Recovery Of Viable Cells Research Articles

Long-Life Inoculant: Bradyrhizobium Stored in Biodegradable Beads for Four Years Shows Optimal Cell Vitality, Interacts with Peanut Roots, and Promotes Early Growth

Currently, bacterial inoculant technology focuses on improving long-term storage conditions to ensure adequate rhizobia numbers and their effectiveness as plant growth promoters. This study aimed to investigate whether storage at 4 °C for four years of alginate beads immobilizing Bradyrhizobium sp. SEMIA6144 maintains bacterial vitality, efficacy in growth promotion, and ability to establish early interactions with Arachis hypogaea L. The recovery of viable SEMIA6144 cells decreased over time (10% at six months, 1% at one year, and 0.01% at four years), while cell vitality remained high at 94.1%, 90.2%, and 93.4%, respectively. The unsaturated/saturated fatty acid ratio declined during storage, reducing membrane fluidity and metabolic activity. Mobility and root adhesion of SEMIA6144 decreased after one and four years. However, growth promotion in peanuts inoculated with SEMIA6144 beads was observed through increased biomass, total chlorophyll, leaf number, leaf area, and decreased chlorophyll fluorescence compared to non-inoculated plants. Although nodulation was low in plants inoculated with four-year-old beads, leghemoglobin levels were maintained. These results demonstrate that Bradyrhizobium sp. SEMIA6144 can be stored for four years in alginate beads at 4 °C, maintaining its vitality and ability to establish a symbiosis that stimulates early peanut growth. Understanding these physiological changes could be valuable for the future improvement of long-lasting inoculants.

Inhibitory effect of helium cold atmospheric plasma on cariogenic biofilms

ABSTRACT This study aimed to determine the effects of low-temperature plasma jet produced in gas helium (LTP-helium) on cariogenic biofilms composedby Streptococcusmutans, Streptococcus sanguinis and Streptococcus gordonii, and also by the combination of Candida albicans, Lactobacillus acidophilus and S. mutans. Biofilms were treated for 1, 3, 5, and 7 minutes. A 0.12% chlorhexidine solution was used as the positive control and sterile physiologic solution was the negative control. Biofilm viability was analyzed by viable cell recovery, scanning electron microscopy, and confocal laser scanning microscopy. All assays were performed intriplicate in three independent experiments. Multispecies biofilms exposed to LTP-helium had a significant reduction in viability when compared to the negative control (p < 0.0001). For biofilm formedby S. mutans, S. sanguinis, and S. gordonii, LTP treatments for 5 and 7 minutes caused similar reduction of morethan 2 log10. Also, a significant reduction in the viability of biofilms formedby C. albicans, L. acidophilus, and S. mutans was detected (p < 0.0001). In conclusion, LTP-helium reduced theviability of cariogenic biofilms with different microbial compositions, which indicates that LTP-helium is a potential tool for developing new protocols for dental caries prevention and treatment.

Establishing a method for the cryopreservation of viable peripheral blood mononuclear cells in the International Space Station

The analysis of cells frozen within the International Space Station (ISS) will provide crucial insights into the impact of the space environment on cellular functions and properties. The objective of this study was to develop a method for cryopreserving blood cells under the specific constraints of the ISS. In a ground experiment, mouse blood was directly mixed with a cryoprotectant and gradually frozen at −80 °C. Thawing the frozen blood sample resulted in the successful recovery of viable mononuclear cells when using a mixed solution of dimethylsulfoxide and hydroxyethyl starch as a cryoprotectant. In addition, we developed new freezing cases to minimize storage space utilization within the ISS freezer. Finally, we confirmed the recovery of major mononuclear immune cell subsets from the cryopreserved blood cells through a high dimensional analysis of flow cytometric data using 13 cell surface markers. Consequently, this ground study lays the foundation for the cryopreservation of viable blood cells on the ISS, enabling their analysis upon return to Earth. The application of this method in ISS studies will contribute to understanding the impact of space environments on human cells. Moreover, this method may find application in the cryopreservation of blood cells in situations where research facilities are inadequate.

