Cell Recovery Research Articles

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.

The interim analysis of a first-in-human phase 1 trial of ET-901, a CRISPR edited allogeneic immune-cloaked anti-CD19 CAR-T cell therapy in patients with r/r B-NHL.

e19010 Background: Allogeneic CAR T-cell therapy faces challenges like limited persistence and host rejection. Current research has improved its viability by modifying genes like B2M, despite new challenges like NK cell targeting have emerged. Through CRISPR screening, we discovered a novel gene, named GeneX, and its role in enhancing T cell persistence against allogeneic attacks and AICD. GeneX influences glycan composition, affecting immune responses and AICD initiation. Engineered GeneXKO/TCRKO/anti-CD19 CAR T cells (ET-901) resisted allogeneic killing and maintained anti-tumor effects without causing GvHD, marking a significant advancement in CAR T-cell therapy. Methods: ET-901 is being tested in a phase 1 trial for r/r B-NHL at a single center, utilizing a 3+3 dose escalation scheme and planning for expansion at the recommended Phase 2 dose. Participants must have an ECOG status of 0–2, without a history of allo-HSCT or CAR-T therapy. The study's main goals are to assess ET-901's safety and establish the optimal Phase 2 dosage. Patients undergo a 3-day lymphodepletion with fludarabine (30-50 mg/m2/d) and cyclophosphamide (500-1000 mg/m2/d), followed by a single-dose infusion of ET-901 at escalating doses (DL1:1*10e6/kg, DL2:3*10e6/kg, and DL3:10*10e6/kg). Pharmacokinetic monitoring occurs during follow-up. Results: From September 2023, a total of six patients were enrolled in the escalation cohort, including 4 patients with LBCL and 2 with FL. In these patients (3 males), the median age was 53 years (range: 46–55 years), and the median number of prior treatment lines was 5. The median SPD was 7,650 mm2 (range: 2,700-43,400 mm2). Notably, two cases in DL2 exhibited unusual lymphoma infiltration sites: patient No.4 in the forearm muscle and patient No.5 in the spleen. Update to February 2024, we have completed the safety evaluation of Dose Level (DL) 1 and DL2. No DLTs were observed. The most common TRAEs of any grade included leukopenia (6/6), granulocytopenia (6/6), anemia (6/6), thrombocytopenia (6/6), CRS (6/6), infection (2/6), ICANS (2/6), and GvH like reaction (1/6). All patients achieved objective responses, with 2 cases of PR and 4 cases of CR (1 in DL1, 3 in DL2). The peak values of CAR+ cells count and VCN in peripheral blood were 36.0/μl (range: 14.0-48.0/μl, median: day 7), and 9.6e4 copies/μg (range: 7.7e4-1.2e5 copies/μg, median: day 7), respectively. In all patients, a second expansion of CAR-T cells was observed alongside B cell recovery. This confirms that GeneX knockout enables allo CAR-T cells to maintain long-term immune privilege while preserving their biological function. Conclusions: The study demonstrated the safety and feasibility of ET-901 in patients with r/r B-NHL. These findings highlight the potential of ET-901 as an effective therapeutic approach for improving outcomes for these patients. Clinical trial information: NCT06014073 .

Successful transport across continents of GMP-manufactured and cryopreserved culture-expanded human fetal liver-derived mesenchymal stem cells for use in a clinical trial

