Density Gradient Separation Research Articles

P-2219. Demonstrating a Novel Immune Cell Isolation Strategy for Scalable Single-Cell RNA Sequencing Studies in Patients with Sepsis

Abstract Background Though immune dysregulation often plays a key role in disease, characterizing circulating immune cells in critical illnesses is challenging. Studies using single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) reveals the type and function of patients' immune cells (cell substates) that could advance diagnostics and therapeutics, particularly in diseases defined by immune response heterogeneity such as sepsis. However, PBMC isolation from blood is required for scRNA-seq, and the gold-standard method (density gradient separation, or ‘Ficoll’) requires two hours of onsite processing, precluding large-scale sample collection at most clinical sites. Simplifying sample collection could enable participation of more health care centers, as well as the over-enrollment of patients for later retrospective adjudication, better reflecting the true patient heterogeneity in diseases.Figure 1.Workflow depicting the process from patient presentation in a health care center to sequencing results. The WB cryo method expedites sample collection in clinical settings by eliminating the need for Ficoll PBMC separation, a time-intensive process requiring laboratory equipment and trained operators. Differences in methods are highlighted in yellow. Methods We developed an alternative PBMC isolation method from cryopreserved whole blood (WB-cryo) using magnetic- and fluorescence- activated cell sorting (Fig. 1). We isolated PBMCs using the WB-cryo and Ficoll methods from patients with sepsis (n = 5) who sought evaluation at an Emergency Department and one healthy control. We performed scRNA-seq to identify and compare cell substate proportions and marker genes for key substates.Figure 2.Results of scRNA-seq on PBMCs isolated using Ficoll and WB cryo methods (n = 5 patients with sepsis, 1 healthy control). a) Uniform Manifold Approximation and Projection (UMAP) of separately clustered and annotated scRNA-seq data from each method; Bm denotes memory B cells, Bn: naive B cells, NK: natural killer cells, Mono: monocytes, MS1: monocyte substate 1, DC: dendritic cells, Tn: naive T cells, Tm: memory T cells, gdT: gamma delta T cells, Treg: regulatory T cells. b) correlation plot of resulting cell substate proportions (R = 0.87, p < 0.0001). Results Analysis included 9,996 cells isolated by Ficoll and 15,482 cells from WB-cryo. Sequencing quality was comparable between methods, as were cell substate fractions (Pearson correlation 0.87, p < 0.0001) (Fig. 2), and marker genes for a key monocyte substate enriched in patients with sepsis (19 of top 20 marker genes shared). WB-cryo reduced onsite sample processing time from 2 hours to < 10 minutes. Conclusion PBMCs isolated by WB-cryo retained similar transcriptional states when compared to PBMCs isolated using Ficoll, with > 10-fold less onsite hands-on time. With further validation, WB-cryo may expand scRNA-seq study enrollment in sepsis and other diseases. Ongoing work in a larger cohort (n = 23 patients with sepsis) processed across two clinical sites is underway to validate our isolation method as a way to extend the utility of scRNA-seq in multisite studies of complex and heterogeneous conditions like sepsis. Disclosures Michael R. Filbin, MD, Day Zero Diagnostics: Grant/Research Support|Quidel: Grant/Research Support

Impact of Post-Thaw Enrichment of Primary Human Hepatocytes on Steatosis, Inflammation, and Fibrosis in the TruVivo® System

Background: Liver diseases are a global health concern. Many in vitro liver models utilize cryopreserved primary human hepatocytes (PHHs), which commonly undergo post-thaw processing through colloidal silica gradients to remove debris and enrich for a viable PHH population. Post-thaw processing effects on healthy PHHs are partially understood, but the consequences of applying disease-origin PHHs to post-thaw density gradient separation have not been described. Methods: Using the TruVivo® system, diseased, type 2 diabetes mellitus (T2DM), and fibrotic PHHs were cultured for 14 days after initially being subjected to either low-density (permissive) or high-density (selective) gradients using Percoll-based thawing medium. Results: Changes in functionality, including albumin and urea secretion and CYP3A4 activity, were measured in diseased, T2DM, and fibrotic PHHs enriched in low Percoll compared to PHHs enriched in high Percoll. Lipogenesis increased in the PHHs enriched in low Percoll. Higher expression of CK18 and TGF-β, two fibrotic markers, and changes in expression of the macrophage markers CD68 and CD163 were also measured. Conclusions: The use of Percoll for the enrichment of PHHs post-thaw results in differences in attachment and functionality, along with changes in diseased phenotypes, in the TruVivo® system.

