Cell Harvest Research Articles

Good practices for the industrial cultivation of the cyanobacterium Arthrospira platensis under a greenhouse in a temperate zone (northern Italy)

Arthrospira, Spirulina, and Limnospira are cyanobacteria widely known as food supplements or additives and cultivated worldwide under the commercial name of spirulina. Many studies have been focused on the improvement of operational conditions for optimizing cell growth and harvesting. At present, greater attention is paid to obtaining a good-quality, possibly food-grade, product that can be added to different food formulations and to reducing the environmental impact by saving water and avoiding or minimizing the release of mineral salts in the environment. A few studies have addressed these aspects although, in most cases, through laboratory experiments or pilot plants. This study focused on the effects of medium recycling, monitored at each harvesting step, and nutrient replenishment on Arthrospira platensis growth, biomass, biochemical composition, and extracellular polysaccharide (EPS) production, in a production plant located in a temperate zone (northeastern Italy). Four out of the seven largest ponds (11,000 L each) of the plant were followed for a three-month semi-continuous cultivation, which included the period with the highest biomass productivity. Recycling the culture medium after biomass harvest effectively allowed the best nutritional status of the cells. Biomass productivity increased from June to August 2023 (mean values: 0.029 and 0.038 g L-1 d-1 at 25–26 °C and 27–29 °C, respectively). Protein, polysaccharides, and C-phycocyanin contents were 62, 22, and 12%, respectively, while EPS was < 7 mg L-1. The biochemical composition did not vary during the cultivation period, differently from previous studies performed with small culture volumes and for a short time.

Development of Thermosensitive Hydrogels with Tailor-Made Geometries to Modulate Cell Harvesting of Non-Flat Cell Cultures

Considering the complexity in terms of design that characterizes the different tissues of the human body, it is necessary to study and develop more precise therapies. In this sense, this article presents the possibility of fabricating photocurable thermosensitive hydrogels with free geometry and based on N-Vinyl Caprolactam (VCL) with the aim of modulating the adhesion of non-planar cell cultures. The fabrication process is based on the use as a mold of two-layer thick water-soluble polyvinyl alcohol (PVA) previously printed by Extrusion Material (MatEx). From this technology it has been possible to obtain hydrogels with different 3D geometries and different crosslinking percentages (2, 4 and 6 mol%). Studies have shown that networks reduce their thermosensitivity not only when the percentage of crosslinking in the formulation increases, but also when the thickness of the hydrogel obtained increases. Based on this reduction in thermosensitivity, the less crosslinked (2 mol%) hydrogels have been evaluated to carry out a novel direct application in which hydrogels with curved geometry have allowed cell adhesion and proliferation at 37 °C with the endothelial cell line C166-GFP; likewise, non-aggressive cell detachment was observed when the hydrogel temperature was reduced to values of 20 °C. Therefore, the present manuscript shows a novel application for the synthesis of free-form thermosensitive hydrogels that allows modulation of non-planar cell cultures.

Intra-Articular Injection of Stem Cells for the Regeneration of Knee Joint Cartilage: a Therapeutic Option for Knee Osteoarthritis - a Narrative Review.

Current approaches to regulating osteoarthritis primarily focus on symptom management; however, these methods often have significant side effects and may not be suitable for long-term care. As an alternative to conventional treatments, injecting stem cells into knee joint cartilage is a promising option for repairing damaged cartilage. In this review, we outline the general procedure for stem cell treatment of knee joint cartilage regeneration, emphasizing the potential of intra-articular stem cell injections as a therapeutic option for osteoarthritis. We examined and summarized patient evaluation and preparation for knee joint stem cell therapy, stem cell harvesting, stem cell preparation, injection procedures for stem cell therapy, post-injection care and monitoring, potential outcomes of stem cell therapy, and considerations and risks associated with stem cell therapy. Overall, stem cell injections for knee joint cartilage damage represent a promising frontier in orthopedic care. They offer potential benefits such as pain and inflammation reduction, promotion of cartilage repair and regeneration, and the possibility of avoiding more invasive treatments such as knee surgery. Ongoing collaboration among researchers, clinicians, and regulatory organizations is crucial for advancing this field and translating scientific discoveries into effective clinical applications.