Phytocosmetic potential of Blumea balsamifera oil in mitigating UV-induced photoaging: Evidence from cellular and mouse models

Ethnopharmacological relevanceBlumea balsamifera (L.) DC. (BB), the source of Blumea balsamifera oil (BBO), is an aromatic medicinal plant, renowned for its pharmacological properties and its traditional use in Southeast Asian countries such as China, Thailand, Vietnam, Malaysia, and the Philippines for centuries. Traditionally, BB has been used as a raw herbal medicine for treating various skin conditions like eczema, dermatitis, athlete's foot, and wound healing for skin injuries. Aim of the studyThis research aimed to explore the inhibitory effects of BBO on skin aging using two models: in vitro analysis with human dermal fibroblasts (HDF) under UVB-induced stress, and in vivo studies on UVA-induced dorsal skin aging in mice. The study sought to uncover the mechanisms behind BBO's anti-aging effects, specifically, its impact on cellular and tissue responses to UV-induced skin aging. Materials and methodsWe applied doses of 10–20 μL/mL of BBO to HDF cells that had been exposed to UVB radiation to simulate skin aging. We measured cell viability, and levels of reactive oxygen species (ROS), SA-β-gal, pro-inflammatory cytokines, and matrix metalloproteinases (MMPs). In addition, we investigated the involvement of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) signaling pathways in mediating the anti-aging effects of BBO. Histopathological and biochemical analyses were conducted in a mouse model to examine the effects of BBO on UV-induced photoaging. ResultsUV exposure accelerated aging, and caused cellular damage and inflammatory responses through ROS-mediated pathways. In HDF cells, BBO treatment countered the UVB-induced senescence, and the recovery of cell viability was correlated to notable reductions in SA-β-gal, ROS, pro-inflammatory cytokines, and MMPs. Mechanistically, the anti-aging effect of BBO was associated with the downregulation of the JNK/NF-κB signaling pathways. In the in vivo mouse model, BBO exhibited protective capabilities against UV-induced photoaging, which were manifested by the enhanced antioxidant enzyme activities and tissue remodeling. ConclusionsBBO effectively protects fibroblasts from UV-induced photoaging through the JNK/NF-κB pathway. Recovery from photoaging involves an increase in dermal fibroblasts, alleviation of inflammation, accelerated synthesis of antioxidant enzymes, and slowed degradation of ECM proteins. Overall, BBO enhances the skin's defensive capabilities against oxidative stress, underscoring its potential as a therapeutic agent for oxidative stress-related skin aging.

Age and sex effects on histological features and invitro culturing of Antillean manatee (Trichechus manatus manatus, Linnaeus 1758) dermis.

The biobanks from dermal biopsies represent an interesting strategy for biodiversity conservation. Nevertheless, the morphological and cellular patterns of the dermis can be influenced by the age and sex of the individual. Therefore, evaluating these factors is interesting for forming biobanks of Antillean manatees. These animals, representatives of marine fauna, have had their population reduced, and biobanks are essential for their conservation. Then, we evaluated the effects of age (3.5 years vs. 3.6-16 years vs. 23.6 years) and sex (males vs. females) on morphological and cellular parameters using histological and invitro culture techniques. Regardless of age, no differences were observed for dermal thickness, collagen fibres, tissue proliferative activity and viable cell recovery. Nonetheless, fibroblast reduction was observed in groups aged 23.6 years compared to other animals (p < 0.05). Additionally, cells from animals aged 3.6-16 years showed more significant mitochondrial damage than the other groups (p < 0.05). Regardless of sex, no differences were observed for dermal thickness, collagen fibres, tissue proliferative activity and viable cell recovery; however, females had fewer fibroblasts than males (p < 0.05). Cells from females showed lower mitochondrial damage when compared to cells from males. In summary, although age and sex do not influence dermal thickness and cell recovery, variations in the number of fibroblasts and mitochondrial characteristics were observed among the groups. These differences may be significant for understanding the dermis aspects to be correlated to biobank systems.