Efficient Manufacturing of CAR-T Cells from Whole Blood: A Scalable Approach to Reduce Costs and Enhance Accessibility in Cancer Therapy

BackgroundChimeric antigen receptor T (CAR-T) cells have significantly advanced the treatment of cancers such as leukemia and lymphoma. Traditionally, T cells are collected from patients through leukapheresis, an expensive and potentially invasive process that requires specialized equipment and trained personnel. Although whole blood collections are much more technically straightforward, whole blood starting material has not been widely utilized for clinical CAR-T cell manufacturing, in part due to lack of manufacturing processes designed for use in a good manufacturing practice (GMP) environment. Collecting cellular starting material from whole blood without leukapheresis could reduce manufacturing complexity and cost, thereby improving accessibility to CAR-T cell therapy. MethodsWhole blood samples were collected from eight healthy donors and one pediatric B-cell acute lymphoblastic leukemia (B-ALL) patient. These samples were processed using the Sepax C-Pro (Cytiva) instrument to isolate mononuclear cells (MNCs) via density gradient separation. CAR-T cells were then manufactured from the isolated MNCs using a GMP-compliant 7-day protocol, whereby T cells were activated with anti-CD3 and IL-2, transduced with GMP lentiviral vector encoding a CD19/CD22 bispecific CAR, and expanded in gas permeable cell culture bags. The resulting CAR-T cells were then evaluated for their phenotypic and functional properties using flow cytometry, cytokine release, and cytotoxicity assays. ResultsFrom an average 77.7 mL of whole blood from healthy donors (range = 29 - 96 mL), we isolated an average of 42.2 × 106 CD3⁺ T cells (range 7.3 - 63.0) post processing. CAR-T cell cultures were initiated from thaw using 1 - 10 × 106 starting CD3+ T cells, yielding a median T cell number of 105 × 106 cells on day 7 (range 61 - 188 × 106). We observed 66 ± 11% mean transduction efficiency and produced a mean of 77.4 × 106 transduced CAR-T cells (range 30.8 - 143.5 × 106). Similar results were obtained when using a blood sample (28mL) obtained from a patient with relapsed B-ALL who had received recent chemotherapy. ConclusionsTherapeutically relevant doses of CD19/CD22 CAR-T cells can be successfully manufactured from whole blood. On average, 80 mL of whole blood yields enough CAR-T cells to create a single dose for a pediatric patient (50 kg) at a dosage of 1 × 106 CAR-T cells/kg. For larger patients, scaling up is straightforward by collecting a larger blood volume. This method also demonstrates a cost-effective approach to T cell activation and expansion which, alongside a more straightforward collection of whole blood, makes it more widely accessible especially for middle- and low-income countries. By reducing costs and labor, this strategy has the potential to significantly expand global access to CAR-T cell therapy.

Cryo-PRO facilitates whole blood cryopreservation for single-cell RNA sequencing of immune cells from clinical samples.

Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) has enhanced our understanding of host immune mechanisms in small cohorts, particularly in diseases with complex and heterogeneous immune responses such as sepsis. However, PBMC isolation from blood requires technical expertise, training, and approximately two hours of onsite processing using Ficoll density gradient separation ('Ficoll') for scRNA-seq compatibility, precluding large-scale sample collection at most clinical sites. To minimize onsite processing, we developed Cryo-PRO (Cryopreservation with PBMC Recovery Offsite), a method of PBMC isolation from cryopreserved whole blood that allows immediate onsite sample cryopreservation and subsequent PBMC isolation in a central laboratory prior to sequencing. We compared scRNA-seq results from samples processed using Cryo-PRO versus standard onsite Ficoll separation in 23 patients with sepsis. Critical scRNA-seq outputs including cell substate fractions and marker genes were similar for each method across multiple cell types and substates, including an important monocyte substate enriched in patients with sepsis (Pearson correlation 0.78, p<0.001; 70% of top marker genes shared). Cryo-PRO reduced onsite sample processing time from >2 hours to <15 minutes and was reproducible across two enrollment sites, thus demonstrating potential for expanding scRNA-seq in multicenter studies of sepsis and other diseases.

Proteomic analysis of carbapenem-resistant Klebsiella pneumoniae outer membrane vesicles under the action of phages combined with tigecycline

BackgroundKlebsiella pneumoniae is the most commonly encountered pathogen in clinical practice. Widespread use of broad-spectrum antibiotics has led to the current global dissemination of carbapenem-resistant K. pneumoniae, which poses a significant threat to antibacterial treatment efficacy and public health. Outer membrane vesicles (OMVs) have been identified as carriers capable of facilitating the transfer of virulence and resistance genes. However, the role of OMVs in carbapenem-resistant K. pneumoniae under external pressures such as antibiotic and phage treatments remains unclear.MethodsTo isolate and purify OMVs under the pressure of phages and tigecycline, we subjected K. pneumoniae 0692 harboring plasmid-mediated blaNDM-1 and blaKPC-2 genes to density gradient separation. The double-layer plate method was used to isolate MJ1, which efficiently lysed K. pneumoniae 0692 cells. Transmission electron microscopy (TEM) was used to characterize the isolated phages and extract OMV groups for relevant morphological identification. Determination of protein content of each OMV group was conducted through bicinchoninic acid assay (BCA) and proteomic analysis.ResultsK. pneumoniae 0692 released OMVs in response to different environmental stimuli, which were characterized through TEM as having the typical structure and particle size of OMVs. Phage or tigecycline treatment alone resulted in a slight increase in the mean protein concentration of OMVs secreted by K. pneumoniae 0692 compared to that in the untreated group. However, when phage treatment was combined with tigecycline, there was a significant reduction in the average protein concentration of OMVs compared to tigecycline treatment alone. Proteomics showed that OMVs encapsulated numerous functional proteins and that under different external stresses of phages and tigecycline, the proteins carried by K. pneumoniae 0692-derived OMVs were significantly upregulated or downregulated compared with those in the untreated group.ConclusionsThis study confirmed the ability of OMVs to carry abundant proteins and highlighted the important role of OMV-associated proteins in bacterial responses to phages and tigecycline, representing an important advancement in microbial resistance research.