IntroductionCell therapy has been increasingly considered to treat diseases, but it has been proven difficult to manufacture the same product at multiple manufacturing sites. Thus, for a wider implementation an alternative is to have one manufacturing site with a wide distribution to clinical sites. To ensure administration of a good quality cell therapy product with maintained functional characteristics, several obstacles must be overcome, which includes for example transfer of knowledge, protocols and procedures, site assessment, transportation and preparation of the product. MethodsAs the preparatory work for a clinical trial in India using fetal mesenchymal stem cells (fMSCs) developed and manufactured in Sweden, we performed a site assessment of the receiving clinical site, transferred methods, developed procedures and provided training of operators for handling of the cell therapy product. We further developed a Pharmacy Manual to cover the management of the product, from ordering it from the manufacturer, through transport, reconstitution, testing and administration at the clinical site. Lastly, the effect of long-distance transport on survival and function of, as well as the correct handling of the cell therapy product, was evaluated according to the pre-determined and approved Product Specification. ResultsFour batches of cryopreserved human fetal liver-derived fMSCs manufactured according to Good Manufacturing Practice and tested according to predetermined release criteria in Sweden, were certified and transported in a dry shipper at −150 °C to the clinical site in India. The transport was temperature monitored and took three–seven days to complete. The thawed and reconstituted cells showed more than 80% viability up to 3 h post-thawing, the cell recovery was more than 94%, the cells displayed the same surface protein expression pattern, differentiated into bone, had stable chromosomes and were sterile, which conformed with the data from the manufacturing site in Sweden. ConclusionsOur study shows the feasibility of transferring necessary knowledge and technology to be able to carry out a clinical trial with a cell therapy product in distant country. It also shows that it is possible to transport a cryopreserved cell therapy product over long distances and borders with retained quality. This extends the use of cryopreserved cell therapy products in the future.

Effect of Hydroxyurea on Morphology, Proliferation, and Protein Expression on Taenia crassiceps WFU Strain.

Flatworms are known for their remarkable regenerative ability, one which depends on totipotent cells known as germinative cells in cestodes. Depletion of germinative cells with hydroxyurea (HU) affects the regeneration of the parasite. Here, we studied the reduction and recovery of germinative cells in T. crassiceps cysticerci after HU treatment (25 mM and 40 mM of HU for 6 days) through in vitro assays. Viability and morphological changes were evaluated. The recovery of cysticerci's mobility and morphology was evaluated at 3 and 6 days, after 6 days of treatment. The number of proliferative cells was evaluated using EdU. Our results show morphological changes in the size, shape, and number of evaginated cysticerci at the 40 mM dose. The mobility of cysticerci was lower after 6 days of HU treatment at both concentrations. On days 3 and 6 of recovery after 25 mM of HU treatment, a partial recovery of the proliferative cells was observed. Proteomic and Gene Ontology analyses identified modifications in protein groups related to DNA binding, DNA damage, glycolytic enzymes, cytoskeleton, skeletal muscle, and RNA binding.

Mitochondria-engine with self-regulation to restore degenerated intervertebral disc cells via bioenergetic robust hydrogel design

Previous studies have confirmed that intervertebral disc degeneration (IDD) is closely associated with inflammation-induced reactive oxygen species (ROS) and resultant cell mitochondrial membrane potential (MMP) decline. Clearance of ROS in an inflammatory environment is essential for breaking the vicious cycle of MMP decline. Additionally, re-energizing the mitochondria damaged in the inflammatory milieu to restore their function, is equally important. Herein, we proposed an interesting concept of mitochondrion-engine equipped with coolant, which enables first to “cool-down” the inflammatory environment, next to restore the MMP, finally to allow cells to regain normal energy metabolism through materials design. As such, we developed a multi-functional composite composed of a reactive oxygen species (ROS)-responsive sodium alginate/gelatin hydrogel infused into a rigid 3D-printed thermoplastic polyurethane (TPU) scaffold. The TPU scaffold was coated with conductive polypyrrole (PPy) to electrophoretically deposit l-arginine, which could upregulate the Mammalian target of rapamycin (mTOR) pathway, thus increasing MMP and energy metabolism to stimulate extracellular matrix synthesis for IVD repair. While the ROS-responsive hydrogel acting as the “mito-engine coolant” could scavenge the excessive ROS to create a favorable environment for IVD cells recovery. Demonstrated by in vitro and in vivo evaluations, the mito-engine system markedly promoted the proliferation and collagen synthesis of nucleus pulposus cells while enhancing the mitochondrial respiration and MMP under oxidative stress. Radiological and histological assessments in vivo revealed the efficacy of this system in IVD repair. This unique bioinspired design integrated biomaterial science with mitochondrial biology, presents a promising paradigm for IDD treatment.