Impact of cryoprotective agents on human gut microbes and invitro stabilized artificial gut microbiota communities.

The availability of microbial biobanks for the storage of individual gut microbiota members or their derived and artificially assembled consortia has become fundamental for invitro investigation of the molecular mechanisms behind microbe-microbe and/or microbe-host interactions. However, to preserve bacterial viability, adequate storage and processing technologies are required. In this study, the effects on cell viability of seven different combinations of cryoprotective agents were evaluated by flow cytometry for 53 bacterial species representing key members of the human gut microbiota after one and 3 months of cryopreservation at -80°C. The obtained results highlighted that no universal cryoprotectant was identified capable of guaranteeing effective recovery of intact cells after cryopreservation for all tested bacteria. However, the presence of inulin or skimmed milk provided high levels of viability protection during cryoexposure. These results were further corroborated by cryopreserving 10 artificial gut microbiota produced through invitro continuous fermentation system technology. Indeed, in this case, the inclusion of inulin or skimmed milk resulted in a high recovery of viable cells, while also allowing consistent and reliable preservation of the artificial gut microbiota biodiversity. Overall, these results suggest that, although the efficacy of various cryoprotective agents is species-specific, some cryoprotectants based on glycerol and the addition of inulin or skimmed milk are preferable to retain viability and biodiversity for both single bacterial species and artificial gut microbiota.

Impact of different tissue dissociation protocols on endothelial cell recovery from developing mouse lungs.

Flow cytometry and fluorescence-activated cell sorting are widely used to study endothelial cells, for which the generation of viable single-cell suspensions is an essential first step. Two enzymatic approaches, collagenase A and dispase, are widely employed for endothelial cell isolation. In this study, the utility of both enzymatic approaches, alone and in combination, for endothelial cell isolation from juvenile and adult mouse lungs was assessed, considering the number, viability, and subtype composition of recovered endothelial cell pools. Collagenase A yielded an 8-12-fold superior recovery of viable endothelial cells from lung tissue from developing mouse pups, compared to dispase, although dispase proved superior in efficiency for epithelial cell recovery. Single-cell RNA-Seq revealed that the collagenase A approach yielded a diverse endothelial cell subtype composition of recovered endothelial cell pools, with broad representation of arterial, capillary, venous, and lymphatic lung endothelial cells; while the dispase approach yielded a recovered endothelial cell pool highly enriched for one subset of general capillary endothelial cells, but poor representation of other endothelial cells subtypes. These data indicate that tissue dissociation markedly influences the recovery of endothelial cells, and the endothelial subtype composition of recovered endothelial cell pools, as assessed by single-cell RNA-Seq.

Antibacterial activity of Cymbopogon species essential oils against Xanthomonas citri and their use in post-harvest treatment for citrus canker management.

Citrus canker is a disease caused by the gram-negative bacterium Xanthomonas citri subp. citri (X. citri), which affects all commercially important varieties of citrus and can lead to significant losses. Fruit sanitization with products such as chlorine-based ones can reduce the spread of the disease. While effective, their use raises concerns about safety of the workers. This work proposes essential oils (EOs) as viable alternatives for fruit sanitization. EOs from Cymbopogon species were evaluated as to their antibacterial activity, their effect on the bacterial membrane, and their ability to sanitize citrus fruit. The in vitro assays revealed that the EOs from C. schoenanthus and C. citratus had a lower bactericidal concentration at 312mg L-1, followed by 625mg L-1 for C. martini and C. winterianus. Microscopy assay revealed that the bacterial cell membranes were disrupted after 15min of contact with all EOs tested. Regarding the sanitizing potential, the EOs with higher proportions of geraniol were more effective in sanitizing acid limes. Fruit treated with C. shoenanthus and C. martini showed a reduction of ∼68% in the recovery of viable bacterial cells. Therefore, these EOs can be used as viable natural alternatives in citrus fruit disinfection.