Advancing Diabetes Research: A Novel Islet Isolation Method from Living Donors.

Pancreatic islet isolation is critical for type 2 diabetes research. Although -omics approaches have shed light on islet molecular profiles, inconsistencies persist; on the other hand, functional studies are essential, but they require reliable and standardized isolation methods. Here, we propose a simplified protocol applied to very small-sized samples collected from partially pancreatectomized living donors. Islet isolation was performed by digesting tissue specimens collected during surgery within a collagenase P solution, followed by a Lympholyte density gradient separation; finally, functional assays and staining with dithizone were carried out. Isolated pancreatic islets exhibited functional responses to glucose and arginine stimulation mirroring donors' metabolic profiles, with insulin secretion significantly decreasing in diabetic islets compared to non-diabetic islets; conversely, proinsulin secretion showed an increasing trend from non-diabetic to diabetic islets. This novel islet isolation method from living patients undergoing partial pancreatectomy offers a valuable opportunity for targeted study of islet physiology, with the primary advantage of being time-effective and successfully preserving islet viability and functionality. It enables the generation of islet preparations that closely reflect donors' clinical profiles, simplifying the isolation process and eliminating the need for a Ricordi chamber. Thus, this method holds promises for advancing our understanding of diabetes and for new personalized pharmacological approaches.

Extracellular and intracellular inflammatory response of immune cells to radiation exposure

Background: Exposure to low doses of ionizing radiation is associated with increased risk of cardiovascular disease (CVD) in a dose-dependent manner. A person exposed to low doses of radiation have a 27-29% risk of developing and dying from CVD. Low doses of radiation initiate an inflammatory process that can increase the risk of developing CVD. Inflammation initiates atherosclerosis via immune cell infiltration subsequent to endothelial cell damage. We hypothesized that CD14+ monocytes will exhibit an amplified inflammatory response, as demonstrated by significant increases in the expression of inflammatory cytokines at 48 and 72 hours post radiation exposure compared to controls which would be magnified by the inflammatory stimulus LPS. Methods: Human venous peripheral blood was collected from 5 healthy young adults (24 ± 3 years, 22 ± 1 BMI, 5 Male) using an institutional review board (IRB)-approved protocol. Women were studied in days 1-5 of menstrual cycle. Peripheral blood mononuclear cells (PBMCs) were harvested by density gradient separation and re-suspended and incubated with anti-human CD14 microbeads. CD14+ immune cells were incubated in plates, exposed to 4 gray (Gy) of x-ray radiation, and then incubated for 48 and 72 hours. CD14+ immune cells were exposed to 1:500 dilution of 0.1 mg/mL of lipopolysaccharide (LPS) for the final 8 hours of incubation. The culture media was recovered, and assayed for secreted factors, specifically for IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IFN-γ, and TNF-α. Additionally, flow cytometry analysis was performed to quantify intracellular content of IL-10, IL-8, IFN-g, IL-4, receptor for advanced glycation end products (RAGE), and damage-associated molecular pattern (DAMP) molecules (S100A8/9) involved in inflammatory activation. All data were analyzed with two-way repeated measures ANOVA. Results: Secreted Cytokines: Cytokine expression at 48 and 72 hours did not differ between the control, radiation (4Gy), and 4Gy+LPS groups. At 48 and 72 hours, there were no significant differences observed in the expression levels of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α in control, 4Gy, and 4Gy+LPS cells. Intracellular Content: Compared to control IL-4 content was increased at 48 and 72 hours, after exposure to 4Gy (48h: p=0.013 and 72h: p=0.008) and 4Gy+LPS (48h: p=0.022 and 72h: p=0.002). Furthermore, IL-4 was again increased at 72 hours after exposure to 4Gy+LPS compared to 48 hours (p=0.014). IL-10 content was also increased in after exposure to 4Gy and 4Gy+LPS at 72 hours compared to control (p=0.046 and p=0.007, respectively). S100A8/9 also showed a trending increase at 4Gy compared to control at 72 hours (p=0.11). No other content changed significantly. Conclusion: These data suggest that acute radiation exposure (4 Gy x-ray) has no effect on the secretome of CD14+ immune cells. However, the intracellular content of IL-4 was increased at both 48 and 72 hours, while IL-10 increased at 72 hours, indicating a shift from pro-inflammatory to anti-inflammatory intracellular content the further in time monocytes are from radiation exposure. Future work needs to test increased and repeated doses of radiation, as well as examine both x-ray and proton exposure. TRISH through NASA Cooperative Agreement NNX16AO69A and NIH HL150361. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Isolated bovine pancreatic islets as an alternate in vitro model for diabetes research.