A multiband NIR upconversion core-shell design for enhanced light harvesting of silicon solar cells

Exploring lanthanide light upconversion (UC) has emerged as a promising strategy to enhance the near-infrared (NIR) responsive region of silicon solar cells (SSCs). However, its practical application under normal sunlight conditions has been hindered by the narrow NIR excitation bandwidth and the low UC efficiency of conventional materials. Here, we report the design of an efficient multiband UC system based on Ln3+/Yb3+-doped core-shell upconversion nanoparticles (Ln/Yb-UCNPs, Ln3+ = Ho3+, Er3+, Tm3+). In our design, Ln3+ ions are incorporated into distinct layers of Ln/Yb-UCNPs to function as near-infrared (NIR) absorbers across different spectral ranges. This design achieves broad multiband absorption withtin the 1100 to 2200 nm range, with an aggregated bandwidth of ~500 nm. We have identified a synthetic electron pumping (SEP) effect involving Yb3+ ions, facilitated by the synergistic interplay of energy transfer and cross-relaxation between Yb3+ and other ions Ln3+ (Ho3+, Er3+, Tm3+). This SEP effect enhances the UC efficiency of the nanomaterials by effectively transferring electrons from the low-excited states of Ln3+ to the excited state of Yb3+, resulting in intense Yb3+ luminescence at ~980 nm within the optimal response region for SSCs, thus markedly improving their overall performance. The SSCs integrated with Ln/Yb-UCNPs with multiband excitation demonstrate the largest reported NIR response range up to 2200 nm, while enabling the highest improvement in absolute photovoltaic efficiency reported, with an increase of 0.87% (resulting in a total efficiency of 19.37%) under standard AM 1.5 G irradiation. Our work tackles the bottlenecks in UCNP-coupled SSCs and introduces a viable approach to extend the NIR response of SSCs.

Side-by-Side Economic Process Model for the Comparison and Evaluation of Magnetic Bead-Based Processes and Legacy Process for the Manufacturing of Monoclonal Antibodies

This study models two alternative downstream processes based on magnetic separation with the objective of understanding the economic feasibility of these processes compared to the traditional mAb process. The key focus lies in the economic understanding of the cell harvest and capture steps in the models. Here, the models revealed that integrating cell removal and product capture in a single operation is the main factor driving the unified productivity between USP and the magnetic bead-based processes. This results in significant economic benefits, such as savings in both the cost of goods per gram of mAb and fixed costs, as well as increasing annual facility output. The predicted savings potential approaches 38% for COGs, 17% for capital investment, and 40% for annual facility output. For mammalian cell-based manufacturing, the magnetic separation-based DSP provides a highly valuable option due to its integration of several individual unit operations compared to the traditional process both in reducing process time and cost and accommodating higher demands.

Customizing H2O‐Poor Electric Double Layer and Boosting Texture Exposure of Zn (101) Plane towards Super‐High Areal Capacity Zinc Metal Batteries

AbstractThe catastrophic dendrite hyperplasia and parasitic reactions severely impede the future deployment of aqueous Zn‐ion batteries. Controlling zinc orientation growth is considered to be an effective method to overcome the aforementioned concerns, especially for regulating the (002) plane of deposited Zn. Unfortunately, Zn (002) texture is difficult to obtain stable cycling under high deposition capacity resulting from its large lattice distortion and nonuniform distribution in electric field. Herein, different from traditional cognition, a crystallization orientation regulation tactic is proposed to boost Zn (101) texture exposure and inhibit zinc dendrite proliferation during plating/stripping. Experimental results and theoretical calculations demonstrate the malate molecules preferentially adsorb on the Zn (002) facet, leading to the texture exposure of distinctive Zn (101) plane. Meanwhile, the −COOH and −OH groups of malate molecules exhibit strong adsorption on the Zn anode surface and chelate with Zn2+, achieving H2O‐poor electrical double layer. Very impressively, the multifunctional malate additive enlists zinc anode to survive for 600 h under a harsh condition of 15 mA cm−2/15 mAh cm−2. Moreover, the symmetric cell harvests highly‐reversible cycling life of 6600 h at 5 mA cm−2/1.25 mAh cm−2, remarkably outperforming the ZnSO4 electrolyte. The assembled Zn//MnO2 full cells also demonstrate prominent electrochemical reversibility.

In Vitro Assessment of Thermo-Responsive Scaffold as a 3D Synthetic Matrix for CAR-T Potency Testing Against Glioblastoma Spheroids.