Unveiling the Nexus of CD38 Overactivation, NAD+ Depletion, and Mitochondrial Dysfunction in Immunological Failure Among Virologically Suppressed HIV Patients

Introduction With the advent of antiretroviral therapy (ART), HIV has become a manageable chronic disease. Despite effective virologic suppression, approximately 30% of people living with HIV (PLWH) experience immunological failure, characterized by inadequate CD4+ T cell recovery. This study explores the hypothesis that overactivation of the CD38 receptor leads to NAD+ depletion and subsequent mitochondrial dysfunction, contributing to immunological failure in virologically suppressed HIV patients. Methods A comprehensive review of existing literature was conducted to investigate the roles of CD38, NAD+, and mitochondrial function in HIV pathogenesis. Data were collected from studies on CD38 expression, NAD+ metabolism, and mitochondrial dysfunction in the context of HIV and aging. The integrative approach included examining immune cell activation, metabolic pathways, and potential therapeutic interventions. Results CD38, a type II transmembrane glycoprotein, is overexpressed in PLWH and serves as a predictor of HIV progression. Its enzymatic activities deplete NAD+, a crucial coenzyme involved in energy metabolism, DNA repair, and cell signaling. NAD+ depletion impairs mitochondrial oxidative phosphorylation (OXPHOS), leading to reduced ATP production and increased reliance on glycolysis, which promotes inflammation. Overactivation of CD38 also activates the kynurenine pathway through IDO-1, further depleting NAD+ and generating toxic metabolites that damage mitochondria. This cascade results in persistent immune activation, immune exhaustion, and CD4+ T cell apoptosis. Conclusion The overactivation of CD38 and subsequent NAD+ depletion are central to the pathogenesis of immunological failure in virologically suppressed HIV patients. This mechanism links chronic immune activation, metabolic dysfunction, and accelerated aging. Therapeutic interventions targeting CD38 inhibition, NAD+ supplementation, and mitochondrial function enhancement could potentially reverse immunological failure and improve health outcomes in PLWH. Further experimental validation and clinical trials are necessary to confirm these findings and develop effective treatments.

Circular RNA circ_0006168 accelerates the development of hepatocellular carcinoma through sponging microRNA-125b.

This study explored the role of circular RNA circ_0006168 in the progression of hepatocellular carcinoma (HCC) and its interaction with microRNA-125b. The expression of circ_0006168 was examined in 42 pairs of HCC tumor and adjacent tissue specimens using quantitative polymerase chain reaction (qPCR). Elevated circ_0006168 expression in HCC tissues was significantly associated with advanced pathological staging and lower overall survival rates. Lentivirus-mediated circ_0006168 knockdown in HCC cell lines (Hep3B and Huh7) demonstrated a notable reduction in cell proliferation and an increase in apoptosis. MicroRNA-125b expression exhibited a marked reduction in HCC tissues, negatively correlating with circ_0006168 levels. Luciferase reporting assays indicated that circ_0006168 was a direct target of microRNA-125b. Additionally, cell recovery experiments suggested a reciprocal regulation between circ_0006168 and microRNA-125b, contributing to the accelerated malignant progression of HCC. The study underscored the significantly increased expression of circ_0006168 in both HCC tissues and cell lines, highlighting its association with advanced pathological stages and poor prognosis in HCC patients. Furthermore, circ_0006168 appeared to play a pivotal role in elevating the proliferation rate of HCC cells through its modulation of microRNA-125b. These findings contribute to a deeper understanding of the molecular mechanisms underlying HCC development and may offer potential therapeutic targets for intervention.

A hybrid demultiplexing strategy that improves performance and robustness of cell hashing.

Cell hashing, a nucleotide barcode-based method that allows users to pool multiple samples and demultiplex in downstream analysis, has gained widespread popularity in single-cell sequencing due to its compatibility, simplicity, and cost-effectiveness. Despite these advantages, the performance of this method remains unsatisfactory under certain circumstances, especially in experiments that have imbalanced sample sizes or use many hashtag antibodies. Here, we introduce a hybrid demultiplexing strategy that increases accuracy and cell recovery in multi-sample single-cell experiments. This approach correlates the results of cell hashing and genetic variant clustering, enabling precise and efficient cell identity determination without additional experimental costs or efforts. In addition, we developed HTOreader, a demultiplexing tool for cell hashing that improves the accuracy of cut-off calling by avoiding the dominance of negative signals in experiments with many hashtags or imbalanced sample sizes. When compared to existing methods using real-world datasets, this hybrid approach and HTOreader consistently generate reliable results with increased accuracy and cell recovery.