Background & objectives Isolation of functional pancreatic islets for diabetes research and clinical islet transplantation stands as a big challenge despite the advancements in the field. In this context, the non-availability of human/animal tissues is one of the major impediments to islet-based research, which has tremendous scope for translation. The current study explores the feasibility of using the bovine pancreas as an alternative source to isolate pancreatic islets and assess its functionality for in vitro studies. Methods The bovine pancreas was collected from a registered slaughterhouse and transported in an ice-cold medium - Hank's Balanced Salt Solution (HBSS) to the laboratory. Islets were isolated by sequential collagenase digestion followed by a two-step filtration and purification by density gradient separation method. After isolation, islets were identified with dithizone staining and the islet function was assayed in vitro for assessing the dynamic insulin secretory function by monitoring the glucose-stimulated insulin secretion (GSIS), in response to low and high glucose. Staining techniques were also used to understand the cytoarchitecture of the bovine pancreas. Results The islet yield was 157±23 islets per gram of pancreas and was viable. The cold ischaemia time was reduced to 60-75 min. The islets released insulin with glucose stimulation. The insulin release was observed more with high glucose (28 mM) than with low glucose (2.8 mM). Dithizone staining confirmed the presence of islets after isolation and the size of islets ranged from 50 to 600 µm size. The mantled islets (islets with acinar tissue) were also noted with the pure islets in culture. Hematoxylin and eosin (H&E) and aldehyde- fuchsin showed islets interspersed in the acinar tissue of the bovine pancreas. Special stain defined the islets better than regular staining. Fluorescent and diaminobenzidine (DAB) staining with insulin, glucagon and somatostatin revealed the arrangement of the cells in each islet. The beta cells were majorly found in the islet core with alpha cells interspersed with the delta cells in the periphery. Interpretation & conclusions The isolation procedure described in this study yielded viable islets for in vitro studies which showed a differential response to glucose challenge, confirming their viability. We provide a simple and reproducible method for small-scale isolation of functional islets from the bovine pancreas. This model proffers the beginner a hands-on in islet experiments and helps to re-iterate the process that could be extrapolated to other pancreatic tissues as well as to expand on diabetes research.

SAP-expressing T peripheral helper cells identify systemic lupus erythematosus patients with lupus nephritis.

T follicular (TFH) and peripheral helper (TPH) cells have been increasingly recognized as a pathogenic subset of CD4 T cells in systemic lupus erythematosus (SLE). The SLAM Associated Protein (SAP) regulates TFH and TPH function by binding to the co-stimulatory signaling lymphocyte activation molecule family (SLAMF) receptors that mediate T cell - B cell interactions. SAP and SLAMF are critical for TPH-dependent B cell maturation into autoantibody-producing plasma cells that characterize SLE pathogenesis. We hypothesized that SAP-expressing TPH cells are involved in the pathogenesis of lupus nephritis (LN). Peripheral blood mononuclear cells (PBMC) were isolated using density gradient separation from whole blood. Cells were stained for cell surface markers, followed by permeabilization and staining of intracellular SAP for spectral flow cytometry analysis. We also analyzed SAP expression from renal infiltrating LN T cells using the available single-cell RNA sequencing (scRNA seq) Accelerated Medicines Partnership (AMP) SLE dataset. PBMC from 30 patients with SLE (34 ± 10 years old, 83% female), including 10 patients with LN, were analyzed. We found an increase in total SAP-positive CD4 and CD8 T cells in SLE compared with controls (55.5 ± 2.6 vs. 41.3 ± 3.4, p=0.007, and 52.5 ± 3.0 vs. 39.2 ± 2.8, p=0.007 respectively). In CD4 T cells, the highest SAP expression was in the TPH subset. The frequency of SAP+TPH in circulation correlated with disease activity; SLE patients with renal disease had higher levels of circulating SAP+TPH that remained significant after adjusting for age, sex, race, low complements, and elevated anti-dsDNA (p=0.014). scRNA-seq data of renal infiltrating T cells in LN identified SAP expression to localize to the TFH-like CD4 cluster and GZMK+ CD8 cluster. Increased SAP expression in LN was associated with the differential expression of SLAMF3 and SLAMF7 and granzyme K and EOMES. The existence of two predominant SAP-expressing subsets, the TFH-like CD4 T cells, and GZMK+ effector CD8 T cells, was verified using scRNA-seq data from a human transcriptomic atlas of fifteen major organs. The expansion of SAP-expressing T helper cells was associated with LN in our cohort and verified using scRNA-seq data of renal infiltrating T cells. Improved SLAM and SAP signaling understanding can identify new therapeutic targets in LN.

An evaluation of microplastic contamination in the marine waters and species in the coastal region of the South Yellow Sea, China

Microplastics (MPs) contamination of marine environments poses a significant ecological risk, although impacts on species’ realized niche spaces remain unclear. The current study investigates MPs distribution across pelagic habitats, benthic sediments, and key biota in the South Yellow Sea, China. Samples were collected via trawling across estuarine transects, and tissues were digested to extract MPs. Density gradient separations and vacuum-filtrations prepared particle extracts for ATR-FTIR and Micro-Raman spectroscopic characterization. Sampling along industrialized river transects reveals ubiquitous plastic particle presence, with concentrations ranging from 0 to 51.68 item/L seawater. Contamination levels reach their peak at station estuaries before dispersing offshore, indicating significant waste stream inputs. Importantly, MPs detected in demersal and pelagic fish species, as well as in bivalves, confirm exposure across trophic niches. Gastrointestinal tract and gill concentrations reached 0.6 items/g fresh tissue, reflecting significant biological uptake and in vivo retention. The greatest population of organisms occurred adjacent to polluted areas. Overall, distribution of MPs from polluted rivers to coastal food webs was evident, suggesting potential negative impacts on key ecological functions in this system. These findings underscore the need to develop upstream mitigation efforts so as to minimize MPs contamination in areas where nearshore and offshore niches intersect.