Chimeric antigen receptor (CAR) T cell immunotherapy has demonstrated exceptional efficacy against hematological malignancies, but notably less against solid tumors. To overcome this limitation, it is critical to investigate antitumor CAR-T cell potency in synthetic 3D microenvironments that can simulate the physical barriers presented by solid tumors. The overall goal of this study was the preliminary assessment of a synthetic thermo-responsive material as a substrate for invitro co-cultures of anti-disialoganglioside (GD2) CAR-T cells and patient-derived glioblastoma (GBM) spheroids. Independent co-culture experiments demonstrated that the encapsulation process did not adversely affect the cell cycle progression of glioma stem cells (GSCs) or CAR-T cells. GSC spheroids grew over time within the terpolymer scaffold, when seeded in the same ratio as the suspension control. Co-cultures of CAR-T cells in suspension with hydrogel-encapsulated GSC spheroids demonstrated that CAR-T cells could migrate through the hydrogel and target the encapsulated GSC spheroids. CAR-T cells killed approximately 80% of encapsulated GSCs, while maintaining effective CD4:CD8 T cell ratios and secreting inflammatory cytokines after interacting with GD2-expressing GSCs. Importantly, the scaffolds also facilitated cell harvesting for downstream cellular analysis. This study demonstrated that a synthetic 3D terpolymer hydrogel can serve as an artificial scaffold to investigate cellular immunotherapeutic potency against solid tumors.

Microalgae aggregation induced by thermoresponsive polymers

Algal biomass harvesting is one of key technical hurdles impeding the commercialization of algae-based biorefinery. The goal of this work is to develop an innovative technology for algae cell harvesting. Thermoresponsive polymers (TRPs) such as poly(N-isopropylacrylamide) (PNIPAM) and its derivatives were studied on their properties and potential applications for microalgae harvesting. Various PNIPAM was synthesized, and the effects of charge, molecular weight (MW), amine content, and polymer concentration on the polymer phase transition temperature, the degree of phase separation, and the harvesting of microalgae (Chlorella vulgaris) were investigated. The lower critical solution temperature (LCST) of PNIPAM decreased with the increase of polymer concentration, while the decline rate reduced under high MW. The amine content didn’t significantly affect the LCST of TRPs. Approx. 92 % of algae cells were harvested by PNIPAM-300 kDa. Modified TRPs showed few benefits in enhancing algae harvesting. TRPs are a promising class of polymers for microalgae harvesting.

Thermo-responsible PNIPAM-grafted polystyrene microspheres for mesenchymal stem cells culture and detachment.

The preparation of cells is a critical step in cell therapy. To ensure the effectiveness of cells used for clinical treatments, it is essential to harvest adherent cells from the culture media in a way that preserves their high viability and full functionality. In this study, we developed temperature-responsive poly(N-isopropylacrylamide) (PNIPAM)-grafted polystyrene (PS) microspheres using reversible addition-fragmentation chain transfer polymerization. These microspheres allow for the non-destructive harvesting of cultured cells through temperature changes. The composition and physicochemical properties of the PNIPAM-grafted PS microspheres were confirmed using infrared spectroscopy, elemental analysis, dynamic light scattering, and thermogravimetric analysis.In vitroexperiments demonstrated that these microspheres exhibit excellent biocompatibility, supporting the adhesion and proliferation of various cells. Moreover, the microspheres showed good temperature responsiveness in thermosensitive detachment experiments with GFP-HepG2cells and umbilical cord mesenchymal stem cells (UC-MSCs). Additionally, through orthogonal experiments, we identified a cell detachment aid mixture that significantly improved the dispersibility of cells detached from the microspheres, enhancing the efficiency of thermosensitive cell detachment by approximately 40%. The harvested UC-MSCs retained their capacity for re-proliferation and trilineage differentiation. Consequently, the temperature-responsive microspheres developed in this study, combined with the cell detachment aid mixtures, hold great potential for large-scale culture and harvesting of therapeutic cells in clinical applications.

Customizing H2O-Poor Electric Double Layer and Boosting Texture Exposure of Zn (101) Plane towards Super-High Areal Capacity Zinc Metal Batteries.