Abstract 2930: Recovery of limited viable cells from low-quality precious samples for scRNA-seq

Abstract Biomarker discovery is a critical milestone in pursuing personalized and targeted cancer treatments, yet it faces formidable challenges hindering success rates. Limited access to high-quality samples restricts in-depth biological investigation, with scarce and compromised viable cells acting as barriers to progress. Levitation Technology™ addresses these challenges by significantly improving sample quality and preserving maximum viable cells for single-cell RNA sequencing (scRNA-seq). This innovative approach overcomes limitations in sample quality and availability and heralds a new era of possibilities, propelling significant advancements in biomarker discovery for personalized cancer treatment. To assess the performance of Levitation Technology using the LeviCell® 1.0 system, 105 cryopreserved samples derived from diseased human tissues were processed by Discovery Life Sciences™. This diverse sample set covered 20 disease indications, including 17 types of cancer. A staggering 70% of the samples (n=74) exhibited cell viability ranging between 14-69%, underscoring the need for enhanced viability for effective scRNA-seq analysis. Notably, 45 of these samples presented fewer than 500,000 cells, a scenario marked by low quality and limited viable cells. Traditional methods fail to recover viable cells from such samples without considerable or complete viable cell loss. After processing the samples with the LeviCell 1.0 system, all samples had improved quality, with an impressive 90% of the most difficult samples (n=40) presenting a viability increase to 70-94%, meeting the minimum quality criteria for scRNA-seq. A notable success story was a particular breast cancer sample with only 31,000 cells and 14% viability. Processing with the LeviCell 1.0 system yielded 13,300 viable cells and a substantial 4.5-fold increase in viability to 64%. Such enhancements in sample quality translate into significant improvements in scRNA-seq data quality, enabling more single-cell data extraction from each invaluable sample. Levitation Technology represents a paradigm shift in sample processing, rescuing even the most challenging samples. This groundbreaking technology maximizes the retention of viable cells while efficiently eliminating dead cells and debris, factors that have traditionally compromised scRNA-seq data quality. By revolutionizing this aspect of sample preparation, Levitation Technology opens up unparalleled opportunities to explore deeper into disease biology, providing a leap forward in biomarker discovery. Citation Format: Camila Egidio, Dinesh Bhosale, Lanxin Shen, Susana Jett, Amir Malek, Teddy de Groot, Suzanne Weaver, Gang Sun, Kevin Travers, Geoff Facer. Recovery of limited viable cells from low-quality precious samples for scRNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2930.

Surviving process and transit: Controlled freeze drying, storage and enteric coated capsules for targeted delivery of probiotic Lactobacillusacidophilus

Viability loss of probiotics often occur during processing, storage and gastrointestinal transit. In this study, the viability of freeze-dried Lactobacillus acidophilus LA-5® was assessed after controlled freeze drying and storage at 4 °C and 25 °C over six months using glycerol, skim milk and trehalose as protectants. The freeze-dried probiotic was filled into hard gelatin capsules and enteric coated with the co-polymer Eudragit L100-55 using a fluidised bed coater to determine if the freeze-dried probiotic will survive the enteric coating process and remain viable during gastric transit. Empty hard gelatin capsules were also enteric coated by dipping in the co-polymer solution. These were dried, filled with microcrystalline cellulose and tested for their resistance to simulated gastric condition. The results showed that controlled freezing of the probiotic bacteria did not cause significant loss in viability when the cells were cryopreserved in the protectants. Viable cell loss was greater during the drying stage. Relatively better cell survival was recorded when the freeze-dried samples that were cryopreserved with skim milk were stored over six months at 4 °C. Freeze-dried samples that were preserved with trehalose stored better at 25 °C. The results also demonstrated that capsules coated with Eudragit L100-55 did not disintegrate in simulated gastric fluid. However, the capsules disintegrated in a simulated intestinal fluid. The enteric coating process resulted in about 95% recovery of viable cells. The high viable cell recovery after the coating process is likely due to the coating solution and conditions impacting the capsule body and cap rather than the cells directly. The study highlights that enteric coated capsules can offer gastric protection whilst minimizing viability losses associated with the enteric coating process.