Polycarbonate Ultracentrifuge Tube Re-use in Proteomic Analyses of Extracellular Vesicles.

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins. This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples. Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (≤20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs.

Non-graphitic carbon as sodium-ion battery anode materials with improved ratio of plateau to sloping capacities prepared from lignite-based organic maceral

Herein, huminite concentrate is obtained from lignite with high yield of 68.9 % by density gradient separation, subsequently converted to non-graphitic carbon (HM-H) by carbonization at 1400 °C for 2 h. Huminite concentrate possesses relatively high oxygen content and considerable amount of aromatic component, with modest yield of volatile matter during pyrolysis, resulting in optimized carbon phase distribution and enhanced electrical conductivity of HM-H. Compared with HM prepared by direct carbonization of lignite, HM-H has slightly higher contents of pseudo-graphitic and graphite-like carbons, and lower content of highly disordered one. Notably, HM-H exhibits remarkably improved electrical conductivity (1773 S m−1) than HM (1406 S m−1). Compared with HM anode for SIBs, HM-H one delivers higher plateau capacity (170.1 mAh g−1) meanwhile lower sloping capacity (91.8 mAh g−1), with significantly improved rate and cycling performances. Full cell assorted with NaFe1/3Ni1/3Mn1/3O2 cathode and HM-H anode demonstrates higher energy density of 210.5 Wh kg−1 than its counterpart (193.8 Wh kg−1) at 20 mA g−1, with superior rate performance (208.6 mAh g−1 at 200 mA g−1) and a capacity retention ratio of 89.7 % after 100 cycles. This work provides a facile and effective strategy for preparing non-graphitic carbon as high-performance anode materials for SIBs.

Dissociation protocols influence the phenotypes of lymphocyte and myeloid cell populations isolated from the neonatal lymph node.

Frequencies and phenotypes of immune cells differ between neonates and adults in association with age-specific immune responses. Lymph nodes (LN) are critical tissue sites to quantify and define these differences. Advances in flow cytometry have enabled more multifaceted measurements of complex immune responses. Tissue processing can affect the immune cells under investigation that influence key findings. To understand the impact on immune cells in the LN after processing for single-cell suspension, we compared three dissociation protocols: enzymatic digestion, mechanical dissociation with DNase I treatment, and mechanical dissociation with density gradient separation. We analyzed cell yields, viability, phenotypic and maturation markers of immune cells from the lung-draining LN of neonatal and adult mice two days after intranasal respiratory syncytial virus (RSV) infection. While viability was consistent across age groups, the protocols influenced the yield of subsets defined by important phenotypic and activation markers. Moreover, enzymatic digestion did not show higher overall yields of conventional dendritic cells and macrophages from the LN. Together, our findings show that the three dissociation protocols have similar impacts on the number and viability of cells isolated from the neonatal and adult LN. However, enzymatic digestion impacts the mean fluorescence intensity of key lineage and activation markers that may influence experimental findings.

Placental Trophoblast Cell Isolation from the Term Placenta.

Multiple cell lines have been utilized over time in studying placental biology. Still, most of them rely on choriocarcinoma cells or immortalized trophoblast cells that may not be entirely comparable with actual human placental trophoblast cells. Term placentas can be a source of primary villous trophoblasts. However, challenges remain in isolating them and maintaining them in extended culture. This manuscript describes our three-phase protocol utilizing enzymatic/mechanical digestion, modified Percoll gradient density separation, and immunopurification using magnetic beads. The resulting trophoblast culture remains viable for an extended period and highly pure after initial passaging.

Advances in the axenic isolation methods of Blastocystis sp. and their applications.

Blastocystis sp. is a prevalent protistan parasite found globally in the gastrointestinal tract of humans and various animals. This review aims to elucidate the advancements in research on axenic isolation techniques for Blastocystis sp. and their diverse applications. Axenic isolation, involving the culture and isolation of Blastocystis sp. free from any other organisms, necessitates the application of specific media and a series of axenic treatment methods. These methods encompass antibiotic treatment, monoclonal culture, differential centrifugation, density gradient separation, micromanipulation and the combined use of culture media. Critical factors influencing axenic isolation effectiveness include medium composition, culture temperature, medium characteristics, antibiotic type and dosage and the subtype (ST) of Blastocystis sp. Applications of axenic isolation encompass exploring pathogenicity, karyotype and ST analysis, immunoassay, characterization of surface chemical structure and lipid composition and understanding drug treatment effects. This review serves as a valuable reference for clinicians and scientists in selecting appropriate axenic isolation methods.

