSs) have been conceived and evaluated in vitro and in vivo showing promising results in the preclinical stage. However, only a few constructs have been used in the clinic. The lack of standardization in evaluation methods employed may in part be responsible for this discrepancy. This review covers the most well-known and up-to-date methods for evaluating the optimization of new TESSs and orientative guidelines for the evaluation of TESSs are proposed.

Spotlight on the Compositional Quality of Probiotic Formulations Marketed Worldwide

On the worldwide market, a great number of probiotic formulations are available to consumers as drugs, dietary supplements, and functional foods. For exerting their beneficial effects on host health, these preparations should contain a sufficient amount of the indicated living microbes and be pathogen-free to be safe. Therefore, the contained microbial species and their amount until product expiry are required to be accurately reported on the labels. While commercial formulations licensed as drugs are subjected to rigorous quality controls, less stringent regulations are generally applied to preparations categorized as dietary supplements and functional foods. Many reports indicated that the content of several probiotic formulations does not always correspond to the label claims in terms of microbial identification, number of living organisms, and purity, highlighting the requirement for more stringent quality controls by manufacturers. The main focus of this review is to provide an in-depth overview of the microbiological quality of probiotic formulations commercialized worldwide. Many incongruences in the compositional quality of some probiotic formulations available on the worldwide market were highlighted. Even if manufacturers carry at least some of the responsibility for these inconsistencies, studies that analyze probiotic products should be conducted following recommended and up-to-date methodologies.

Regional Features of Long-Term Exposure to PM2.5 Air Quality over Asia under SSP Scenarios Based on CMIP6 Models

This study investigates changes in fine particulate matter (PM2.5) concentration and air-quality index (AQI) in Asia using nine different Coupled Model Inter-Comparison Project 6 (CMIP6) climate model ensembles from historical and future scenarios under shared socioeconomic pathways (SSPs). The results indicated that the estimated present-day PM2.5 concentrations were comparable to satellite-derived data. Overall, the PM2.5 concentrations of the analyzed regions exceeded the WHO air-quality guidelines, particularly in East Asia and South Asia. In future SSP scenarios that consider the implementation of significant air-quality controls (SSP1-2.6, SSP5-8.5) and medium air-quality controls (SSP2-4.5), the annual PM2.5 levels were predicted to substantially reduce (by 46% to around 66% of the present-day levels) in East Asia, resulting in a significant improvement in the AQI values in the mid-future. Conversely, weak air pollution controls considered in the SSP3-7.0 scenario resulted in poor AQI values in China and India. Moreover, a predicted increase in the percentage of aged populations (>65 years) in these regions, coupled with high AQI values, may increase the risk of premature deaths in the future. This study also examined the regional impact of PM2.5 mitigations on downward shortwave energy and surface air temperature. Our results revealed that, although significant air pollution controls can reduce long-term exposure to PM2.5, it may also contribute to the warming of near- and mid-future climates.