The catastrophic dendrite hyperplasia and parasitic reactions severely impede the future deployment of aqueous Zn-ion batteries. Controlling zinc orientation growth is considered to be an effective method to overcome the aforementioned concerns, especially for regulating the (002) plane of deposited Zn. Unfortunately, Zn (002) texture is difficult to obtain stable cycling under high deposition capacity resulting from its large lattice distortion and nonuniform distribution in electric field. Herein, different from traditional cognition, a crystallization orientation regulation tactic is proposed to boost Zn (101) texture exposure and inhibit zinc dendrite proliferation during plating/stripping. Experimental results and theoretical calculations demonstrate the malate molecules preferentially adsorb on the Zn (002) facet, leading to the texture exposure of distinctive Zn (101) plane. Meanwhile, the -COOH and -OH groups of malate molecules exhibit strong adsorption on the Zn anode surface and chelate with Zn2+, achieving H2O-poor electrical double layer. Very impressively, the multifunctional malate additive enlists zinc anode to survive for 600 h under a harsh condition of 15 mA cm-2/15 mAh cm-2. Moreover, the symmetric cell harvests highly-reversible cycling life of 6600 h at 5 mA cm-2/1.25 mAh cm-2, remarkably outperforming the ZnSO4 electrolyte. The assembled Zn//MnO2 full cells also demonstrate prominent electrochemical reversibility.

Preparation of Thermosensitive Lignocellulose Hollow Fiber Membrane Grafted With PNIPAAm and Its Application as a Cell Culture Carrier in a RSOC Dynamic Culture.

Currently, the cells, which are urgently required for large-scale application in biomedical-related fields, harvested by traditional trypsin digestion are usually subject to repeated digestion, leading to a reduction of cell activity. In this study, poly (N-isopropylacrylamide) (PNIPAAm) was grafted onto the lignocellulose hollow fiber membranes (HFMs) with cerium ammonium nitrate (CAN) as the initiator to prepare thermosensitive HFMs, which was combined with a rotation system of culture (RSOC) to achieve dynamic culture and non-destructive harvesting of cells from the HFMs. The results of ATR-FTIR, elemental analysis, and SEM confirmed the successful preparation of PNIPAAm-grafted-HFMs, which also showed good biocompatibility to apply for cell culture carriers. In cooling detachment, the HFMs-0.01 group could completely detach the cells within 1h with a cell separation efficiency of more than 90%. The laminin (LN) and fibronectin (FN) harvested by cooling detachment of P8 generation PC12 cells reached 0.0531±0.0032 and 2.5045±0.0001pg/cell, respectively, which were significantly higher than that by trypsin digestion. In addition, the cells on the thermosensitive HFMs proliferated fastest in RSOC at 30rpm with higher glucose consumption and lactate metabolism than in static conditions. Moreover, the cells that had dynamic detachment at 20rpm had the highest cell density and activity. Therefore, the thermosensitive HFMs could be applied as cell culture carriers in RSOC for cell culturing at 30rpm and harvesting at 20rpm, which would provide considerable potential for large-scale cell culture in vitro.

Significant Impact of let-7d MicroRNA on Breast Cancer Cell Lines Post-Radiation Treatment

Introduction: Radiotherapy is a common treatment for breast cancer treatment, that induces DNA damage. These DNA damages are addressed through various repair pathways, which regulate the DNA repair systems and confer radio-resistance. Non-coding RNAs are a big proportion of genome transcripts without the potential to encode proteins. Related studies demonstrated that radiation affects the expression of non-coding RNAs. Let-7d, a tumor suppressor in numerous cancers, has some target genes that play a role in the DNA repair system. Materials and Methods: Human breast cancer cell lines MDA-MB-231 and MCF-7 were cultured in a Dulbecco's Modified Eagle Medium. The exponentially growing cells were treated with some doses of X-rays. After radiation treatment and cell harvesting, RNA was extracted, and cDNA synthesis was done. The let-7d miRNA expression changes were calculated with real-time quantitative reverse transcription polymerase chain reaction. Results: The results implied that radiation caused increased let-7d expression in breast cancer cell lines after radiation treatment. In addition, the results showed that 24 h after radiation, the expression of let-7d in the radioresistant cell line was higher than the radiosensitive one; 48 h after radiation, the expression of let-7d in the radiosensitive cell line was higher than the other one. Conclusions: The results demonstrated that radiation treatment increased let-7d miRNA expression in both radiosensitive and radio-resistant breast cancer cell lines. Therefore, let-7d might be involved in the radiosensitivity of breast cancer.