Cationic Vitamin E-TPGS Mixed Micelles of Berberine to Neutralize Doxorubicin-Induced Cardiotoxicity via Amelioration of Mitochondrial Dysfunction and Impeding Apoptosis.

Anthracycline antibiotics, namely, doxorubicin (DOX) and daunorubicin, are among the most widely used anticancer therapies, yet are notoriously associated with severe myocardial damage due to oxidative stress and mitochondrial damage. Studies have indicated the strong pharmacological properties of Berberine (Brb) alkaloid, predominantly mediated via mitochondrial functions and nuclear networks. Despite the recent emphasis on Brb in clinical cardioprotective studies, pharmaceutical limitations hamper its clinical use. A nanoformulation for Brb was developed (mMic), incorporating a cationic lipid, oleylamine (OA), into the TPGS-mixed corona of PEGylated-phosphatidylethanolamine (PEG-PE) micelles. Cationic TPGS/PEG-PE mMic with superior Brb loading and stability markedly enhanced both intracellular and mitochondria-tropic Brb activities in cardiovascular muscle cells. Sub-lethal doses of Brb via cationic OA/TPGS mMic, as a DOX co-treatment, resulted in significant mitochondrial apoptosis suppression. In combination with an intense DOX challenge (up to ~50 µM), mitochondria-protective Brb-OA/TPGS mMic showed a significant 24 h recovery of cell viability (p ≤ 0.05-0.01). Mechanistically, the significant relative reduction in apoptotic caspase-9 and elevation of antiapoptotic Bcl-2 seem to mediate the cardioprotective role of Brb-OA/TPGS mMic against DOX. Our report aims to demonstrate the great potential of cationic OA/TPGS-mMic to selectively enhance the protective mitohormetic effect of Brb to mitigate DOX cardiotoxicity.

Formulation of Culture Medium for the Enumeration of Lactic Streptococci and Lactobacilli from Fermented Milk Products

Aims: The present study is focused to formulate a single selective differential medium for the enumeration of mixed lactic flora such as lactic streptococci and lactobacilli in domestic and market fermented milk products.&#x0D; Study Design: In order to enumerate lactic streptococci and lactobacilli in a mixed flora of fermented milk, formulation of single selective medium was optimized using inhibitors and indicator instead of separate selective medium for lactic streptococci and lactobacilli.&#x0D; Place and Duration of Study: The present study was conducted in the Department of dairy Microbiology, Dairy Science College, Karnataka Veterinary, Animal and Fisheries Sciences University (KVAFSU), Hebbal, Bengaluru-24, Karnataka from February 2020 to February 2021.&#x0D; Methodology: Fermented milk samples like curd, yoghurt were collected, serially diluted and pour plated using Readymade M17 and MRS agar media and formulated selective yeast glucose agar medium. Lactic colonies obtained were enumerated, expressed as log10cfu/g and critical difference was calculated to declare significance and non-significance among the microbial parameters.&#x0D; Results: Readymade M17 and MRS agar media gave better recovery of viable cells of lactic streptococci and lactobacilli from market samples of curd, yoghurt and domestic curd samples. The study helped to convert a general purpose medium yeast glucose agar as selective differential medium with addition of calcium propionate of 0.8 per cent to avoid Bacillus spore formers and sodium benzoate of 1.2 per cent to inhibit yeast, the common contaminants in fermented milk with 0.05 per cent BCP that led to formation of purple colonies of lactic streptococci and yellow colonies of lactobacilli.&#x0D; Conclusion: The selective yeast glucose agar with 0.8 per cent and sodium benzoate of 1.2 per cent with 0.05 per cent BCP with purple colonies of lactic streptococci and yellow colonies of lactobacilli can be successfully used. This study verified the enumeration of mixed lactic flora in fermented milk products instead of M17 medium for lactic streptococci and MRS for lactobacilli, separately.