Enteric nervous system regeneration and functional cure of experimental digestive Chagas disease with trypanocidal chemotherapy

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. There is a lack of evidence on the mechanism of pathogenesis and rationales for treatment. We used a female C3H/HeN mouse model that recapitulates key clinical manifestations to study how infection dynamics shape DCD pathology and the impact of treatment with the front-line, anti-parasitic drug benznidazole. Curative treatment 6 weeks post-infection resulted in sustained recovery of gastrointestinal transit function, whereas treatment failure led to infection relapse and gradual return of DCD symptoms. Neuro/immune gene expression patterns shifted from chronic inflammation to a tissue repair profile after cure, accompanied by increased cellular proliferation, glial cell marker expression and recovery of neuronal density in the myenteric plexus. Delaying treatment until 24 weeks post-infection led to partial reversal of DCD, suggesting the accumulation of permanent tissue damage over the course of chronic infection. Our study shows that murine DCD pathogenesis is sustained by chronic T. cruzi infection and is not an inevitable consequence of acute stage denervation. The risk of irreversible enteric neuromuscular tissue damage and dysfunction developing highlights the importance of prompt diagnosis and treatment. These findings support the concept of treating asymptomatic, T. cruzi-infected individuals with benznidazole to prevent DCD development.

Model-Based Antithymocyte Globulin in αβhaplo-Hematopoietic Stem Cell Transplantation Facilitates Engraftment, Expedites T Cell Recovery, and Mitigates the Risk of Acute Graft-versus-Host Disease

In αβ T-cell/CD19 B-cell depleted hematopoietic stem cell transplantation (αβhaplo-HSCT) recipients, antithymocyte globulin (ATG; Thymoglobulin) is used for preventing graft rejection and graft-versus-host disease (GVHD). The optimal dosing remains to be established, however. Here we present the first comparative analysis of 3 different ATG dosing strategies and their impact on immune reconstitution and GVHD. Our study aimed to evaluate the effects of 3 distinct dosing strategies of ATG on engraftment success, αβ+ and γδ+ T cell immune reconstitution, and the incidence and severity of acute GVHD in recipients of αβhaplo-HSCT. This comparative analysis included 3 cohorts of pediatric patients with malignant (n = 36) or nonmalignant (n = 8) disease. Cohorts 1 and 2 were given fixed ATG doses, whereas cohort 3 received doses via a new nomogram, based on absolute lymphocyte count (ALC) and body weight (BW). Cohort 3 showed a 0% incidence of day 100 grade II-IV acute GVHD, compared to 48% in cohort 1 and 27% in cohort 2. Furthermore, cohort 3 (the ALC/BW-based cohort) had a significant increase in CD4+ and CD8+ naïve T cells by day 90 (P = .04 and .03, respectively). Additionally, we found that the reconstitution and maturation of γδ+ T cells post-HSCT was not impacted across all 3 cohorts. Cumulative ATG exposure in all cohorts was lower than previously reported in T cell-replete settings, with a lower pre-HSCT exposure (<40 AU*day/mL) correlating with engraftment failure (P = .007). Conversely, a post-HSCT ATG exposure of 10 to 15 AU*day/mL was optimal for improving day 100 CD4+ (P = .058) and CD8+ (P = .03) immune reconstitution without increasing the risk of relapse or nonrelapse mortality. This study represents the first comparative analysis of ATG exposure in αβhaplo-HSCT recipients. Our findings indicate that (1) a 1- to 2-fold ATG to ATLG bioequivalence is more effective than previously established standards, and (2) ATG exposure post-HSCT does not adversely affect γδ+ T cell immune reconstitution. Furthermore, a model-based ATG dosing strategy effectively reduces graft rejection and day 100 acute GVHD while also promoting early CD4+/CD8+ immune reconstitution. These insights suggest that further optimization, including more distal administration of higher ATG doses within an ALC/BW-based strategy, will yield even greater improvements in outcomes.

Optimization of electroporation method and promoter evaluation for type-1 methanotroph, Methylotuvimicrobium alcaliphilum.