Osmotic energy conversion in serpentinite-hosted deep-sea hydrothermal vents

Cells harvest energy from ionic gradients by selective ion transport across membranes, and the same principle is recently being used for osmotic power generation from salinity gradients at ocean-river interfaces. Common to these ionic gradient conversions is that they require intricate nanoscale structures. Here, we show that natural submarine serpentinite-hosted hydrothermal vent (HV) precipitates are capable of converting ionic gradients into electrochemical energy by selective transport of Na+, K+, H+, and Cl-. Layered hydroxide nanocrystals are aligned radially outwards from the HV fluid channels, constituting confined nanopores that span millimeters in the HV wall. The nanopores change the surface charge depending on adsorbed ions, allowing the mineral to function as a cation- and anion-selective ion transport membrane. Our findings indicate that chemical disequilibria originating from flow and concentration gradients in geologic environments generate confined nanospaces which enable the spontaneous establishment of osmotic energy conversion.

Engineering Thermoresponsive Poly(N-isopropylacrylamide)-Based Films with Enhanced Stability and Reusability for Efficient Bone Marrow Mesenchymal Stem Cell Culture and Harvesting.

Poly(N-isopropylacrylamide) (PNIPAM) offers a promising platform for non-invasive and gentle cell detachment. However, conventional PNIPAM-based substrates often suffer from limitations including limited stability and reduced reusability, which hinder their widespread adoption in biomedical applications. In this study, PNIPAM copolymer films were formed on the surfaces of glass slides or silicon wafers using a two-step film-forming method involving coating and grafting. Subsequently, a comprehensive analysis of the films' surface wettability, topography, and thickness was conducted using a variety of techniques, including contact angle analysis, atomic force microscopy (AFM), and ellipsometric measurements. Bone marrow mesenchymal stem cells (BMMSCs) were then seeded onto PNIPAM copolymer films prepared from different copolymer solution concentrations, ranging from 0.2 to 10 mg·mL-1, to select the optimal culture substrate that allowed for good cell growth at 37 °C and effective cell detachment through temperature reduction. Furthermore, the stability and reusability of the optimal copolymer films were assessed. Finally, AFM and X-ray photoelectron spectroscopy (XPS) were employed to examine the surface morphology and elemental composition of the copolymer films after two rounds of BMMSC adhesion and detachment. The findings revealed that the surface properties and overall characteristics of PNIPAM copolymer films varied significantly with the solution concentration. Based on the selection criteria, the copolymer films derived from 1 mg·mL-1 solution were identified as the optimal culture substrates for BMMSCs. After two rounds of cellular adhesion and detachment, some proteins remained on the film surfaces, acting as a foundation for subsequent cellular re-adhesion and growth, thereby implicitly corroborating the practicability and reusability of the copolymer films. This study not only introduces a stable and efficient platform for stem cell culture and harvesting but also represents a significant advance in the fabrication of smart materials tailored for biomedical applications.

Abstract PR-12: The circadian rhythm gene Dec2 regulates multiple components of the antigen presentation pathway to promote pancreatic cancer dormancy by immune evasion

Abstract Metastatic recurrence following curative-intent pancreatic surgery is a major clinical problem as nearly all die of this at both early and latent periods following surgery. While latent recurrences are due to the reactivation of disseminated dormant tumor cells, the mechanisms that regulate dormancy remain unclear. Using a novel model of early-stage resectable pancreatic cancer that mimics early and latent outcomes in patients, we identified Dec2 (bhlh41) a transcriptional repressor and circadian regulator, as upregulated in dormant tumor cells harvested from the liver. Disseminated tumor cells harvest from the livers of latent recurrent mice were Dec2 hi/Ki67lo indicating that these cells are dormant. We confirmed the relationship of Dec2 and Ki67 in human pancreatic cancer using liver tissue specimens from patients with metastatic disease whereby small clusters of metastatic cells were Dec2hi/Ki67lo and larger metastatic tumors were Dec2lo/Ki67hi. To understand the mechanism of Dec2 in dormancy we deleted Dec2 from the tumor cells evaluated them in the resection model and observed a substantial increase in survival after resection due to an immune mediated mechanism as the survival benefit was abrogated in immunodeficient conditions indicating that Dec2 promotes tumor progression through immune mediated mechanism. Dec2 promotes immune evasion by downregulating multiple components of the antigen presentation pathway including proteosome and immunoproteosome genes, MHCI and MHCI chaperones. As Dec2 is a circadian regulator, we evaluated several components of the antigen presentation pathway including proteosome and surface MHCI and found these to display oscillation that was not only anti-phase with Dec2 but resulted in differential T-cell mediated tumor cell killing depending on the time of day. These studies elucidate a new mechanism by which Dec2 regulates cancer dormancy through immune evasion and identifies several novel therapeutic strategies to restore anti-tumor immunity. They also provide the first evidence that antigen presentation is under circadian regulation in pancreatic cancer. Citation Format: Chris Harris, Crissy Dudgeon, Lan Wang, Orjola Prela, Juliana Cazarin de Menezes, Brian Altman, Brian Haab, Chongfeng Gao, Zachary Klamer, Anna Repesh, Subhajyoti De, Darren R Carpizo. The circadian rhythm gene Dec2 regulates multiple components of the antigen presentation pathway to promote pancreatic cancer dormancy by immune evasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-12.