Efficacy of Hyperthermic Pressurized Intraperitoneal Aerosol Chemotherapy in an In Vitro Model Using a Human Gastric Cancer AGS Cell Line and an Abdominal Cavity Model.

Peritoneal carcinomatosis (PC) presents a major challenge in the treatment of late-stage, solid tumors, with traditional therapies limited by poor drug penetration. We evaluated a novel hyperthermic pressurized intraperitoneal aerosol chemotherapy (HPIPAC) system using a human abdominal cavity model for its efficacy against AGS gastric cancer cells. A model simulating the human abdominal cavity and AGS gastric cancer cell line cultured dishes were used to assess the efficacy of the HPIPAC system. Cell viability was measured to evaluate the impact of HPIPAC under 6 different conditions: heat alone, PIPAC with paclitaxel (PTX), PTX alone, normal saline (NS) alone, heat with NS, and HPIPAC with PTX. Results showed a significant reduction in cell viability with HPIPAC combined with PTX, indicating enhanced cytotoxic effects. Immediately after treatment, the average cell viability was 66.6%, which decreased to 49.2% after 48 hours and to a further 19.6% after 120 hours of incubation, demonstrating the sustained efficacy of the treatment. In contrast, control groups exhibited a recovery in cell viability; heat alone showed cell viability increasing from 90.8% to 94.4%, PIPAC with PTX from 82.7% to 89.7%, PTX only from 73.3% to 74.8%, NS only from 90.9% to 98.3%, and heat with NS from 74.4% to 84.7%. The HPIPAC system with PTX exhibits a promising approach in the treatment of PC in gastric cancer, significantly reducing cell viability. Despite certain limitations, this study highlights the system's potential to enhance treatment outcomes. Future efforts should focus on refining HPIPAC and validating its effectiveness in clinical settings.

Optimizing the cryopreservation and post-thaw recovery of natural killer cells is critical for the success of off-the-shelf platforms.

Natural killer (NK) cells are a promising allogeneic, off-the-shelf, cellular immunotherapy product. These cells can be manipulated ex vivo, genetically edited to enhance tumor targeting and expanded to produce large cell banks for multiple patient infusions. Therapeutic efficacy of these products depends on the recovery of viable and functional cells post-thaw. Post-thaw loss of viability and cytolytic activity results in large, and often variable, discrepancies between the intended cell dose (based on counts at cryopreservation) and the actual dose administered. Compared to their highly activated state in fresh culture, post-thaw NK cells demonstrate critical changes in cytokine production, cytotoxic activity, in vivo proliferation and migration. When these NK cells are introduced into the highly immunosuppressive tumor microenvironment, the functional changes induced by cryopreservation further limits the clinical potential of these products. This report will review the impact of cryopreservation on ex vivo expanded NK cells and outlines strategies described in published studies to recover post-thaw function.

Quantitative Determination of Antibacterial Activity During Bacterial Coculture.

Antibacterial activity assays are an important tool in the assessment of the ability of one bacterium to kill or inhibit the growth of another, for example, during the study of the Type VI secretion system (T6SS) and the antibacterial toxins it secretes. The method we describe here can detect the ability of a bacterial strain to kill or inhibit other bacterial cells in a contact-dependent manner when cocultured on an agar surface. It is particularly useful since it enumerates the recovery of viable target cells and thus enables quantification of the antibacterial activity. We provide a detailed description of how to measure the T6SS-dependent antibacterial activity of a bacterium such as Serratia marcescens against a competitor prokaryotic organism, Escherichia coli, and describe possible variations in the method to allow adaptation to other attacker and target organisms.