Methanotrophic bacteria are promising hosts for methane bioconversion to biochemicals or bioproducts. However, due to limitations associated with long genetic manipulation timelines and, lack of choice in genetic tools required for strain engineering, methanotrophs are currently not employed for bioconversion technologies. In this study, a rapid and reproducible electroporation protocol is developed for type 1 methanotroph, Methylotuvimicrobium alcaliphilum using common laboratory solutions, analyzing optimal electroshock voltages and post-shock cell recovery time. Successful reproducibility of the developed method was achieved when different replicative plasmids were assessed on lab adapted vs. wild-type M. alcaliphilum strains (DASS vs. DSM19304). Overall, a ∼ 3-fold decrease in time is reported with use of electroporation protocol developed here, compared to conjugation, which is the traditionally employed approach. Additionally, an inducible (3-methyl benzoate) and a constitutive (sucrose phosphate synthase) promoter is characterized for their strength in driving gene expression.

Oral quercetin nanoparticles in hydrogel microspheres alleviate high-altitude sleep disturbance based on the gut-brain axis

High-altitude sleep disturbance is a common symptom of acute mountain sickness, which can be alleviated via modulation of the gut-brain axis. Quercetin (Que) is used to modulate gut microbiota and serves as a potential drug to regulate the gut-brain axis, but the poor solubility and bioavailability affect its biological functions. Here, Que nanoparticles (QNPs) were prepared with zein using an antisolvent method, and QNP-loaded calcium alginate hydrogel microspheres (QNP@HMs) were prepared using electrospinning technology to improve the gastrointestinal stability and intestinal adhesion of QNPs. In the mouse model of high-altitude sleep disturbance, oral administration of QNP@HMs before the mice entering high altitude prolonged sleep duration, improved blood cell recovery, spontaneous behavior and short-term memory, and reduced such inflammation factors as TNF-α and iNOS. Moreover, QNP@HMs enhanced the abundance of probiotics in the gut, including Lactobacillus and Lachnospira, and reduced intestinal inflammation. However, in the mice after gut sterilization by long-term oral antibiotics, QNP@HMs showed no therapeutic effect. QNP@HMs are a promising medication for the prevention of high-altitude sleep disturbance based on the gut-brain axis.

Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation

Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.

Aqueous two‐phase systems as a novel alternative in the primary recovery of human mesenchymal cells obtained from adipose tissue

AbstractIn recent years, therapies based on regenerative medicine have gained wide interest owing to their potential in organ transplantation and in the treatment of multiple diseases. These cell‐based therapies rely mainly on the therapeutic potential of stem cells as a result of their self‐renewal, differentiation, capacities and immunosuppressive properties. Adipose tissue contains a large number of mesenchymal stem cells compared to other tissues and is easily accessible through noninvasive techniques; hence the primary recovery of adipose‐derived mesenchymal stem cells (ASC) has become a critical step. Aqueous two‐phase systems (ATPS) have shown promise as a separation technique for mesenchymal stem cells (MSC) as a result of their scalability, cost‐effectiveness and compatibility. In this study, we investigated the partitioning behavior of the stromal vascular fraction cells, including ASC, in different ATPS compositions [polyethylene glycol, dextran (DEX), Ficoll and Ucon). Among the three ATPS tested, most of the ASC consistently migrates to the top phase as indicated by immunostaining studies for CD73, CD90 and CD105 stem cell markers. Nevertheless, the Ficoll–DEX system retrieves the greatest quantity of ASC recovered in the top phase (89% of total ASC), whereas the Ucon–DEX system effectively eliminates the most contaminating cells (38%). Our findings provide insights into the conditions under which ASC and contaminant cells concentrate in opposite phases in different ATPS, which can contribute to the development of efficient and scalable separation systems for MSC in regenerative medicine and tissue engineering applications. © 2024 Society of Chemical Industry (SCI).

Electrochemical cytosensor utilizing tetrahedral DNA/bimetallic AuPd holothurian-shaped nanoparticles for ultrasensitive non-destructive detection of circulating tumor cells.