Development of a Companion Diagnostic for a Targeted Protein Degradation Therapy: Ultrasensitive and Minimally-Invasive Detection of SALL4 Oncoproteins for Therapeutic Monitoring

Abstract Introduction/Objective Spalt-Like Transcription Factor 4 (SALL4), a member of the SALL family, is a regulator of embryonic stem cell development that plays a crucial role in cell renewal and proliferation. SALL4 expression is normally repressed in most adult organs but is reactivated in a multitude of cancers and upon treatment with hypomethylating agents (HMA). Research in the past 20 years established SALL4 as a novel, druggable cancer target. SALL4 targeting therapies (inhibitors and degraders) are under pre-clinical development. There are currently no FDA/CLIA approved SALL4 diagnostic tests beyond tissue immunohistochemistry (IHC). Therefore, there is outstanding clinical need for the development and validation of a less invasive and sufficiently-sensitive SALL4 expression assay which can be used for selecting, and monitoring patients on SALL4-targeting therapy such as molecular glue degraders, as well as monitoring patients on HMA treatment. Methods/Case Report Using the Simoa (Single Molecule Array) platform, a bead-based digital enzyme-linked immunosorbent assay, we cross-tested 5 commercial SALL4 antibodies and 13 antibody pair combinations, using the best antibody pair to test the matrix effects of human plasma, buffy coats, and bone marrow aspirates. For quantitative assay development, we successfully expressed and purified full-length SALL4 which will be used to calibrate the standard curve and determine the assay’s limit of quantification. Results (if a Case Study enter NA) The best antibody pair can detect both SALL4A and SALL4B isoforms, with stronger signal detected with the SALL4A isoform. We successfully optimized detection of SALL4 in bone marrow and peripheral blood matrix, and tested the utility of the assay for quantifying the efficacy of SALL4 degrader drugs and with hypomethylating agent treatment, using cell culture system. We also optimized multiple pre-analytic steps, including our cell lysis protocol, isolation of buffy coats using density gradient separation, and sample collection method comparison. Conclusion We developed an ultrasensitive SALL4 expression assay will be useful for monitoring patients on HMA treatment and SALL4-targeting therapy such as molecular glue degraders. There is ongoing effort to use MGB Biobank peripheral blood samples to validate SALL4 Simoa for patients with myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML), and liver cancer.

Determinants of Response to Anti CD-19 CAR-T Cells for Diffuse Large B-Cell Lymphoma in Pre-Treatment Peripheral Blood Mononuclear Cells Using Single-Cell RNA-Seq

Background: Chimeric antigen receptor T (CAR-T) cell therapy has become the standard of care in patients with relapsed/refractory diffuse large cell lymphoma (R/R DLBCL), providing durable remission in 40% of patients. Lack of response is likely to be attributed to inter- and intra-patient variability, including PBMC heterogeneity and apheresis material composition, CAR-T infusion product, in-vivo expansion and activity of CAR-T cells and the development of tumor-intrinsic mechanisms. Current methods for evaluating CAR-T cell activation and potency have limited predictive value for clinical outcomes. To address this challenge, we performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) obtained from patients with R/R LBCL patients at the time of apheresis for the manufacturing of anti-CD19 CAR-T cell therapy. Methods: Between 10/2020 and 03/2022, 31 patients who underwent lymphopheresis were enrolled. Samples were obtained - 1) At day of lymphopheresis from peripheral blood; 2) On the day of infusion of CAR-T from the product; and 3) On day +7 after infusion from peripheral blood. All patients had day +30 PET-CT. In addition, PBMCs samples from healthy donors were used as control to analyze baseline characteristics compared to the DLBCL patients. PBMCs from patients of healthy donors were purified from frozen blood samples by density gradient separation, stained using human CD45 antibody and loaded onto a 10x Chromium system. libraries were generated using the 10X Genomics Chromium Single Cell 3' Kit. The data was processed and analyzed using the Seurat R package. The study was approved by the local Ethic committee. Results: Patient characteristics: 31 RR DLBCL patients (42% females; median age 72, range 47-81 years), all treated with CAR-T cells therapy as third line of therapy, participated in the study. All patients underwent lymphopheresis, followed by CAR-T production. Disease status at lymphodepletion was complete remission (CR, n=4, 13%), partial remission (n=6, 19%), and progressive disease (n=21, 68%) and patients received either tisagenlecleucel (n=20, 65%) or axicabtagene ciloleucel (n=11, 35%) . PET scan performed on day 30 post treatment revealed that 68% and 32% obtained CR, and PD, respectively. scRNA analysis revealed differences in PBMC composition between healthy donors and LBCL patients (Figure 1A), with lower levels of naïve and memory T cells and elevated levels of activated monocytes in LBCL patients, suggesting a shift towards an inflamed and pre-dysfunctional immune state. Categorization of LBCL patients into responders (PR/CR on day +30 PET-CT) and non-responders (PD on day +30 PET-CT) revealed differences in immune cell subsets between responders and non-responders (Figure 2A), with enriched inflammation-related pathways in non-responders, In contrast, responders exhibited a distinct healthy-like immune profiles, particularly high levels of naive T cells, similar to that obtained in their age matched healthy controls. Of note, non-responders showed alterations in immune cell subsets, including increased levels of T regulatory cells and effector NK cells. Furthermore, non-responders demonstrated a prominent inflammatory immune profile, characterized by gene pathways related to inflammation and activation, such as type I and III IFN-G, and TNFb response (Figure 1B). Notably, an IFN-G score was significantly higher in non-responder's monocytes subpopulations, compared to responders. Conclusions: These findings indicate that the success of CAR-T therapy could be determined by the molecular characteristics and subpopulation distribution of each patient's PBMC. The diverse immune profiles of DLBCL patients might impact the effectiveness of their immune cells in generating successful CAR-T products or providing a conducive environment for CAR T cells to combat cancer. Furthermore, these findings emphasize the potential of scRNA-seq in evaluating PBMC from CAR-T patients prior to treatment, allowing the potential calculation of an “immune potency score” associated with the probability of a positive outcome from CAR-T treatment of DLBCL patients. This score could potentially be used by oncologists in decision-making processes to determine the appropriate candidates for CAR-T therapy.