Assessment of Live Lactobacilli Recovery from Probiotic Products for Vaginal Application

The interest in the use of probiotics to treat and prevent vaginal infections is known. The new regulation of medical devices by the European Medical Agency (EMA) introduced big changes in Europe regarding probiotic products for vaginal application, as they are no longer considered as medical devices. As the future classification will be as drugs, it will stress the need to define robust and reliable pre-clinical in vitro testing in order to assess the quality, safety and efficacy of probiotics for human use. Before discussing the efficacy in human pathology, it is mandatory to evaluate the survival and multiplication potential of probiotic strains when brought into contact with vaginal fluid. In this work, our objective was to assess the recovery and stability profile of lactobacilli from six vaginal probiotic formulations brought in contact with specific culture media or vaginal fluid simulants (VFS). Overall, the recovery of viable lactobacilli cells from a modified vaginal fluid simulant (MVFS) solution was comparable to the recovery pattern obtained in standard culture medium. Therefore, we conclude that the MVFS seems to better simulate the conditions of the human vaginal fluid, in contrast with other simulants, and may be used to predict the viability of probiotics over time in the normal vaginal milieu. We discovered that each probiotic product has a unique profile that requires stand-alone studies in conditions that mimic the in vivo status in order to assess their preclinical effectiveness and promote their differential use by the medical community.

Liquid Storage of Peripheral Blood Stem Cell Products: Effects of Time and Temperature on Product Quality

Unrelated donor peripheral blood stem cell (PBSC) products often require transport to distant locations, which may take up to 72 hours. Temperature is an important variable that can be controlled during PBSC storage or transport; therefore, we studied the impact of temperature on prolonged storage of clinical-grade, mobilized PBSC products. PBSC products were collected by apheresis from 3 granulocyte colony-stimulating factor-mobilized donors, split into 2 PVC blood bags of equal volume, and stored at room temperature (RT) (18°C to 25 ºC) or 4 °C (2°C to 8 ºC) for 96 hours. Samples were obtained at 24-hour intervals for pH, cell counts, flow cytometry phenotyping and viability (7AAD), and hematopoietic colony-forming units (CFU). Starting PBSC products contained 52, 65, and 38×109 total nucleated cells (TNCs), with cell concentrations of 125, 263, and 94.6×106 TNCs/mL, respectively. Product pH dropped during storage, with significantly lower values for RT stored products than for 4 ºC stored products, and was greatest in the product with the highest TNC count. The percent recovery of viable CD34+ progenitor cells, CD3+ T cells, CD4+ T helper cells, CD8+ cytotoxic T cells, CD19+ B cells, CD15+ granulocytes, CD14+ monocytes, and CD16+/56+ natural killer (NK) cells all decreased over 96 hours but decreased more dramatically in the RT group. Cell recovery differences were statistically significant at most time points for all cell populations except CD15+ granulocytes. For CD34+ cells stored at 4 °C, mean recovery from prestorage values were 97 ± 3% at 24 hours, 87 ± 4% at 48 hours, 88 ± 10% at 72 hours, and 78 ± 1% at 96 hours, compared to RT product values of 45 ± 11%, 19 ± 19%, 2 ± 2%, and 0 ± 0%, respectively. CFUs were well preserved through 96 hours at 4 ºC but not at RT. During PBSC storage, pH and content of viable CD34+ cells, T cells, B cells, monocytes, NK cells, and CFU all declined. However, at 4 ºC, viable cell recoveries are relatively well preserved, even at 72 hours, whereas RT storage resulted in rapid product deterioration. PBSC products requiring prolonged liquid storage or transport before cryopreservation or infusion should be maintained at 4 ºC.

Hydrogel capsules as new approach for increasing drying survival of plant biostimulant gram-negative consortium