As a real-time fluid biopsy method, the detection of circulating tumor cells (CTCs) provides important information for the early diagnosis, precise treatment, and prognosis of cancer. However, the low density of CTCs in the peripheral blood hampers their capture and detection with high sensitivity and selectivity using currently available methods. Hence, we designed a sandwich-type electrochemical aptasensor that utilizes holothurian-shaped AuPd nanoparticles (AuPd HSs), tetrahedral DNA nanostructures (TDNs), and CuPdPt nanowire networks (NWs) interwoven with a graphdiyne (GDY) sheet for ultrasensitive non-destructive detection of MCF-7 breast cancer cells. CuPdPt NW-GDY effectively enhanced the electron transfer rate and coupled with the loaded TDNs. The TDNs could capture MCF-7 cells with precision and firmness, and the resulting composite complex was combined with AuPd HSs to form a sandwich-type structure. This novel aptasensor showed a linear range between 10 and 106 cells mL-1 and an ultralow detection limit of 7 cells mL-1. The specificity, stability, and repeatability of the measurements were successfully verified. Moreover, we used benzonase nuclease to achieve non-destructive recovery of cells for further clinical studies. According to the results, our aptasensor was more sensitive measuring the number of CTCs than other approaches because of the employment of TDNs, CuPdPt NW-GDY, and AuPd HSs. We designed a reliable sensor system for the detection of CTCs in the peripheral blood, which could serve as a new approach for cancer diagnosis at an early stage.

Efficiencies of filtration sampling and extraction for recovery of viable Staphylococcus aureus bioaerosols

Airborne Staphylococcus aureus is detected in various locations and linked to human infection. Reliable quantification of viable S. aureus bioaerosols by filter-based samplers helps characterize personal exposure, requiring efficient sampling and post-sampling processing. However, the efficiencies of filtration sampling and cell extraction are undetermined for viable S. aureus. In coupled with quantitative PCR and propidium monoazide, the performance of three widely-used samplers (IOM, Button, and Cassette) loaded with polycarbonate (PC) or gelatin filters was evaluated over 30–270 min of sampling and compared to that of BioSampler containing deionized water (DW). Effects of sampler type, filter type, and sampling time on cell recovery efficiency of sampling methods were assessed. Methods to extract cells from filters were also studied. The 1.5-min vortexing in peptone-Tween mixture and 10-min heating in DW were respectively granted optimal for cell extraction from PC and gelatin filters with extraction efficiencies averaged 1.0–1.76 (n = 4). Both Button and IOM with 3-μm gelatin filter performed best to capture S. aureus, significantly greater than Button with 0.8-μm PC by a factor of 9–11 (P < 0.05) and Cassette or IOM with 0.2-μm PC by a factor of 15–79 (P < 0.05). Cassette and IOM with 0.2-μm PC also showed less efficiencies than BioSampler/DW by a factor of 4–16 (P < 0.05). Cell recovery efficiency was not affected by sampling time except for the Button with 0.8-μm PC. Overall, filter type is the most critical factor governing cell recovery efficiency. Button and IOM with gelatin filter and 10-min heating in DW are considered the most efficient filtration sampling and extraction methods for viable S. aureus.

Persistent Impairment in Immune Reconstitution and Worse Survival Outcomes in Allogeneic Stem Cell Transplantation Patients with Early Coronavirus Disease 2019 Infection

Patients undergoing allogenic hematopoietic stem cell transplantation (HSCT) are at an increased risk of mortality due to transplantation-related complications in the first year post-transplantation, owing in part to the profound immune dysregulation with T cell and B cell lymphopenia and functional impairment. Although several large studies have reported higher mortality rates from Coronavirus disease 2019 (COVID-19) in HSCT recipients, to date no study has focused on the impact of early COVID-19 infection on immune reconstitution post-transplantation and the correlation with transplantation outcomes. We retrospectively analyzed 61 consecutive adult patients who underwent their first allogeneic HSCT at our institution. Thirteen patients (21.3%) experienced early COVID-19 infection, with a median time to diagnosis of 100 days post-transplantation. In multivariable analysis, patients with early COVID-19 infection had significantly worse overall survival (adjusted hazard ratio [aHR], 4.06; 95% confidence interval [CI], 1.26 to 13.05; P = .019) and progression-free survival (aHR, 6.68; 95% CI, 2.11 to 21.11; P = .001). This was attributed mainly to higher nonrelapse mortality (NRM) among early COVID-19 patients (P = .042). Allogeneic HSCT recipients with early COVID-19 infection had significant delays in absolute lymphocyte count (95% CI, -703.69 to -56.79; P = .021), CD3+CD4+ cell (95% CI, -105.35 to -11.59; P = .042), CD3+CD8+ cell (95% CI, -324.55 to -57.13; P = .038), and CD3-CD56+ cell (95% CI, -193.51 to -47.31; P = .014) recovery compared to those without early COVID-19 infection. Our findings suggest that patients with early COVID-19 infection after allogeneic HSCT have higher NRM and worse survival, at least in part due to impaired immune reconstitution post-transplantation.