Lipidomics profiles of human spermatozoa: insights into capacitation and acrosome reaction using UPLC-MS-based approach.

Lipidomics elucidates the roles of lipids in both physiological and pathological processes, intersecting with many diseases and cellular functions. The maintenance of lipid homeostasis, essential for cell health, significantly influences the survival, maturation, and functionality of sperm during fertilization. While capacitation and the acrosome reaction, key processes before fertilization, involve substantial lipidomic alterations, a comprehensive understanding of the changes in human spermatozoa's lipidomic profiles during these processes remains unknown. This study aims to explicate global lipidomic changes during capacitation and the acrosome reaction in human sperm, employing an untargeted lipidomic strategy using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Twelve semen specimens, exceeding the WHO reference values for semen parameters, were collected. After discontinuous density gradient separation, sperm concentration was adjusted to 2 x 106 cells/ml and divided into three groups: uncapacitated, capacitated, and acrosome-reacted. UPLC-MS analysis was performed after lipid extraction from these groups. Spectral peak alignment and statistical analysis, using unsupervised principal component analysis (PCA), bidirectional orthogonal partial least squares discriminant analysis (O2PLS-DA) analysis, and supervised partial least-squares-latent structure discriminate analysis (PLS-DA), were employed to identify the most discriminative lipids. The 1176 lipid peaks overlapped across the twelve individuals in the uncapacitated, capacitated, and acrosome-reacted groups: 1180 peaks between the uncapacitated and capacitated groups, 1184 peaks between the uncapacitated and acrosome-reacted groups, and 1178 peaks between the capacitated and acrosome-reacted groups. The count of overlapping peaks varied among individuals, ranging from 739 to 963 across sperm samples. Moreover, 137 lipids had VIP values > 1.0 and twenty-two lipids had VIP > 1.5, based on the O2PLS-DA model. Furthermore, the identified twelve lipids encompassed increases in PI 44:10, LPS 20:4, LPA 20:5, and LPE 20:4, and decreases in 16-phenyl-tetranor-PGE2, PC 40:6, PS 35:4, PA 29:1, 20-carboxy-LTB4, and 2-oxo-4-methylthio-butanoic acid. This study has been the first time to investigate the lipidomics profiles associated with acrosome reaction and capacitation in human sperm, utilizing UPLC-MS in conjunction with multivariate data analysis. These findings corroborate earlier discoveries on lipids during the acrosome reaction and unveil new metabolites. Furthermore, this research highlights the effective utility of UPLC-MS-based lipidomics for exploring diverse physiological states in sperm. This study offers novel insights into lipidomic changes associated with capacitation and the acrosome reaction in human sperm, which are closely related to male reproduction.

Ultrasensitive Detection of SALL4 Oncoproteins in Peripheral Blood and Bone Marrow Aspirate: Companion Diagnostics and Therapeutic Monitoring