Several plant growth–promoting bacteria (PGPB) are gram-negative, and their cell viability is affected during the bio-inoculant production. Hence, formulation-drying processes provide challenges that limit the adoption of these beneficial microorganisms in sustainable agricultural production. Among delivery system strategies for gram-negative PGPB, the encapsulating cells in biopolymeric materials are emerging as a promising alternative. This research aims to evaluate the effect of additives and crosslinking agents on the survival of the consortium of Herbaspirillum frisingense AP21, Azospirillum brasilense D7, and Rhizobium leguminosarum T88 in hydrogel capsules. Three crosslinkers and diverse potential drying protectors were tested. Calcium gluconate provides notable consortium survival advantages regarding colony-forming units (CFUs) (losses of up to 4 log CFU) compared to calcium lactate and calcium chloride (up to 6 log CFU). Additives such as skimmed milk, whey protein, and Gelita® EC improve the recovery of viable cells after the drying process, demonstrating an increase in cell survival of the three bacteria by up to 4 log CFU. The combination of these substances into a capsule prototype extends the storage stability of bacterial consortium up to 3 months at 18 ± 2 °C. This study expands the knowledge for formulating gram-negative PGPB consortium, regarding the crosslinker and drying protector relationship on encapsulation processes with drying survival and further storage stability performance.Key points• Hydrogel immobilization formulation approach for PGPB consortium• Enhancing drying survival of gram-negative PGPB consortium• Increasing storage stability of PGPB consortium at 18 °C

A novel cryopreservation and biobanking strategy to study lymphoid tissue stromal cells in human disease.

Nonhematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, the study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils and lymph nodes (LN), lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable nonhematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LN stromal cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and LN. The presence and spatial distribution of transcriptionally defined cell types were confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSCs in human disease.

Abstract 5461: Gain early insights from single cell RNA-sequencing of clinical trial needle biopsy cores

Abstract Single cell RNA-sequencing (scRNA-seq) yields valuable insights into the molecular heterogeneity of multiple cell types in normal and cancer tissues. scRNA-seq approach has the potential to not only answer basic research questions but also enhance clinical studies and clinical trials and accelerate drug development and testing. However, a consistent and reproducible method for isolating viable single cells and generating high quality single cell data from needle biopsy cores has been challenging to develop. Based on our experience isolating single cells from a large number of surgical tumor tissues, we set out to optimize a dissociation and processing protocol to generate viable single cell suspensions in adequate quantity for scRNA-seq from needle biopsy cores. A total of 17 de-identified 18-gauge needle biopsy samples were collected in collaboration with MD Anderson Cancer Center investigators. Samples were from liver, lymph node, pelvis, abdominal, neck, and lung tissues and included matched pre- and post-treatment samples. Patient biopsies were kept in RPMI medium on ice and transferred to EMPIRI (time to capture ranged from 3 to 6.5 hours after biopsy collection). Samples were dissociated using mechanical and enzymatic methods to achieve a single cell suspension. Cells were washed and viability and cell counts were verified before capture via 10x Chromium Connect. Captured cells were processed through the 10x Genomics Chromium Next GEM Single Cell workflow before sequencing on Illumina NovaSeq 6000 sequencer. Raw sequencing data were processed through EMPIRI’s computational pipeline that includes QC steps, doublet removal, and Seurat and other downstream analyses. Recovery of viable cells from individual 18-gauge biopsy cores ranged from 2,000-300,000 cells per core, depending on the tissue type and cellularity. Cell viability was strictly monitored and was maintained above 80% for 16 of 17 samples (most samples &amp;gt;90%). Cell counts after sequencing and pipeline analysis averaged 6,466 cells per sample. The median number of UMI counts per cell was 5,580, and an average of 1,699 genes were identified per cell. Average sequencing saturation was 73%. Here, we demonstrate the feasibility of integrating scRNA-seq analysis into clinical trials or clinical studies to obtain rapid insights into on-target drug effects and anticipated cellular responses to therapies, including immunotherapies, within 3 weeks of biopsy collection. We have established a method for isolating high quality single cells for scRNA-seq from tissue biopsy core, as small as a single 18-gauge needle core. We tested our protocol on 17 human samples and showed successful isolation of highly viable cells from lung, lymph node, pelvis, abdominal, neck, and lung tissues. We were able to generate scRNA-seq data sets from all samples for analysis. Acknowledgements: We thank our MDACC collaborators, particularly Drs. Scott Kopetz and Van Morris. Citation Format: Thomas Gallup, Sang Yun, Dave Gallup, Kyuson Yun. Gain early insights from single cell RNA-sequencing of clinical trial needle biopsy cores. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5461.