Band gap engineering of novel double perovskites Na2LiYX6 (X=Cl, Br) through halide ion replacement for solar cell and energy harvesting applications

Using the Perdew − Burke − Ernzerhof-generalized-gradient approximation (PBE-GGA) and PBE+SOC exchange − correlation functional, this work investigates the physical properties and energy storage capacity of novel double perovskite Halides Na2LiYX6 (X=Cl and Br) through Density Functional Theory (DFT) and semiclassical transport theory under ideal conditions. Thermal stability has been verified through the computation of thermodynamic parameters and formation energies. Brittle and anisotropic nature has been confirmed by the calculation of elastic and mechanical parameters. With PBE+SOC, the band gaps (Eg) for Na2LiYCl6 and Na2LiYBr6 are 2.90 and 3.76 eV, respectively, whereas with PBE-GGA, they are 3.061 and 3.824 eV, respectively. The analysis of the computed optical characteristics determines whether double perovskite halides are suitable for use in photovoltaic applications. The calculations for the transport properties were carried out using the BoltzTraP code. The effectiveness and transport mechanism for thermal energy conversion have been studied by determining the thermoelectric properties. To assess the stability at high temperatures and suitability for industrial applications, thermal properties such as heat capacity, entropy, Debye temperature, and thermal expansion are investigated. Overall, evaluations of the compounds’ thermoelectric characteristics show that they have a high-power factor and remarkably low thermal conductivity, which makes them ideal for energy harvesting devices.

Metal Oxide vs Organic Semiconductor Charge Extraction Layers for Halide Perovskite Indoor Photovoltaics

Halide perovskite indoor photovoltaics (PVs) are highly promising to autonomously power the billions of microelectronic sensors in the emerging and disruptive technology of the Internet of Things (IoT). However, how the wide range of different types of hole extraction layers (HELs) impacts the indoor light harvesting of perovskite solar cells is still elusive, which hinders the material selection and industrial‐scale fabrication of indoor perovskite photovoltaics. In the present study, new insights are provided regarding the judicial selection of HELs at the buried interface of halide perovskite indoor photovoltaics. This study unravels the detrimental and severe light‐soaking effect of metal oxide transport layer‐based PV devices under the indoor lighting effect for the first time, which then necessitates the interface passivation/engineering for their reliant performance. This is not a stringent criterion under 1 sun illumination. By systematically investigating the charge carrier dynamics and sequence of measurements from dark, light‐soaked, interlayer‐passivated device, the bulk and interface defects are decoupled and reveal the gradual defect passivation from shallow to deep level traps. Thus, the present study puts forward a useful design strategy to overcome the deleterious effect of metal oxide HELs and employ them in halide perovskite indoor PVs.

FixNCut: A Practical Guide to Sample Preservation by Reversible Fixation for Single Cell Assays.