Characterization and Determinants of Long-Term Immune Recovery Under Suppressive Antiretroviral Therapy.

We developed a robust characterization of immune recovery trajectories in people living with HIV on antiretroviral treatment (ART) and relate our findings to epidemiological risk factors and bacterial pneumonia. Using data from the Swiss HIV Cohort Study and the Zurich Primary HIV Infection Cohort Study (n = 5907), we analyzed the long-term trajectories of CD4 cell and CD8 cell counts and their ratio in people living with HIV on ART for at least 8 years by fitting nonlinear mixed-effects models. The determinants of long-term immune recovery were investigated using generalized additive models. In addition, prediction accuracy of the modeled trajectories and their impact on the fit of a model for bacterial pneumonia was assessed. Overall, our population showed good immune recovery (median plateau [interquartile range]-CD4: 718 [555-900] cells/μL, CD8: 709 [547-893] cells/μL, CD4/CD8: 1.01 [0.76-1.37]). The following factors were predictive of recovery: age, sex, nadir/zenith value, pre-ART HIV-1 viral load, hepatitis C, ethnicity, acquisition risk, and timing of ART initiation. The fitted models proved to be an accurate and efficient way of predicting future CD4 and CD8 cell recovery dynamics: Compared with carrying forward the last observation, mean squared errors of the fitted values were lower by 1.3%-18.3% across outcomes. When modeling future episodes of bacterial pneumonia, using predictors derived from the recovery dynamics improved most model fits. We described and validated a method to characterize individual immune recovery trajectories of people living with HIV on suppressive ART. These trajectories accurately predict long-term immune recovery and the occurrence of bacterial pneumonia.

A Comparative Study of B Cell Blast Isolation Methods from Bone Marrow Aspirates of Pediatric Leukemia Patients.

Density gradient centrifugation is a conventional technique widely utilized to isolate bone marrow mononuclear cells (BM-MNC) from bone marrow (BM) aspirates obtained from pediatric B-cell acute lymphoblastic leukemia (B-ALL) patients. Nevertheless, this technique achieves incomplete recovery of mononuclear cells and is relatively time-consuming and expensive. Given that B-ALL is the most common childhood malignancy, alternative methods for processing B-ALL samples may be more cost-effective. In this pilot study, we use several readouts, including immune phenotype, cell viability, and leukemia-initiating capacity in immune-deficient mice, to directly compare the density gradient centrifugation and buffy coat processing methods. Our findings indicate that buffy coat isolation yields comparable BM-MNC product in terms of both immune and leukemia cell content and could provide a viable, lower cost alternative for biobanks processing pediatric leukemia samples.

Single-Cell Screening through Cell Encapsulation in Photopolymerized Gelatin Methacryloyl

This study evaluated the potential of gelatin methacryloyl (GelMA) for single-cell screening compared to polyethylene glycol diacrylate (PEGDA). GelMA photopolymerized at 1000–2000 mJ/cm2 produced consistent patterns and supported HeLa cell viability. GelMA (5%w/v) facilitated better cell collection within 2 days due to its shape retention. GelMA demonstrated biocompatibility with HeLa cells exhibiting exponential proliferation and biodegradation over 5 days. The average cell displacement over 2 days was 16 µm. Two targeted cell recovery strategies using trypsin were developed: one for adherent cells encapsulated at 800 mJ/cm2, and another for floating cells encapsulated at 800 mJ/cm2, enabling the selective removal of unwanted cells. These findings suggest GelMA as a promising biomaterial for single-cell screening applications, offering advantages over PEGDA in cell encapsulation and targeted recovery.