Spalt-Like Transcription Factor 4 (SALL4), a member of the SALL family, is a regulator of embryonic stem cell development that plays a crucial role in cell renewal and proliferation. SALL4 is a tumor driver, as shown by gain-of-function and loss-of-function studies; high SALL4 expression invariably correlates with worse prognosis. These studies established SALL4 as a novel, druggable target, such as in liver, lung, and blood cancers. In myelodysplastic syndrome (MDS), SALL4 can be upregulated by hypomethylating agents (HMAs), correlating with worse patient outcomes. Despite its well-established function on oncogenesis, SALL4 targeting therapies (inhibitors and degraders) are under pre-clinical development. There are currently no FDA/CLIA approved SALL4 diagnostic tests beyond tissue immunohistochemistry. Therefore, there is outstanding clinical need for the development and validation of a less invasive and sensitive SALL4 expression assay via peripheral blood, which can be used for monitoring patients on HMA treatment and SALL4-targeting therapy such as molecular glue degraders. We developed a protein based SALL4 assay, compatible with peripheral blood and bone marrow aspirates samples, using the SIMOA (Single Molecule Array) technology, a bead-based digital enzyme-linked immunosorbent assay (aka digital ELISA). Previously, circulating SALL4 oncoproteins have been detected in the peripheral blood of patients with liver cancer using a conventional ELISA kit, but it is only semi-quantitative with limited sensitivity. We cross-tested 5 commercial SALL4 antibodies and 13 antibody pair combinations. After antibody screening, we used the best antibody pair to test the matrix effects of human plasma, buffy coats, and bone marrow aspirates. For quantitative assay development, we successfully expressed and purified full-length SALL4 for the first time, which was used to calibrate the standard curve and determine the assay's limit of quantification. The top bead and detector antibody pair could detect both SALL4A and SALL4B isoforms, with stronger signal detected with the SALL4A isoform in two different cell lysis buffers. Assay sensitivity was then tested. The Simoa assay captured endogenous SALL4 expression with greater sensitivity compared to the Western blot. The Western blot's limit of detection for SALL4A protein was 0.08 µg/µL whereas the Simoa assay continued until 3 ng/µL. Pre-analytic steps were then optimized, including our cell lysis protocol, isolation of buffy coats using density gradient separation, and sample collection method comparison. We assessed the effect of sample age for downstream processing and signal recovery as well as the possible matrix effect and interference from the buffy coat and plasma components of the peripheral blood and bone marrow samples. Our data showed no evidence of interference from the peripheral blood components, and interference from bone marrow aspirate can be further optimized. Antibody validation was then conducted. The preliminary data showed that the SALL4 Simoa assay can be used as a proof of principle to monitor SALL4 upregulation on HMA treatment for blood cancers and for SALL4 targeting therapy with two cell lines (SNU398 and H661) for solid cancers. To validate assay accuracy and precision, a retrospective cohort analysis is being conducted from patient peripheral blood samples. We obtained MGB Biobank samples for MDS, acute myeloid leukemia (AML), and liver cancer. We will use these samples to test for digital SALL4 protein detection. Further plans include expanding these analyses to breast and lung cancer. Additionally, we will conduct prospective studies to explore the role of the SALL4 protein biomarker in risk stratification and treatment monitoring, especially in MDS/AML patients undergoing HMA treatments. In conclusion, we have developed an ultrasensitive SALL4 protein expression diagnostic assay for use in monitoring patient treatment response and for guiding targeted therapy by analyzing peripheral blood for both solid and liquid tumors. Future steps include validating the diagnostic assay for specificity and sensitivity in a retrospective cohort analysis and conducting prospective studies to explore the role of the SALL4 protein biomarker for risk stratification, monitoring on HMA treatment, and guiding targeted therapy, ideally filing for approval as a laboratory developed test (LDT) in the near future

Umbilical Cord Blood Processing Techniques and Their Comparative Advantages: A Review

Background: Umbilical Cord Blood (UCB) has steadily gained prominence in haematopoietic stem cell transplantation (HSCT). Despite UCB advantages, the main disadvantage of UCB in haematopoietic stem cell transplantation (HSCT) is its limited cell dose. Initially, UCB used to be processed and then made to undergo cryopreservation as whole cord blood banking leading to the problem of storing sufficiently large number of cryoprotected UCB units which requires vast amounts of costly storage space in liquid nitrogen. The sole purpose of processing is to concentrates the stem cells and reduce the volume for storage. Different UCB processing methods have been developed. &#x0D; Aim: This review is aimed at bringing together the literature on the different processing methods and highlighting the underlying principles of each method, the relative efficiency and advantages of the methods.&#x0D; Methodology: The work involved mainly the critical review of all available academic, professional and industry documents on cord blood processing. The relevant information was obtained from textbooks, academic journals, conference proceedings, the internet among others. The major UCB processing methods include Plasma Depletion, Density Gradient Centrifugation (DGC), Hetastarch, PrepaCyte-CB and Sepax. A study of the potential impact of Hetastarch and PrepaCyte processing methods on transplantation outcomes revealed no difference that was significant was observed between patients receiving cells after the processing regimens were compared.&#x0D; Results: A comparison of the engraftment time of PrepaCyte-CB with five other processing methods revealed a quicker engraftment time for PrepaCyte-CB processed cord blood units compared to other processing methods. PrepaCyte-CB also recovers significantly more viable stem cells than AutoXpress (AXP) and hydroxyethyl starch (HES) processing methods. Other workers demonstrated that Sepax depletion produces higher recovery of cells that are nucleated. The effect initial volume of cord blood had on the recovery of nucleated cells for the different method of processing were also compared. Recovery when using Sepax is reduced as the unit size processed increases. Hetastarch, which is a density gradient, and plasma depletion separation is also affected in like manner, however, processing done using PrepaCyte-CB was not affected by the initial volume of the collected unit. The advantage of Sepax is that it is fully automated and this allows for mass processing of samples, suitable for bigger cord blood banks. For erythrocyte removal, density gradient separation is a better method that is effective. PrepaCyte-CB is the second most efficient method for removing RBC. The result of Total Nucleated Cells (TNC) and Mono Nucleated Cells (MNC) recovery rate of Hespan and Sepax against AXP processing methods shows that both Hespan and Sepax reproducibly recover greater than 95% of the cord blood stem cells in a typical collection and result in a reduced final volume for final storage.&#x0D; Conclusion: The five most popular processing techniques are Plasma Depletion, Density Gradient, Hetastarch, PrepaCyte-CB and Automated Centrifugal Machine (Sepax). Most methods involve centrifugation, sedimentation and/or filtration for reducing the red cell content, plasma volume, or both. The different UCB processing methods each has its advantages and disadvantages.