The quality of standard single-cell experiments often depends on the immediate processing of cells or tissues post-harvest to preserve fragile and vulnerable cell populations, unless the samples are adequately fixed and stored. Despite the recent rise in popularity of probe-based and aldehyde-fixed RNA assays, these methods face limitations in species and target availability and are not suitable for immunoprofiling or assessing chromatin accessibility. Recently, a reversible fixation strategy known as FixNCut has been successfully deployed to separate sampling from downstream applications in a reproducible and robust manner, avoiding stress or necrosis-related artifacts. In this article, we present an optimized and robust practical guide to the FixNCut protocol to aid the end-to-end adaptation of this versatile method. This protocol not only decouples tissue or cell harvesting from single-cell assays but also enables a flexible and decentralized workflow that unlocks the potential for single-cell analysis as well as unconventional study designs that were previously considered unfeasible. Key features • Reversible fixation: Preserves cellular and molecular structures with the option to later reverse the fixation for downstream applications, maintaining cell integrity • Compatibility with single-cell assays: Supports single-cell genomic assays such as scRNA-seq and ATAC-seq, essential for high-resolution analysis of cell function and gene expression • Flexibility in sample handling: Allows immediate fixation post-collection, decoupling sample processing from analysis, beneficial in settings where immediate processing is impractical • Preservation of RNA and DNA integrity: Effectively preserves RNA and DNA, reducing degradation to ensure accurate transcriptomic and genomic profiling • Suitability for various biological samples: Applicable to a wide range of biological samples, including tissues and cell suspensions, whether freshly isolated or post-dissociated • Enables multi-center studies: Facilitates collaborative research across multiple centers by allowing sample fixation at the point of collection, enhancing research scale and diversity • Avoidance of artifacts: Minimizes stress or necrosis-related artifacts, preserving the natural cellular physiology for accurate genomic and transcriptomic analysis.

Mosaic chromosomal alterations in hematopoietic cells and clinical outcomes in patients with multiple myeloma.

Mosaic chromosomal alterations (mCAs) in hematopoietic cells increase mortality and risk of hematological cancers and infections. We investigated the landscape of mCAs and their clinical consequences in 976 patients with multiple myeloma undergoing high-dose chemotherapy and autologous stem cell support (ASCT) with median 6.4 years of follow-up. mCAs were detected in the stem cell harvest product of 158 patients (16.2%). Autosomal aberrations were found in 60 patients (6.1%) and affected all chromosomes. Loss of chromosome X was found in 51 females (12.7%) and loss of chromosome Y in 55 males (9.6%). Overall survival and progression were similar between carriers of autosomal mCAs and non-carriers. In contrast, female patients with loss of the X chromosome had longer overall survival (age-adjusted[a.a.] HR 0.54, 95% CI 0.32-0.93, p = 0.02), lower risk of progression (a.a. HR 0.55, 95% CI 0.35-0.87; p = 0.01), and better post-transplant response (higher degree of complete response (CR) or very good partial response (VGPR)). The reason for this substantial effect is unknown. Additionally, myeloma clones in the stem cell product was confirmed by mCA analysis in the few patients with multiple mCAs (n = 12 patients). Multiple mCAs conferred inferior overall survival (a.a. HR 2.0, 95% CI 1.02-3.84; p = 0.04) and higher risk of myeloma progression (a.a. HR 3.36, 95% CI 1.67-6.81; p < 0.001), which is presumed to be driven by suspected myeloma contaminants.

Effective and durable repigmentation for stable vitiligo: A randomized within-subject controlled trial assessing treatment with autologous skin cell suspension transplantation

BackgroundVitiligo lesions are often challenging to repigment with conventional medical therapies. Surgical autologous melanocyte transfer methods can be utilized for stable vitiligo but demand specialized skills and equipment. A point-of-care autologous cell harvesting device was designed enabling simple preparation of autologous skin cell suspension (ASCS) containing melanocytes, keratinocytes, and fibroblasts providing a straightforward approach for cellular transplantation. ObjectiveTo evaluate the safety and effectiveness of ASCS for repigmentation of stable vitiligo lesions among adults. MethodsA US multicenter, randomized, within-subject controlled trial compared ASCS to NB-UVB only (Control) in similar vitiligo lesions. ASCS was applied after laser skin resurfacing and followed by NB-UVB treatment. The primary effectiveness endpoint was the proportion of lesions achieving ≥80% repigmentation at week-24. Repigmentation durability was assessed at week-52. ResultsAmong 25 subjects, 36% of ASCS-treated lesions achieved ≥80% repigmentation at week-24 compared to 0% for Control (p<0.025), with durability through week-52. The safety profile of ASCS was acceptable, with favorable patient- and investigator-reported outcomes. LimitationsStudy sample size limited robust subgroup analyses. ConclusionApplication of ASCS is a safe and effective treatment for repigmentation of stable vitiligo lesions with the potential to improve health-related quality of life and reduce burden of disease.